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Ankyrin repeat-rich membrane spanning (ARMS) plays roles in neural development, neuropathies, and tumor formation. Such pleiotropic function of ARMS is often attributed to diverse ARMS-interacting molecules in different cell context. However, it might be achieved by ARMS' effect on global biological mediator like reactive oxygen species (ROS). We established ARMS -knockdown in melanoma cells (si ARMS ) and in Drosophila eyes ( GMR>dARMS RNAi ) and challenged them with H 2 O 2 . Decreased ARMS in both systems compromises nuclear translocation of NF-κB and induces ROS, which in turn augments autophagy flux and confers susceptibility to H 2 O 2 -triggered autophagic cell death. Resuming NF-κB activity or reducing ROS by antioxidants in si ARMS cells and GMR>dARMS RNAi fly decreases intracellular peroxides level concurrent with reduced autophagy and attenuated cell death. Conversely, blocking NF-κB activity in wild-type flies/melanoma enhances ROS and induces autophagy with cell death. We thus uncover intracellular ROS modulated by ARMS-NFκB signaling primes autophagy for autophagic cell death upon oxidative stress., Competing Interests: We report no potential conflict of interest., (© 2023 The Author(s).)

Background: Genome-wide association studies (GWAS) provide a powerful means to identify associations between genetic variants and phenotypes. However, GWAS techniques for detecting epistasis, the interactions between genetic variants associated with phenotypes, are still limited. We believe that developing an efficient and effective GWAS method to detect epistasis will be a key for discovering sophisticated pathogenesis, which is especially important for complex diseases such as Alzheimer's disease (AD)., Results: In this regard, this study presents GenEpi, a computational package to uncover epistasis associated with phenotypes by the proposed machine learning approach. GenEpi identifies both within-gene and cross-gene epistasis through a two-stage modeling workflow. In both stages, GenEpi adopts two-element combinatorial encoding when producing features and constructs the prediction models by L1-regularized regression with stability selection. The simulated data showed that GenEpi outperforms other widely-used methods on detecting the ground-truth epistasis. As real data is concerned, this study uses AD as an example to reveal the capability of GenEpi in finding disease-related variants and variant interactions that show both biological meanings and predictive power., Conclusions: The results on simulation data and AD demonstrated that GenEpi has the ability to detect the epistasis associated with phenotypes effectively and efficiently. The released package can be generalized to largely facilitate the studies of many complex diseases in the near future.

Trichostatin A (TSA), an antifungal antibiotic derived from Streptomyces , inhibits mammalian histone deacetylases, and especially, selectively inhibits class I and II histone deacetylase (HDAC) families of enzymes. TSA reportedly elicits an antiproliferative response in multifarious tumors. This study investigated the antitumor effects of TSA alone and in combination with paclitaxel when applied to two high-grade urothelial carcinoma (UC) cell lines (BFTC-905 and BFTC-909). Fluorescence-activated cell sorting, flow cytometry, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium assay were used to assess TSA's cytotoxicity and effects on apoptosis induction. TSA induced synergistic cytotoxicity, when combined with paclitaxel (combination index < 1), resulted in concomitant suppression of paclitaxel-induced activation of phospho-extracellular signal-regulated kinase (ERK) 1/2. A xenograft nude mouse model confirmed that TSA enhances the antitumor effects of paclitaxel. These findings demonstrate that the administration of TSA in combination with paclitaxel elicits a synergistic cytotoxic response. The results of this study indicate that the chemoresistance of UC could be circumvented by combining HDAC inhibitors to target the ERK pathway.

In this study, we aimed to investigate the antitumor effects of trichostatin A (TSA), an antifungal antibiotic that inhibits histone deacetylase (HDAC) family of enzymes, alone or in combination with anyone of the three chemotherapeutic agents (cisplatin, gemcitabine, and doxorubicin) for the treatment of human urothelial carcinoma (UC). Two high-grade human UC cell lines (T24 and NTUB1) were used. Cytotoxicity and apoptosis were assessed by MTT assay and flow cytometry, respectively. The expression of phospho-c-Raf, phospho-MEK1/2, and phospho-ERK1/2 was measured by western blotting. ERK siRNA knockdown and the specific MEK inhibitor U0126 were used to examine the role of Raf/MEK/ERK signaling pathway in combined cytotoxicity of TSA and chemotherapy. TSA co-treatment with any one of the three chemotherapeutic agents induced synergistic cytotoxicity (combination index < 1) and concomitantly suppressed chemotherapeutic drug-induced activation of Raf-MEK-ERK pathway. Combination of ERK siRNA knockdown and treatment with the specific MEK inhibitor (U0126) enhanced the cytotoxic effects of the chemotherapy on UC cells. These observations were confirmed in a xenograft nude mouse model. Moreover, activated Raf/MEK/ERK pathway was observed in human bladder UC specimens from patients with chemoresistant status. In conclusion, TSA elicits a synergistic cytotoxic response in combination with chemotherapy via targeting the Raf/MEK/ERK pathway. TSA elicits synergistic cytotoxic response in combination with three DNA-damaging drugs (cisplatin, gemcitabine, and doxorubicin). Activated Raf/MEK/ERK pathway is involved in chemoresistant mechanism of UC. Combining chemotherapeutic agents with HDAC inhibitor (TSA) or with targeting Raf/MEK/ERK pathway is promising to circumvent chemoresistance in UCs.

Transit amplifying cells (TACs) are highly proliferative in nature and tend to be sensitive to ionizing radiation. Due to the abundance of TACs that support the elongation of hair shafts, growing hair follicles are highly sensitive to radiation injury. How hair follicles repair themselves after radiation injury is unclear. In this study, we observed that in 4 Gy irradiated mice, hair follicle dystrophy was induced with apoptosis-driven loss of hair matrix cells, which are the TACs that fuel hair growth. The dystrophy was repaired within 96 h without significant hair loss, indicating that a regenerative attempt successfully restored the TAC population to resume anagen growth. Soon after irradiation, mTORC1 signaling was activated in the TAC compartment and its activation was maintained until the regeneration process was completed. Inhibition of mTORC1 by rapamycin treatment increased radiation-induced cell apoptosis, reduced cell proliferation and delayed restoration of Wnt signaling in the hair matrix after radiation injury, leading to prolonged dystrophy and hair loss. These results demonstrate that mTORC1 signaling is activated after irradiation and is required for timely regeneration of the TAC pool of hair follicles, so that hair growth can resume after radiation injury.

Breast carcinoma amplified sequence 2 (BCAS2) is a core component of the hPrP19 complex that controls RNA splicing. Here, we performed an exon array assay and showed that β-catenin is a target of BCAS2 splicing regulation. The regulation of dendrite growth and morphology by β-catenin is well documented. Therefore, we generated conditional knockout (cKO) mice to eliminate the BCAS2 expression in the forebrain to investigate the role of BCAS2 in dendrite growth. BCAS2 cKO mice showed a microcephaly-like phenotype with a reduced volume in the dentate gyrus (DG) and low levels of learning and memory, as evaluated using Morris water maze analysis and passive avoidance, respectively. Golgi staining revealed shorter dendrites, less dendritic complexity and decreased spine density in the DG of BCAS2 cKO mice. Moreover, the cKO mice displayed a short dendrite length in newborn neurons labeled by DCX, a marker of immature neurons, and BrdU incorporation. To further examine the mechanism underlying BCAS2-mediated dendritic malformation, we overexpressed β-catenin in BCAS2-depleted primary neurons and found that the dendritic growth was restored. In summary, BCAS2 is an upstream regulator of β-catenin gene expression and plays a role in dendrite growth at least partly through β-catenin.

To maintain a particular cell fate, a unique set of genes should be expressed while another set is repressed. One way to repress gene expression is through Polycomb group (PcG) proteins that compact chromatin into a silent configuration. In addition to cell fate maintenance, PcG proteins also maintain normal cell physiology, for example cell cycle. In the absence of PcG, ectopic activation of the PcG-repressed genes leads to developmental defects and malignant tumors. Little is known about the molecular nature of ectopic gene expression; especially what differentiates expression of a given gene in the orthotopic tissue (orthotopic expression) and the ectopic expression of the same gene due to PcG mutations. Here we present that ectopic gene expression in PcG mutant cells specifically requires dBRWD3, a negative regulator of HIRA/Yemanuclein (YEM)-mediated histone variant H3.3 deposition. dBRWD3 mutations suppress both the ectopic gene expression and aberrant tissue overgrowth in PcG mutants through a YEM-dependent mechanism. Our findings identified dBRWD3 as a critical regulator that is uniquely required for ectopic gene expression and aberrant tissue overgrowth caused by PcG mutations., Competing Interests: The authors have declared that no competing interests exist.

Judiciously tuning heart rates is critical for regular cardiovascular function. The fractal pattern of heartbeats - a multiscale regulation in instantaneous fluctuations - is well known for vertebrates. The most primitive heart system of the Drosophila provides a useful model to understand the evolutional origin of such a fractal pattern as well as the alterations of fractal pattern during diseased statuses. We developed a non-invasive visible optical heart rate recording system especially suitable for long-term recording by using principal component analysis (PCA) instead of fluorescence recording system to avoid the confounding effect from intense light irradiation. To deplete intracellular Ca(2+) levels, the expression of sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA) was tissue-specifically knocked down. The SERCA group shows longer heart beat intervals (Mean ± SD: 1009.7 ± 151.6 ms) as compared to the control group (545.5 ± 45.4 ms, p < 0.001). The multiscale correlation of SERCA group (scaling exponent: 0.77 ± 0.07), on the other hand, is weaker than that of the control Drosophila (scaling exponent: 0.85 ± 0.03) (p = 0.016).

Background: Recent advances in sequencing technology have opened a new era in RNA studies. Novel types of RNAs such as long non-coding RNAs (lncRNAs) have been discovered by transcriptomic sequencing and some lncRNAs have been found to play essential roles in biological processes. However, only limited information is available for lncRNAs in Drosophila melanogaster, an important model organism. Therefore, the characterization of lncRNAs and identification of new lncRNAs in D. melanogaster is an important area of research. Moreover, there is an increasing interest in the use of ChIP-seq data (H3K4me3, H3K36me3 and Pol II) to detect signatures of active transcription for reported lncRNAs., Results: We have developed a computational approach to identify new lncRNAs from two tissue-specific RNA-seq datasets using the poly(A)-enriched and the ribo-zero method, respectively. In our results, we identified 462 novel lncRNA transcripts, which we combined with 4137 previously published lncRNA transcripts into a curated dataset. We then utilized 61 RNA-seq and 32 ChIP-seq datasets to improve the annotation of the curated lncRNAs with regards to transcriptional direction, exon regions, classification, expression in the brain, possession of a poly(A) tail, and presence of conventional chromatin signatures. Furthermore, we used 30 time-course RNA-seq datasets and 32 ChIP-seq datasets to investigate whether the lncRNAs reported by RNA-seq have active transcription signatures. The results showed that more than half of the reported lncRNAs did not have chromatin signatures related to active transcription. To clarify this issue, we conducted RT-qPCR experiments and found that ~95.24% of the selected lncRNAs were truly transcribed, regardless of whether they were associated with active chromatin signatures or not., Conclusions: In this study, we discovered a large number of novel lncRNAs, which suggests that many remain to be identified in D. melanogaster. For the lncRNAs that are known, we improved their characterization by integrating a large number of sequencing datasets (93 sets in total) from multiple sources (lncRNAs, RNA-seq and ChIP-seq). The RT-qPCR experiments demonstrated that RNA-seq is a reliable platform to discover lncRNAs. This set of curated lncRNAs with improved annotations can serve as an important resource for investigating the function of lncRNAs in D. melanogaster.

In this study, we demonstrate that an E3-ubiquitin ligase associated with human X-linked intellectual disability, CUL4B, plays a crucial role in post-meiotic sperm development. Initially, Cul4b(Δ)/Y male mice were found to be sterile and exhibited a progressive loss in germ cells, thereby leading to oligoasthenospermia. Adult Cul4b mutant epididymides also contained very low numbers of mature spermatozoa, and these spermatazoa exhibited pronounced morphological abnormalities. In post-meiotic spermatids, CUL4B was dynamically expressed and mitosis of spermatogonia and meiosis of spermatocytes both appeared unaffected. However, the spermatids exhibited significantly higher levels of apoptosis during spermiogenesis, particularly during the acrosome phase through the cap phase. Comparative proteomic analyses identified a large-scale shift between wild-type and Cul4b mutant testes during early post-meiotic sperm development. Ultrastructural pathology studies further detected aberrant acrosomes in spermatids and nuclear morphology. The protein levels of both canonical and non-canonical histones were also affected in an early spermatid stage in the absence of Cul4b. Thus, X-linked CUL4B appears to play a critical role in acrosomal formation, nuclear condensation, and in regulating histone dynamics during haploid male germ cell differentiation in relation to male fertility in mice. Thus, it is possible that CUL4B-selective substrates are required for post-meiotic sperm morphogenesis.

Cisplatin-based chemotherapy is the primary treatment for metastatic bladder urothelial carcinoma. However, the response rate is only 40-65%. This study investigated the anti-tumor effect and underlying mechanisms of the combination of cisplatin and the NEDD8-activating enzyme inhibitor MLN4924 in human bladder urothelial carcinoma. The combination of cisplatin and MLN4924 exerted synergistic cytotoxicity on two high-grade bladder urothelial carcinoma cell lines, NTUB1 and T24 (combination index <1). MLN4924 also potentiated the cisplatin-induced apoptosis and activation of caspase-3 and -7, phospho-histone H2A.X and PARP. c-Jun N-terminal kinase (JNK) activation and a down-regulation of B-cell lymphoma-extra large (Bcl-xL) were also observed during cisplatin and MLN4924 treatment. Inhibition of JNK activation partially restored cell viability and Bcl-xL expression. Bcl-xL overexpression also rescued cell viability. MLN4924 significantly potentiated cisplatin-induced tumor suppression in urothelial carcinoma xenograft mice. In summary, MLN4924 synergistically enhanced the anti-tumor effect of cisplatin via an increase in DNA damage, JNK activation and down-regulation of Bcl-xL in urothelial carcinoma cells. These findings provide a new therapeutic strategy for the treatment of bladder cancer.

During development, certain Drosophila sensory neurons undergo dendrite pruning that selectively eliminates their dendrites but leaves the axons intact. How these neurons regulate pruning activity in the dendrites remains unknown. Here, we identify a coiled-coil protein Spindle-F (Spn-F) that is required for dendrite pruning in Drosophila sensory neurons. Spn-F acts downstream of IKK-related kinase Ik2 in the same pathway for dendrite pruning. Spn-F exhibits a punctate pattern in larval neurons, whereas these Spn-F puncta become redistributed in pupal neurons, a step that is essential for dendrite pruning. The redistribution of Spn-F from puncta in pupal neurons requires the phosphorylation of Spn-F by Ik2 kinase to decrease Spn-F self-association, and depends on the function of microtubule motor dynein complex. Spn-F is a key component to link Ik2 kinase to dynein motor complex, and the formation of Ik2/Spn-F/dynein complex is critical for Spn-F redistribution and for dendrite pruning. Our findings reveal a novel regulatory mechanism for dendrite pruning achieved by temporal activation of Ik2 kinase and dynein-mediated redistribution of Ik2/Spn-F complex in neurons.

MLN4924, an inhibitor of NEDD8 activating enzyme (NAE), has been reported to have activity against various malignancies. Here, we investigated the antitumor properties of MLN4924 and MLN4924 in combination with cisplatin on human cervical carcinoma (CC) in vitro and in vivo. Two human CC cell lines, ME-180 and HeLa, were used in this study. The cytotoxic effects of MLN4924 and/or cisplatin were measured by cell viability (MTT), proliferation (BrdU incorporation), apoptosis (flow cytometry with annexin V-FITC labeling), and the expression of cell apoptosis-related proteins (Western blotting). In vivo efficacy was determined in Nu/Nu nude mice with ME-180 and HeLa xenografts. The results showed that MLN4924 elicited viability inhibition, anti-proliferation and apoptosis in human CC cells, accompanied by activations of apoptosis-related molecules and Bid, Bcl-2 phosphorylation interruption, and interference with cell cycle regulators. Moreover, MLN4924 caused an endoplasmic reticulum stress response (caspase-4, ATF-4 and CHOP activations) and expression of other cellular stress molecules (JNK and c-Jun activations). Additionally, MLN4924 suppressed growth of CC xenografts in nude mice. Furthermore, we demonstrated that MLN4924 potentiated cisplatin-induced cytotoxicity in CC cells with activation of caspases. Consistently with this, MLN4924 significantly enhanced cisplatin-induced growth inhibition of CC xenografts. Together, these findings suggest that MLN4924 alone or in combination with cisplatin is of value in treating human CCs.

Organs are composed of heterotypic cells with patterned architecture that enables intercellular interaction to perform specific functions. In tissue engineering, the ability to pattern heterotypic cells into desired arrangement will allow us to model complex tissues in vitro and to create tissue equivalents for regeneration. This study was aimed at developing a method for fast heterotypic cell patterning with controllable topological manipulation on a glass chip. We found that poly(vinyl alcohol)-coated glass showed a biphasic change in adhesivity to cells in vitro: low adhesivity in the first 24 h and higher adhesivity at later hours due to increased serum protein adsorption. Combining programmable CO2 laser ablation to remove poly(vinyl alcohol) and glass, we were able to create arrays of adhesive microwells of adjustable patterns. We tested whether controllable patterns of epithelial-mesenchymal interaction could be created. When skin dermal papilla cells and fibroblasts were seeded respectively 24 h apart, we were able to pattern these two cells into aggregates of dermal papilla cells in arrays of microwells in a background of fibroblasts sheet. Seeded later, keratinocytes attached to these mesenchymal cells. Keratinocytes contacting dermal papilla cells started to differentiate toward a hair follicle fate, demonstrating patternable epithelial-mesenchymal interaction. This method allows fast adjustable heterotypic cell patterning and surface topology control and can be applied to the investigation of heterotypic cellular interaction and creation of tissue equivalent in vitro.

MLN4924, a small molecule inhibitor of NEDD8 activating enzyme (NAE), has been reported to elicit an anti-tumor effect on various malignancies. In this study, we investigated the anti-tumor effect of MLN4924 in human urothelial carcinoma (UC) in vitro and in vivo by using three human UC cell lines of various grading (T24, NTUB1 and RT4). The impact of MLN4924 on UC cells was determined by measuring viability (MTT), proliferation (BrdU incorporation), cell cycle progression (flow cytometry with propidium iodide staining) and apoptosis (flow cytometry with annexin V-FITC labeling). The cell cycle regulatory molecules, apoptosis-related molecules, and cell stress-related proteins were examined by Western blotting. The influence of tumor cell migration and invasion was analyzed by Transwell and wound healing assays. We also evaluated the effects of MLN4924 on tumor growth by a SCID xenograft mouse model. The data show that MLN4924 induced dose-dependent cytotoxicity, anti-proliferation, anti-migration, anti-invasion and apoptosis in human UC cells, accompanied by activations of Bad, phospho-histone H2A.X, caspase-3, 7 and PARP, decreased level of phospho-Bcl2, and caused cell cycle retardation at the G2M phase. Moreover, MLN4924 activated endoplasmic reticulum stress-related molecules (caspase-4, phospho-eIF2α, ATF-4 and CHOP) and other stress responses (JNK and c-Jun activations). Finally, we confirmed MLN4924 inhibited tumor growth in a UC xenograft mouse model with minimal general toxicity. We concluded that MLN4924 induces apoptosis and cell cycle arrest, as well as activation of cell stress responses in human UC. These findings imply MLN4924 provides a novel strategy for the treatment of UC., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Previously, we showed that BCAS2 is essential for Drosophila viability and functions in pre-mRNA splicing. In this study, we provide strong evidence that BCAS2 regulates the activity of Delta-Notch signaling via Delta pre-mRNA splicing. Depletion of dBCAS2 reduces Delta mRNA expression and leads to accumulation of Delta pre-mRNA, resulting in diminished transcriptions of Delta-Notch signaling target genes, such as cut and E(spl)m8. Furthermore, ectopic expression of human BCAS2 (hBCAS2) and Drosophila BCAS2 (dBCAS2) in a dBCAS2-deprived fly can rescue dBCAS2 depletion-induced wing damage to the normal phenotypes. These rescued phenotypes are correlated with the restoration of Delta pre-mRNA splicing, which affects Delta-Notch signaling activity. Additionally, overexpression of Delta can rescue the wing deformation by deprivation of dBCAS2; and the depletion of dBCAS2 can restore the aberrant eye associated with Delta-overexpressing retinas; providing supporting evidence for the regulation of Delta-Notch signaling by dBCAS2. Taken together, dBCAS2 participates in Delta pre-mRNA splicing that affects the regulation of Delta-Notch signaling in Drosophila wing development.

Voltage-gated CLC-1 chloride channels play a critical role in controlling the membrane excitability of skeletal muscles. Mutations in human CLC-1 channels have been linked to the hereditary muscle disorder myotonia congenita. We have previously demonstrated that disease-associated CLC-1 A531V mutant protein may fail to pass the endoplasmic reticulum quality control system and display enhanced protein degradation as well as defective membrane trafficking. Currently the molecular basis of protein degradation for CLC-1 channels is virtually unknown. Here we aim to identify the E3 ubiquitin ligase of CLC-1 channels. The protein abundance of CLC-1 was notably enhanced in the presence of MLN4924, a specific inhibitor of cullin-RING E3 ligases. Subsequent investigation with dominant-negative constructs against specific subtypes of cullin-RING E3 ligases suggested that CLC-1 seemed to serve as the substrate for cullin 4A (CUL4A) and 4B (CUL4B). Biochemical examinations further indicated that CUL4A/B, damage-specific DNA binding protein 1 (DDB1), and cereblon (CRBN) appeared to co-exist in the same protein complex with CLC-1. Moreover, suppression of CUL4A/B E3 ligase activity significantly enhanced the functional expression of the A531V mutant. Our data are consistent with the idea that the CUL4A/B-DDB1-CRBN complex catalyses the polyubiquitination and thus controls the degradation of CLC-1 channels.

Many causal mutations of intellectual disability have been found in genes involved in epigenetic regulations. Replication-independent deposition of the histone H3.3 variant by the HIRA complex is a prominent nucleosome replacement mechanism affecting gene transcription, especially in postmitotic neurons. However, how HIRA-mediated H3.3 deposition is regulated in these cells remains unclear. Here, we report that dBRWD3, the Drosophila ortholog of the intellectual disability gene BRWD3, regulates gene expression through H3.3, HIRA, and its associated chaperone Yemanuclein (YEM), the fly ortholog of mammalian Ubinuclein1. In dBRWD3 mutants, increased H3.3 levels disrupt gene expression, dendritic morphogenesis, and sensory organ differentiation. Inactivation of yem or H3.3 remarkably suppresses the global transcriptome changes and various developmental defects caused by dBRWD3 mutations. Our work thus establishes a previously unknown negative regulation of H3.3 and advances our understanding of BRWD3-dependent intellectual disability., (© 2015 The Authors.)

During development, neural competence is conferred and maintained by integrating spatial and temporal regulations. The Drosophila sensory bristles that detect mechanical and chemical stimulations are arranged in stereotypical positions. The anterior wing margin (AWM) is arrayed with neuron-innervated sensory bristles, while posterior wing margin (PWM) bristles are non-innervated. We found that the COP9 signalosome (CSN) suppresses the neural competence of non-innervated bristles at the PWM. In CSN mutants, PWM bristles are transformed into neuron-innervated, which is attributed to sustained expression of the neural-determining factor Senseless (Sens). The CSN suppresses Sens through repression of the ecdysone signaling target gene broad (br) that encodes the BR-Z1 transcription factor to activate sens expression. Strikingly, CSN suppression of BR-Z1 is initiated at the prepupa-to-pupa transition, leading to Sens downregulation, and termination of the neural competence of PWM bristles. The role of ecdysone signaling to repress br after the prepupa-to-pupa transition is distinct from its conventional role in activation, and requires CSN deneddylating activity and multiple cullins, the major substrates of deneddylation. Several CSN subunits physically associate with ecdysone receptors to represses br at the transcriptional level. We propose a model in which nuclear hormone receptors cooperate with the deneddylation machinery to temporally shutdown downstream target gene expression, conferring a spatial restriction on neural competence at the PWM.

Background: Drosophila heart tube is a feasible model for cardiac physiological research. However, obtaining Drosophila electrocardiograms (ECGs) is difficult, due to the weak signals and limited contact area to apply electrodes. This paper presents a non-invasive Gallium-Indium (GaIn) based recording system for Drosophila ECG measurement, providing the heart rate and heartbeat features to be observed. This novel, high-signal-quality system prolongs the recording time of insect ECGs, and provides a feasible platform for research on the molecular mechanisms involved in cardiovascular diseases., Methods: In this study, two types of electrode, tungsten needle probes and GaIn electrodes, were used respectively to noiselessly conduct invasive and noninvasive ECG recordings of Drosophila. To further analyze electrode properties, circuit models were established and simulated. By using electromagnetic shielded heart signal acquiring system, consisted of analog amplification and digital filtering, the ECG signals of three phenotypes that have different heart functions were recorded without dissection., Results and Discussion: The ECG waveforms of different phenotypes of Drosophila recorded invasively and repeatedly with n value (n>5) performed obvious difference in heart rate. In long period ECG recordings, non-invasive method implemented by GaIn electrodes acts relatively stable in both amplitude and period. To analyze GaIn electrode, the correctness of GaIn electrode model established by this paper was validated, presenting accuracy, stability, and reliability., Conclusions: Noninvasive ECG recording by GaIn electrodes was presented for recording Drosophila pupae ECG signals within a limited contact area and signal strength. Thus, the observation of ECG changes in normal and SERCA-depleted Drosophila over an extended period is feasible. This method prolongs insect survival time while conserving major ECG features, and provides a platform for electrophysiological signal research on the molecular mechanism involved in cardiac arrhythmia, as well as research related to drug screening and development.

The heart chamber of an adult Drosophila is approximately 2 mm long and 0.5 mm wide, and the interwall separation of different heart portions during systole and diastole range from tens of micrometers to hundreds of micrometers. Furthermore, the heart chamber has a curved structure, which results in the larger differences in depth between the different heart portions. However, applying the wavelength calibration process before Fourier transform in an optical coherence tomography (OCT) system may cause degradation in system sensitivity and longitudinal resolution when the optical path difference between the reference and sample arms increases, which makes imaging the entire heart chamber difficult with OCT system. Additionally, since the heartbeat rate of Drosophila is approximately 6 beats/s, a high-speed OCT system is necessary to record the dynamics of the heat beats. In this study, we propose a new approach to visualize the entire heart chamber including the conical chamber and four ostia portions, and to observe the retrograde and anterograde beats. A buffered Fourier-domain mode-locked (FDML) laser is implemented to provide a high imaging speed. Two output ports of the buffered FDML laser are used simultaneously to scan the different heart portions of Drosophila, and the effective A-scan rate of the OCT system can be doubled. Then, the two scanned images are merged into a single B-mode scan. Furthermore, with dual-beam OCT system, the beating behaviors of the different heart portions from 7-day-old and 21-day-old flies are compared., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Reinnervation of target tissues determines functional outcomes after nerve grafting, which is important in traumatic injury caused by accidents or consequences resulting from surgical removal of tumors. Previous studies documented the influences of nerve repair mainly based on nerve morphometry but rarely compared the final outcomes according to target reinnervation patterns by nerve fibers of different categories., Methods: In a mouse model of nerve grafting, the authors analyzed the innervation indexes of different target tissues after transection-reimplantation on the sciatic nerve, which were defined as a parameter on the operated side normalized to that on the control side., Results: Muscle reinnervation appeared to be the best compared with skin reinnervation (p < 0.0001) and sweat gland reinnervation (p < 0.0001) at postoperative month 3. The sudomotor reinnervation was relatively higher than the cutaneous reinnervation (p = 0.014). The abundance of trophin transcripts for brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), and neurotrophin 3 (NT3) was higher in plantar muscles on the operated side than those on the control side. In contrast, transcripts of BDNF, GDNF, nerve growth factor, and NT3 were all similar in the footpad skin between the operated and control sides., Conclusions: The results suggested that, compared with the skin, muscles achieved the best reinnervation after nerve grafting, which was related to higher expression of BDNF, GDNF, and NT3 in muscles than in the skin.

Here, we show that dBCAS2 (CG4980, human Breast Carcinoma Amplified Sequence 2 ortholog) is essential for the viability of Drosophila melanogaster. We find that ubiquitous or tissue-specific depletion of dBCAS2 leads to larval lethality, wing deformities, impaired splicing, and apoptosis. More importantly, overexpression of hBCAS2 rescues these defects. Furthermore, the C-terminal coiled-coil domain of hBCAS2 binds directly to CDC5L and recruits hPrp19/PLRG1 to form a core complex for splicing in mammalian cells and can partially restore wing damage induced by knocking down dBCAS2 in flies. In summary, Drosophila and human BCAS2 share a similar function in RNA splicing, which affects cell viability.

To improve our understanding of lipid metabolism, Drosophila is used as a model animal, and its lipid homeostasis is monitored by coherent anti-Stokes Raman scattering microscopy. We are able to achieve in vivo imaging of larval fat body (analogous to adipose tissue in mammals) and oenocytes (analogous to hepatocytes) in Drosophila larvae at subcellular level without any labeling. By overexpressing two lipid regulatory proteins--Brummer lipase (Bmm) and lipid storage droplet-2 (Lsd-2)--we found different phenotypes and responses under fed and starved conditions. Comparing with the control larva, we observed more lipid droplet accumulation by ≈ twofold in oenocytes of fat-body-Bmm-overexpressing (FB-Bmm-overexpressing) mutant under fed condition, and less lipid by ≈ fourfold in oenocytes of fat-body-Lsd-2-overexpressing (FB-Lsd-2-overexpressing) mutant under starved condition. Moreover, together with reduced size of lipid droplets, the lipid content in the fat body of FB-Bmm-overexpressing mutant decreases much faster than that of the control and FB-Lsd-2-overexpressing mutant during starvation. From long-term starvation assay, we found FB-Bmm-overexpressing mutant has a shorter lifespan, which can be attributed to faster consumption of lipid in its fat body. Our results demonstrate in vivo observations of direct influences of Bmm and Lsd-2 on lipid homeostasis in Drosophila larvae.

In order to investigate the influence of light/dark cycle on the biosynthesis of metabolites during oogenesis, here we demonstrate a simple experimental protocol which combines in-vivo isotopic labeling of primary metabolites with mass spectrometric analysis of single eggs of fruit fly (Drosophila melanogaster). First, fruit flies were adapted to light/dark cycle using artificial white light. Second, female flies were incubated with an isotopically labeled sugar ((13)C(6)-glucose) for 12 h--either during the circadian day or the circadian night, at light or at dark. Third, eggs were obtained from the incubated female flies, and analyzed individually by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS): this yielded information about the extent of labeling with carbon-13. Since the incorporation of carbon-13 to uridine diphosphate glucose (UDP-glucose) in fruit fly eggs is very fast, the labeling of this metabolite was used as an indicator of the biosynthesis of metabolites flies/eggs during 12-h periods, which correspond to circadian day or circadian night. The results reveal that once the flies adapted to the 12-h-light/12-h-dark cycle, the incorporation of carbon-13 to UDP-glucose present in fruit fly eggs was not markedly altered by an acute perturbation to this cycle. This effect may be due to a relationship between biosynthesis of primary metabolites in developing eggs and an alteration to the intake of the labeled substrate - possibly related to the change of the feeding habit. Overall, the study shows the possibility of using MALDI-MS in conjunction with isotopic labeling of small metazoans to unravel the influence of environmental cues on primary metabolism.

Acetylation of histone and nonhistone proteins is an important posttranslational modification affecting many cellular processes. Here, we report that NuA4 acetylation of Sip2, a regulatory β subunit of the Snf1 complex (yeast AMP-activated protein kinase), decreases as cells age. Sip2 acetylation, controlled by antagonizing NuA4 acetyltransferase and Rpd3 deacetylase, enhances interaction with Snf1, the catalytic subunit of Snf1 complex. Sip2-Snf1 interaction inhibits Snf1 activity, thus decreasing phosphorylation of a downstream target, Sch9 (homolog of Akt/S6K), and ultimately leading to slower growth but extended replicative life span. Sip2 acetylation mimetics are more resistant to oxidative stress. We further demonstrate that the anti-aging effect of Sip2 acetylation is independent of extrinsic nutrient availability and TORC1 activity. We propose a protein acetylation-phosphorylation cascade that regulates Sch9 activity, controls intrinsic aging, and extends replicative life span in yeast., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Time-resolved optical coherence tomography (OCT) scanning images of wild-type and mutant fruit flies (Drosophila melanogaster), illustrating the heartbeat patterns for evaluating their cardiac functions, are demonstrated. Based on the heartbeat patterns, the beat rate and the relative phase between the first two heart segments can be evaluated. The OCT scanning results of mutant flies with impaired proteasome function in cardiac muscles show irregular heartbeat patterns and systematically decreased average beat rates, when compared with the regular patterns of ~4.97 beats/s in average beat rate of the wild-type. In both wild-type and proteasome mutant flies, the beatings at different locations in the same heart segment are essentially synchronized. However, between different heart segments, although the beating in the second segment shows a lag in phase behind that of the first segment in a wild-type, in a proteasome mutant, the beating in the second segment becomes significantly leading that of the first segment. Besides the comparison between the wild-type and proteasomal mutant flies, the influences of using different methods for immobilizing flies during OCT scanning on the heart functions are demonstrated., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Fruit fly (Drosophila melanogaster) is a standard model organism used in genetics and molecular biology. Phospholipids are building blocks of cellular membranes, and components of a complex signaling network. Here, we present a facile method, based on matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS), for molecular imaging of phospholipid distributions in submillimeter-sized components of the fruit fly reproductive system. Individual egg chambers were deposited on a specially prepared MALDI target comprising an aluminum slide with a rough surface created by ablation with a microsecond-laser: this helped to immobilize biological specimens, remove excess of saline solution by adhesive forces, carry out microscopic observations, and facilitated distribution of the MALDI matrix. A continuous-flow ultrasound-assisted spray was used for the deposition of MALDI matrix (9-aminoacridine) onto the sample. The upper surface of the specimen was then scanned with a 355-nm solid-state laser with a preset beam focus of 10 μm to obtain negative-ion mode MALDI-MS images. Overall, this provided sufficient spatial resolution to reveal micrometer-scale gradient-like patterns of phospholipids along the anterior/posterior axis of egg chambers. Several phosphatidylinositols are seen to be segregated according to the number of unsaturated bonds, with an elevated abundance of polyunsaturated phosphatidylinositols within the oocyte compartment.

The Hedgehog (Hh) morphogen directs distinct cell responses according to its distinct signalling levels. Hh signalling stabilizes transcription factor cubitus interruptus (Ci) by prohibiting SCF(Slimb)-dependent ubiquitylation and proteolysis of Ci. How graded Hh signalling confers differential SCF(Slimb)-mediated Ci proteolysis in responding cells remains unclear. Here, we show that in COP9 signalosome (CSN) mutants, in which deneddylation of SCF(Slimb) is inactivated, Ci is destabilized in low-to-intermediate Hh signalling cells. As a consequence, expression of the low-threshold Hh target gene dpp is disrupted, highlighting the critical role of CSN deneddylation on low-to-intermediate Hh signalling response. The status of Ci phosphorylation and the level of E1 ubiquitin-activating enzyme are tightly coupled to this CSN regulation. We propose that the affinity of substrate-E3 interaction, ligase activity and E1 activity are three major determinants for substrate ubiquitylation and thereby substrate degradation in vivo.

Drosophila is one of the most valuable model organisms for studying genetics and developmental biology. The fat body in Drosophila, which is analogous to the liver and adipose tissue in human, stores lipids that act as an energy source during its development. At the early stages of metamorphosis, the fat body remodeling occurs involving the dissociation of the fat body into individual fat cells. Here we introduce a combination of coherent anti-Stokes Raman scattering (CARS) and two-photon excitation autofluorescence (TPE-F) microscopy to achieve label-free imaging of Drosophila in vivo at larval and pupal stages. The strong CARS signal from lipids allows direct imaging of the larval fat body and pupal fat cells. In addition, the use of TPE-F microscopy allows the observation of other internal organs in the larva and autofluorescent globules in fat cells. During the dissociation of the fat body, the findings of the degradation of lipid droplets and an increase in autofluorescent globules indicate the consumption of lipids and the recruitment of proteins in fat cells. Through in vivo imaging and direct monitoring, CARS microscopy may help elucidate how metamorphosis is regulated and study the lipid metabolism in Drosophila.

Histiocytoid Sweet syndrome is a recently described variant of acute febrile neutrophilic dermatosis. The histiocytoid cells are easily misinterpreted as histiocytes, when in fact they are immature myeloid cells. The recognition of myeloperoxidase expression in these cells is important in avoiding confusion with histiocyte-rich dermatoses. Herein, we report a case of histiocytoid Sweet syndrome with neutropenia that had an unusual presentation. The recognition of this rare combination helps expand the spectrum of Sweet syndrome with histiocytoid infiltrate.

In higher eukaryotes, the pre-replication complex (pre-RC) component Cdt1 is the major regulator in licensing control for DNA replication. The Cul4-DDB1-based ubiquitin ligase mediates Cdt1 ubiquitylation for subsequent proteolysis. During the initiation of chorion gene amplification, Double-parked (Dup), the Drosophila ortholog of Cdt1, is restricted to chorion gene foci. We found that Dup accumulated in nuclei in Cul4 mutant follicle cells, and the accumulation was less prominent in DDB1 mutant cells. Loss of Cul4 or DDB1 activity in follicle cells also compromised chorion gene amplification and induced ectopic genomic DNA replication. The focal localization of Orc2, a subunit of the origin recognition complex, is frequently absent in Cul4 mutant follicle cells. Therefore, Cul4 and DDB1 have differential functions during chorion gene amplification.

The ubiquitin-proteasome system plays a major role in the rhythmic accumulation and turnover of molecular clock components. In turn, these approximately 24 h molecular rhythms drive circadian rhythms of behavior and physiology. In Drosophila, the ubiquitin-proteasome system also plays a critical role in light-dependent degradation of the clock protein Timeless (TIM), a key step in the entrainment of the molecular clocks to light-dark cycles. Here, we investigated the role of the COP9 signalosome (CSN), a general regulator of protein degradation, in fly circadian rhythms. We found that null mutations in the genes encoding the CSN4 and CSN5 subunits prevent normal TIM degradation by light in the pacemaker lateral neurons (LNs) as does LN-specific expression of a dominant-negative CSN5 transgene. These defects are accompanied by strong reductions in behavioral phase shifts of adult flies lacking normal CSN5 activity in LNs. Defects in TIM degradation and resetting of behavioral phases were rescued by overexpression of Jetlag (JET), the F-box protein required for light-mediated TIM degradation. Flies lacking normal CSN activity in all clock neurons are rhythmic in constant light, a phenotype previously associated with jet mutants. Together, these data indicate that JET and the CSN lie in a common pathway leading to light-dependent TIM degradation. Surprisingly, we found that manipulations that strongly inhibit CSN activity had minimal effects on circadian rhythms in constant darkness, indicating a specific role for the CSN in light-mediated TIM degradation.

The ubiquitin-like protein Nedd8/Rub1 covalently modifies and activates cullin ubiquitin ligases. However, the repertoire of Nedd8-modified proteins and the regulation of protein neddylation status are not clear. The cysteine protease DEN1/NEDP1 specifically processes the Nedd8 precursor and has been suggested to deconjugate Nedd8 from cullin proteins. By characterizing the Drosophila DEN1 protein and DEN1 null (DEN1(null)) mutants, we provide in vitro and in vivo evidence that DEN1, in addition to processing Nedd8, deneddylates many cellular proteins. Although purified DEN1 protein efficiently deneddylates the Nedd8-conjugated cullin proteins Cul1 and Cul3, neddylated Cul1 and Cul3 protein levels are not enhanced in DEN1(null). Strikingly, many cellular proteins are highly neddylated in DEN1 mutants and are deneddylated by purified DEN1 protein. DEN1 deneddylation activity is distinct from that of the cullin-deneddylating CSN. Genetic analyses indicate that a balance between neddylation and deneddylation maintained by DEN1 is crucial for animal viability.

The fruit fly Drosophila melanogaster is one of the most valuable organisms in studying genetics and developmental biology. To gain insight into Drosophila development, we successfully acquired label-free, in vivo images of both developing muscles and internal organs in a stage 2 larva using the minimally invasive imaging modality of multiphoton autofluorescence (MAF) and second harmonic generation (SHG) microscopy. We found that although MAF is useful in identifying structures such as the digestive system, trachea, and intestinal track, it is the SHG signal that allowed the investigation of the muscular architecture within the developing larva. Our results suggest that multiphoton microscopy is a powerful in vivo, label-free imaging technique to examine Drosophila physiology and may be used for developmental studies.

Neddylation, a process that conjugates the ubiquitin-like polypeptide NEDD8 to cullin proteins, activates cullin-RING ubiquitin ligases (CRLs). Deneddylation, in which the COP9 signalosome (CSN) removes NEDD8 from cullins, inactivates CRLs. However, genetic studies of CSN function conclude that deneddylation also promotes CRL activity. It has been proposed that a cyclic transition through neddylation and deneddylation is required for the regulation of CRL activity in vivo. Recent discoveries suggest that an additional level of complexity exists, whereby CRL components are targets for degradation, mediated either by autocatalytic ubiquitination or by unknown mechanisms. Deneddylation by CSN and deubiquitylation by CSN-associated ubiquitin-specific protease 12 protect CRL components from cellular depletion, thus maintaining the physiological CRL activities.

The molecular chaperone heat shock protein 90 (Hsp90) and its accessory cochaperones function by facilitating the structural maturation and complex assembly of client proteins, including steroid hormone receptors and selected kinases. By promoting the activity and stability of these signaling proteins, Hsp90 has emerged as a critical modulator in cell signaling. Here, we present evidence that Hsp90 chaperone activity is regulated by reversible acetylation and controlled by the deacetylase HDAC6. We show that HDAC6 functions as an Hsp90 deacetylase. Inactivation of HDAC6 leads to Hsp90 hyperacetylation, its dissociation from an essential cochaperone, p23, and a loss of chaperone activity. In HDAC6-deficient cells, Hsp90-dependent maturation of the glucocorticoid receptor (GR) is compromised, resulting in GR defective in ligand binding, nuclear translocation, and transcriptional activation. Our results identify Hsp90 as a target of HDAC6 and suggest reversible acetylation as a unique mechanism that regulates Hsp90 chaperone complex activity.

Biphasic Th1/Th2 development plays a central role in the pathogenesis of atopic dermatitis. In the sensitization phase after protein antigen exposure, an immune response polarized toward Th2 differentiation, which is due to the hosts' genetic proneness to the disease, initiates the skin lesions. Th1/Th2 antagonism is a potential mechanism that could be manipulated to suppress the initial Th2 deviation. IL-12 is the key cytokine for Th1 differentiation. Interferon gamma (IFN-gamma) can assist Th1 development through several mechanisms and suppress Th2 differentiation. We took advantage of a recently developed murine model of atopic dermatitis elicited by epicutaneous sensitization with protein antigen through patch application to examine the effects of different routes of IFN-gamma administration on Th1/Th2 differentiation during the sensitization phase of antigen exposure. Our data showed that systemic administration of IFN-gamma during the sensitization phase could not promote serum levels of specific IgG(2a). However, local administration (intradermal injection or patch application) of IFN-gamma during the sensitization phase could promote serum levels of specific IgG(2a) and suppress serum levels of specific IgE. Moreover, pretreatment of local IFN-gamma with protein antigen has a long-term modulatory effect on serum levels of specific IgG(2a) and IgE after repeated antigen immunization. Our results demonstrate that local but not systemic administration of IFN-gamma during the sensitization phase of protein antigen immunization could suppress the Th2 deviation in this murine model of atopic dermatitis. Thus, this may represent a novel strategy for the treatment and prevention of atopic dermatitis., (Copyright 2002 Elsevier Science Ltd.)