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Magnesium and its related preparations are already in medical use and have recognized therapeutic effects on sleep disorders. However, its underlying molecular mechanisms remain unclear. Here, using Drosophila as a model, we found that RNAi-mediated knockdown of Uex, the homologous gene of magnesium transporters of the Cyclin M family (CNNM) causes increased daily total sleep. Ectopic-expression of CNNM1 can rescue the sleep phenotype in Uex knockdown flies. UEX exhibits rhythmic oscillations in the brain and affects the efflux of cellular Mg2+. Knockdown of Uex in the nervous system influences Ca2+-mediated CREB signaling and neuroplasticity. Additionally, Uex physically interacts with CNK, the upstream regulator of ERK pathway. Similar effects on sleep are observed with knockdown of Cnk in flies. We propose that the UEX regulates sleep through its downstream Ca2+-dependent CREB signaling and a CNK-ERK pathway. Our findings may provide new insight into mechanisms of magnesium and magnesium transporter related sleep disorder.

Background Neural stem cell fate is regulated for the systematic production of a precise quantity of neurons during the development of the central nervous system. Results Here we found that two Drosophila pipsqueak family genes, distal antenna (dan) and distal antenna-related (danr), promote the proliferation of neural stem cells, termed neuroblasts (NBs), in third instar larval brains. In the absence of Dan and Danr (Dan/Danr), the NBs produce fewer daughter cells with smaller MARCM clone (lineage) sizes. The larval brain NBs in dan/danr clones show premature accumulation of nuclear Prospero (Pros), which only appears in the wildtype (wt) terminating NBs at early pupal stage. The premature nuclear Pros leads to NBs cell cycle defects and NB marker loss. Removal of Pros from dan/danr MARCM clones prevents lineage size shrinkage and rescues the loss of NB marker expression. Our data show that the appearance of nuclear Pros is behind the downregulation of Dan/Danr in the wt terminating NBs. We demonstrate that Dan/Danr and nuclear Pros are mutually exclusive in NBs. In addition, Dan/Danr are partially required for the late temporal regulator, Grainyhead (Grh), in third instar larval brain NBs. Conclusion Together our study uncovers the novel function of Dan/Danr in NB cell fate maintenance. Dan/Danr antagonize nuclear Pros, preventing NBs from premature decommission in Drosophila third instar larval brains.

In this paper, the three-dimensional segmentation of pulmonary honeycomb lesions and the function of image biomarkers are integrated into a quantitative analysis module of the lung, and integrated into the PACS image diagnosis workstation for the quantitative analysis of doctors. It is difficult to segment honeycomb lesions in lung CT images because of its diffuse and blurred edges, but it has good texture characteristics. Based on this feature, this paper converts honeycomb lesions into a texture classification problem. Firstly, the lung parenchyma is extracted by automatic threshold segmentation and region growing method, and then the lung parenchyma is divided into several small regions according to the texture by watershed method. Then, according to the texture characteristics of each small area, the trained support vector machine is used to classify. Finally, the correlation between gray level and spatial position of slice data is used to correct the classification results, so as to reduce false positive areas. In order to expand the study of imaging biomarkers of chronic lung diseases to more extensive major diseases, a sample database was established for a wide range of multiple lesions. Through feature extraction and feature analysis of multiple lesions in database, potential feature differences can be excavated, which lays a solid foundation for further study of image biomarkers of multiple lesions.

N-6-Methyladenosine (m

Summary Neuroprotection is essential for the maintenance of normal physiological functions in the nervous system. This is especially true under stress conditions. Here, we demonstrate a novel protective function of PRL-1 against CO2 stimulation in Drosophila. In the absence of PRL-1, flies exhibit a permanent held-up wing phenotype upon CO2 exposure. Knockdown of the CO2 olfactory receptor, Gr21a, suppresses the phenotype. Our genetic data indicate that the wing phenotype is due to a neural dysfunction. PRL-1 physically interacts with Uex and controls Uex expression levels. Knockdown of Uex alone leads to a similar wing held-up phenotype to that of PRL-1 mutants. Uex acts downstream of PRL-1. Elevated Uex levels in PRL-1 mutants prevent the CO2-induced phenotype. PRL-1 and Uex are required for a wide range of neurons to maintain neuroprotective functions. Expression of human homologs of PRL-1 could rescue the phenotype in Drosophila, suggesting a similar function in humans., Graphical Abstract, Highlights • PRL-1 functions to protect the nervous system against olfactory CO2 stimulation • PRL-1 physically interacts with Uex and controls Uex expression levels • PRLs may retain a similar neuroprotective function in humans, Molecular Genetics; Molecular Neuroscience

CD133 (AC133/prominin-1) has been identified as a stem cell marker and a putative cancer stem cell marker in many solid tumors. Its biologic function and molecular mechanisms remain largely elusive. Here, we show that a fly mutant for prominin-like, a homolog of mammalian CD133, shows a larger body size and excess weight accompanied with higher fat deposits as compared with the wild type. The expression levels of prominin-like are mediated by ecdysone signaling where its protein levels increase dramatically in the fat body during metamorphosis. Prominin-like mutants exhibit higher Drosophila insulin-like peptide 6 (di lp6) levels during nonfeeding stages and increased Akt/ Drosophila target of rapamycin (dTOR) signaling. On an amino acid-restricted diet, prominin-like mutants exhibit a significantly larger body size than the wild type does, similar to that which occurs upon the activation of the dTOR pathway in the fat body. Our data suggest that prominin-like functions by suppressing TOR and dilp6 signaling to control body size and weight. The identification of the physiologic function of prominin-like in Drosophila may provide valuable insight into the understanding of the metabolic function of CD133 in mammals.-Zheng, H., Zhang, Y., Chen, Y., Guo, P., Wang, X., Yuan, X., Ge, W., Yang, R., Yan, Q., Yang, X., Xi, Y. Prominin-like, a homolog of mammalian CD133, suppresses di lp6 and TOR signaling to maintain body size and weight in Drosophila.

Background Drosophila Prominin-like is a homolog of mammalian CD133, which is recognized as a biomarker for stem cells. The interacting proteins of CD133 and their biological functions remain elusive. Results In this study, we using yeast two-hybrid assays, GST pull-down assay and co-immunoprecipitation (Co-IP) methods found that Drosophila Prominin-like interacts with ND20, a subunit of mitochondrial respiratory complex I. Bioinformatics analysis suggests that Prominin-like is a six-transmembrane glycoprotein which localizes on cellular membranes. Immunostaining and mitochondrial fractionation indicate that Drosophila Prominin-like could localize in the mitochondria. The knockdown of prominin-like in S2 cells resulted in transient mitochondrial dysfunctions as evidenced by reduced ATP production, elevated ROS generation and an accompanied reduction in mitochondrial proteins. Mitochondrial dysfunctions were detected in aged prominin-like mutant flies. Conclusion Our data indicates that Prominin-like acts to maintain mitochondrial function through its interaction with ND20 which, itself, is active in the mitochondrial electron transport chain. Our study provides insights into a novel molecular mechanism of Drosophila prominin-like and suggests a similar function of CD133 in mammals.

Atg101 is an autophagy-related gene identified in worms, flies, mice, and mammals, which encodes a protein that functions in autophagosome formation by associating with the ULK1-Atg13-Fip200 complex. In the last few years, the critical role of Atg101 in autophagy has been well-established through biochemical studies and the determination of its protein structure. However, Atg101's physiological role, both during development and in adulthood, remains less understood. Here, we describe the generation and characterization of an Atg101 loss-of-function mutant in Drosophila and report on the roles of Atg101 in maintaining tissue homeostasis in both adult brains and midguts. We observed that homozygous or hemizygous Atg101 mutants were semi-lethal, with only some of them surviving into adulthood. Both developmental and starvation-induced autophagy processes were defective in the Atg101 mutant animals, and Atg101 mutant adult flies had a significantly shorter lifespan and displayed a mobility defect. Moreover, we observed the accumulation of ubiquitin-positive aggregates in Atg101 mutant brains, indicating a neuronal defect. Interestingly, Atg101 mutant adult midguts were shorter and thicker and exhibited abnormal morphology with enlarged enterocytes. Detailed analysis also revealed that the differentiation from intestinal stem cells to enterocytes was impaired in these midguts. Cell type–specific rescue experiments disclosed that Atg101 had a function in enterocytes and limited their growth. In summary, the results of our study indicate that Drosophila Atg101 is essential for tissue homeostasis in both adult brains and midguts. We propose that Atg101 may have a role in age-related processes.

The dynamic process of spermatogenesis shows little variation between invertebrate models such as Drosophila, and vertebrate models such as mice and rats. In each case, germ stem cells undergo mitotic division to proliferate and then continue, via meiosis, through various stages of elongation and individualization from spermatogonia to spermatid to finally to form mature sperm. Mature sperm are then stored in the seminal vesicles for fertilization. Errors in any of these stages can lead to male infertility. Here, we identify that Drosophila Pif1A acts as a key regulator for sperm individualization. Loss of Pif1A leads to male sterility associated with irregular individualization complex and empty seminal vesicles without mature sperm. Pif1A is highly expressed in the testes of mated male adult flies and the Pif1A protein is expressed at a higher level in male than in female flies. Pif1A is homologous to mammalian coiled-coil domain-containing protein 157 (CCDC157), which is also enriched in the testes of humans and mice. Human CCDC157, with unknown function, was identified to be downregulated in men with idiopathic non-obstructive azoospermia (NOA). We map the function of Drosophila Pif1A during spermatogenesis, showing that Pif1A is essential for spermatide individualization and involved in the regulation of the lipid metabolism genes. Our findings might be applicable for studying the function of CCDC157 in spermatogenesis and other aspects of human male fertility.

Additional file 1: Figure S1. GST pull-down assays. GST or GST-PL-1 fusion protein generated by E. coli BL21 cells was purified by glutathione agarose resin, followed by incubation of the resin with HA-TSG101(A) or HA-Hrb27C (B) protein expressed in S2 cells. After washing with PBS, the bound proteins were analyzed by Western blotting with anti-HA antibodies. Data showed that neither TSG101 nor HRB27C could be pulled down by Prominin-like. Figure S2. Images of S2 cells of prominin-like knock down and control cells. Scale bar: 50μm. Upon dsRNA treatment, the morphology of the treated cells seemed have no obvious differences between prominin-like knock down and control cells from day 1 to day 4, except for the cell density.

By the end of neurogenesis in Drosophila pupal brain neuroblasts (NBs), nuclear Prospero (Pros) triggers cell cycle exit and terminates NB lifespan. Here, we reveal that in larval brain NBs, an intrinsic mechanism facilitates import and export of Pros across the nuclear envelope via a Ran‐mediated nucleocytoplasmic transport system. In rangap mutants, the export of Pros from the nucleus to cytoplasm is impaired and the nucleocytoplasmic transport of Pros becomes one‐way traffic, causing an early accumulation of Pros in the nuclei of the larval central brain NBs. This nuclear Pros retention initiates NB cell cycle exit and leads to a premature decrease of total NB numbers. Our data indicate that RanGAP plays a crucial role in this intrinsic mechanism that controls NB lifespan during neurogenesis. Our study may provide insights into understanding the lifespan of neural stem cells during neurogenesis in other organisms.

Biosensors can be used in applications ranging from identifying disease biomarkers to detecting spatial and temporal distributions of specific molecules in living cells. A major challenge facing biosensor development is how to functionally couple a biological recognition domain to an output module so that the binding event can be transduced to a visible and quantifiable signal [e.g., Förster resonance energy transfer (FRET)]. Most designs achieve coupling by means of a binding protein that changes conformation upon interacting with its target. This approach is limited by the fact that few proteins possess such natural allosteric mechanisms, and for those that do, the conformational change is frequently not extensive enough to produce a large change in distance between FRET donor and acceptor groups. Here, we introduce protein fragment exchange (FREX) to address both problems. FREX employs two components: a folded binding protein and a fragment duplicated from it, the latter of which can be chosen from many possible fragments. The system is rationally tuned so that addition of ligand induces a conformational change in which the fragment exchanges positions with the corresponding segment of the binding protein. Placing fluorescent donor and acceptor groups on the binding protein and fragment reduces the background level of FRET of the unbound sensor, resulting in a ratiometric FRET response that is expected to be strong and reproducible from protein to protein. FREX is demonstrated using fibronectin III, a monobody binding scaffold that has been tailored to recognize multiple targets. Sensors labeled with Alexa FRET pairs exhibit ratiometric FRET changes of up to 8.6-fold and perform equally well in buffer and serum. A genetically encoded variant of this sensor is shown to be functional in cell lysates and in mammalian cell cultures.

Remarkable advances have been made in recent years in our understanding of the Drosophila fat body and its functions in energy storage, immune response and nutrient sensing. The fat body interplays with other tissues to respond to the physiological needs of the body's growth and coordinates various metabolic processes at different developmental stages and under different environmental conditions. The identification of various conserved genetic functions and signaling pathways relating to the Drosophila fat body may provide clues to lipometabolic disease and other aspects of tissue remodeling in humans. Here, we discuss recent insights into how regulation of fat body remodeling contributes to hemostasis with a special focus on how signaling networks and internal physiological states shape different aspects of the lipid metabolism in Drosophila.

In yeast cells, subunit a of the vacuolar proton pump (V-ATPase) is encoded by two organelle-specific isoforms, VPH1 and STV1. V-ATPases containing Vph1 and Stv1 localize predominantly to the vacuole and the Golgi apparatus/endosomes, respectively. Ratiometric measurements of vacuolar pH confirm that loss of STV1 has little effect on vacuolar pH. Loss of VPH1 results in vacuolar alkalinization that is even more rapid and pronounced than in vma mutants, which lack all V-ATPase activity. Cytosolic pH responses to glucose addition in the vph1Δ mutant are similar to those in vma mutants. The extended cytosolic acidification in these mutants arises from reduced activity of the plasma membrane proton pump, Pma1p. Pma1p is mislocalized in vma mutants but remains at the plasma membrane in both vph1Δ and stv1Δ mutants, suggesting multiple mechanisms for limiting Pma1 activity when organelle acidification is compromised. pH measurements in early prevacuolar compartments via a pHluorin fusion to the Golgi protein Gef1 demonstrate that pH responses of these compartments parallel cytosolic pH changes. Surprisingly, these compartments remain acidic even in the absence of V-ATPase function, possibly as a result of cytosolic acidification. These results emphasize that loss of a single subunit isoform may have effects far beyond the organelle where it resides.

Biosensors couple binding events with a readily quantifiable optical output signal. Proteins are excellent recognition modules because of their inherent ability to bind ligands with high affinity and specificity. FRET is a powerful detection method because it can be ratiometric and genetically encoded. Researchers who wish to convert ordinary binding proteins into FRET-based biosensors are faced with two main challenges: (i) how to establish a binding-dependent conformational change, and (ii) how to make this change sufficiently large to produce robust FRET output. Here we develop the FRagment EXchange (FREX) methodology to address those two challenges. In the FREX approach, point mutations are introduced into the full-length parent protein to thermodynamically destabilize the protein and to knock out ligand binding. A polypeptide segment that covers both mutation sites, but contains the wild-type residues at those positions, is synthesized by partial sequence duplication of the parent protein. In the absence of ligand, the full-length protein is sufficiently stable to assume its native conformation. Addition of ligand induces a conformational change where a portion of the binding-incompetent full-length protein is swapped by the corresponding peptide. Ternary complex formation is driven by ligand binding energy and the gain in stability afforded by restoring wild-type side chain interactions. Placement of fluorescent reporter groups onto the full-length protein and the peptide fragment directly links ligand binding to a consistent and robust FRET increase. We demonstrate the method using the tenth human fibronectin type III domain (FN3), a versatile binding scaffold that has been previously modified to recognize a variety of targets. FREX establishes a customizable platform for a conformational switch in which ligand binding is coupled to a high-output reporter signal.