Osiris gene family defines the cuticle nanopatterns of Drosophila.

Nanostructures of pores and protrusions in the insect cuticle modify molecular permeability and surface wetting, and help insects sense various environmental cues. However, the cellular mechanisms that modify cuticle nanostructures are poorly understood. Here, we elucidate how insect-specific Osiris family genes are expressed in various cuticle-secreting cells in the Drosophila head during the early stages of cuticle secretion and cover nearly the entire surface of the head epidermis. Furthermore, we demonstrate how each sense organ cell with various cuticular nanostructures expressed a unique combination of Osiris genes. Osiris gene mutations cause various cuticle defects in the corneal nipples and pores of the chemosensory sensilla. Thus, our study emphasizes on the importance of Osiris genes for elucidating cuticle nanopatterning in insects.

Novel missense variants cause intermediate phenotypes in the phenotypic spectrum of SLC5A6-related disorders.

SLC5A6 encodes the sodium-dependent multivitamin transporter, a transmembrane protein that uptakes biotin, pantothenic acid, and lipoic acid. Biallelic SLC5A6 variants cause sodium-dependent multivitamin transporter deficiency (SMVTD) and childhood-onset biotin-responsive peripheral motor neuropathy (COMNB), which both respond well to replacement therapy with the above three nutrients. SMVTD usually presents with various symptoms in multiple organs, such as gastrointestinal hemorrhage, brain atrophy, and global developmental delay, at birth or in infancy. Without nutrient replacement therapy, SMVTD can be lethal in early childhood. COMNB is clinically milder and has a later onset than SMVTD, at approximately 10 years of age. COMNB symptoms are mostly limited to peripheral motor neuropathy. Here we report three patients from one Japanese family harboring novel compound heterozygous missense variants in SLC5A6, namely NM_021095.4:c.[221C>T];[642G>C] p.[(Ser74Phe)];[(Gln214His)]. Both variants were predicted to be deleterious through multiple lines of evidence, including amino acid conservation, in silico predictions of pathogenicity, and protein structure considerations. Drosophila analysis also showed c.221C>T to be pathogenic. All three patients had congenital brain cysts on neonatal cranial imaging, but no other morphological abnormalities. They also had a mild motor developmental delay that almost completely resolved despite no treatment. In terms of severity, their phenotypes were intermediate between SMVTD and COMNB. From these findings we propose a new SLC5A6-related disorder, spontaneously remitting developmental delay with brain cysts (SRDDBC) whose phenotypic severity is between that of SMVTD and COMNB. Further clinical and genetic evidence is needed to support our suggestion.

Three-dimensional evaluation of sleep bruxism-related splint wear using a dental laboratory scanner: A preliminary clinical study.

BACKGROUND: The wear depth on the occlusal splint (OS) is reportedly associated with the sleep bruxism (SB) level, as evaluated using portable polysomnography (PSG) recordings. However, the OS is deformed owing to SB forces, possibly preventing the accurate quantification of the wear facets. OBJECTIVES: We aimed to introduce a newly developed system to quantify the wear facets on the OS using a dental laboratory scanner (D810) and investigate the association between the wear facets, as evaluated with this system, and the SB level. METHODS: Ten healthy individuals who were diagnosed with SB based on portable PSG recordings participated in this study. They were asked to wear the OS for 2 months. The first day after a 2-week adaptation period was defined as the reference day, and sequential scanning of the OS surface was performed on days 15, 30, and 45. Changes in the OS surface from the reference day allowed dimensional evaluation of the wear facets in terms of maximum wear depth, wear area, and wear volume. Multiple regression analyses were conducted to test whether each of these variables could be predicted by any of the SB-related variables. RESULTS: The total duration of SB episodes per hour of sleep and the maximum muscle activity were significantly associated with the wear area, as measured with our system (adjusted R-squared was .78, p < .01). CONCLUSION: Our system allows dimensional analysis of the wear facets on the OS surface in association with the SB level.

Lint-O cooperates with L(3)mbt in target gene suppression to maintain homeostasis in fly ovary and brain.

Loss-of-function mutations in Drosophila lethal(3)malignant brain tumor [l(3)mbt] cause ectopic expression of germline genes and brain tumors. Loss of L(3)mbt function in ovarian somatic cells (OSCs) aberrantly activates germ-specific piRNA amplification and leads to infertility. However, the underlying mechanism remains unclear. Here, ChIP-seq for L(3)mbt in cultured OSCs and RNA-seq before and after L(3)mbt depletion shows that L(3)mbt genomic binding is not necessarily linked to gene regulation and that L(3)mbt controls piRNA pathway genes in multiple ways. Lack of known L(3)mbt co-repressors, such as Lint-1, has little effect on the levels of piRNA amplifiers. Identification of L(3)mbt interactors in OSCs and subsequent analysis reveals CG2662 as a novel co-regulator of L(3)mbt, termed "L(3)mbt interactor in OSCs" (Lint-O). Most of the L(3)mbt-bound piRNA amplifier genes are also bound by Lint-O in a similar fashion. Loss of Lint-O impacts the levels of piRNA amplifiers, similar to the lack of L(3)mbt. The lint-O-deficient flies exhibit female sterility and tumorous brains. Thus, L(3)mbt and its novel co-suppressor Lint-O cooperate in suppressing target genes to maintain homeostasis in the ovary and brain.

Amelioration of a neurodevelopmental disorder by carbamazepine in a case having a gain-of-function GRIA3 variant.

GRIA3 at Xq25 encodes glutamate ionotropic receptor AMPA type 3 (GluA3), a subunit of postsynaptic glutamate-gated ion channels mediating neurotransmission. Hemizygous loss-of-function (LOF) variants in GRIA3 cause a neurodevelopmental disorder (NDD) in male individuals. Here, we report a gain-of-function (GOF) variant at GRIA3 in a male patient. We identified a hemizygous de novo missense variant in GRIA3 in a boy with an NDD: c.1844C > T (p.Ala615Val) using whole-exome sequencing. His neurological signs, such as hypertonia and hyperreflexia, were opposite to those in previous cases having LOF GRIA3 variants. His seizures and hypertonia were ameliorated by carbamazepine, inhibiting glutamate release from presynapses. Patch-clamp recordings showed that the human GluA3 mutant (p.Ala615Val) had slower desensitization and deactivation kinetics. A fly line expressing a human GluA3 mutant possessing our variant and the Lurcher variant, which makes ion channels leaky, showed developmental defects, while one expressing a mutant possessing either of them did not. Collectively, these results suggest that p.Ala615Val has GOF effects. GRIA3 GOF variants may cause an NDD phenotype distinctive from that of LOF variants, and drugs suppressing glutamatergic neurotransmission may ameliorate this phenotype. This study should help in refining the clinical management of GRIA3-related NDDs.

Effect of assistive devices on the precision of digital impressions for implants placed in edentulous maxilla: an in vitro study.

PURPOSE: To examine the effect of assistive devices on the precision of digital impression for multiple implants placed in the edentulous maxilla. METHODS: A reference model representing an edentulous maxilla with four implants was developed. The digital impression group included three settings: Type 0, without an assistive device; Type 1, with an assistive device connecting only neighboring implants; and Type 2, with an assistive device connecting not only neighboring implants but also the two posterior implants, with perpendicular branches from this bar towards the anterior implants. Digital impressions were made five times for each type using three intraoral scanners (IOSs). For conventional method, silicone impressions and verification jigs were prepared; fabricated plaster models were scanned using a laboratory scanner/industrial 3D scanner. In analysis 1, two-way ANOVA analyzed the effect of IOSs and assistive devices on the precision of digital impressions. In analysis 2, one-way ANOVA compared the silicone impressions, the verification jigs, and the most precise group of digital impressions from analysis 1. RESULTS: In analysis 1, the IOS and assistive device type (F = 25.22, p < .0001) effects and the interaction between these two factors (F = 5.64, p = .0005) were statistically significant. In analysis 2, CON, VJ, and digital impression with Type 2 devices (most precise devices in analysis 1) were compared; better precision was obtained by digital impression with Type 2 device than by CON and VJ (F = 30.08, p < .0001). CONCLUSIONS: For implants placed in an edentulous maxilla, digital impressions with assistive devices can provide better precision compared to silicone impressions and verification jigs.

Trueness and precision of digital implant impressions by intraoral scanners: a literature review.

BACKGROUND: With the development of intraoral scanners, their trueness and precision have been evaluated in various studies. Through these studies, the amount of accuracy that can be expected from intraoral scanners has gradually been disclosed, at the same time, it was difficult to integrate the results of individual studies due to differences in evaluation methods between studies. The purpose of this article was to review the currently available evidence, summarise what is currently known about IOS, analyse the evaluation methods of each study, and list points to note when interpreting the results. MAIN TEXT: Most of the studies were conducted in vitro. The accuracy is evaluated in situations such as single missing teeth, partially edentulous ridges with multiple missing teeth, and fully edentulous jaws. To evaluate the accuracy, direct measurement of distance or angle by coordinate measuring machines and calculation of surface deviation by superimposing surface data were predominantly performed. The influence of parameters such as the number of implants, distance between implants, angle between implants, and experience of the operator was evaluated. Many studies have shown that trueness tends to decrease as the distance between the implants and the scan range increases. It was agreed that the implant angle did not affect either trueness or precision. Regarding other factors, the results varied among studies. Therefore, the effects of these parameters are not clear. CONCLUSIONS: Heterogeneity in the research methodology was prevalent among the studies considered in this review. Therefore, we cannot make a decisive statement regarding the trueness and precision of digital implant impressions by IOSs. So far, the comparison of the numerical values of error between studies has yet to elucidate any clear answers, despite small methodological differences.

Effects of different types of intraoral scanners and scanning ranges on the precision of digital implant impressions in edentulous maxilla: An in vitro study.

OBJECTIVE: This study aimed to evaluate the precision of digital implant impressions in comparison with conventional impressions and assess the impact of the scanning range on precision. MATERIALS AND METHODS: An edentulous maxilla model with six implants was scanned with four intraoral scanners (IOSs) and a dental laboratory scanner five times each, and stereolithography (STL) data were generated. A conventional silicone impression was made, and a model was fabricated, which was scanned using the laboratory scanner. This procedure was also repeated five times. Nine different ranges of interest (ROIs) were defined, and the average discrepancies of the measurement points between each pair of STL images out of five for each ROI were calculated. The effects of "impression method" and "ROI" on precision, as evaluated by the averaged discrepancy, were tested by two-way analysis of variance (p < .05). RESULTS: The effects of "impression methods" and "ROI" and their interactions were statistically significant. The discrepancies in the scanned datasets of the dental laboratory scanner were significantly lower than those in the other impression methods. The discrepancies of the IOSs were comparable with those of the laboratory scanner when the ROI was limited, however; the discrepancies deteriorated when the ROI expanded across the arch, while those of the laboratory scanner remained stable irrespective of the ROI. CONCLUSIONS: Within the limitation of this in vitro study, digital implant impressions by IOSs may show clinically acceptable precision when the scan range is limited, such as in 3-unit superstructure supported by two implants.

NMD-degradome sequencing reveals ribosome-bound intermediates with 3'-end non-templated nucleotides.

Nonsense-mediated messenger RNA decay (NMD) controls mRNA quality and degrades physiologic mRNAs to fine-tune gene expression in changing developmental or environmental milieus. NMD requires that its targets are removed from the translating pool of mRNAs. Since the decay steps of mammalian NMD remain unknown, we developed assays to isolate and sequence direct NMD decay intermediates transcriptome-wide based on their co-immunoprecipitation with phosphorylated UPF1, which is the active form of this essential NMD factor. We show that, unlike steady-state UPF1, phosphorylated UPF1 binds predominantly deadenylated mRNA decay intermediates and activates NMD cooperatively from 5'- and 3'-ends. We leverage method modifications to characterize the 3'-ends of NMD decay intermediates, show that they are ribosome-bound, and reveal that some are subject to the addition of non-templated nucleotide. Uridines are added by TUT4 and TUT7 terminal uridylyl transferases and removed by the Perlman syndrome-associated exonuclease DIS3L2. The addition of other non-templated nucleotides appears to inhibit decay.

Publisher Correction: NMD-degradome sequencing reveals ribosome-bound intermediates with 3'-end non-templated nucleotides.

In the version of this paper originally published, in the PDF references 48-55 appeared in the reference list for the Methods section although they should have been in the reference list for the main text. The error has been corrected in the PDF now available.

Transcriptional coactivator PGC-1α contains a novel CBP80-binding motif that orchestrates efficient target gene expression.

Although peroxisome proliferator-activated receptor-γ (PPARγ) coactivator 1α (PGC-1α) is a well-established transcriptional coactivator for the metabolic adaptation of mammalian cells to diverse physiological stresses, the molecular mechanism by which it functions is incompletely understood. Here we used in vitro binding assays, X-ray crystallography, and immunoprecipitations of mouse myoblast cell lysates to define a previously unknown cap-binding protein 80 (CBP80)-binding motif (CBM) in the C terminus of PGC-1α. We show that the CBM, which consists of a nine-amino-acid α helix, is critical for the association of PGC-1α with CBP80 at the 5' cap of target transcripts. Results from RNA sequencing demonstrate that the PGC-1α CBM promotes RNA synthesis from promyogenic genes. Our findings reveal a new conduit between DNA-associated and RNA-associated proteins that functions in a cap-binding protein surveillance mechanism, without which efficient differentiation of myoblasts to myotubes fails to occur.

Evidence for convergent evolution of SINE-directed Staufen-mediated mRNA decay.

Primate-specific Alu short interspersed elements (SINEs) as well as rodent-specific B and ID (B/ID) SINEs can promote Staufen-mediated decay (SMD) when present in mRNA 3'-untranslated regions (3'-UTRs). The transposable nature of SINEs, their presence in long noncoding RNAs, their interactions with Staufen, and their rapid divergence in different evolutionary lineages suggest they could have generated substantial modification of posttranscriptional gene-control networks during mammalian evolution. Some of the variation in SMD regulation produced by SINE insertion might have had a similar regulatory effect in separate mammalian lineages, leading to parallel evolution of the Staufen network by independent expansion of lineage-specific SINEs. To explore this possibility, we searched for orthologous gene pairs, each carrying a species-specific 3'-UTR SINE and each regulated by SMD, by measuring changes in mRNA abundance after individual depletion of two SMD factors, Staufen1 (STAU1) and UPF1, in both human and mouse myoblasts. We identified and confirmed orthologous gene pairs with 3'-UTR SINEs that independently function in SMD control of myoblast metabolism. Expanding to other species, we demonstrated that SINE-directed SMD likely emerged in both primate and rodent lineages >20-25 million years ago. Our work reveals a mechanism for the convergent evolution of posttranscriptional gene regulatory networks in mammals by species-specific SINE transposition and SMD.

UPF1 helicase promotes TSN-mediated miRNA decay.

While microRNAs (miRNAs) regulate the vast majority of protein-encoding transcripts, little is known about how miRNAs themselves are degraded. We recently described Tudor-staphylococcal/micrococcal-like nuclease (TSN)-mediated miRNA decay (TumiD) as a cellular pathway in which the nuclease TSN promotes the decay of miRNAs that contain CA and/or UA dinucleotides. While TSN-mediated degradation of either protein-free or AGO2-loaded miRNAs does not require the ATP-dependent RNA helicase UPF1 in vitro, we report here that cellular TumiD requires UPF1. Results from experiments using AGO2-loaded miRNAs in duplex with target mRNAs indicate that UPF1 can dissociate miRNAs from their mRNA targets, making the miRNAs susceptible to TumiD. miR-seq (deep sequencing of miRNAs) data reveal that the degradation of ∼50% of candidate TumiD targets in T24 human urinary bladder cancer cells is augmented by UPF1. We illustrate the physiological relevance by demonstrating that UPF1-augmented TumiD promotes the invasion of T24 cells in part by degrading anti-invasive miRNAs so as to up-regulate the expression of proinvasive proteins.

Tudor-SN-mediated endonucleolytic decay of human cell microRNAs promotes G1/S phase transition.

MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression. The pathways that mediate mature miRNA decay are less well understood than those that mediate miRNA biogenesis. We found that functional miRNAs are degraded in human cells by the endonuclease Tudor-SN (TSN). In vitro, recombinant TSN initiated the decay of both protein-free and Argonaute 2-loaded miRNAs via endonucleolytic cleavage at CA and UA dinucleotides, preferentially at scissile bonds located more than five nucleotides away from miRNA ends. Cellular targets of TSN-mediated decay defined using microRNA sequencing followed this rule. Inhibiting TSN-mediated miRNA decay by CRISPR-Cas9 knockout of TSN inhibited cell cycle progression by up-regulating a cohort of miRNAs that down-regulates mRNAs that encode proteins critical for the G1-to-S phase transition. Our study indicates that targeting TSN nuclease activity could inhibit pathological cell proliferation.

Purification of dFMR1-containing complexes using tandem affinity purification.

Fragile X syndrome results from the lack of FMR1 expression. To understand how the lack of FMR1 function leads to the syndrome, we are studying the Drosophila FMR1 related protein (dFMR1). We performed affinity purification of dFMR1-associated complexes from cultured Drosophila S2 cells and found that dFMR1 associates with a key component of RNA interference, AGO2. Our finding suggests cross talk between the fragile X syndrome protein and RNA interference. In this chapter, we describe a tandem affinity purification method to isolate the protein components and small RNAs in the dFMR1-associated complexes.

Roles of R2D2, a cytoplasmic D2 body component, in the endogenous siRNA pathway in Drosophila.

Endogenous small interfering RNAs (endo-siRNAs) in Drosophila are processed by Dicer-2 (Dcr-2) and loaded onto Ago2 by the Dcr-2/R2D2 heterodimer. In r2d2 mutants, the level of endo-siRNAs is unchanged, but endo-siRNAs are misloaded onto Ago1. However, the mechanism underlying the control of endo-siRNA sorting by R2D2 remains unknown. Here, we show that R2D2 controls endo-siRNA sorting by localizing Dcr-2, and presumably endo-siRNA duplexes, to cytoplasmic foci, D2 bodies. Ago2, but not Ago1, localized to D2 bodies. dsRNA-binding-deficient mutant, but not wild-type, R2D2 failed to localize D2 bodies and caused endo-siRNA misdirection to Ago1 in R2D2-depleted cells. However, R2D2 was dispensable for sorting miRNAs and exogenous siRNAs onto Ago1 and Ago2, respectively, in vivo. Endo- and exo-siRNA guide selection also occurred R2D2 independently. The functions of R2D2 are required to avoid endo-siRNA misdirection to Ago1, because Ago1 is capable of loading incompletely complementary miRNA duplexes and endo-siRNA duplexes.

Up-regulation of Imp3 confers in vivo tumorigenicity on murine osteosarcoma cells.

Osteosarcoma is a high-grade malignant bone tumor that manifests ingravescent clinical behavior. The intrinsic events that confer malignant properties on osteosarcoma cells have remained unclear, however. We previously established two lines of mouse osteosarcoma cells: AX cells, which are able to form tumors in syngeneic mice, and AXT cells, which were derived from such tumors and acquired an increased tumorigenic capacity during tumor development. We have now identified Igf2 mRNA-binding protein3 (Imp3) as a key molecule responsible for this increased tumorigenicity of AXT cells in vivo. Imp3 is consistently up-regulated in tumors formed by AX cells, and its expression in these cells was found to confer malignant properties such as anchorage-independent growth, loss of contact inhibition, and escape from anoikis in vitro. The expression level of Imp3 also appeared directly related to tumorigenic ability in vivo which is the critical determination for tumor-initiating cells. The effect of Imp3 on tumorigenicity of osteosarcoma cells did not appear to be mediated through Igf2-dependent mechanism. Our results implicate Imp3 as a key regulator of stem-like tumorigenic characteristics in osteosarcoma cells and as a potential therapeutic target for this malignancy.

Chromatin-associated RNA interference components contribute to transcriptional regulation in Drosophila.

RNA interference (RNAi) pathways have evolved as important modulators of gene expression that operate in the cytoplasm by degrading RNA target molecules through the activity of short (21-30 nucleotide) RNAs. RNAi components have been reported to have a role in the nucleus, as they are involved in epigenetic regulation and heterochromatin formation. However, although RNAi-mediated post-transcriptional gene silencing is well documented, the mechanisms of RNAi-mediated transcriptional gene silencing and, in particular, the role of RNAi components in chromatin dynamics, especially in animal multicellular organisms, are elusive. Here we show that the key RNAi components Dicer 2 (DCR2) and Argonaute 2 (AGO2) associate with chromatin (with a strong preference for euchromatic, transcriptionally active, loci) and interact with the core transcription machinery. Notably, loss of function of DCR2 or AGO2 showed that transcriptional defects are accompanied by the perturbation of RNA polymerase II positioning on promoters. Furthermore, after heat shock, both Dcr2 and Ago2 null mutations, as well as missense mutations that compromise the RNAi activity, impaired the global dynamics of RNA polymerase II. Finally, the deep sequencing of the AGO2-associated small RNAs (AGO2 RIP-seq) revealed that AGO2 is strongly enriched in small RNAs that encompass the promoter regions and other regions of heat-shock and other genetic loci on both the sense and antisense DNA strands, but with a strong bias for the antisense strand, particularly after heat shock. Taken together, our results show that DCR2 and AGO2 are globally associated with transcriptionally active loci and may have a pivotal role in shaping the transcriptome by controlling the processivity of RNA polymerase II.

Biochemical analyzes of endogenous argonaute complexes immunopurified with anti-Argonaute monoclonal antibodies.

Argonaute proteins are key factors in RNA silencing. After association with small RNAs of 20-30 -nucleotides, Argonaute proteins are targeted to homologous RNA molecules that are to be silenced. To understand the functional contributions of Argonaute proteins to RNA silencing at a biochemical level, immunoisolation of Argonaute proteins from living cells of various organisms has been performed. This has enabled the analysis of Argonaute-associated proteins and RNAs. Identifying the small RNAs that associate with individual Argonaute proteins, for instance, could help to elucidate the silencing pathways in which particular Argonaute proteins are involved. However, it is also necessary to note that the results obtained through such biochemical analyzes are greatly affected by the quality and properties of the antibodies used, as well as by the immunoprecipitation conditions employed, including buffer contents and/or salt concentration. In this chapter, we describe fundamental methods for immunoprecipitating Argonaute proteins using monoclonal antibodies as well as for detecting associated proteins and small RNAs. Furthermore, we will also explain how various parameters, such as antibody properties and buffer conditions, can alter the production and interpretation of experimental data.

Isolation and partial characterization of the Chlamydomonas reinhardtii γ-glutamyl kinase.

We isolated a Chlamydomonas gene encoding putative γ-glutamyl kinase (GK), an enzyme that catalyzes the first step of proline biosynthesis. Using an Escherichia coli auxotroph and a purified recombinant protein, we show that Chlamydomonas GK is a functional GK. The sensitivity of this kinase to feedback inhibition by proline was lower than in that of microbial GKs previously reported.