Chimera RNA transcribed from integrated HPV18 genome with adjacent host genomic region promotes oncogenic gene expression through condensate formation.

Most cervical cancers are caused by human papillomavirus (HPV) infection. In HeLa cells, the HPV18 viral genome is integrated at chromosome 8q24.21 and activates transcription of the proto-oncogene c-Myc. However, the mechanism of how the integrated HPV genome and its transcribed RNAs exhibit transcription activation function has not been fully elucidated. In this study, we found that HPV18 transcripts contain an enhancer RNA-like function to activate proximal genes including CCAT1-5L and c-Myc. We showed that the human genome-integrated HPV18 genes are activated by transcription coregulators including BRD4 and Mediator. The transcribed HPV18 RNAs form a liquid-like condensate at chromosome 8q24.21 locus, which in turn accumulates RNA polymerase II. Moreover, we focused on a relatively uncharacterized transcript from the upstream region of CCAT1, named URC. The URC RNA is transcribed as a chimera RNA with HPV18 and is composed of the 3'-untranslated region of the HPV18 transcript. We experimentally showed that the URC contributes to stabilization of HPV18 RNAs by supplying a polyadenylation site for the HPV18 transcript. Our findings suggest that integrated HPV18 at 8q24.21 locus produces HPV18-URC chimera RNA and promotes tumorigenesis through RNA-based condensate formation.

MED26-containing Mediator may orchestrate multiple transcription processes through organization of nuclear bodies.

Mediator is a coregulatory complex that plays essential roles in multiple processes of transcription regulation. One of the human Mediator subunits, MED26, has a role in recruitment of the super elongation complex (SEC) to polyadenylated genes and little elongation complex (LEC) to non-polyadenylated genes, including small nuclear RNAs (snRNAs) and replication-dependent histone (RDH) genes. MED26-containing Mediator plays a role in 3' Pol II pausing at the proximal region of transcript end sites in RDH genes through recruitment of Cajal bodies (CBs) to histone locus bodies (HLBs). This finding suggests that Mediator is involved in the association of CBs with HLBs to facilitate 3' Pol II pausing and subsequent 3'-end processing by supplying 3'-end processing factors from CBs. Thus, we argue the possibility that Mediator is involved in the organization of nuclear bodies to orchestrate multiple processes of gene transcription.

La(III)-Catalysed degradation of polyesters to monomers via transesterifications.

Tris(acetylacetonato)lanthanum(III) (1 mol%) catalyses the degradation of poly(butylene succinate) (Mw = 90 700) by transesterification in MeOH at 90 °C for 4 h, thus affording dimethyl succinate (>99% yield) and 1,4-butanediol (98% yield). Moreover, the quantitative degradations of poly(ethylene adipate), poly(ethylene terephthalate) and poly(butylene terephthalate) are also reported.

Dual function of SF3B2 on chromatin and RNA to regulate transcription in head and neck squamous cell carcinoma.

RNA is spliced concomitantly with transcription and the process is organized by RNA splicing factors, transcriptional regulators, and chromatin regulators. RNA is spliced in close proximity to transcription machinery. Hence, some RNA splicing factors may play a role in transcription. Here, we show that the splicing factor SF3B2 binds to gene regulatory elements and mRNA to modulate transcription and RNA stability in head and neck squamous cell carcinoma cells. High SF3B2 expression leads to poor prognosis in patients with head and neck squamous cell carcinoma and to progression of tumor growth in mice. SF3B2 promotes tumor growth, owing to its involvement in activation of gene expression associated with mitochondrial electron transport and transcription regulatory region DNA binding. SF3B2 is enriched around the promoter element on chromatin and the transcription termination site on RNA. SF3B2 is involved in the regulation of RNA stability. According to the SF3B2-binding profile, SF3B2 regulates RNA polymerase II activity, in addition to regulating RNA splicing. Mechanistically, SF3B2 promotes the binding of structural maintenance of chromosomes 1A and CCCTC-binding factor (CTCF) to the SF3B2-binding genomic regions. SF3B2 also modulates CTCF transcriptional activity. Our findings indicate that SF3B2 has a dual function in both transcription and RNA stability, leading to head and neck squamous cell carcinoma progression.

The 3' Pol II pausing at replication-dependent histone genes is regulated by Mediator through Cajal bodies' association with histone locus bodies.

Non-polyadenylated mRNAs of replication-dependent histones (RDHs) are synthesized by RNA polymerase II (Pol II) at histone locus bodies (HLBs). HLBs frequently associate with Cajal bodies (CBs), in which 3'-end processing factors for RDH genes are enriched; however, this association's role in transcription termination of RDH genes remains unclear. Here, we show that Pol II pauses immediately upstream of transcript end sites of RDH genes and Mediator plays a role in this Pol II pausing through CBs' association with HLBs. Disruption of the Mediator docking site for Little elongation complex (LEC)-Cap binding complex (CBC)-Negative elongation factor (NELF), components of CBs, interferes with CBs' association with HLBs and 3' Pol II pausing, resulting in increased aberrant unprocessed RDH gene transcripts. Our findings suggest Mediator's involvement in CBs' association with HLBs to facilitate 3' Pol II pausing and subsequent 3'-end processing of RDH genes by supplying 3'-end processing factors.

Digold(I) Thianthrenyl Complexes. Effect of Diphosphine Ligands on Molecular Structures in the Solid State and in Solution.

A series of digold complexes possessing two thianthrenyl ligands, Au2(Thi)2(Ph2P(CH2) n PPh2) (Thi: 1-thianthrenyl; 1: n = 1, 2: n = 2, 3: n = 3, 4: n = 4), were prepared and characterized by crystallographic and spectroscopic measurements. X-ray crystallography of complexes 1 and 3 revealed U-shaped structures with short Au-Au distances [3.2171(3) Å and 3.0735(2) Å]. Complex 2 and three of the four structure-determined molecules of complex 4 showed structures without Au-Au contacts. UV-vis spectroscopic measurements of 1-4 and TD-DFT calculations of the two conformers of 1 revealed that complexes 1 and 3 in the solution phase contained conformers with Au(I)-Au(I) interactions in a much higher proportion than complexes 2 and 4. As a result, complexes with diphosphine ligands containing an odd number of methylene groups preferred structures with Au-Au interactions in the solid state and in solution. Oxidation of 1 with 2 equiv of PhICl2 yielded a mixture of monomeric and dimeric thianthrenes and its dimer via ligand elimination and C-C coupling, respectively.

A Macrocyclic Gold(I)-Biphenylene Complex: Triangular Molecular Structure with Twisted Au2 (diphosphine) Corners and Reductive Elimination of [6]Cycloparaphenylene.

The digold(I) complex [Au2 Cl2 (Cy2 PCH2 PCy2 )] reacts with 4,4'-diphenylene diboronic acid to form a triangular macrocyclic complex with twisted Au-P-C-P-Au groups at the three corners. The synthesis of the complex and its chemical oxidation produced [6]cycloparaphenylene ([6]CPP) in 59 % overall yield.

The role of Mediator and Little Elongation Complex in transcription termination.

Mediator is a coregulatory complex that regulates transcription of Pol II-dependent genes. Previously, we showed that human Mediator subunit MED26 plays a role in the recruitment of Super Elongation Complex (SEC) or Little Elongation Complex (LEC) to regulate the expression of certain genes. MED26 plays a role in recruiting SEC to protein-coding genes including c-myc and LEC to small nuclear RNA (snRNA) genes. However, how MED26 engages SEC or LEC to regulate distinct genes is unclear. Here, we provide evidence that MED26 recruits LEC to modulate transcription termination of non-polyadenylated transcripts including snRNAs and mRNAs encoding replication-dependent histone (RDH) at Cajal bodies. Our findings indicate that LEC recruited by MED26 promotes efficient transcription termination by Pol II through interaction with CBC-ARS2 and NELF/DSIF, and promotes 3' end processing by enhancing recruitment of Integrator or Heat Labile Factor to snRNA or RDH genes, respectively.

Gain-of-Function MN1 Truncation Variants Cause a Recognizable Syndrome with Craniofacial and Brain Abnormalities.

MN1 was originally identified as a tumor-suppressor gene. Knockout mouse studies have suggested that Mn1 is associated with craniofacial development. However, no MN1-related phenotypes have been established in humans. Here, we report on three individuals who have de novo MN1 variants that lead to a protein lacking the carboxyl (C) terminus and who presented with severe developmental delay, craniofacial abnormalities with specific facial features, and structural abnormalities in the brain. An in vitro study revealed that the deletion of the C-terminal region led to increased protein stability, an inhibitory effect on cell proliferation, and enhanced MN1 aggregation in nuclei compared to what occurred in the wild type, suggesting that a gain-of-function mechanism is involved in this disease. Considering that C-terminal deletion increases the fraction of intrinsically disordered regions of MN1, it is possible that altered phase separation could be involved in the mechanism underlying the disease. Our data indicate that MN1 participates in transcriptional regulation of target genes through interaction with the transcription factors PBX1, PKNOX1, and ZBTB24 and that mutant MN1 impairs the binding with ZBTB24 and RING1, which is an E3 ubiquitin ligase. On the basis of our findings, we propose the model that C-terminal deletion interferes with MN1's interaction molecules related to the ubiquitin-mediated proteasome pathway, including RING1, and increases the amount of the mutant protein; this increase leads to the dysregulation of MN1 target genes by inhibiting rapid MN1 protein turnover.

Total Syntheses and Configuration Assignments of JBIR-06 and Related Depsipeptides.

The first total syntheses of JBIR-06 and two analogous depsipeptides, 12-membered antimycin-class antibiotics, have been accomplished via Shiina macrolactonization. Comparison of the spectroscopic data of the synthesized compounds with those reported for natural products verified that the absolute configutation of the natural products was (2 S,4 S,6 S,7 R,14 S).

Association between third molar agenesis patterns and agenesis of other teeth in a Japanese orthodontic population.

The purpose of this study was to investigate the patterns of third molar agenesis and incidence of agenesis of other permanent teeth in a Japanese orthodontic patient group. A total of 417 Japanese subjects (134 males and 283 females) with agenesis of one or more third molars were divided into four groups according to the agenesis pattern, and 874 other Japanese subjects (302 males and 572 females) without third molar agenesis were assigned to a control group. Panoramic radiographs and medical and dental records were used to examine for tooth agenesis. The Chi-square test and odds ratio were used to make statistical comparisons. The prevalence of third molar agenesis worked out at 32.3% with no statistically significant gender difference. A high prevalence rate of agenesis of third molars, unilateral or bilateral, could be considered characteristic of the Japanese orthodontic population. Significant increases in occurrence of oligodontia, and unilateral or bilateral agenesis of other teeth, including maxillary lateral incisors and maxillary and mandibular second premolars, were observed in all or almost all of the third molar agenesis groups, compared with the controls. A significantly increased prevalence rate of mandibular lateral incisor agenesis was observed in almost all of the third molar agenesis groups. The Japanese patients with third molar agenesis had a significantly increased occurrence of oligodontia, and unilateral or bilateral agenesis of maxillary and mandibular lateral incisors and second premolars, except for bilateral agenesis of mandibular lateral incisors.

Bidirectional binding property of high glycine-tyrosine keratin-associated protein contributes to the mechanical strength and shape of hair.

Since their first finding in wool 50years ago, keratin-associated proteins (KAPs), which are classified into three groups; high sulfur (HS) KAPs, ultra high sulfur (UHS) KAPs, and high glycine-tyrosine (HGT) KAPs, have been the target of curiosity for scientists due to their characteristic amino acid sequences. While HS and UHS KAPs are known to function in disulfide bond crosslinking, the function of HGT KAPs remains unknown. To clarify the function as well as the binding partners of HGT KAPs, we prepared KAP8.1 and other KAP family proteins, the trichocyte intermediate filament proteins (IFP) K85 and K35, the head domain of K85, and the C subdomain of desmoplakin C-terminus (DPCT-C) and investigated the interactions between them in vitro. Western blot analysis and isothermal titration calorimetry (ITC) indicate that KAP8.1 binds to the head domain of K85, which is helically aligned around the axis of the intermediate filament (IF). From these results and transmission electron microscopy (TEM) observations of bundled filament complex in vitro, we propose that the helical arrangement of IFs found in the orthocortex, which is uniquely distributed on the convex fiber side of the hair, is regulated by KAP8.1. Structure-dependent binding of DPCT-C to trichocyte IFP was confirmed by Western blotting, ITC, and circular dichroism. Moreover, DPCT-C also binds to some HGT KAPs. It is probable that such bidirectional binding property of HGT KAPs contribute to the mechanical robustness of hair.

Initial shear bond strength of orthodontic brackets bonded to bleached teeth with a self-etching adhesive system.

OBJECTIVES: To examine the initial shear bond strength of orthodontic brackets bonded to bleached teeth with a self-etching adhesive system, as well as the effect of the length of time after bleaching on the initial bond strength. METHOD AND MATERIALS: Ninety premolars were collected and divided equally among a control group (CG) of unbleached teeth with brackets bonded, an immediate group (IG) with teeth bonded soon after bleaching, and a delayed group (DG) consisting of teeth bleached and then immersed in artificial saliva for 7 days before bracket bonding. Subsequently, the teeth of each group were equally assigned to two different primer adhesive systems: the acid-etch-and-rinse adhesive system (35% phosphoric acid gel and Transbond XT primer) and the self-etching adhesive system (Transbond Plus self-etching primer). Initial bond strength was measured 24 hours after bracket bonding. RESULTS: IG registered the lowest mean initial bond strength when the self-etching adhesive system was in use, followed by DG and CG (in that order). As for the acid-etch-and-rinse adhesive system, the mean initial bond strength was significantly lower in IG than in CG and DG. Significant differences in mean initial bond strength were noted between the two adhesive systems in IG and DG. CONCLUSION: In trials using a self-etching adhesive system, the mean initial bond strength that declined immediately after bleaching returned to a clinically acceptable level of 6 MPa after the specimens were immersed in artificial saliva for 7 days but fell short of the level of unbleached teeth.

Mapping ultra-weak protein-protein interactions between heme transporters of Staphylococcus aureus.

Iron is an essential nutrient for the proliferation of Staphylococcus aureus during bacterial infections. The iron-regulated surface determinant (Isd) system of S. aureus transports and metabolizes iron porphyrin (heme) captured from the host organism. Transportation of heme across the thick cell wall of this bacterium requires multiple relay points. The mechanism by which heme is physically transferred between Isd transporters is largely unknown because of the transient nature of the interactions involved. Herein, we show that the IsdC transporter not only passes heme ligand to another class of Isd transporter, as previously known, but can also perform self-transfer reactions. IsdA shows a similar ability. A genetically encoded photoreactive probe was used to survey the regions of IsdC involved in self-dimerization. We propose an updated model that explicitly considers self-transfer reactions to explain heme delivery across the cell wall. An analogous photo-cross-linking strategy was employed to map transient interactions between IsdC and IsdE transporters. These experiments identified a key structural element involved in the rapid and specific transfer of heme from IsdC to IsdE. The resulting structural model was validated with a chimeric version of the homologous transporter IsdA. Overall, our results show that the ultra-weak interactions between Isd transporters are governed by bona fide protein structural motifs.

Catalytic activity of MsbA reconstituted in nanodisc particles is modulated by remote interactions with the bilayer.

ATP-binding cassette (ABC) transporters couple hydrolysis of ATP with vectorial transport across the cell membrane. We have reconstituted ABC transporter MsbA in nanodiscs of various sizes and lipid compositions to test whether ATPase activity is modulated by the properties of the bilayer. ATP hydrolysis rates, Michaelis-Menten parameters, and dissociation constants of substrate analog ATP-γ-S demonstrated that physicochemical properties of the bilayer modulated binding and ATPase activity. This is remarkable when considering that the catalytic unit is located ~50Šfrom the transmembrane region. Our results validated the use of nanodiscs as an effective tool to reconstitute MsbA in an active catalytic state, and highlighted the close relationship between otherwise distant transmembrane and ATPase modules.

Stepwise characterization of the thermodynamics of trichocyte intermediate filament protein supramolecular assembly.

Trichocyte intermediate filament protein (IFP) is a heterodimeric complex that plays a pivotal role in the hair shaft for its mechanical strength, hair shape, and so on. Trichocyte IFP consists of acidic-type IFP and basic-type IFP, and the well-studied supramolecular assembly process of the complex occurs via the following steps: dimer formation, tetramer formation, formation of the lateral 32mer, and the elongation of the 32mer. Among these interactions, only the dimer formation, owing to coiled-coil interaction, has been described in detail; the nature of other interactions remains unspecified. For each assembly step, we report interaction isotherms obtained by means of isothermal titration calorimetry at various urea and NaCl concentrations. Decreasing the urea concentration generally promotes protein refolding, and we therefore expected to observe endothermic interactions owing to the refolding process. However, exothermic interactions were observed at 4 and 2 M urea, along with various characteristic endothermic interactions at the other urea concentrations as well as NaCl titration. The thermal responses described herein enabled us to analyze the protein supramolecular assembly process in a stepwise manner.

Effects of tooth bleaching on shear bond strength of brackets rebonded with a self-etching adhesive system.

The purpose of this study was to ascertain the effects of tooth bleaching on the shear bond strength of orthodontic brackets rebonded with a self-etching adhesive system. A total of 39 premolars were collected and divided into three equal groups: in group 1 bracket bonding was performed without bleaching treatment; specimens in group 2 were bonded immediately after bleaching; and group 3 teeth were bleached, then immersed in artificial saliva and left for 7 days before bonding. The shear bond strength was measured, with the bonding/debonding procedures repeated once after the first debonding, and the bracket/adhesive failure modes were evaluated by the adhesive remnant index after each debonding. Excepting the mean shear bond strength for group 2 after the first debonding, the overall mean values reached the minimum clinical requirement of 6 MPa. The mean values at the first and second debondings were significantly higher in groups 1 and 3 than in group 2. Between groups 1 and 3, significant differences were noted at the first debonding, but not at the second debonding. Group 2 showed significant differences in mean shear bond strength between the first and second debondings. Bond failure at the enamel-adhesive interface occurred more frequently in group 2 than in groups 1 or 3 after the first debonding. The bracket-rebonding procedure can recover the reduced shear bond strength caused by immediate bonding after bleaching to a clinically acceptable level, but not to the prebleaching level.

Maxillary first molar agenesis and other dental anomalies.

OBJECTIVE: To explore the association of maxillary first molar agenesis with other dental anomalies in Japanese orthodontic patients. MATERIALS AND METHODS: A total of 32 subjects with one or two congenitally missing maxillary first molars (group M) were selected and divided into group 1M (12 subjects with one maxillary first molar missing) and group 2M (20 subjects with two maxillary first molars missing). As controls, 32 sex-matched subjects without agenesis of maxillary first molars were collected (group C). Panoramic and periapical radiographs, cephalograms, study models, intraoral photographs, and anamnestic data were used to identify anomalies of permanent teeth. Chi-square, Fisher's exact, Kruskal-Wallis, and Steel-Dwass tests were used to make statistical comparisons. RESULTS: The prevalence rates of tooth agenesis other than the maxillary first molars and advanced tooth agenesis, with third molars excluded, were significantly higher in group 2M than in group C. The absence of second premolars was most common. The prevalence rate of third molar agenesis was significantly higher in groups 1M and 2M than in group C. The occurrence of symmetrical agenesis of the mandibular third molars was particularly notable in group 2M as compared to group 1M, in which maxillary third molar agenesis was predominant. There was no significant association between maxillary first molar agenesis and other dental anomalies, except for agenesis of teeth other than maxillary first molars. CONCLUSION: Agenesis of maxillary first molars is associated with a higher prevalence of other permanent tooth agenesis and advanced tooth agenesis.

Solubilization of membrane proteins with novel N-acylamino acid detergents.

N-Acylamino acids are a new family of versatile biological surfactants capable of extracting integral membrane proteins of various topologies from the biological membrane, in many instances surpassing the efficiency of commercial detergents.

Non-denaturing solubilization of inclusion bodies.

It has been a conventional notion that cytoplasmic recombinant expression leads to either soluble protein or inclusion bodies. In the latter case, it was always assumed that proteins in inclusion bodies (IBs) are more or less unfolded and hence require complete denaturing condition for solubilization, which uses strong detergents, urea or guanidine hydrochloride. However, we often observe distribution of expressed proteins in both soluble and insoluble fractions. In such expression, IBs are often loose and of flocculate morphology. We believe that such distribution is due to association of near native structures of the expressed proteins, which cause either aggregation into insoluble fractions or unstable soluble proteins. In our experience, although not reported by others, interleukin-1alpha, interferon-gamma, tumor necrosis factors, fibroblast growth factors, His-tagged fyn kinase and many other proteins showed such behavior. If this occurs, we have experienced problems of instability, low yield and insolubility whether purification is done from the soluble fraction or by refolding of IBs. Arginine has shown great promise in non-denaturating solubilization of some of these proteins we have tested.