Structures of Vac8-containing protein complexes reveal the underlying mechanism by which Vac8 regulates multiple cellular processes.

Vac8, a yeast vacuolar protein with armadillo repeats, mediates various cellular processes by changing its binding partners; however, the mechanism by which Vac8 differentially regulates these processes remains poorly understood. Vac8 interacts with Nvj1 to form the nuclear-vacuole junction (NVJ) and with Atg13 to mediate cytoplasm-to-vacuole targeting (Cvt), a selective autophagy-like pathway that delivers cytoplasmic aminopeptidase I directly to the vacuole. In addition, Vac8 associates with Myo2, a yeast class V myosin, through its interaction with Vac17 for vacuolar inheritance from the mother cell to the emerging daughter cell during cell divisions. Here, we determined the X-ray crystal structure of the Vac8-Vac17 complex and found that its interaction interfaces are bipartite, unlike those of the Vac8-Nvj1 and Vac8-Atg13 complexes. When the key amino acids present in the interface between Vac8 and Vac17 were mutated, vacuole inheritance was severely impaired in vivo. Furthermore, binding of Vac17 to Vac8 prevented dimerization of Vac8, which is required for its interactions with Nvj1 and Atg13, by clamping the H1 helix to the ARM1 domain of Vac8 and thereby preventing exposure of the binding interface for Vac8 dimerization. Consistently, the binding affinity of Vac17-bound Vac8 for Nvj1 or Atg13 was markedly lower than that of free Vac8. Likewise, free Vac17 had no affinity for the Vac8-Nvj1 and Vac8-Atg13 complexes. These results provide insights into how vacuole inheritance and other Vac8-mediated processes, such as NVJ formation and Cvt, occur independently of one another.

MSH2-MSH3 promotes DNA end resection during homologous recombination and blocks polymerase theta-mediated end-joining through interaction with SMARCAD1 and EXO1.

DNA double-strand break (DSB) repair via homologous recombination is initiated by end resection. The extent of DNA end resection determines the choice of the DSB repair pathway. Nucleases for end resection have been extensively studied. However, it is still unclear how the potential DNA structures generated by the initial short resection by MRE11-RAD50-NBS1 are recognized and recruit proteins, such as EXO1, to DSB sites to facilitate long-range resection. We found that the MSH2-MSH3 mismatch repair complex is recruited to DSB sites through interaction with the chromatin remodeling protein SMARCAD1. MSH2-MSH3 facilitates the recruitment of EXO1 for long-range resection and enhances its enzymatic activity. MSH2-MSH3 also inhibits access of POLθ, which promotes polymerase theta-mediated end-joining (TMEJ). Collectively, we present a direct role of MSH2-MSH3 in the initial stages of DSB repair by promoting end resection and influencing the DSB repair pathway by favoring homologous recombination over TMEJ.

Tuning Hot Carrier Dynamics of InP/ZnSe/ZnS Quantum Dots by Shell Morphology Control.

Isotropic InP/ZnSe/ZnS quantum dots (QDs) are prepared at a high reaction temperature, which facilitates ZnSe shell growth on random facets of the InP core. Fast crystal growth enables stacking faults elimination, which induces anisotropic growth, and as a result, improves the photoluminescence (PL) quantum yield by nearly 20%. Herein, the effect of the QD morphology on photophysical properties is investigated by observing the PL blinking and ultrafast charge carrier dynamics. It is found that hot hole trapping is considerably suppressed in isotropic InP QDs, indicating that the stacking faults in the anisotropic InP/ZnSe structures act as defects for luminescence. These results highlight the importance of understanding the correlation between QD shapes and hot carrier dynamics, and present a way to design highly luminescent QDs for further promising display applications.

The structure of human EXD2 reveals a chimeric 3' to 5' exonuclease domain that discriminates substrates via metal coordination.

EXD2 (3'-5' exonuclease domain-containing protein 2) is an essential protein with a conserved DEDDy superfamily 3'-5' exonuclease domain. Recent research suggests that EXD2 has two potential functions: as a component of the DNA double-strand break repair machinery and as a ribonuclease for the regulation of mitochondrial translation. Herein, electron microscope imaging analysis and proximity labeling revealed that EXD2 is anchored to the mitochondrial outer membrane through a conserved N-terminal transmembrane domain, while the C-terminal region is cytosolic. Crystal structures of the exonuclease domain in complex with Mn2+/Mg2+ revealed a domain-swapped dimer in which the central α5-α7 helices are mutually crossed over, resulting in chimeric active sites. Additionally, the C-terminal segments absent in other DnaQ family exonucleases enclose the central chimeric active sites. Combined structural and biochemical analyses demonstrated that the unusual dimeric organization stabilizes the active site, facilitates discrimination between DNA and RNA substrates based on divalent cation coordination and generates a positively charged groove that binds substrates.

Electrochemical Charging Effect on the Optical Properties of InP/ZnSe/ZnS Quantum Dots.

Semiconductor quantum dots (QDs) are spotlighted as a key type of emissive material for the next generation of light-emitting diodes (LEDs). This work presents the investigation of the electrochemical charging effect on the absorption and emission of the InP/ZnSe/ZnS QDs with different mid-shell thicknesses. The excitonic peak is gradually bleached during electrochemical charging, which is caused by 1Se (or 1Sh ) state filling when the electron (or hole) is injected into the InP core. Additional charges also lead to photoluminescence (PL) intensity reduction, however, it is greatly mitigated as the mid-shell thickness increases. Various PL measurements reveal that the PL reduction under electrochemical charging is attributed to the acoustic phonon-assisted Auger recombination. Here, the Auger recombination in QDs with a thick mid-shell is reduced under the electrochemically charged condition, indicating that QDs with larger volume are more stable emitters in charge-injecting devices such as LEDs. Furthermore, the negative and positive trion Auger recombination rate constants are estimated, respectively, via electrochemical charging. The negative trion Auger rate constants decrease with an increase in the mid-shell thickness increases, whereas the positive trion Auger rate constants are not heavily reliant on the mid-shell thickness.

Crystal structures of Mmm1 and Mdm12-Mmm1 reveal mechanistic insight into phospholipid trafficking at ER-mitochondria contact sites.

The endoplasmic reticulum (ER)-mitochondria encounter structure (ERMES) comprises mitochondrial distribution and morphology 12 (Mdm12), maintenance of mitochondrial morphology 1 (Mmm1), Mdm34, and Mdm10 and mediates physical membrane contact sites and nonvesicular lipid trafficking between the ER and mitochondria in yeast. Herein, we report two crystal structures of the synaptotagmin-like mitochondrial lipid-binding protein (SMP) domain of Mmm1 and the Mdm12-Mmm1 complex at 2.8 Å and 3.8 Å resolution, respectively. Mmm1 adopts a dimeric SMP structure augmented with two extra structural elements at the N and C termini that are involved in tight self-association and phospholipid coordination. Mmm1 binds two phospholipids inside the hydrophobic cavity, and the phosphate ion of the distal phospholipid is specifically recognized through extensive H-bonds. A positively charged concave surface on the SMP domain not only mediates ER membrane docking but also results in preferential binding to glycerophospholipids such as phosphatidylcholine (PC), phosphatidic acid (PA), phosphatidylglycerol (PG), and phosphatidylserine (PS), some of which are substrates for lipid-modifying enzymes in mitochondria. The Mdm12-Mmm1 structure reveals two Mdm12s binding to the SMP domains of the Mmm1 dimer in a pairwise head-to-tail manner. Direct association of Mmm1 and Mdm12 generates a 210-Å-long continuous hydrophobic tunnel that facilitates phospholipid transport. The Mdm12-Mmm1 complex binds all glycerophospholipids except for phosphatidylethanolamine (PE) in vitro.

Mechanistic insight into the nucleus-vacuole junction based on the Vac8p-Nvj1p crystal structure.

Formation of the nucleus-vacuole junction (NVJ) is mediated by direct interaction between the vacuolar protein Vac8p and the outer nuclear endoplasmic reticulum membrane protein Nvj1p. Herein we report the crystal structure of Vac8p bound to Nvj1p at 2.4-Å resolution. Vac8p comprises a flexibly connected N-terminal H1 helix followed by 12 armadillo repeats (ARMs) that form a right-handed superhelical structure. The extended 80-Å-long loop of Nvj1p specifically binds the highly conserved inner groove formed from ARM1-12 of Vac8p. Disruption of the Nvj1p-Vac8p interaction results in the loss of tight NVJs, which impairs piecemeal microautophagy of the nucleus in Saccharomyces cerevisiae Vac8p cationic triad (Arg276, Arg317, and Arg359) motifs interacting with Nvj1p are also critical to the recognition of Atg13p, a key component of the cytoplasm-to-vacuole targeting (CVT) pathway, indicating competitive binding to Vac8p. Indeed, mutation of the cationic triad abolishes CVT of Ape1p in vivo. Combined with biochemical data, the crystal structure reveals a Vac8p homodimer formed from ARM1, and this self-association, likely regulated by the flexible H1 helix and the C terminus of Nvj1p, is critical for Vac8p cellular functions.

Pancreatic endocrine-like cells differentiated from human umbilical cords Wharton's jelly mesenchymal stem cells using small molecules.

Following success of pancreatic islet transplantation in the treatment of Type I diabetes mellitus, there is a growing interest in using cell-based treatment approaches. However, severe shortage of donor islets-pancreas impeded the growth, and made researchers to search for an alternative treatment approaches. In this context, recently, stem cell-based therapy has gained more attention. The current study demonstrated that epigenetic modification improves the in vitro differentiation of Wharton's jelly mesenchymal stem cells (WJMSCs) into pancreatic endocrine-like cells. Here we used two histone deacetylase (HDAC) inhibitors namely trichostatin A (TSA) and TMP269. TSA inhibits both class I and II HDACs whereas TMP269 inhibits only class IIa HDACs. WJMSCs were differentiated using a multistep protocol in a serum-free condition with or without TSA pretreatment. A marginal improvement in differentiation was observed after TSA pretreatment though it was not significant. However, exposing endocrine precursor-like cells derived from WJMSCs to TMP269 alone has significantly improved the differentiation toward insulin-producing cells. Further, increase in the expression of paired box 4 (PAX4), insulin, somatostatin, glucose transporter 2 (GLUT2), MAF bZIP transcription factor A (MAFA), pancreatic duodenal homeobox 1 (PDX-1), and NKX6.1 was observed both at messenger RNA and protein levels. Nevertheless, TMP269-treated cells secreted higher insulin upon glucose challenge, and demonstrated increased dithizone staining. These findings suggest that TMP269 may improve the in vitro differentiation of WJMSCs into insulin-producing cells.

Identification of extremely rare mitochondrial disorders by whole exome sequencing.

Whole exome sequencing (WES) is an effective tool for the genetic diagnosis of mitochondrial disorders due to various nuclear genetic defects. In this study, three patients affected by extremely rare mitochondrial disorders caused by nuclear genetic defects are described. The medical records of each patient were reviewed to obtain clinical symptoms, results of biochemical and imaging studies, and muscle biopsies. WES and massive parallel sequencing of whole mtDNA were performed for each patient. The oxygen consumption rate (OCR) and complex activity I and IV was measured. Patients 1 and 2 had exhibited global developmental delay and seizure since early infancy. Blood lactate, the lactate-to-pyruvate ratio, and urinary excretion of Krebs cycle intermediates were markedly elevated. Patient 1 also was noted for ophthalmoplegia. Patient 2 had left ventricular hypertrophy and ataxia. Patient 3 developed dysarthria, gait disturbance, and right-side weakness at age 29. Brain magnetic resonance imaging demonstrated abnormal signal intensity involving the bilateral thalami, midbrain, or pons. Based on WES, patient 1 had p.Glu415Gly and p.Arg484Trp variants in MTO1. In patient 2, p.Gln111ThrfsTer5 and RNA mis-splicing were identified in TSFM. Patient 3 carried p.Met151Thr and p.Met246Lys variants in AARS2. Skin fibroblasts of three patients exhibited decreased OCRs and complex 1 activity, and mitochondrial DNA was normal. These results demonstrate the utility of WES for identifying the genetic cause of extremely rare mitochondrial disorders, which has implications for genetic counseling.

Crystal structure of Mdm12 reveals the architecture and dynamic organization of the ERMES complex.

The endoplasmic reticulum-mitochondria encounter structure (ERMES) is a protein complex that plays a tethering role in physically connecting ER and mitochondria membranes. The ERMES complex is composed of Mdm12, Mmm1, and Mdm34, which have a SMP domain in common, and Mdm10. Here, we report the crystal structure of S. cerevisiae Mdm12. The Mdm12 forms a dimeric SMP structure through domain swapping of the ß1-strand comprising residues 1-7. Biochemical experiments reveal a phospholipid-binding site located along a hydrophobic channel of the Mdm12 structure and that Mdm12 might have a binding preference for glycerophospholipids harboring a positively charged head group. Strikingly, both full-length Mdm12 and Mdm12 truncated to exclude the disordered region (residues 74-114) display the same organization in the asymmetric unit, although they crystallize as a tetramer and hexamer, respectively. Taken together, these studies provide a novel understanding of the overall organization of SMP domains in the ERMES complex, indicating that Mdm12 interacts with Mdm34 through head-to-head contact, and with Mmm1 through tail-to-tail contact of SMP domains.

Prosthodontic treatment of a retrognathic edentulous maxilla demonstrating limited interarch distance: 3.5-year results with fixed and removable implant prostheses.

The prosthodontic treatment of patients with a retrognathic edentulous maxilla should consider the restoration of the lower facial profile and access for oral hygiene. This clinical report describes prosthodontic treatments of a patient with edentulism who presented with repeated fractures of the denture teeth of a maxillary implant-supported complete fixed dental prosthesis (ICFDP) and a mandibular implant-supported overdenture. Considerable plaque accumulation was noted on the ICFDP, which was replaced with an open palatal design implant-supported overdenture. However, the patient experienced difficulty managing the 2 removable prostheses. The patient's mandible was eventually restored with a milled titanium alloy framework ICFDP with metal occlusal surfaces. This combined approach of fixed and removable prostheses was stable at 3.5-year follow-up appointment, without compromising the patient's oral hygiene or comfort.

Cardiomyogenic Differentiation of Human Dental Follicle-derived Stem Cells by Suberoylanilide Hydroxamic Acid and Their In Vivo Homing Property.

The purpose of the present study was to investigate the in vitro cardiomyogenic differentiation potential of human dental follicle-derived stem cells (DFCs) under the influence of suberoylanilide hydroxamic acid (SAHA), a member of the histone deacetylase inhibitor family, and analyze the in vivo homing capacity of induced cardiomyocytes (iCMs) when transplanted systemically. DFCs from extracted wisdom teeth showed mesenchymal stem cell (MSC) characteristics such as plate adherent growing, expression of MSC markers (CD44, CD90, and CD105), and mesenchymal lineage-specific differentiation potential. Adding SAHA to the culture medium induced the successful in vitro differentiation of DFCs into cardiomyocytes. These iCMs expressed cardiomyogenic markers, including alpha-smooth muscle actin (α-SMA), cardiac muscle troponin T (TNNT2), Desmin, and cardiac muscle alpha actin (ACTC1), at both the mRNA and protein level. For the assessment of homing capacity, PKH26 labeled iCMs were intraperitoneally injected (1×106 cells in 100 µL of PBS) into the experimental mice, and the ratios of PKH26 positive cells to the total number of injected cells, in multiple organs were determined. The calculated homing ratios, 14 days after systemic cell transplantation, were 5.6 ± 1.0%, 3.6 ± 1.1%, and 11.6 ± 2.7% in heart, liver, and kidney respectively. There was no difference in the serum levels of interleukin-2 and interleukin-10 at 14 days after transplantation, between the experimental (iCM injected) and control (no injection or PBS injection) groups. These results demonstrate that DFCs can be an excellent source for cardiomyocyte differentiation and regeneration. Moreover, the iCMs can be delivered into heart muscle via systemic administration without eliciting inflammatory or immune response. This can serve as the pilot study for further investigations into the in vitro cardiomyogenic differentiation potential of DFCs under the influence of SAHA and the in vivo homing capacity of the iCMs into the heart muscle, when injected systemically.

Impact of implant support on mandibular free-end base removable partial denture: theoretical study.

OBJECTIVES: This study investigated the impact of implant support on the development of shear force and bending moment in mandibular free-end base removable partial dentures (RPDs). MATERIAL AND METHODS: Three theoretical test models of unilateral mandibular free-end base RPDs were constructed to represent the base of tooth replacement, as follows: Model 1: first and second molars (M1 and M2); Model 2: second premolar (P2), M1, and M2; and Model 3: first premolar (P1), P2, M1, and M2. The implant support located either at M1 or M2 sites. The occlusal loading was concentrated at each replacement tooth to calculate the stress resultants developed in the RPD models using the free-body diagrams of shear force and bending moment. RESULTS: There was a trend of reduction in the peak shear force and bending moment when the base was supported by implant. However, the degree of reduction varied with the location of implant support. The moment reduced by 76% in Model 1, 58% in Model 2, and 42% in Model 3, when the implant location shifted from M1 to M2 sites. CONCLUSIONS: The shear forces and bending moments subjected to mandibular free-end base RPDs were found to decrease with the addition of implant support. However, the impact of implant support varied with the location of implant in this theoretical study.

Expedited Record Base Fabrication Using an Irreversible Hydrocolloid Cast.

The registration of a maxillomandibular relationship requires additional clinical and laboratory procedures when the mouth presents with loss of occlusal support. This procedure can be a challenge for a patient who needs urgent care or resides in a remote area. This article describes a procedure for expediting the mounting of a master cast for the fabrication of a maxillary immediate complete denture. The technique presented describes the use of a silicone record base made on an irreversible hydrocolloid cast generated from the final impression.

Indirect method of base adaptation against supporting element of tooth root for a partial overdenture prosthesis.

The base of a partial overdenture prosthesis should be fitted intraorally against the supporting element of a tooth root. Chairside relining is a common method; however, an autopolymerizing acrylic resin presents high porosity when polymerized intraorally. This article describes an indirect method where an impression is made with a silicone occlusion registration material to create a replica of the supporting elements of the residual ridge and the tooth root in a high-viscosity polyvinyl siloxane impression material.

Charge Injection and Energy Transfer of Surface-Engineered InP/ZnSe/ZnS Quantum Dots.

Surface passivation is a critical aspect of preventing surface oxidation and improving the emission properties of nanocrystal quantum dots (QDs). Recent studies have demonstrated the critical role of surface ligands in determining the performance of QD-based light-emitting diodes (QD-LEDs). Herein, the underlying mechanism by which the capping ligands of InP/ZnSe/ZnS QDs influence the brightness and lifetime of the QD-LEDs is investigated. The electrochemical results demonstrate that highly luminescent InP/ZnSe/ZnS QDs exhibit modulated charge injection depending on the length of the surface ligand chains: short alkyl chains on the ligands are favorable for charge transport to the QDs. In addition, the correlation between the spectroscopic and XRD analyses suggests that the length of the ligand chain tunes the ligand-ligand coupling strength, thereby controlling the inter-QD energy transfer dynamics. The present findings shed new light on the crucial role of surface ligands for InP/ZnSe/ZnS QD-LED applications.

Overcoming Charge Confinement in Perovskite Nanocrystal Solar Cells.

The small nanoparticle size and long-chain ligands in colloidal metal halide perovskite quantum dots (PeQDs) cause charge confinement, which impedes exciton dissociation and carrier extraction in PeQD solar cells, so they have low short-circuit current density Jsc , which impedes further increases in their power conversion efficiency (PCE). Here, a re-assembling process (RP) is developed for perovskite nanocrystalline (PeNC) films made of colloidal perovskite nanocrystals to increase Jsc in PeNC solar cells. The RP of PeNC films increases their crystallite size and eliminates long-chain ligands, and thereby overcomes the charge confinement in PeNC films. These changes facilitate exciton dissociation and increase carrier extraction in PeNC solar cells. By use of this method, the gradient-bandgap PeNC solar cells achieve a Jsc = 19.30 mA cm-2 without compromising the photovoltage, and yield a high PCE of 16.46% with negligible hysteresis and good stability. This work provides a new strategy to process PeNC films and pave the way for high performance PeNC optoelectronic devices.

Limitations of gene editing assessments in human preimplantation embryos.

Range of DNA repair in response to double-strand breaks induced in human preimplantation embryos remains uncertain due to the complexity of analyzing single- or few-cell samples. Sequencing of such minute DNA input requires a whole genome amplification that can introduce artifacts, including coverage nonuniformity, amplification biases, and allelic dropouts at the target site. We show here that, on average, 26.6% of preexisting heterozygous loci in control single blastomere samples appear as homozygous after whole genome amplification indicative of allelic dropouts. To overcome these limitations, we validate on-target modifications seen in gene edited human embryos in embryonic stem cells. We show that, in addition to frequent indel mutations, biallelic double-strand breaks can also produce large deletions at the target site. Moreover, some embryonic stem cells show copy-neutral loss of heterozygosity at the cleavage site which is likely caused by interallelic gene conversion. However, the frequency of loss of heterozygosity in embryonic stem cells is lower than in blastomeres, suggesting that allelic dropouts is a common whole genome amplification outcome limiting genotyping accuracy in human preimplantation embryos.

Simple method of record base fabrication for a bar-retained implant overdenture.

A record base should be stable and accurately transferable from the cast to the mouth. This article describes a simple and practical method of fabricating a record base for mounting a master cast used to fabricate an implant connecting bar for an implant-retained overdenture.