Meta-analysis of human and mouse ALS astrocytes reveals multi-omic signatures of inflammatory reactive states.

Astrocytes contribute to motor neuron death in amyotrophic lateral sclerosis (ALS), but whether they adopt deleterious features consistent with inflammatory reactive states remains incompletely resolved. To identify inflammatory reactive features in ALS human induced pluripotent stem cell (hiPSC)-derived astrocytes, we examined transcriptomics, proteomics, and glutamate uptake in VCP-mutant astrocytes. We complemented this by examining other ALS mutations and models using a systematic meta-analysis of all publicly-available ALS astrocyte sequencing data, which included hiPSC-derived astrocytes carrying SOD1, C9orf72, and FUS gene mutations as well as mouse ALS astrocyte models with SOD1G93A mutation, Tardbp deletion, and Tmem259 (also known as membralin) deletion. ALS astrocytes were characterized by up-regulation of genes involved in the extracellular matrix, endoplasmic reticulum stress, and the immune response and down-regulation of synaptic integrity, glutamate uptake, and other neuronal support processes. We identify activation of the TGFB, Wnt, and hypoxia signaling pathways in both hiPSC and mouse ALS astrocytes. ALS changes positively correlate with TNF, IL1A, and complement pathway component C1q-treated inflammatory reactive astrocytes, with significant overlap of differentially expressed genes. By contrasting ALS changes with models of protective reactive astrocytes, including middle cerebral artery occlusion and spinal cord injury, we uncover a cluster of genes changing in opposing directions, which may represent down-regulated homeostatic genes and up-regulated deleterious genes in ALS astrocytes. These observations indicate that ALS astrocytes augment inflammatory processes while concomitantly suppressing neuronal supporting mechanisms, thus resembling inflammatory reactive states and offering potential therapeutic targets.

Trueness and intaglio fit of custom-made polyetheretherketone post-and-cores fabricated using different techniques.

STATEMENT OF PROBLEM: Applications of polyetheretherketone (PEEK) as a promising alternative to ceramic materials have extended to include post-and-cores. However, information regarding the available fabrication techniques for producing custom-made PEEK post-and-cores and their effect on accuracy is still scarce. PURPOSE: The purpose of this in vitro study was to evaluate the trueness and intaglio fit of custom-made PEEK post-and-cores fabricated using different techniques. MATERIAL AND METHODS: Ten mandibular second premolars were collected, decoronated, and endodontically treated. For the fabrication of custom-made post-and-cores, each post space was prepared, scanned, and designed by using the exocad software program. Each reference design was used to fabricate PEEK post-and-cores with 3 different techniques (N=30): group P, heat pressing; group S, subtractive manufacturing, and group A, additive manufacturing. All the fabricated restorations were scanned, providing the test data as standard tessellation language (STL) files, which were imported with the reference data into a reverse engineering software program to evaluate the 3D trueness of each fabrication technique. To evaluate the intaglio fit, restorations coated with silicone material were scanned to provide the test data as STL files, which were superimposed on the STL files of the scanned restorations. One-way analysis of variance (ANOVA) and 2-way mixed model ANOVA tests were used to analyze the data (α=.05). RESULTS: For 3D trueness, root mean square (RMS) values representing the deviations between the scans of the fabricated restorations and the reference designs varied significantly among different groups (P<.001), with the highest mean deviation found in group A (102.8 ±31 µm), while the lowest mean deviation was found in group S (42.2 ±11 µm). For the intaglio fit, the deviations between the scans of the fabricated restorations and the scans of the coated restorations varied significantly among different groups (P<.001), with the highest mean deviation found in group A (228.4 ±47 µm), while the lowest mean deviation was found in group S (96.2 ±14 µm). Simple effects comparisons showed the highest value of deviation in the apical area in group A (P<.001). CONCLUSIONS: Custom-made PEEK post-and-cores fabricated by using subtractive manufacturing and heat pressing techniques showed better trueness and intaglio fit compared with additively manufactured restorations. Before printed PEEK restorations can be considered a suitable alternative to pressed or milled ones, improvements are required to confirm their reliability.

Astrocytes display cell autonomous and diverse early reactive states in familial amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis is a rapidly progressive and fatal disease. Although astrocytes are increasingly recognized contributors to the underlying pathogenesis, the cellular autonomy and uniformity of astrocyte reactive transformation in different genetic forms of amyotrophic lateral sclerosis remain unresolved. Here we systematically examine these issues by using highly enriched and human induced pluripotent stem cell-derived astrocytes from patients with VCP and SOD1 mutations. We show that VCP mutant astrocytes undergo cell-autonomous reactive transformation characterized by increased expression of complement component 3 (C3) in addition to several characteristic gene expression changes. We then demonstrate that isochronic SOD1 mutant astrocytes also undergo a cell-autonomous reactive transformation, but that this is molecularly distinct from VCP mutant astrocytes. This is shown through transcriptome-wide analyses, identifying divergent gene expression profiles and activation of different key transcription factors in SOD1 and VCP mutant human induced pluripotent stem cell-derived astrocytes. Finally, we show functional differences in the basal cytokine secretome between VCP and SOD1 mutant human induced pluripotent stem cell-derived astrocytes. Our data therefore reveal that reactive transformation can occur cell autonomously in human amyotrophic lateral sclerosis astrocytes and with a striking degree of early molecular and functional heterogeneity when comparing different disease-causing mutations. These insights may be important when considering astrocyte reactivity as a putative therapeutic target in familial amyotrophic lateral sclerosis.

Reactive astrocytes in ALS display diminished intron retention.

Reactive astrocytes are implicated in amyotrophic lateral sclerosis (ALS), although the mechanisms controlling reactive transformation are unknown. We show that decreased intron retention (IR) is common to human-induced pluripotent stem cell (hiPSC)-derived astrocytes carrying ALS-causing mutations in VCP, SOD1 and C9orf72. Notably, transcripts with decreased IR and increased expression are overrepresented in reactivity processes including cell adhesion, stress response and immune activation. This was recapitulated in public-datasets for (i) hiPSC-derived astrocytes stimulated with cytokines to undergo reactive transformation and (ii) in vivo astrocytes following selective deletion of TDP-43. We also re-examined public translatome sequencing (TRAP-seq) of astrocytes from a SOD1 mouse model, which revealed that transcripts upregulated in translation significantly overlap with transcripts exhibiting decreased IR. Using nucleocytoplasmic fractionation of VCP mutant astrocytes coupled with mRNA sequencing and proteomics, we identify that decreased IR in nuclear transcripts is associated with enhanced nonsense mediated decay and increased cytoplasmic expression of transcripts and proteins regulating reactive transformation. These findings are consistent with a molecular model for reactive transformation in astrocytes whereby poised nuclear reactivity-related IR transcripts are spliced, undergo nuclear-to-cytoplasmic translocation and translation. Our study therefore provides new insights into the molecular regulation of reactive transformation in astrocytes.

Accuracy of computer-aided design trial restorations fabricated with different digital workflows.

STATEMENT OF PROBLEM: Trial restorations are a versatile tool for visualizing the esthetic treatment plan and should be an accurate replica of the planned smile design. The accuracy of conventionally fabricated trial restorations has been investigated; however, studies on the accuracy of different digital workflows for trial restoration fabrication are lacking. PURPOSE: The purpose of this in vitro study was to evaluate the accuracy of computer-aided design (CAD) trial restorations fabricated with different digital workflows. MATERIAL AND METHODS: A patient in need of additive esthetic treatment was examined, and intraoral and extraoral photographs and intraoral digital scans were made, followed by 3D digital smile design using the exocad software program. The reference virtual design was converted into trial restorations (N=40) by using different digital workflows: subtractive manufacturing, additive manufacturing, additive manufacturing of 3D designed index, and silicone index on an additively manufactured cast. Fabricated trial restorations of all groups were scanned with a desktop scanner, providing a standard tessellation language (STL) file serving as the test data for each trial restoration. All the STL files of the test and reference data were imported into a reverse engineering software program to evaluate the 3D trueness of each workflow. Also, analog and digital 2D linear measurements (maxillary left central incisor height, maxillary left central incisor width, and intercanine width) were made to assess any dimensional alterations between the fabricated trial restorations and the reference digital smile design. A 1-way analysis of variance (ANOVA) was used to analyze the data followed by the Tukey post hoc test (α=.05). RESULTS: For 3D trueness measurements, root mean square (RMS) values representing the deviations between the fabricated trial restorations and the reference digital smile design varied significantly among the different groups (P<.001), with the highest mean deviation found in the group of additive manufacturing of 3D designed index (0.21 ±0.01 mm), while the lowest mean value of deviation was found in the group of subtractive manufacturing (0.11 ±0.02 mm). For both the digital and analog 2D linear measurements, post hoc pairwise comparisons showed the group of additive manufacturing of 3D designed index to have significantly higher values of deviation than the other groups in all assigned measurements (P<.001). CONCLUSIONS: The accuracy of CAD trial restorations was affected by the fabrication technique, and implementing conventional steps in the digital workflows of trial restoration fabrication may result in discrepancies that affect accuracy when compared with the reference design.

Aberrant cytoplasmic intron retention is a blueprint for RNA binding protein mislocalization in VCP-related amyotrophic lateral sclerosis.

We recently described aberrantly increased cytoplasmic SFPQ intron-retaining transcripts (IRTs) and concurrent SFPQ protein mislocalization as new hallmarks of amyotrophic lateral sclerosis (ALS). However, the generalizability and potential roles of cytoplasmic IRTs in health and disease remain unclear. Here, using time-resolved deep sequencing of nuclear and cytoplasmic fractions of human induced pluripotent stem cells undergoing motor neurogenesis, we reveal that ALS-causing VCP gene mutations lead to compartment-specific aberrant accumulation of IRTs. Specifically, we identify >100 IRTs with increased cytoplasmic abundance in ALS samples. Furthermore, these aberrant cytoplasmic IRTs possess sequence-specific attributes and differential predicted binding affinity to RNA binding proteins. Remarkably, TDP-43, SFPQ and FUS-RNA binding proteins known for nuclear-to-cytoplasmic mislocalization in ALS-abundantly and specifically bind to this aberrant cytoplasmic pool of IRTs. Our data are therefore consistent with a novel role for cytoplasmic IRTs in regulating compartment-specific protein abundance. This study provides new molecular insight into potential pathomechanisms underlying ALS and highlights aberrant cytoplasmic IRTs as potential therapeutic targets.

Effect of combining different surface treatments on the surface characteristics of polyetheretherketone-based core materials and shear bond strength to a veneering composite resin.

STATEMENT OF PROBLEM: High-performance polyetheretherketone (PEEK) has been used in dentistry because of its excellent physical and chemical properties. However, its lack of translucency requires the use of a veneering composite resin for esthetic applications. Data on bonding a veneering composite resin to PEEK are still scarce. PURPOSE: The purpose of this in vitro study was to assess the effect of using and combining different surface treatments on the surface roughness and wettability of PEEK core materials and the shear bond strength (SBS) to a veneering composite resin. MATERIAL AND METHODS: Sixty square PEEK specimens were divided according to surface treatments into 6 groups: group N, no surface treatment; group A, airborne-particle abrasion; group L, ER:YAG laser treatment; group LA, laser treatment and airborne-particle abrasion; group P, plasma treatment; and group PA, plasma treatment and airborne-particle abrasion. Surface roughness (Ra) and wettability (contact angle θ) were measured followed by evaluation of the surface topography. After veneering, the SBS was measured. A 1-way analysis of variance (ANOVA) was used to assess all interactions with multiple pairwise comparisons (α=.05). RESULTS: The highest mean Ra values were obtained for the combination treatment groups (LA 2.94 ±0.1 and PA 2.7 ±0.1 µm) (P<.001). While the laser (1.3 ±0.1 µm) and plasma treatment (1.4 ±0.1 µm) groups had the lowest Ra values, LA and PA had the highest wetting of PEEK surfaces (LA 6.9 ±0.7 and PA 4.9 ±0.2 degrees) (P<.001). The highest mean SBS values were demonstrated on PEEK surfaces with combination treatments (LA 22 ±1.3 and PA 21.2 ±0.8 MPa) (P<.001). CONCLUSIONS: Combining airborne-particle abrasion with laser or plasma treatments increased the surface roughness and wettability of PEEK surfaces favorably compared with applying each of the 3 treatments individually. The combinations enhanced the bond strength of PEEK to the veneering composite resin.

Regionally encoded functional heterogeneity of astrocytes in health and disease: A perspective.

Increasing evidence has suggested that astrocytes demonstrate striking regionally allocated functional heterogeneity. Here, we discuss how this spatiotemporally encoded diversity determines the astrocytic phenotype along a finely grained spectrum from neuroprotective to deleterious states. With increasing recognition of their diverse and evolving roles in the central neuraxis, astrocytes now represent a tractable cellular target for therapies aiming to restore neural circuit integrity in a broad range of neurodegenerative disorders. Understanding the determinants of astrocyte physiology along with the true extent of heterogeneity in their regional and subregional functions will ultimately inform therapeutic strategy in neurodegenerative diseases.

Distinct responses of neurons and astrocytes to TDP-43 proteinopathy in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal and incurable neurodegenerative disease caused by motor neuron loss, resulting in muscle wasting, paralysis and eventual death. A key pathological feature of ALS is cytoplasmically mislocalized and aggregated TDP-43 protein in >95% of cases, which is considered to have prion-like properties. Historical studies have predominantly focused on genetic forms of ALS, which represent ∼10% of cases, leaving the remaining 90% of sporadic ALS relatively understudied. Additionally, the role of astrocytes in ALS and their relationship with TDP-43 pathology is also not currently well understood. We have therefore used highly enriched human induced pluripotent stem cell (iPSC)-derived motor neurons and astrocytes to model early cell type-specific features of sporadic ALS. We first demonstrate seeded aggregation of TDP-43 by exposing human iPSC-derived motor neurons to serially passaged sporadic ALS post-mortem tissue (spALS) extracts. Next, we show that human iPSC-derived motor neurons are more vulnerable to TDP-43 aggregation and toxicity compared with their astrocyte counterparts. We demonstrate that these TDP-43 aggregates can more readily propagate from motor neurons into astrocytes in co-culture paradigms. We next found that astrocytes are neuroprotective to seeded aggregation within motor neurons by reducing (mislocalized) cytoplasmic TDP-43, TDP-43 aggregation and cell toxicity. Furthermore, we detected TDP-43 oligomers in these spALS spinal cord extracts, and as such demonstrated that highly purified recombinant TDP-43 oligomers can reproduce this observed cell-type specific toxicity, providing further support to a protein oligomer-mediated toxicity hypothesis in ALS. In summary, we have developed a human, clinically relevant, and cell-type specific modelling platform that recapitulates key aspects of sporadic ALS and uncovers both an initial neuroprotective role for astrocytes and the cell type-specific toxic effect of TDP-43 oligomers.

In vitro evaluation of material dependent force damping behavior of implant-supported restorations using different CAD-CAM materials and luting conditions.

STATEMENT OF PROBLEM: Although force-damping behavior that matches natural teeth may be unobtainable, an optimal combination of crown material and luting agent might have a beneficial effect on the force absorption capacity of implant-supported restorations. However, the force-absorbing behavior of various restorative materials has not yet been satisfactorily investigated. PURPOSE: The purpose of this in vitro study was to evaluate the material dependent force-damping behavior of implant-supported crowns fabricated from different computer-aided design and computer-aided manufacturing (CAD-CAM) materials luted to implant abutments under different conditions. MATERIAL AND METHODS: Titanium inserts (N=84) were screwed to implant analogs, scanned to design zirconia abutments, and divided into 4 groups to receive CAD-CAM fabricated crowns in 4 materials: zirconia, polyetheretherketone (PEEK), polymer-infiltrated ceramics (VITA ENAMIC), and lithium disilicate (e.max). The crowns were subdivided as per the luting agent: none, interim cement, and adhesive resin cement. Measurements were performed by loading specimens in a universal testing machine with an increasing force and measuring the resulting force with a digital forcemeter, followed by image processing and data acquisition. Two-way multivariate analysis of variance (MANOVA) was used to assess all interactions with multiple pairwise comparisons (α=.05). RESULTS: The curve progression of the applied and resulting forces varied significantly among the investigated materials, resulting in differently inclined slopes for each material (P<.001). With no cementation, the mean slope values of the resulting force curves ranged from 77.5 ±0.03 degrees for zirconia, followed by 71.8 ±0.03 degrees for lithium disilicate, 56.2 ±0.1 degrees for polymer-infiltrated ceramics, and 51.1 ±0.01 degrees for polyetheretherketone. With interim cementation, the mean slope values ranged from 75.4 ±0.01 degrees for zirconia, followed by 70.05 ±0.02 degrees for lithium disilicate, 56.1 ±0.02 degrees for polymer-infiltrated ceramics, and 52.2 ±0.1 degrees for polyetheretherketone. As with adhesive cementation, curve slopes ranged from 73.2 ±0.02 degrees for zirconia, followed by 70.5 ±0.2 degrees for lithium disilicate, 55.9 ±0.04 degrees for polymer-infiltrated ceramics, and 52.3 ±0.1 degrees for polyetheretherketone. Slope loss was significant after the cementation of zirconia and lithium disilicate crowns but less significant for polymer-infiltrated ceramics and polyetheretherketone. CONCLUSIONS: Force damping is generally material dependent, yet implant-supported crowns fabricated from resilient materials such as polymer-infiltrated ceramics and PEEK show better force absorption than rigid materials such as zirconia and lithium disilicate ceramics. Furthermore, cementation of rigid materials significantly increased slope loss, indicating enhancement in their force-damping behavior, whereas less-rigid materials benefit less from cementation. Further studies are essential to investigate the effect of prosthetic materials on the stress distribution to the peri-implant bone in the crown-abutment-implant complex.

Minimally invasive prosthodontic correction of pseudo class III malocclusion by implementing a systematic digital workflow: A clinical report.

This clinical report describes the digital workflow for implementing different virtual and digital aids for the restoration of tooth wear and loss of occlusal vertical dimension (OVD). This conservative approach was implemented in an adult with pseudo class III malocclusion by using computer-aided design and computer-aided manufacturing (CAD-CAM) monolithic lithium disilicate ceramic restorations.

Widespread FUS mislocalization is a molecular hallmark of amyotrophic lateral sclerosis.

Mutations causing amyotrophic lateral sclerosis (ALS) clearly implicate ubiquitously expressed and predominantly nuclear RNA binding proteins, which form pathological cytoplasmic inclusions in this context. However, the possibility that wild-type RNA binding proteins mislocalize without necessarily becoming constituents of cytoplasmic inclusions themselves remains relatively unexplored. We hypothesized that nuclear-to-cytoplasmic mislocalization of the RNA binding protein fused in sarcoma (FUS), in an unaggregated state, may occur more widely in ALS than previously recognized. To address this hypothesis, we analysed motor neurons from a human ALS induced-pluripotent stem cell model caused by the VCP mutation. Additionally, we examined mouse transgenic models and post-mortem tissue from human sporadic ALS cases. We report nuclear-to-cytoplasmic mislocalization of FUS in both VCP-mutation related ALS and, crucially, in sporadic ALS spinal cord tissue from multiple cases. Furthermore, we provide evidence that FUS protein binds to an aberrantly retained intron within the SFPQ transcript, which is exported from the nucleus into the cytoplasm. Collectively, these data support a model for ALS pathogenesis whereby aberrant intron retention in SFPQ transcripts contributes to FUS mislocalization through their direct interaction and nuclear export. In summary, we report widespread mislocalization of the FUS protein in ALS and propose a putative underlying mechanism for this process.

The Effect of Different Wax Pattern Fabrication Techniques on the Marginal Fit of Customized Lithium Disilicate Implant Abutments.

PURPOSE: To evaluate the effect of different wax pattern fabrication techniques on the fit of customized pressed lithium disilicate implant abutments on titanium inserts before and after pressing. The marginal fit results of pressed lithium disilicate implant abutments were then compared with those of milled lithium disilicate abutments. MATERIALS AND METHODS: After scanning the titanium inserts and designing an implant abutment, wax patterns were fabricated with three techniques (n = 15 each): computer-aided design/computer-aided manufacturing (CAD/CAM) milling, 3D printing and conventional layering. The marginal fit (µm) was measured using a stereomicroscope for all the wax patterns before pressing them into the lithium disilicate abutments. The pressed implant abutments were measured again for marginal fit, and the results were compared to those of the milled lithium disilicate abutments. One-way analysis of variance (ANOVA) was used to assess different wax pattern fabrication techniques in each stage before and after pressing. One-way ANOVA was also used to compare the groups of pressed and milled lithium disilicate abutments. Multiple pairwise comparisons were performed using the Tukey post hoc test in each stage. RESULTS: There were statistically significant differences between the marginal fit of the three wax patterns groups (p < 0.001; f = 123.33), wherein the mean marginal fit was the highest for conventionally layered wax patterns (30 ± 13.09) µm. Furthermore, after pressing, there were statistically significant differences between the marginal fit of the three pressed abutments groups (p < 0.001; f = 518.62), wherein the mean marginal fit was the highest for pressed e.max abutments fabricated from conventionally layered wax patterns (25.26 ± 3.9) µm. There was no statistically significant difference between the mean marginal fit of the pressed abutments fabricated from conventional layered wax patterns and that of the milled CAD/CAM abutments. However, the mean marginal fit of the milled CAD/CAM abutments was higher than that of the pressed abutments fabricated from both CAD/CAM wax and 3D printed wax. CONCLUSION: All the tested fabrication methods provided degrees of accuracy that lie well within accepted limits. The use of pressed lithium disilicate abutments fabricated from conventional layering wax pattern technique should provide a more consistent better marginal fit between the titanium insert and the abutment and may therefore be the preferable fabrication method.

Assessment of marginal adaptation and fracture resistance of endocrown restorations utilizing different machinable blocks subjected to thermomechanical aging.

OBJECTIVE: This in vitro study was conducted to assess the marginal adaptation and fracture resistance of computer aided design/computer aided manufacturer (CAD-CAM) fabricated endocrowns restoring endodontically treated molars using different machinable blocks with thermomechanical loading protocols. MATERIALS AND METHODS: Devitalized mandibular molars were prepared in a standardized way and divided into 4 groups (n = 10) to receive CAD/CAM fabricated endocrowns using four materials (Lithium disilicate ceramics, polymer infiltrated ceramics, zirconia-reinforced lithium silicate ceramics and resin nanoceramics. Marginal gaps (µm) were measured using stereomicroscope before cementation and after cementation. After thermomechanical aging, marginal gap measurements were repeated, and then fracture resistance test was performed. Two-way analysis of variance (ANOVA) and Tukey HSD multiple comparisons were used to assess the effect of material on the marginal gap before, after cementation, and after thermomechanical aging. One Way ANOVA was used to assess the effect of material on the fracture resistance. RESULTS: The difference between marginal gaps values of the tested materials was statistically insignificant but with significant increase after cementation and after thermomechanical aging. Cerasmart endocrowns showed the highest mean fracture load value (1508.5 ± 421.7N) with statistically significant difference than Vita Enamic endocrowns and Celtra Duo. CONCLUSION: The tested materials showed marginal vertical gap readings within the limits of clinically acceptable standards. Resin nanoceramics and lithium disilicate showed the highest values of fracture resistance followed by polymer infiltrated ceramics favoring their use for endocrown restorations. CLINICAL SIGNIFICANCE: The mechanical behavior of ceramic materials varies with the variation of their structure and mechanical properties. Accordingly, further investigation is always needed to explore the biomechanical behavior of recent materials when used as endocrowns before clinical trials.

Grain Boundary Structures and Collective Dynamics of Inversion Domains in Binary Two-Dimensional Materials.

Understanding and controlling the properties and dynamics of topological defects is a lasting challenge in the study of two-dimensional materials, and is crucial to achieve high-quality films required for technological applications. Here grain boundary structures, energies, and dynamics of binary two-dimensional materials are investigated through the development of a phase field crystal model that is parametrized to match the ordering, symmetry, energy, and length scales of hexagonal boron nitride. Our studies reveal some new dislocation core structures for various symmetrically and asymmetrically tilted grain boundaries, in addition to those obtained in previous experiments and first-principles calculations. We also identify a defect-mediated growth dynamics for inversion domains governed by the collective atomic migration and defect core transformation at grain boundaries and junctions, a process that is related to inversion symmetry breaking in binary lattice.

R1 motif is the major actin-binding domain of TRIOBP-4.

TRIOBP is an actin-bundling protein. Mutations of TRIOBP are associated with human deafness DFNB28. In vitro, TRIOBP isoform 4 (TRIOBP-4) forms dense F-actin bundles resembling the inner ear hair cell rootlet structure. Deletion of TRIOBP isoforms 4 and 5 leads to hearing loss in mice due to the absence of stereocilia rootlets. The mechanism of actin bundle formation by TRIOBP is not fully understood. The amino acid sequences of TRIOBP isoforms 4 and 5 contain two repeated motifs, referred to here as R1 and R2. To examine the potential role of R1 and R2 motifs in F-actin binding, we generated TRIOBP-4 mutant proteins deleted for R1 and/or R2, and then assessed their actin-binding activity and bundle formation in vitro using actin cosedimentation assays, and fluorescence and electron microscopy. Cellular distributions of the TRIOBP-4 mutants were examined by confocal microscopy. We showed that deletion of both R1 and R2 motifs completely disrupted the actin binding/bundling activities of TRIOBP-4 and impaired its localization to cellular actin cytoskeleton structures. By contrast, TRIOBP-4, lacking only R2 motif, retained its F-actin bundling ability and remained localized to actin filaments in cells, similar to full length TRIOBP-4. On the contrary, the R1 motif-deleted TRIOBP-4 mutant, which mainly consists of the R2 motif, formed thin F-actin bundles in vitro but failed to colocalize to actin filaments in cells. These results indicate that R1 motif is the major actin-binding domain of TRIOBP-4, and the binding of R2 motif with actin filaments is nonspecific.

Effect of vitamin E, D-limonene, and their combination on nulliparous rabbit reproductive performance.

Oxidative status is important in reproductive performance and using two natural antioxidants is more beneficial than one in nulliparous rabbits. The goal, effect of vitamin E (VitE), D-limonene (DL), and VitE+DL on maternal LBW (MLBW), conception (CR), pregnancy (PR), and kindling rates (KR), gestation length (GL), total litter size at birth (TLSB) and weaning (TLSW), live kits at birth (LKB) and weaning (LKW), dead kits at birth (DKB) and weaning (DKW), kits mortality rate at weaning (KMRW), Kit weight at birth (KWB) and weaning (KWW), total kit weight at birth (TKWB) and weaning (TKWW), and concentrations of progesterone (P4) and Malondialdehyde (MDA), during first two pregnancies. A total of 24 healthy female WNZ rabbits were randomly selected and assigned into four groups (6/each). Control (animals injected with 1.0 mL propylene glycol), VitE (60 mg IM injection/animal, 2X/week pre-mating and 3X post-mating until mid-pregnancy, DL (20 mg IM injection/animal, 2X/week pre-mating and 1X at mating, and VitE+DL (IM injection/animal with the same doses and times applied in VitE and DL groups. All animals were treated during 1st pregnancy only. The results confirmed that animals treated with VitE+DL gained significant maternal LBW in 1st pregnancy, reduced dead kit number at birth and kit mortality rate at weaning, increased live kits and total kit weight at birth and weaning in the two pregnancies, and also increased significantly kit weight at birth and weaning in the treatments than controls in the two pregnancies, and DL was greater in 1st pregnancy. Progesterone concentrations in mid-pregnancy rose significantly in VitE+DL during 1st pregnancy and DL in 2nd pregnancy. Malondialdehyde concentrations dropped significantly in VitE and VitE+DL in mid-pregnancy in the two pregnancies. Eventually, the integration of VitE and DL displayed their unique properties for improving productive and reproductive performance in nulliparous rabbits.

Influence of different combinations of CAD-CAM crown and customized abutment materials on the force absorption capacity in implant supported restorations - In vitro study.

OBJECTIVES: To evaluate the force absorption capacity of implant supported restorations utilizing different CAD-CAM materials for the fabrication of crowns and customized abutments. METHODS: 80 titanium inserts were scanned to design customized abutments and crowns. The specimens were divided into four groups (n = 20/material): (Z): zirconia, (P): PEEK, (V): VITA Enamic, and (E): IPS e.max. Each group was subdivided into two subgroups according to customized abutment material: (Z) zirconia, and (P) for PEEK. For the assessment of force absorption, all specimens were loaded in a universal testing machine, applied loads curves were collected from the machine's software, and resulting loads curves were collected from forcemeter below the assembly. The slopes of all curves were analyzed using Two-way multivariate analysis of variance with pairwise comparisons using Tukey Post Hoc test (p < 0.05). RESULTS: The curve progression of the applied and resulting forces varied among the investigated materials for each specimen. For zirconia abutments, ZZ showed the highest slope values of the applied and resulting force curves, followed by EZ, VZ, and PZ demonstrating statistically significant differences (P < .001). As for PEEK abutments, ZP and EP showed the least slope values, followed by PP then VP demonstrating statistically significant differences (P < .001). For Zirconia and e.max crowns, using PEEK abutments significantly increased slope loss. As for PEEK and Vita Enamic crowns changing abutment material did not significantly affect slope loss. SIGNIFICANCE: Combining rigid crown materials with less rigid abutments might enhance their force absorption capacity. However, with less rigid crown materials a stiff substructure might be mandatory to preserve their force absorption behavior.

Automated and unbiased discrimination of ALS from control tissue at single cell resolution.

Histopathological analysis of tissue sections is invaluable in neurodegeneration research. However, cell-to-cell variation in both the presence and severity of a given phenotype is a key limitation of this approach, reducing the signal to noise ratio and leaving unresolved the potential of single-cell scoring for a given disease attribute. Here, we tested different machine learning methods to analyse high-content microscopy measurements of hundreds of motor neurons (MNs) from amyotrophic lateral sclerosis (ALS) post-mortem tissue sections. Furthermore, we automated the identification of phenotypically distinct MN subpopulations in VCP- and SOD1-mutant transgenic mice, revealing common morphological cellular phenotypes. Additionally we established scoring metrics to rank cells and tissue samples for both disease probability and severity. By adapting this paradigm to human post-mortem tissue, we validated our core finding that morphological descriptors robustly discriminate ALS from control healthy tissue at single cell resolution. Determining disease presence, severity and unbiased phenotypes at single cell resolution might prove transformational in our understanding of ALS and neurodegeneration more broadly.