The Unkempt RNA binding protein reveals a local translation program in centriole overduplication.

Excess centrosomes cause defects in mitosis, cell-signaling, and cell migration, and therefore their assembly is tightly regulated. Plk4 controls centriole duplication at the heart of centrosome assembly, and elevation of Plk4 promotes centrosome amplification (CA), a founding event of tumorigenesis. Here, we investigate the transcriptional consequences of elevated Plk4 and find Unkempt, a gene encoding an RNA binding protein with roles in translational regulation, to be one of only two upregulated mRNAs. Unk protein localizes to centrosomes and Cep131-positive centriolar satellites and is required for Plk4-induced centriole overduplication in an RNA-binding dependent manner. Translation is enriched at centrosomes and centriolar satellites with Unk and Cep131 promoting this localized translation. A transient centrosomal downregulation of translation occurs early in Plk4-induced CA. CNOT9, an Unk interactor and component of the translational inhibitory CCR4-NOT complex, localizes to centrosomes at this time. In summary, centriolar satellites and Unk promote local translation as part of a translational program that ensures centriole duplication.

Accuracy and resolution of conventional and additively manufactured silicone elastomers as applied in maxillofacial therapies.

STATEMENT OF PROBLEM: Silicone elastomers are becoming more readily available for additive manufacturing, which may be advantageous for fabricating maxillofacial prostheses. However, the properties of three-dimensionally (3D) printed silicone as compared with conventionally processed silicone have not been well studied. PURPOSE: The purpose of this in vitro study was to compare the dimensional accuracy and surface resolution of additively manufactured with conventional room-temperature vulcanized (RTV) silicones. MATERIAL AND METHODS: A custom aluminum mold was used to generate hand-spatulated specimens (A103 and VerSilTal-50F, n=20). A computer-aided design and computer-aided manufacturing workflow was used to generate additively manufactured specimens (Sil30 and TrueSil, n=20). Digital surface scans of each specimen were recorded; a scan of the mold served as the control. Surface dimensions were measured with a digital metrology software program, while digital overlays were made using a 3D processing software program. The surface resolution of the specimens was assessed by analyzing 4 topographical landmarks (flat surfaces, raised lines, domes, and scribed lines) with a visual qualitative grading scale. The data were analyzed with 1-way analysis of variance, followed by a Student-Newman-Keuls post hoc test (α=.05). RESULTS: The specimens demonstrated statistical differences in trueness and precision (P<.001). The TrueSil specimens showed the largest deviation in measurements of trueness and precision (up to -1.374%). The other specimens yielded percentage mean differences that were more consistently within the range of the American Dental Association International Organization for Standardization standard for elastomers. The manually fabricated specimens yielded more consistently ideal ratings for resolution than did the additively manufactured ones, with the Sil30 specimens receiving the most Charlie (not clinically acceptable) ratings. CONCLUSIONS: Numerical differences between each specimen and the control were considered negligible for maxillofacial applications. Notable discrepancies related to the quality of resolution, wherein the benchtop-manufactured specimens consistently generated better results compared with additively manufactured ones. Other factors, such as resiliency, odor, and cost, posed limitations in justifying the use of silicones in a direct-to-print workflow.

A specific and portable gene expression program underlies antigen archiving by lymphatic endothelial cells.

Antigens from protein subunit vaccination traffic from the tissue to the draining lymph node, either passively via the lymph or carried by dendritic cells at the local injection site. Lymph node (LN) lymphatic endothelial cells (LEC) actively acquire and archive foreign antigens, and archived antigen can be released during subsequent inflammatory stimulus to improve immune responses. Here, we answer questions about how LECs achieve durable antigen archiving and whether there are transcriptional signatures associated with LECs containing high levels of antigen. We used single cell sequencing in dissociated LN tissue to quantify antigen levels in LEC and dendritic cell populations at multiple timepoints after immunization, and used machine learning to define a unique transcriptional program within archiving LECs that can predict LEC archiving capacity in independent data sets. Finally, we validated this modeling, showing we could predict antigen archiving from a transcriptional dataset of CHIKV infected mice and demonstrated in vivo the accuracy of our prediction. Collectively, our findings establish a unique transcriptional program in LECs that promotes antigen archiving that can be translated to other systems.

Chikungunya virus infection disrupts lymph node lymphatic endothelial cell composition and function via MARCO.

Infection with chikungunya virus (CHIKV) causes disruption of draining lymph node (dLN) organization, including paracortical relocalization of B cells, loss of the B cell-T cell border, and lymphocyte depletion that is associated with infiltration of the LN with inflammatory myeloid cells. Here, we found that, during the first 24 hours of infection, CHIKV RNA accumulated in MARCO-expressing lymphatic endothelial cells (LECs) in both the floor and medullary LN sinuses. The accumulation of viral RNA in the LN was associated with a switch to an antiviral and inflammatory gene expression program across LN stromal cells, and this inflammatory response - including recruitment of myeloid cells to the LN - was accelerated by CHIKV-MARCO interactions. As CHIKV infection progressed, both floor and medullary LECs diminished in number, suggesting further functional impairment of the LN by infection. Consistent with this idea, antigen acquisition by LECs, a key function of LN LECs during infection and immunization, was reduced during pathogenic CHIKV infection.

Chikungunya virus infection disrupts lymph node lymphatic endothelial cell composition and function via MARCO.

Infection with chikungunya virus (CHIKV) causes disruption of draining lymph node (dLN) organization, including paracortical relocalization of B cells, loss of the B cell-T cell border, and lymphocyte depletion that is associated with infiltration of the LN with inflammatory myeloid cells. Here, we find that during the first 24 h of infection, CHIKV RNA accumulates in MARCO-expressing lymphatic endothelial cells (LECs) in both the floor and medullary LN sinuses. The accumulation of viral RNA in the LN was associated with a switch to an antiviral and inflammatory gene expression program across LN stromal cells, and this inflammatory response, including recruitment of myeloid cells to the LN, was accelerated by CHIKV-MARCO interactions. As CHIKV infection progressed, both floor and medullary LECs diminished in number, suggesting further functional impairment of the LN by infection. Consistent with this idea, we find that antigen acquisition by LECs, a key function of LN LECs during infection and immunization, was reduced during pathogenic CHIKV infection.

Zinc transporter 8 haploinsufficiency protects against beta cell dysfunction in type 1 diabetes by increasing mitochondrial respiration.

OBJECTIVE: Zinc transporter 8 (ZnT8) is a major humoral target in human type 1 diabetes (T1D). Polymorphic variants of Slc30A8, which encodes ZnT8, are also associated with protection from type 2 diabetes (T2D). The current study examined whether ZnT8 might play a role beyond simply being a target of autoimmunity in the pathophysiology of T1D. METHODS: The phenotypes of NOD mice with complete or partial global loss of ZnT8 were determined using a combination of disease incidence, histological, transcriptomic, and metabolic analyses. RESULTS: Unexpectedly, while complete loss of ZnT8 accelerated spontaneous T1D, heterozygosity was partially protective. In vivo and in vitro studies of ZnT8 deficient NOD.SCID mice suggested that the accelerated disease was due to more rampant autoimmunity. Conversely, beta cells in heterozygous animals uniquely displayed increased mitochondrial fitness under mild proinflammatory conditions. CONCLUSIONS: In pancreatic beta cells and immune cell populations, Zn2+ plays a key role as a regulator of redox signaling and as an independent secondary messenger. Importantly, Zn2+ also plays a major role in maintaining mitochondrial homeostasis. Our results suggest that regulating mitochondrial fitness by altering intra-islet zinc homeostasis may provide a novel mechanism to modulate T1D pathophysiology.

The pausing zone and control of RNA polymerase II elongation by Spt5: Implications for the pause-release model.

The pause-release model of transcription proposes that 40-100 bases from the start site RNA Pol II pauses, followed by release into productive elongation. Pause release is facilitated by the PTEFb phosphorylation of the RNA Pol II elongation factor, Spt5. We mapped paused polymerases by eNET-seq and found frequent pausing in zones that extend ∼0.3-3 kb into genes even when PTEFb is inhibited. The fraction of paused polymerases or pausing propensity declines gradually over several kb and not abruptly as predicted for a discrete pause-release event. Spt5 depletion extends pausing zones, suggesting that it promotes the maturation of elongation complexes to a low-pausing state. The expression of mutants after Spt5 depletion showed that phosphomimetic substitutions in the CTR1 domain diminished pausing throughout genes. By contrast, mutants that prevent the phosphorylation of the Spt5 RNA-binding domain strengthened pausing. Thus, distinct Spt5 phospho-isoforms set the balance between pausing and elongation.

Precise gene models using long-read sequencing reveal a unique poly(A) signal in Giardia lamblia.

During pre-mRNA processing, the poly(A) signal is recognized by a protein complex that ensures precise cleavage and polyadenylation of the nascent transcript. The location of this cleavage event establishes the length and sequence of the 3' UTR of an mRNA, thus determining much of its post-transcriptional fate. Using long-read sequencing, we characterize the polyadenylation signal and related sequences surrounding Giardia lamblia cleavage sites for over 2600 genes. We find that G. lamblia uses an AGURAA poly(A) signal, which differs from the mammalian AAUAAA. We also describe how G. lamblia lacks common auxiliary elements found in other eukaryotes, along with the proteins that recognize them. Further, we identify 133 genes with evidence of alternative polyadenylation. These results suggest that despite pared-down cleavage and polyadenylation machinery, 3' end formation still appears to be an important regulatory step for gene expression in G. lamblia.

Elevated Detection of Dual Antibody B Cells Identifies Lupus Patients With B Cell-Reactive VH4-34 Autoantibodies.

About 5% of B cells in healthy mice and humans are allelically or isotypically included and hence co-express two different antibodies. In mice, dual antibody B cells (B2R) expand with systemic autoimmunity, co-express autoreactive and non-autoreactive antibodies, and participate in immune responses, but this phenomenon is strain dependent. This study was developed with two goals: 1) to establish the contribution of TLR and IFN receptor signaling to the development of germinal center B cells that express two antibodies in MRL/lpr mice; and 2) to determine whether B2R B cells are increased and particularly activated in a subset of adult patients diagnosed with systemic lupus erythematosus (SLE). Results from the MRL/lpr studies indicate that the enhanced differentiation of dual-κ B cells into germinal center B cells is due to a heightened response to TLR7 and TLR9 signaling, further fueled by an increased response to type II IFN. To understand the clinical and translational implications of our observations in mouse B2R B cells, cohorts of SLE patients and healthy controls were recruited and evaluated for expression of dual BCRs. Results from flow cytometry and microscopy revealed supraphysiological frequencies of κ+λ+ B2R cells in one fourth of the SLE patients. Abnormal numbers of κ+λ+ B cells correlated with higher frequencies of activated naïve B cells and age-associated B cells, and a lower proportion of "B cells that are naïve IgD+" (BND). However, results from single cell V(D)J sequencing demonstrated that these high κ+λ+ SLE patients harbored normal frequencies of κ+λ+ and other B2R B cells. and we further show that their B cells were instead decorated by κ and λ VH4-34 autoantibodies. Thus, our findings indicate that elevated flow cytometric detection of isotypically-included B cells can identify patients with high titers of B cell-reactive VH4-34 autoantibodies and abnormal distribution of B cell subsets relevant to autoimmunity.

MARCO+ lymphatic endothelial cells sequester arthritogenic alphaviruses to limit viremia and viral dissemination.

Viremia in the vertebrate host is a major determinant of arboviral reservoir competency, transmission efficiency, and disease severity. However, immune mechanisms that control arboviral viremia are poorly defined. Here, we identify critical roles for the scavenger receptor MARCO in controlling viremia during arthritogenic alphavirus infections in mice. Following subcutaneous inoculation, arthritogenic alphavirus particles drain via the lymph and are rapidly captured by MARCO+ lymphatic endothelial cells (LECs) in the draining lymph node (dLN), limiting viral spread to the bloodstream. Upon reaching the bloodstream, alphavirus particles are cleared from the circulation by MARCO-expressing Kupffer cells in the liver, limiting viremia and further viral dissemination. MARCO-mediated accumulation of alphavirus particles in the draining lymph node and liver is an important host defense mechanism as viremia and viral tissue burdens are elevated in MARCO-/- mice and disease is more severe. In contrast to prior studies implicating a key role for lymph node macrophages in limiting viral dissemination, these findings exemplify a previously unrecognized arbovirus-scavenging role for lymphatic endothelial cells and improve our mechanistic understanding of viremia control during arthritogenic alphavirus infection.

The SON RNA splicing factor is required for intracellular trafficking structures that promote centriole assembly and ciliogenesis.

Control of centrosome assembly is critical for cell division, intracellular trafficking, and cilia. Regulation of centrosome number occurs through the precise duplication of centrioles that reside in centrosomes. Here we explored transcriptional control of centriole assembly and find that the RNA splicing factor SON is specifically required for completing procentriole assembly. Whole genome mRNA sequencing identified genes whose splicing and expression are affected by the reduction of SON, with an enrichment in genes involved in the microtubule (MT) cytoskeleton, centrosome, and centriolar satellites. SON is required for the proper splicing and expression of CEP131, which encodes a major centriolar satellite protein and is required to organize the trafficking and MT network around the centrosomes. This study highlights the importance of the distinct MT trafficking network that is intimately associated with nascent centrioles and is responsible for procentriole development and efficient ciliogenesis.

Molecular tracking devices quantify antigen distribution and archiving in the murine lymph node.

The detection of foreign antigens in vivo has relied on fluorescent conjugation or indirect read-outs such as antigen presentation. In our studies, we found that these widely used techniques had several technical limitations that have precluded a complete picture of antigen trafficking or retention across lymph node cell types. To address these limitations, we developed a 'molecular tracking device' to follow the distribution, acquisition, and retention of antigen in the lymph node. Utilizing an antigen conjugated to a nuclease-resistant DNA tag, acting as a combined antigen-adjuvant conjugate, and single-cell mRNA sequencing, we quantified antigen abundance in the lymph node. Variable antigen levels enabled the identification of caveolar endocytosis as a mechanism of antigen acquisition or retention in lymphatic endothelial cells. Thus, these molecular tracking devices enable new approaches to study dynamic tissue dissemination of antigen-adjuvant conjugates and identify new mechanisms of antigen acquisition and retention at cellular resolution in vivo.

The lymphatic system is a network of ducts that transports fluid, proteins, and immune cells from different organs around the body. Lymph nodes provide pit stops at hundreds of points along this network where immune cells reside, and lymph fluid can be filtered and cleaned. When pathogens, such as viruses or bacteria, enter the body during an infection, fragments of their proteins can get swept into the lymph nodes. These pathogenic proteins or protein fragments activate resident immune cells and kickstart the immune response. Vaccines are designed to mimic this process by introducing isolated pathogenic proteins in a controlled way to stimulate similar immune reactions in lymph nodes. Once an infection has been cleared by the immune system, or a vaccination has triggered the immune system, most pathogenic proteins get cleared away. However, a small number of pathogenic proteins remain in the lymph nodes to enable immune cells to respond more strongly and quickly the next time they see the same pathogen. Yet it is largely unclear how much protein remains for training and how or where it is all stored. Current techniques are not sensitive or long-lived enough to accurately detect and track these small protein deposits over time. Walsh, Sheridan, Lucas, et al. have addressed this problem by developing biological tags that can be attached to the pathogenic proteins so they can be traced. These tags were designed so the body cannot easily break them down, helping them last as long as the proteins they are attached to. Walsh, Sheridan, Lucas et al. tested whether vaccinating mice with the tagged proteins allowed the proteins to be tracked. The method they used was designed to identify individual cell types based on their genetic information along with the tag. This allowed them to accurately map the complex network of cells involved in storing and retrieving archived protein fragments, as well as those involved in training new immune cells to recognize them. These results provide important insights into the protein archiving system that is involved in enhancing immune memory. This may help guide the development of new vaccination strategies that can manipulate how proteins are archived to establish more durable immune protection. The biological tags developed could also be used to track therapeutic proteins, allowing scientists to determine how long cancer drugs, antibody therapies or COVID19 anti-viral agents remain in the body. This information could then be used by doctors to plan specific and personalized treatment timetables for patients.

Technique to Fabricate Custom Nasal Pillows and Monoblock Mandibular Advancement Device Using A Digital Workflow for Combination Therapy of Obstructive Sleep Apnea.

Combination therapy using continuous positive airway pressure and oral appliance therapy is an effective non-surgical treatment for obstructive sleep apnea. However, the laboratory expense and additional chairside time prevent it from being a preferred option. This article describes a technique for fabricating custom nasal pillows and monoblock mandibular advancement device with potential lower cost and accelerated timeline using a digital workflow.

clustifyr: an R package for automated single-cell RNA sequencing cluster classification.

Assignment of cell types from single-cell RNA sequencing (scRNA-seq) data remains a time-consuming and error-prone process. Current packages for identity assignment use limited types of reference data and often have rigid data structure requirements. We developed the clustifyr R package to leverage several external data types, including gene expression profiles to assign likely cell types using data from scRNA-seq, bulk RNA-seq, microarray expression data, or signature gene lists. We benchmark various parameters of a correlation-based approach and implement gene list enrichment methods. clustifyr is a lightweight and effective cell-type assignment tool developed for compatibility with various scRNA-seq analysis workflows. clustifyr is publicly available at https://github.com/rnabioco/clustifyr.

Effect of high-irradiance light curing on exposure times and pulpal temperature of adequately polymerized composite.

This study investigated the effect of high-irradiance light-curing on exposure time and pulpal temperature of adequately-cured composite. Composite placed in a molar preparation was cured using high-irradiance light-curing units (Flashmax P3, Valo, S.P.E.C. 3 LED, Cybird XD) and tested for hardness occlusal-gingivally. The first group had exposure times set according to manufacturer settings (recommended), second group to yield 80% of maximum hardness at the 2 mm depth (experimental), and third group was set at 20 s (extended). Exposure time necessary to adequately polymerize the composite at 2 mm depth was 9 s for the Cybird XD and Valo and 12 s for S.P.E.C. 3 LED and Flashmax P3. None of the high-irradiance light-curing units adequately polymerized the composite at the manufacturer-recommended minimum-exposure times of 1-3 s. Exposure times necessary to adequately polymerize composite at 2 mm resulted in a maximum pulpal-temperature increase well below the temperature associated with possible pulpal necrosis.

Resident mesenchymal vascular progenitors modulate adaptive angiogenesis and pulmonary remodeling via regulation of canonical Wnt signaling.

Adaptive angiogenesis is necessary for tissue repair, however, it may also be associated with the exacerbation of injury and development of chronic disease. In these studies, we demonstrate that lung mesenchymal vascular progenitor cells (MVPC) modulate adaptive angiogenesis via lineage trace, depletion of MVPC, and modulation of ß-catenin expression. Single cell sequencing confirmed MVPC as multipotential vascular progenitors, thus, genetic depletion resulted in alveolar simplification with reduced adaptive angiogenesis. Following vascular endothelial injury, Wnt activation in MVPC was sufficient to elicit an emphysema-like phenotype characterized by increased MLI, fibrosis, and MVPC driven adaptive angiogenesis. Lastly, activation of Wnt/ß-catenin signaling skewed the profile of human and murine MVPC toward an adaptive phenotype. These data suggest that lung MVPC drive angiogenesis in response to injury and regulate the microvascular niche as well as subsequent distal lung tissue architecture via Wnt signaling.

An Investigation of the Effect of Modifying and Reusing Impression Copings on Transfer of Implant Analog Position and Angulation.

AIM: The aim of this research was to determine whether sterilization and reutilization of impression copings had an impact on the accuracy of casts made for multiimplant restorations. MATERIALS AND METHODS: Four master casts embedded with five implant analogs were fabricated. Polyvinyl siloxane (PVS) impressions of the master cast with copings attached to the analogs were made and poured in dental stone. The impression copings were subjected to cleaning and sterilization. These processes were repeated 30 cycles for each of the two groups of five impression copings: one without modification and one with modification that included air abrasion and PVS adhesive. A coordinate measuring machine (CMM) was used to measure relative angles and distances between the reference analog and analogs. The relative angles and distances measured on the stone casts were compared to the master resin cast to obtain positional and angular displacements. RESULTS: For impression copings that were not modified, a significant difference was detected for both positional and angular displacements. For impression copings that were modified, a significant change was observed only for positional displacement. The maximum discrepancies measured for positional and angular displacements after 30 cycles of reuse were only 81 µm and 0.46°, respectively, regardless of the modification. CONCLUSION: Within the limitations of this study, unmodified impression copings that have undergone 30 cycles of cleaning and sterilization appeared to incur more impression inaccuracy than those impression copings that were modified by airborne-particle abrasion and PVS adhesive. CLINICAL SIGNIFICANCE: Impression copings used in this study can likely be recycled up to 30 times without reducing the accuracy of the impression to a level that may be considered clinically significant.

Targeted Endodontic Microsurgery: Digital Workflow Options.

Targeted endodontic microsurgery (TEMS) combines a precisely designed 3-dimensional (3D)-printed surgical guide with a trephine bur for safe and efficient osteotomy and root-end resection. The TEMS digital workflow converts the patient's anatomy into digital data in 4 steps. First, bone, teeth, and neurovascular spaces are rendered with cone-beam computed tomographic imaging. Next, crowns and soft tissues are rendered with an intraoral optical scan, a benchtop optical scan of an impression or cast, or a cone-beam computed tomographic scan of an impression or cast. Third, these renderings are merged within design software to create a 3D construction containing a virtual model. Finally, guide design is performed on the virtual model for 3D printing. A significant gap in knowledge exists in that digital workflow principles and considerations are not documented in the endodontic literature. The aim of this article is to describe TEMS digital workflow guiding principles.

Monocytic Subclones Confer Resistance to Venetoclax-Based Therapy in Patients with Acute Myeloid Leukemia.

Venetoclax-based therapy can induce responses in approximately 70% of older previously untreated patients with acute myeloid leukemia (AML). However, up-front resistance as well as relapse following initial response demonstrates the need for a deeper understanding of resistance mechanisms. In the present study, we report that responses to venetoclax +azacitidine in patients with AML correlate closely with developmental stage, where phenotypically primitive AML is sensitive, but monocytic AML is more resistant. Mechanistically, resistant monocytic AML has a distinct transcriptomic profile, loses expression of venetoclax target BCL2, and relies on MCL1 to mediate oxidative phosphorylation and survival. This differential sensitivity drives a selective process in patients which favors the outgrowth of monocytic subpopulations at relapse. Based on these findings, we conclude that resistance to venetoclax + azacitidine can arise due to biological properties intrinsic to monocytic differentiation. We propose that optimal AML therapies should be designed so as to independently target AML subclones that may arise at differing stages of pathogenesis. SIGNIFICANCE: Identifying characteristics of patients who respond poorly to venetoclax-based therapy and devising alternative therapeutic strategies for such patients are important topics in AML. We show that venetoclax resistance can arise due to intrinsic molecular/metabolic properties of monocytic AML cells and that such properties can potentially be targeted with alternative strategies.

Effect of Toothbrushing on Surface Color of Ceramic-polymer Materials: An In Vitro Study.

AIM AND OBJECTIVE: The purpose of this study was to examine the effects of toothbrushing on the change in color of extrinsic characterization of ceramic-polymer materials. MATERIALS AND METHODS: Two ceramic-polymer materials (CeraSmart, GC; Enamic, VITA) and one lithium-disilicate material (IPS e.max CAD; Ivoclar Vivadent) were tested. Specimens of each material were prepared, characterized, and glazed per manufacturer's instructions. The treated surface of the blocks were then brushed in a toothpaste slurry with artificial saliva using a toothbrush machine with a soft toothbrush. Commission Internationale de L'Eclairage (CIE) L*a*b* values were recorded with a spectrophotometer at baseline and at 3, 6, 9, and 12 simulated years of brushing (7,300 strokes/year). A mean change in color (ΔE*) and standard deviation was determined for each group and brushing interval. Data were analyzed with a two-way repeated measures ANOVA examining the effects of toothbrushing the ceramic materials on ΔE* over time (α = 0.05). RESULTS: The difference in the ΔE* between CeraSmart and Enamic was significant at 3 years, while the differences between them were not significant at 6, 9, and 12 years of simulated brushing. The ΔE* of IPS e.max CAD was significantly lower than CeraSmart and Enamic at all time points (all p < 0.0001) except for the comparison with Enamic at 3 years. CONCLUSION: The extrinsic stains on the ceramic-polymer materials may be more susceptible to change from simulated toothbrushing compared to the lithium-disilicate material. CLINICAL SIGNIFICANCE: Toothbrushing may change the color of extrinsic characterization of ceramic-polymer materials. However, the change may remain clinically imperceptible to the naked eye (ΔE* > 1.0) for nearly 6 equivalent years of brushing.