Utilizing genomic signatures to gain insights into the dynamics of SARS-CoV-2 through Machine and Deep Learning techniques.

The global spread of the SARS-CoV-2 pandemic, originating in Wuhan, China, has had profound consequences on both health and the economy. Traditional alignment-based phylogenetic tree methods for tracking epidemic dynamics demand substantial computational power due to the growing number of sequenced strains. Consequently, there is a pressing need for an alignment-free approach to characterize these strains and monitor the dynamics of various variants. In this work, we introduce a swift and straightforward tool named GenoSig, implemented in C++. The tool exploits the Di and Tri nucleotide frequency signatures to delineate the taxonomic lineages of SARS-CoV-2 by employing diverse machine learning (ML) and deep learning (DL) models. Our approach achieved a tenfold cross-validation accuracy of 87.88% (± 0.013) for DL and 86.37% (± 0.0009) for Random Forest (RF) model, surpassing the performance of other ML models. Validation using an additional unexposed dataset yielded comparable results. Despite variations in architectures between DL and RF, it was observed that later clades, specifically GRA, GRY, and GK, exhibited superior performance compared to earlier clades G and GH. As for the continental origin of the virus, both DL and RF models exhibited lower performance than in predicting clades. However, both models demonstrated relatively higher accuracy for Europe, North America, and South America compared to other continents, with DL outperforming RF. Both models consistently demonstrated a preference for cytosine and guanine over adenine and thymine in both clade and continental analyses, in both Di and Tri nucleotide frequencies signatures. Our findings suggest that GenoSig provides a straightforward approach to address taxonomic, epidemiological, and biological inquiries, utilizing a reductive method applicable not only to SARS-CoV-2 but also to similar research questions in an alignment-free context.

Atypical Duplex appendix arising from the ascending colon: a case report.

BACKGROUND: Duplex or vermiform appendix refers to the presence of an appendix beside the naturally occurring one. Although, duplex appendix emerges from the caecum most of the time, yet it is encountered in other parts of the colon. Inflammation of duplex appendix may represent not only a clinical, but also a surgical dilemma, and this would be confusing further among patients who already had prior appendectomy. CASE PRESENTATION: We present a case of 29-years old Egyptian male patient with history of appendectomy one and half year before presenting to the emergency department with recurrent acute abdominal pain that was linked to duplex appendicitis abnormally emerged from the mid-ascending colon. The first episode was treated conservatively considering atypical right colon diverticulitis as a potential differential diagnosis. Seven months later the patient was treated by laparoscopic appendectomy and experienced an uneventful pot-operative course. CONCLUSION: Duplex appendicitis, though rare, should be considered in the differential diagnosis of recurrent acute abdomen even after appendectomy.

Phage susceptibility determinants of the opportunistic pathogen Staphylococcus epidermidis.

Staphylococcus epidermidis is a common member of the human skin and nose microbiomes and a frequent cause of invasive infections. Transducing phages accomplish the horizontal transfer of resistance and virulence genes by mispackaging of mobile-genetic elements, contributing to severe, therapy-refractory S. epidermidis infections. Lytic phages on the other hand can be interesting candidates for new anti-S. epidermidis phage therapies. Despite the importance of phages, we are only beginning to unravel S. epidermidis phage interactions. Recent studies shed new light on S. epidermidis phage diversity, host range, and receptor specificities. Modulation of cell wall teichoic acids, the major phage receptor structures, along with other phage defense mechanisms, are crucial determinants for S. epidermidis susceptibility to different phage groups.

A microcomputed tomography analysis of adaptation in premolars with flared root canals restored with different digitally custom fabricated post materials.

STATEMENT OF PROBLEM: Preserving and strengthening the remaining tooth structure of compromised flared root canals after endodontic treatment is challenging. PURPOSE: The purpose of this in vitro study was to compare the adaptation of milled polymer- infiltrated ceramic, fiber-reinforced composite resin, and high-performance semicrystalline thermoplastic resin posts as used to restore mandibular premolars with flared root canals. MATERIAL AND METHODS: Forty sound mandibular premolars were randomly divided into 4 groups: custom Vita Enamic (CV), custom fiber-reinforced composite resin (CF), custom polyetherketoneketone (CP), and prefabricated fiber (RF) posts. After endodontic treatment, each tooth was sectioned 1.5 mm occlusal to the cementoenamel junction. Then, the post space was prepared and flared, except the RF group, to a depth of 9 mm. The post space in RF group was prepared with a post drill. For the CV, CF, and CP groups, the posts were milled, finished, and cemented to their corresponding teeth. Each tooth was scanned using a microcomputed tomography device, and the reconstructed images were analyzed in mesiodistal, buccolingual, and horizontal planes. The cement thickness, cement volume, and volume of voids were measured. The data were analyzed using 3-way ANOVA (cement thickness) and 2-way ANOVA (cement volume and voids volume) tests followed by the post hoc Tukey test (α=.05). RESULTS: The 3-way ANOVA test revealed a significant interaction (P<.001) between material type, section, and surface on the cement thickness. The mean cement thickness in the RF group was significantly higher than in the CV group (P=.001) and CF group (P=.005). The least mean cement thickness was at the apical section followed by the cervical and middle sections. Regarding cement volume, the 2-way ANOVA test showed statistically significant interaction between material type and section. The mean cement volume in the RF group was significantly lower than in the CV group (P=.001), CF group (P=.001), and CP group (P=.001). The highest mean cement volume was in the cervical section followed by the middle and apical sections. The 2-way ANOVA test showed statistically significant interaction (P<.001) between material type and section on the volume of voids. Significant differences were found between the mean volume of voids at the cervical and middle sections (P=.001) and the cervical and apical sections (P=.002). CONCLUSIONS: Compared with prefabricated fiber posts, digitally fabricated polymer-infiltrated ceramic and fiber-reinforced composite resin posts had a thinner cement layer with minimal thickness at the apical section. The digitally fabricated posts had higher cement volume, especially at the cervical section, than prefabricated fiber posts. High volumes of voids were related to the cervical section of all tested posts.

Commensal production of a broad-spectrum and short-lived antimicrobial peptide polyene eliminates nasal Staphylococcus aureus.

Antagonistic bacterial interactions often rely on antimicrobial bacteriocins, which attack only a narrow range of target bacteria. However, antimicrobials with broader activity may be advantageous. Here we identify an antimicrobial called epifadin, which is produced by nasal Staphylococcus epidermidis IVK83. It has an unprecedented architecture consisting of a non-ribosomally synthesized peptide, a polyketide component and a terminal modified amino acid moiety. Epifadin combines a wide antimicrobial target spectrum with a short life span of only a few hours. It is highly unstable under in vivo-like conditions, potentially as a means to limit collateral damage of bacterial mutualists. However, Staphylococcus aureus is eliminated by epifadin-producing S. epidermidis during co-cultivation in vitro and in vivo, indicating that epifadin-producing commensals could help prevent nasal S. aureus carriage. These insights into a microbiome-derived, previously unknown antimicrobial compound class suggest that limiting the half-life of an antimicrobial may help to balance its beneficial and detrimental activities.

Secretory IgA impacts the microbiota density in the human nose.

BACKGROUND: Respiratory mucosal host defense relies on the production of secretory IgA (sIgA) antibodies, but we currently lack a fundamental understanding of how sIgA is induced by contact with microbes and how such immune responses may vary between humans. Defense of the nasal mucosal barrier through sIgA is critical to protect from infection and to maintain homeostasis of the microbiome, which influences respiratory disorders and hosts opportunistic pathogens. METHODS: We applied IgA-seq analysis to nasal microbiota samples from male and female healthy volunteers, to identify which bacterial genera and species are targeted by sIgA on the level of the individual host. Furthermore, we used nasal sIgA from the same individuals in sIgA deposition experiments to validate the IgA-seq outcomes. CONCLUSIONS: We observed that the amount of sIgA secreted into the nasal mucosa by the host varied substantially and was negatively correlated with the bacterial density, suggesting that nasal sIgA limits the overall bacterial capacity to colonize. The interaction between mucosal sIgA antibodies and the nasal microbiota was highly individual with no obvious differences between potentially invasive and non-invasive bacterial species. Importantly, we could show that for the clinically relevant opportunistic pathogen and frequent nasal resident Staphylococcus aureus, sIgA reactivity was in part the result of epitope-independent interaction of sIgA with the antibody-binding protein SpA through binding of sIgA Fab regions. This study thereby offers a first comprehensive insight into the targeting of the nasal microbiota by sIgA antibodies. It thereby helps to better understand the shaping and homeostasis of the nasal microbiome by the host and may guide the development of effective mucosal vaccines against bacterial pathogens. Video Abstract.

Genomic Analysis of Bacteriocin-Producing Staphylococci: High Prevalence of Lanthipeptides and the Micrococcin P1 Biosynthetic Gene Clusters.

Bacteriocins are antimicrobial peptides produced by bacteria. This study aimed to in silico analyze the presence of bacteriocin gene clusters (BGCs) among the genomes of 22 commensal Staphylococcus isolates from different origins (environment/human/food/pet/wild animals) previously identified as bacteriocin producers. The resistome and plasmidome were studied in all isolates. Five types of BGC were detected in 18 genomes of the 22 bacteriocin-producing staphylococci included in this study: class I (Lanthipeptides), class II, circular bacteriocins, the non-ribosomal-peptide lugdunin and the thiopeptide micrococcin P1 (MP1). A high frequency of lanthipeptides was detected in this collection: BGC variants of BSA, bacCH91, and epilancin15X were identified in two Staphylococcus aureus and one Staphylococcus warneri isolates from food and wild animals. Moreover, two potentially new lanthipeptide-like BGCs with no identity to database entries were found in Staphylococcus epidermidis and Staphylococcus simulans from food and wild animal, respectively. Interestingly, four isolates (one S. aureus and one Staphylococcus hominis, environmental origin; two Staphylococcus sciuri, food) carried the MP1 BGC with differences to those previously described. On the other hand, seven of the 22 genomes (~32%) lacked known genes related with antibiotic or disinfectant-acquired resistance mechanisms. Moreover, the potential carriage of plasmids was evaluated, and several Rep-proteins were identified (~73% of strains). In conclusion, a wide variety of BGCs has been observed among the 22 genomes, and an interesting relationship between related Staphylococcus species and the type of bacteriocin has been revealed. Therefore, bacteriocin-producing Staphylococcus and especially coagulase-negative staphylococci (CoNS) can be considered good candidates as a source of novel bacteriocins.

Inhibition of acid sphingomyelinase reduces reactive astrocyte secretion of mitotoxic extracellular vesicles and improves Alzheimer's disease pathology in the 5xFAD mouse.

In Alzheimer's disease (AD), reactive astrocytes produce extracellular vesicles (EVs) that affect mitochondria in neurons. Here, we show that Aß-induced generation of the sphingolipid ceramide by acid sphingomyelinase (A-SMase) triggered proinflammatory cytokine (C1q, TNF-α, IL-1α) release by microglia, which induced the reactive astrocytes phenotype and secretion of EVs enriched with ceramide. These EVs impeded the capacity of neurons to respond to energy demand. Inhibition of A-SMase with Arc39 and Imipramine reduced the secretion of cytokines from microglia, prompting us to test the effect of Imipramine on EV secretion and AD pathology in the 5xFAD mouse model. Brain derived-EVs from 5xFAD mice treated with Imipramine contained reduced levels of the astrocytic marker GFAP, ceramide, and Aß and did not impair mitochondrial respiration when compared to EVs derived from untreated 5xFAD brain. Consistently, Imipramine-treated 5xFAD mice showed reduced AD pathology. Our study identifies A-SMase inhibitors as potential AD therapy by preventing cyotokine-elicited secretion of mitotoxic EVs from astrocytes.

The S1P receptor 1 antagonist Ponesimod reduces TLR4-induced neuroinflammation and increases Aß clearance in 5XFAD mice.

BACKGROUND: Previously, we showed that the sphingosine-1-phosphate (S1P) transporter spinster 2 (Spns2) mediates activation of microglia in response to amyloid ß peptide (Aß). Here, we investigated if Ponesimod, a functional S1P receptor 1 (S1PR1) antagonist, prevents Aß-induced activation of glial cells and Alzheimer's disease (AD) pathology. METHODS: We used primary cultures of glial cells and the 5XFAD mouse model to determine the effect of Aß and Ponesimod on glial activation, Aß phagocytosis, cytokine levels and pro-inflammatory signaling pathways, AD pathology, and cognitive performance. FINDINGS: Aß42 increased the levels of TLR4 and S1PR1, leading to their complex formation. Ponesimod prevented the increase in TLR4 and S1PR1 levels, as well as the formation of their complex. It also reduced the activation of the pro-inflammatory Stat1 and p38 MAPK signaling pathways, while activating the anti-inflammatory Stat6 pathway. This was consistent with increased phagocytosis of Aß42 in primary cultured microglia. In 5XFAD mice, Ponesimod decreased the levels of TNF-α and CXCL10, which activate TLR4 and Stat1. It also increased the level of IL-33, an anti-inflammatory cytokine that promotes Aß42 phagocytosis by microglia. As a result of these changes, Ponesimod decreased the number of Iba-1+ microglia and GFAP+ astrocytes, and the size and number of amyloid plaques, while improving spatial memory as measured in a Y-maze test. INTERPRETATION: Ponesimod targeting S1PR1 is a promising therapeutic approach to reprogram microglia, reduce neuroinflammation, and increase Aß clearance in AD. FUNDING: NIHR01AG064234, RF1AG078338, R21AG078601, VAI01BX003643.

Novel Isolation Method Reveals Sex-Specific Composition and Neurotoxicity of Small Extracellular Vesicles in a Mouse Model of Alzheimer's Disease.

We developed a new method to isolate small extracellular vesicles (sEVs) from male and female wild-type and 5xFAD mouse brains to investigate the sex-specific functions of sEVs in Alzheimer's disease (AD). A mass spectrometric analysis revealed that sEVs contained proteins critical for EV formation and Aß. ExoView analysis showed that female mice contained more GFAP and Aß-labeled sEVs, suggesting that a larger proportion of sEVs from the female brain is derived from astrocytes and/or more likely to bind to Aß. Moreover, sEVs from female brains had more acid sphingomyelinase (ASM) and ceramide, an enzyme and its sphingolipid product important for EV formation and Aß binding to EVs, respectively. We confirmed the function of ASM in EV formation and Aß binding using co-labeling and proximity ligation assays, showing that ASM inhibitors prevented complex formation between Aß and ceramide in primary cultured astrocytes. Finally, our study demonstrated that sEVs from female 5xFAD mice were more neurotoxic than those from males, as determined by impaired mitochondrial function (Seahorse assays) and LDH cytotoxicity assays. Our study suggests that sex-specific sEVs are functionally distinct markers for AD and that ASM is a potential target for AD therapy.

Influence of resin cement on color stability when luting lithium disilicate and zirconia restorations.

AIM: To evaluate the influence of resin cement on the color stability of lithium disilicate and zirconia restorations immersed in coffee after aging. MATERIALS AND METHODS: Eighty maxillary premolars were classified into eight groups (n = 10) based on restorative material type (lithium disilicate or zirconia), resin cement type (G-CEM LinkForce; GC Corporation or Panavia SA Cement Plus Automix; Kuraray Noritake Dental), and preheating temperature (25°C or 54°C). Following tooth preparation, each restoration was bonded to its corresponding substrate. Using a reflectance spectrophotometer, Commission Internationale de l'Éclairage (CIE) tristimulus values were detected and calculated (D65 standard illumination, 10-degree observer angle). All specimens were aged (240,000 load cycles followed by 10,000 thermal cycles), then immersed in coffee (18 h). Following that, the second measurements of the color coordinates were determined. The total color differences were measured, and the data were statistically analyzed (α = 0.05). RESULTS: The temperature had a significant effect on ΔL΄ (P < 0.001), ΔC΄ (P < 0.001), and ΔH΄ (P < 0.001). The lithium disilicate restorations were more color stable than the zirconia restorations. Also, there was a significant difference (P = 0.047) between the LinkForce (2.28 ± 0.48) and Panavia SA (2.15 ± 0.46) cement. The restorations cemented at a temperature of 54°C (1.76 ± 0.11) showed significant color differences (P < 0.001) compared with those cemented at a temperature of 25°C (2.67 ± 0.15). A three-way analysis of variance (ANOVA) test revealed that the interaction between the ceramic material, cement type, and temperature had no statistically significant effect (P = 0.611) on the color stability of the ceramic restorations. CONCLUSIONS: Cement type has a significant effect on the color stability of lithium disilicate and zirconia restorations. Cement at a temperature of up to 54°C enhances the color stability of lithium disilicate and zirconia restorations.

Horizontal Transfer of Bacteriocin Biosynthesis Genes Requires Metabolic Adaptation To Improve Compound Production and Cellular Fitness.

Biosynthetic gene clusters (BGCs) encoding the production of bacteriocins are widespread among bacterial isolates and are important genetic determinants of competitive fitness within a given habitat. Staphylococci produce a tremendous diversity of compounds, and the corresponding BGCs are frequently associated with mobile genetic elements, suggesting gain and loss of biosynthetic capacity. Pharmaceutical biology has shown that compound production in heterologous hosts is often challenging, and many BGC recipients initially produce small amounts of compound or show reduced growth rates. To assess whether transfer of BGCs between closely related Staphylococcus aureus strains can be instantly effective or requires elaborate metabolic adaptation, we investigated the intraspecies transfer of a BGC encoding the ribosomally synthesized and posttranslationally modified peptide (RiPP) micrococcin P1 (MP1). We found that acquisition of the BGC by S. aureus RN4220 enabled immediate MP1 production but also imposed a metabolic burden, which was relieved after prolonged cultivation by adaptive mutation. We used a multiomics approach to study this phenomenon and found adaptive evolution to select for strains with increased activity of the tricarboxylic acid cycle (TCA), which enhanced metabolic fitness and levels of compound production. Metabolome analysis revealed increases of central metabolites, including citrate and α-ketoglutarate in the adapted strain, suggesting metabolic adaptation to overcome the BGC-associated growth defects. Our results indicate that BGC acquisition requires genetic and metabolic predispositions, allowing the integration of bacteriocin production into the cellular metabolism. Inappropriate metabolic characteristics of recipients can entail physiological burdens, negatively impacting the competitive fitness of recipients within natural bacterial communities. IMPORTANCE Human microbiomes are critically associated with human health and disease. Importantly, pathogenic bacteria can hide in human-associated communities and can cause disease when the composition of the community becomes unbalanced. Bacteriocin-producing commensals are able to displace pathogens from microbial communities, suggesting that their targeted introduction into human microbiomes might prevent pathogen colonization and infection. However, to develop probiotic approaches, strains are needed that produce high levels of bioactive compounds and retain cellular fitness within mixed bacterial communities. Our work offers insights into the metabolic burdens associated with the production of the bacteriocin micrococcin P1 and highlights evolutionary strategies that increase cellular fitness in the context of production. Metabolic adaptations are most likely broadly relevant for bacteriocin producers and need to be considered for the future development of effective microbiome editing strategies.

Effect of ceramic and resin cement type on color stability and translucency of ceramic laminate veneers for diastema closure: an in vitro study.

To investigate the effect of resin cements on the color stability and translucency of ceramic laminate veneers used for diastema closure. Sixty resin abutments were prepared for ceramic laminate veneers and divided into six groups according to the ceramic type (lithium disilicate, zirconia-reinforced lithium silicate, and translucent zirconia) and the cement type (Variolink Esthetic LC and RelyX Veneer). Color coordinates and translucency were analyzed after cementation and after soaking in the coffee solution. Differences in color and translucency were estimated, and results were statistically assessed (α = 0.05). Ceramic materials showed a significant impact on color changes after soaking in coffee within Variolink Esthetic groups. Translucent zirconia showed the highest color change, followed by zirconia reinforced lithium silicate and lithium disilicate. Ceramic materials showed a significant impact among the RelyX Veneer groups. A significant interaction in color changes was found between ceramic types and cement types after cementation, and after soaking in coffee was found. All groups showed a clinically acceptable difference in translucency parameters after soaking in coffee. The resin cement affects the color and translucency of ceramic laminate veneers used for diastema closure, and ceramic laminate veneers bonded with Variolink Esthetic LC resin cement are more translucent, while ceramic laminate veneers bonded with RelyX Veneer resin are more resistant to coffee staining. The lithium disilicate laminate veneer is more resistant to coffee staining than zirconia reinforced lithium silicate and translucent zirconia laminate veneers used for diastema closure.

A rare loss-of-function genetic mutation suggest a role of dermcidin deficiency in hidradenitis suppurativa pathogenesis.

Hidradenitis suppurativa (HS) is a chronic inflammatory skin disease with a multifactorial aetiology that involves a strict interplay between genetic factors, immune dysregulation and lifestyle. Familial forms represent around 40% of total HS cases and show an autosomal dominant mode of inheritance of the disease. In this study, we conducted a whole-exome sequence analysis on an Italian family of 4 members encompassing a vertical transmission of HS. Focusing on rare damaging variants, we identified a rare insertion of one nucleotide (c.225dupA:p.A76Sfs*21) in the DCD gene encoding for the antimicrobial peptide dermcidin (DCD) that was shared by the proband, his affected father and his 11-years old daughter. Since several transcriptome studies have shown a significantly decreased expression of DCD in HS skin, we hypothesised that the identified frameshift insertion was a loss-of-function mutation that might be associated with HS susceptibility in this family. We thus confirmed by mass spectrometry that DCD levels were diminished in the affected members and showed that the antimicrobial activity of a synthetic DCD peptide resulting from the frameshift mutation was impaired. In order to define the consequences related to a decrease in DCD activity, skin microbiome analyses of different body sites were performed by comparing DCD mutant and wild type samples, and results highlighted significant differences between the groins of mutated and wild type groups. Starting from genetic analysis conducted on an HS family, our findings showed, confirming previous transcriptome results, the potential role of the antimicrobial DCD peptide as an actor playing a crucial part in the etio-pathogenesis of HS and in the maintenance of the skin's physiological microbiome composition; so, we can hypothesise that DCD could be used as a novel target for personalised therapeutic approach.

Function of ceramide transfer protein for biogenesis and sphingolipid composition of extracellular vesicles.

The formation of extracellular vesicles (EVs) is induced by the sphingolipid ceramide. How this pathway is regulated is not entirely understood. Here, we report that the ceramide transport protein (CERT) mediates a non-vesicular transport of ceramide between the endoplasmic reticulum (ER) and the multivesicular endosome at contact sites. The process depends on the interaction of CERT's PH domain with PI4P generated by PI4KIIα at endosomes. Furthermore, a complex is formed between the START domain of CERT, which carries ceramide, and the Tsg101 protein, which is part of the endosomal sorting complex required for transport (ESCRT-I). Inhibition of ceramide biosynthesis reduces CERT-Tsg101 complex formation. Overexpression of CERT increases EV secretion while its inhibition reduces EV formation and the concentration of ceramides and sphingomyelins in EVs. In conclusion, we discovered a function of CERT in regulating the sphingolipid composition and biogenesis of EVs, which links ceramide to the ESCRT-dependent pathway.

Neutral Sphingomyelinase 2 Mediates Oxidative Stress Effects on Astrocyte Senescence and Synaptic Plasticity Transcripts.

We have shown that deficiency of neutral sphingomyelinase 2 (nSMase2), an enzyme generating the sphingolipid ceramide, improves memory in adult mice. Here, we performed sphingolipid and RNA-seq analyses on the cortex from 10-month-old nSMase2-deficient (fro/fro) and heterozygous (+ /fro) mice. fro/fro cortex showed reduced levels of ceramide, particularly in astrocytes. Differentially abundant transcripts included several functionally related groups, with decreases in mitochondrial oxidative phosphorylation and astrocyte activation transcripts, while axon guidance and synaptic transmission and plasticity transcripts were increased, indicating a role of nSMase2 in oxidative stress, astrocyte activation, and cognition. Experimentally induced oxidative stress decreased the level of glutathione (GSH), an endogenous inhibitor of nSMase2, and increased immunolabeling for ceramide in primary + /fro astrocytes, but not in fro/fro astrocytes. ß-galactosidase activity was lower in 5-week-old fro/fro astrocytes, indicating delayed senescence due to nSMase2 deficiency. In fro/fro cortex, levels of the senescence markers C3b and p27 and the proinflammatory cytokines interleukin 1ß, interleukin 6, and tumor necrosis factor α were reduced, concurrent with twofold decreased phosphorylation of their downstream target, protein kinase Stat3. RNA and protein levels of the ionotropic glutamate receptor subunit 2B (Grin2b/NR2B) were increased by twofold, which was previously shown to enhance cognition. This was consistent with threefold reduced levels of exosomes carrying miR-223-3p, a micro-RNA downregulating NR2B. In summary, our data show that nSMase2 deficiency prevents oxidative stress-induced elevation of ceramide and secretion of exosomes by astrocytes that suppress neuronal function, indicating a role of nSMase2 in the regulation of neuroinflammation and cognition.

Does Resin Cement Type and Cement Preheating Influence the Marginal and Internal Fit of Lithium Disilicate Single Crowns?

This paper assesses the effect of cement type and cement preheating on the marginal and internal fit of lithium disilicate single crown. Methods: 40 maxillary premolars were selected, restored with lithium disilicate single crowns. Teeth were randomly assigned into four groups (n = 10) based on cement type (Panavia SA or LinkForce) and preheating temperature (25 °C or 54 °C). After fabrication of the restoration, cements were incubated at 25 °C or 54 °C for 24 h, and each crown was cemented to its corresponding tooth. After 24 h, all specimens were thermally aged to (10,000 thermal cycles between 5 °C and 55 °C), then load cycled for 240,000 cycles. Each specimen was then sectioned in bucco-palatal direction and inspected under a stereomicroscope at x45 magnification for marginal and internal fit evaluation. The data were statistically analyzed (significance at p ≤ 0.05 level). Results: At the mid-buccal finish line, mid-buccal wall, palatal cusp, mid-palatal wall, mid-palatal finish line, and palatal margin measuring points, there was a significant difference (p ≤ 0.05) between the lithium disilicate group cemented with Panavia SA at 25 °C and the group cemented with LinkForce at 25 °C, while there was no significant difference (p > 0.05) at the other points. At all measuring points, except at the palatal cusp tip (p = 0.948) and palatal margin (p = 0.103), there was a statistically significant difference (p ≤ 0.05) between the lithium disilicate group cemented with Panavia SA at 54 °C and the group cemented with LinkForce at 54 °C. Regardless of cement preheating, statistically significant differences were found in the buccal cusp tip, central groove, palatal cusp tip, and mid-palatal wall (p ≤ 0.05) in the lithium disilicate group cemented with Panavia SA at 25 °C and 54 °C, as well as the mid-palatal chamfer finish line and palatal margin in the LinkForce group cemented with Panavia SA at 25 °C and 54 °C. At the other measurement points, however, there was no significant difference (p > 0.05). Conclusions: The type of resin cement affects the internal and marginal fit of lithium disilicate crowns. At most measuring points, the cement preheating does not improve the internal and marginal fit of all lithium disilicate crowns.

Extracellular vesicles in pharmacology: Novel approaches in diagnostics and therapy.

Exosomes are nano-sized lipid vesicles that are produced by all eukaryotic cells, and they typically range in size from 30 to 150 nm. Exosomes were discovered almost 40 years ago; however, the last two decades have attracted considerable attention due to exosomes' inherent abilities to shuttle nucleic acids, lipids and proteins between cells, along with their natural affinity to exosome target cells. From a pharmaceutical perspective, exosomes are regarded as naturally produced nanoparticle drug delivery vehicles. The application of exosomes as a means of drug delivery offers critical advantages compared to other nanoparticulate drug delivery systems, such as liposomes and polymeric nanoparticles. These advantages are due to the exosomes' intrinsic features, such as low immunogenicity, biocompatibility, stability, and their ability to overcome biological barriers. Herein, we outline the structure and origin of exosomes, as well as their biological functions. We also touch upon recent advances in exosome labeling, imaging and drug loading. Finally, we discuss exosomes in targeted drug delivery and clinical trial development.

Does Preheating Resin Cements Affect Fracture Resistance of Lithium Disilicate and Zirconia Restorations?

This paper assesses the impact of preheating of adhesive cement on the fracture resistance of lithium disilicate and zirconia restorations. Methods: A total of 80 human maxillary premolar teeth were assigned into 8 groups (n = 10) according to material type (either lithium disilicate or zirconia) and type of resin cement (either LinkForce or Panavia SA) with preheating temperature at 54 °C or at room temperature (25 °C). Teeth were prepared and restored with either lithium disilicate or zirconia restorations. After cementation, specimens were thermal cycled (10,000 cycles, 5 °C/55 °C), then load cycled for 240,000 cycles (50 N). Each specimen was statically loaded until fracture and the load (N) at fracture was recorded, then the failure mode was detected. Statistical analysis of data was performed (p ≤ 0.05). Results: There was no significant difference (p = 0.978) in fracture mean values between LinkForce and Panavia SA. Statistically significant difference (p = 0.001) was revealed between fracture resistance of lithium disilicate restorations cemented with LinkForce at 25 °C and at 54 °C; however there was no significant difference (p = 0.92) between the fracture resistance of lithium disilicate restorations cemented with Panavia SA used at 25 °C and at 54 °C. Regarding the interaction between ceramic material, cement type, and cement preheating, there was no significant effect (p > 0.05) in fracture resistance. The cement type does not influence the fracture resistance of ceramic restorations. Preheating of resin cement has negatively influenced the fracture resistance of all tested groups, except for lithium disilicate cemented using LinkForce cement.

Palmitoylation of acetylated tubulin and association with ceramide-rich platforms is critical for ciliogenesis.

Microtubules are polymers composed of αß-tubulin subunits that provide structure to cells and play a crucial role in in the development and function of neuronal processes and cilia, microtubule-driven extensions of the plasma membrane that have sensory (primary cilia) or motor (motile cilia) functions. To stabilize microtubules in neuronal processes and cilia, α tubulin is modified by the posttranslational addition of an acetyl group, or acetylation. We discovered that acetylated tubulin in microtubules interacts with the membrane sphingolipid, ceramide. However, the molecular mechanism and function of this interaction are not understood. Here, we show that in human induced pluripotent stem cell-derived neurons, ceramide stabilizes microtubules, which indicates a similar function in cilia. Using proximity ligation assays, we detected complex formation of ceramide with acetylated tubulin in Chlamydomonas reinhardtii flagella and cilia of human embryonic kidney (HEK293T) cells, primary cultured mouse astrocytes, and ependymal cells. Using incorporation of palmitic azide and click chemistry-mediated addition of fluorophores, we show that a portion of acetylated tubulin is S-palmitoylated. S-palmitoylated acetylated tubulin is colocalized with ceramide-rich platforms in the ciliary membrane, and it is coimmunoprecipitated with Arl13b, a GTPase that mediates transport of proteins into cilia. Inhibition of S-palmitoylation with 2-bromo palmitic acid or inhibition of ceramide biosynthesis with fumonisin B1 reduces formation of the Arl13b-acetylated tubulin complex and its transport into cilia, concurrent with impairment of ciliogenesis. Together, these data show, for the first time, that ceramide-rich platforms mediate membrane anchoring and interaction of S-palmitoylated proteins that are critical for cilium formation, stabilization, and function.