AglH,a thermophilic UDP-N-acetylglucosamine-1-phosphate:dolichyl phosphate GlcNAc-1-phosphotransferase initiating protein N-glycosylation pathway in Sulfolobus acidocaldarius,is capable of complementing the eukaryal Alg7

AglH, a predicted UDP-GlcNAc-1-phosphate:dolichyl phosphate GlcNAc-1-phosphotransferase, is initiating the protein N-glycosylation pathway in the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius. AglH successfully replaced the endogenous GlcNAc-1-phosphotransferase activity of Alg7 in a conditional lethal Saccharomyces cerevisiae strain, in which the first step of the eukaryal protein N-glycosylation process was repressed. This study is one of the few examples of cross-domain complementation demonstrating a conserved polyprenyl phosphate transferase reaction within the eukaryal and archaeal domain like it was demonstrated for Methanococcus voltae (Shams-Eldin et al. 2008). The topology prediction and the alignment of the AglH membrane protein with GlcNAc-1-phosphotransferases from the three domains of life show significant conservation of amino acids within the different proposed cytoplasmic loops. Alanine mutations of selected conserved amino acids in the putative cytoplasmic loops II (D₁₀₀), IV (F₂₂₀) and V (F₂₆₄) demonstrated the importance of these amino acids for cross-domain AlgH activity in in vitro complementation assays in S. cerevisiae. Furthermore, antibiotic treatment interfering directly with the activity of dolichyl phosphate GlcNAc-1-phosphotransferases confirmed the essentiality of N-glycosylation for cell survival.

     

Aqueous extract of tobacco induces mitochondrial potential dependent cell death and epithelial-mesenchymal transition in gingival epithelial cells

Smokeless tobacco habits are detrimental to oral health. A correlation between tobacco use and local epithelial tissue damage exists. Yet, the underlying cellular mechanism is not precisely characterized. This study assessed the dose-dependent action of Smokeless tobacco extract on gingival epithelial cells. Gingival tissue was taken from 5 healthy donors. Gingival epithelial cells were isolated by an enzymatic method and cultured up to passage 2. The cultured cells were treated with smokeless tobacco extract at 10%, 25%, 50%, and 75% volume concentration. After 48 h of incubation, MTT assay, Annexin V/PI assay, and DiIC1(5) assay were used to evaluate viability, apoptosis, and mitochondrial potential of the cells. RT-qPCR was used to determine the expression of BAX, BCL2, ECAD, NCAD, and TWIST. The Smokeless tobacco extract reduced cell viability by disrupting the mitochondrial potential and inducing apoptosis. Further, the Smokeless tobacco extract induced a dose-dependent epithelial-mesenchymal-transition in gingival epithelial cells. Apoptotic cellular death caused by tobacco extract on the gingival epithelial system was dependant on the mitochondrial potential of the cell. The results demonstrate that smokeless tobacco causes detrimental metabolic alterations of the periodontium.Featured applicationThis study elucidates the mechanism by which Smokeless tobacco products cause cellular damage to the gingival epithelium. The use of Smokeless tobacco products can lead to major cellular and surface changes in the gingiva and its appearance. The consequences of these changes are not limited to oral cancer but also increases a person’s risk for dental and periodontal disease.     

Designing an immunoinformatic vaccine for peri-implantitis using a structural biology approach

ObjectivesPeri-implantitis is a destructive inflammatory process that affects the soft and hard tissues around dental implants. porphyromonas gingivalis, an anaerobic gram-negative bacterium, appears to be the main culprit. Since there is no efficient and specific vaccine to treat peri-implantitis, the goal of our research has been to develop a multi-epitope vaccination utilizing an immunoinformatics approach that targeted P. gingivalis type I fim A.Materials and methodsP. gingivalis peptides 6JKZ and 6KMF are suitable for vaccine development. B- and T-cell epitopes from 6KMF and 6JKZ were detected and evaluated based on critical factors to produce a multi-epitope vaccine construct. It was assessed based on allergenicity, antigenicity, stability. The vaccine's dual major histocompatibility complex (MHC-I and MHC-II) binding epitopes allowed it to reach a larger population. P. gingivalis fimbriae induce immune subversion through TLR -CXCR4 receptor complex pathway. The ClusPro 2.0 server was used to do the molecular docking using TLR2 - CXCR4 and vaccine epitopes as receptor and ligand respectively.ResultsThe designed vaccine was non-allergenic and had a high antigenicity, solubility, and stability. The 3D structure of the vaccine revealed strong interaction with CXCR4(TLR2) using molecular docking. The vaccine-CXCR4 interface was more consistent, possibly because the vaccination has a higher affinity for the CXCR4-TLR2 complex.ConclusionThis study details the vaccine's distinct and sustained interaction with the CXCR4(TLR2) immunological receptor and its consistent and effective utterance in the bacterial system. As a result, our vaccine formulation will evoke a significant memory response and induce an adaptive immune response against P. gingivalis.     

Visualizing the Attack of RNase Enzymes on Dendriplexes and Naked RNA Using Atomic Force Microscopy

Cationic polymers such as poly(amidoamine), PAMAM, dendrimers have been used to electrostatically complex siRNA molecules forming dendriplexes for enhancing the cytoplasmic delivery of the encapsulated cargo. However, excess PAMAM dendrimers is typically used to protect the loaded siRNA against enzymatic attack, which results in systemic toxicity that hinders the in vivo use of these particles. In this paper, we evaluate the ability of G4 (flexible) and G5 (rigid) dendrimers to complex model siRNA molecules at low +/− ratio of 2/1 upon incubation for 20 minutes and 24 hours. We examine the ability of the formed G4 and G5 dendriplexes to shield the loaded siRNA molecules and protect them from degradation by RNase V1 enzymes using atomic force microscopy (AFM). Results show that G4 and G5 dendrimers form similar hexagonal complexes upon incubation with siRNA molecules for 20 minutes with average full width of 43±19.3 nm and 62±8.3 at half the maximum height, respectively. AFM images show that these G4 and G5 dendriplexes were attacked by RNase V1 enzyme leading to degradation of the exposed RNA molecules that increased with the increase in incubation time. In comparison, incubating G4 and G5 dendrimers with siRNA for 24 hours led to the formation of large particles with average full width of 263±60 nm and 48.3±2.5 nm at half the maximum height, respectively. Both G4 and G5 dendriplexes had a dense central core that proved to shield the loaded RNA molecules from enzymatic attack for up to 60 minutes. These results show the feasibility of formulating G4 and G5 dendriplexes at a low N/P (+/−) ratio that can resist degradation by RNase enzymes, which reduces the risk of inducing non-specific toxicity when used in vivo.     

Mandibular symphysis dimensions in different sagittal and vertical skeletal relationships

BackgroundThe aim of this cross-sectional study was to compare the dimensions of mandibular symphysis (MS) between gender and the different sagittal and vertical skeletal relationships.Material and MethodsPre-treatment records of orthodontic patients were divided according to gender, sagittal (Class I, II and III) and vertical (decreased, average and increased mandibular plane [MP] angle) skeletal relationships. Measurements of MS parameters were performed on lateral cephalograms using IMAGEJ software. Comparisons between MS parameters and gender and the different skeletal relationships was performed using multifactorial and one-way ANOVA, and independent sample t-tests.ResultsA total of 104 records (25 males and 79 females) fulfilled the inclusion criteria. Males had significantly greater MS surface area, dentoalveolar length, skeletal symphysis length, total symphysis length, vertical symphysis dimension and symphysis convexity (p < 0.05). Skeletal Class II patients had significantly greater dentoalveolar and skeletal symphysis lengths while Class III had greater chin length, vertical symphysis dimension and symphysis convexity (p < 0.05). Patients with decreased vertical dimension had greater skeletal symphysis length (p = 0.026) and those with an average vertical relationship had greater chin length (p < 0.001).ConclusionsThe morphology of the mandibular symphysis is affected by gender, sagittal and vertical skeletal patterns. Males had increased mandibular symphysis surface area and linear dimensions. Class II patients had greater dentoalveolar length. Chin length was greater in patients with an average MP angle.     

PtxtPME1 and homogalacturonans influence xylem hydraulic properties in poplar

While the xylem hydraulic properties, such as vulnerability to cavitation (VC), are of paramount importance in drought resistance, their genetic determinants remain unexplored. There is evidence that pectins and their methylation pattern are involved, but the detail of their involvement and the corresponding genes need to be clarified. We analyzed the hydraulic properties of the 35S::PME1 transgenic aspen that ectopically under‐ or over‐express a xylem‐abundant pectin methyl esterase, PtxtPME1. We also produced and analyzed 4CL1::PGII transgenic poplars expressing a fungal polygalacturonase, AnPGII, under the control of the Ptxa4CL1 promoter that is active in the developing xylem after xylem cell expansion. Both the 35S::PME1 under‐ and over‐expressing aspen lines developed xylem with lower‐specific hydraulic conductivity and lower VC, while the 4CL1::PGII plants developed xylem with a higher VC. These xylem hydraulic changes were associated with modifications in xylem structure or in intervessel pit structure that can result in changes in mechanical behavior of the pit membrane. This study shows that homogalacturonans and their methylation pattern influence xylem hydraulic properties, through its effect on xylem cell expansion and on intervessel pit properties and it show a role for PtxtPME1 in the xylem hydraulic properties.     

Nanosilica and jasmonic acid as alternative methods for control Tuta absoluta (Meyrick) in tomato crop under field conditions

Recently, Tuta absoluta became one of the major pests that attack commercial tomato globally. Field test was done to evaluate the effect of different concentrations of nanosilica (NS) and jasmonic acid (JA), and compared them with indoxacrb (recommended insecticide) on reduction of damage rate caused by T. absoluta larvae under field conditions. Nanosilica (600 ppm) and indoxacrb (0.25 cm³/L) had the highest efficiency to reduce the rate of mines in the leaves. Jasmonic acid at rate 1.141 μM/plant showed a good reduction of number of fruits damaged. Nanosilica with 600 ppm concentration and Jasmonic acid at rate 1.141 μM/plant is used to control T. absoluta in the field.     

Composition of archaeal,bacterial, and eukaryal RuBisCO genotypes in three Western Pacific arc hydrothermal vent systems

We studied the diversity of all forms of the RuBisCO large subunit-encoding gene cbbL in three RuBisCO uncharacterized hydrothermal vent communities. This diversity included the archaeal cbbL and the forms IC and ID, which have not previously been studied in the deep-sea environment, in addition to the forms IA, IB and II. Vent plume sites were Fryer and Pika in the Mariana arc and the Suiyo Seamount, Izu-Bonin, Japan. The cbbL forms were PCR amplified from plume bulk microbial DNA and then cloned and sequenced. Archaeal cbbL was detected in the Mariana samples only. Both forms IA and II were amplified from all samples, while the form IC was amplified only from the Pika and Suiyo samples. Only the Suiyo sample showed amplification of the form ID. The form IB was not recorded in any sample. Based on rarefaction analysis, nucleotide diversity and average pairwise difference, the archaeal cbbL was the most diverse form in Mariana samples, while the bacterial form IA was the most diverse form in the Suiyo sample. Also, the Pika sample harbored the highest diversity of cbbL phylogenetic lineages. Based on pairwise reciprocal library comparisons, the Fryer and Pika archaeal cbbL libraries showed the most significant difference, while Pika and Suiyo showed the highest similarity for forms IA and II libraries. This suggested that the Fryer supported the most divergent sequences. All archaeal cbbL sequences formed unique phylogenetic lineages within the branches of anaerobic thermophilic archaea of the genera Pyrococcus, Archaeoglobus, and Methanococcus. The other cbbL forms formed novel phylogenetic clusters distinct from any recorded previously in other deep-sea habitats. This is the first evidence for the diversity of archaeal cbbL in environmental samples.     

Novel and diverse integron integrase genes and integron-like gene cassettes are prevalent in deep-sea hydrothermal vents

The lack of information about mobile DNA in deep-sea hydrothermal vents limits our understanding of the phylogenetic diversity of the mobile genome of bacteria in these environments. We used culture-independent techniques to explore the diversity of the integron/mobile gene cassette system in a variety of hydrothermal vent communities. Three samples, which included two different hydrothermal vent fluids and a mussel species that contained essentially monophyletic sulfur-oxidizing bacterial endosymbionts, were collected from Suiyo Seamount, Izu-Bonin, Japan, and Pika site, Mariana arc. First, using degenerate polymerase chain reaction (PCR) primers, we amplified integron integrase genes from metagenomic DNA from each sample. From vent fluids, we discovered 74 new integrase genes that were classified into 11 previously undescribed integron classes. One integrase gene was recorded in the mussel symbiont and was phylogenetically distant from those recovered from vent fluids. Second, using PCR primers targeting the gene cassette recombination site (59-be), we amplified and subsequently identified 60 diverse gene cassettes. In multicassette amplicons, a total of 13 59-be sites were identified. Most of these sites displayed features that were atypical of the features previously well conserved in this family. The Suiyo vent fluid was characterized by gene cassette open reading frames (ORFs) that had significant homologies with transferases, DNA-binding proteins and metal transporter proteins, while the majority of Pika vent fluid gene cassettes contained novel ORFs with no identifiable homologues in databases. The symbiont gene cassette ORFs were found to be matched with DNA repair proteins, methionine aminopeptidase, aminopeptidase N, O-sialoglycoprotein endopeptidase and glutamate synthase, which are proteins expected to play a role in animal/symbiont metabolism. The success of this study indicates that the integron/gene cassette system is common in deep-sea hydrothermal vents, an environment type well removed from anthropogenic disturbance.     

Responses of core and peripheral temperatures to chronic cold stress in transiently goitrous goats

Thyroid hormones play a major role in controlling basal metabolic rate, body temperature and hemodynamics. Responses of core and peripheral body temperatures to transient use of thyreostatic drugs are seldom measured simultaneously under chronic experimental settings. We developed a technique to attain this goal. Eighteen Damascus goat kids (initial Avg. BW=44±0.7 kg) underwent a minor invasive surgical procedure to implant two miniature temperature data loggers, intraperitoneally and subcutaneously, to assess core (T_core) and subcutaneous (T_sq) temperature changes when subjected to chronic (47 days) cold (< 10 °C) environment along with or without oral administration of the reversible goitrogen 6-N-propyl-2-thiouracil (PTU; 20 mg/kg BW/day). Nine animals served as controls whereas the remaining nine were treated with PTU for 31 out of total 47-day trial period. The PTU treatment significantly (P<0.05) depressed circulating free thyroxine (FT4), followed by a rebound after termination. Prior to initiation of PTU treatment, all animals displayed similar daily means of T_core and T_sq. Subsequently, both temperatures precipitously declined (P<0.01) with the gradual drop in ambient temperature (T_a) overtime. However, PTU caused an immediate and further drop in T_sq, suggesting a reduction in cutaneous perfusion. Interestingly, on the other hand, a delayed downregulation of T_core by PTU occurred 14 days post-treatment, indicating a relatively slower depression in metabolic thermogenesis after suppression of circulating thyroxine. Upon termination of PTU, T_core but not T_sq, returned to simulate control levels. Results from the current study show the practicality of our technique to concurrently record core and peripheral temperatures in chronically-instrumented animals, and that PTU causes a marked depression in thermoregulatory ability during cold stress with no indication for a reduction in the temperature set-point.