Identification and characterization of VapBC toxin-antitoxin system in Bosea sp. PAMC 26642 isolated from Arctic lichens.

Toxin-antitoxin (TA) systems are genetic modules composed of a toxin interfering with cellular processes and its cognate antitoxin, which counteracts the activity of the toxin. TA modules are widespread in bacterial and archaeal genomes. It has been suggested that TA modules participate in the adaptation of prokaryotes to unfavorable conditions. The Bosea sp. PAMC 26642 used in this study was isolated from the Arctic lichen Stereocaulon sp. There are 12 putative type II TA loci in the genome of Bosea sp. PAMC 26642. Of these, nine functional TA systems have been shown to be toxic in Escherichia coli The toxin inhibits growth, but this inhibition is reversed when the cognate antitoxin genes are coexpressed, indicating that these putative TA loci were bona fide TA modules. Only the BoVapC1 (AXW83_01405) toxin, a homolog of VapC, showed growth inhibition specific to low temperatures, which was recovered by the coexpression of BoVapB1 (AXW83_01400). Microscopic observation and growth monitoring revealed that the BoVapC1 toxin had bacteriostatic effects on the growth of E. coli and induced morphological changes. Quantitative real time polymerase chain reaction and northern blotting analyses showed that the BoVapC1 toxin had a ribonuclease activity on the initiator tRNAfMet, implying that degradation of tRNAfMet might trigger growth arrest in E. coli Furthermore, the BoVapBC1 system was found to contribute to survival against prolonged exposure at 4°C. This is the first study to identify the function of TA systems in cold adaptation.

Clinical Performance of Implant Crown Retained Removable Partial Dentures for Mandibular Edentulism-A Retrospective Study.

The studies on implant-crown-retained removable partial dentures (IC-RPDs) for edentulism are scarce. The purpose of this study was to evaluate survival rates and marginal bone loss (MBL) of IC-RPDs compared to implant overdentures (IODs) in mandibular edentulism. Variables that influenced survival and marginal bone loss (MBL) of implants in both treatment modalities were analyzed and the functional/esthetic satisfaction of patients as well as prosthetic complications were also observed. Eighteen IC-RPDs with a total of 60 implant-supported survey crowns and 24 IODs with a total 94 implants retained with magnet attachments were observed. After a median observation period of 46.6 months (up to 149 months), we observed 98.3% implant survival rates for IC-RPDs and 92.5% for IODs. Kaplan-Meier survival curves based on the treatment modality showed that, at 96 months, cumulative survival rates were 98.3% in IC-RPD and 83.1% in IOD. For implant survival rates, no statistical differences were observed according to age, sex, opposing dentition, or implant positions (p = 0.515, 0.666, 0.201, 0.749, respectively). The implant MBL measurements for IC-RPD and IOD groups at the final recall check were 0.93 ± 1.22 mm and 2.12 ± 2.09 mm, respectively. Additionally, there were no significant differences between groups (p = 0.554). The implants with peri-implantitis at year 1 showed significantly higher MBL at final check-up (p < 0.001). The MBL of implants showed significant differences based on age (p = 0.008) and opposing dentition (p = 0.003). No significant differences of implant MBL were observed for the position of placed implants (p = 0.621) or sex (p = 0.666). Patient-reported outcome measures (PROMs) on functional and esthetic satisfaction were significantly improved after IC-RPD or IOD treatment (p < 0.001). The most frequent prosthetic complication of IC-RPD was clasp loosening, while for IOD group, it was attachment dislodgement. Within the limitations of this retrospective study, we concluded that IC-RPDs could be considered as a viable treatment option for edentulous patients who need few fixed abutments for satisfaction.

New Rehabilitation Concept for Maxillary Edentulism: A Clinical Retrospective Study of Implant Crown Retained Removable Partial Dentures.

There have been no studies of implant-crown-retained removable partial dentures (IC-RPD) for the treatment of maxillary edentulism. The purpose of this study was to perform clinical and radiographic evaluations of implants in IC-RPD compared to implant overdentures (IOD) in maxillary edentulous patients. Twenty IC-RPDs with 74 splinted implant crowns and 18 IODs with 71 implants retained with magnet attachments were observed in 38 patients. We statistically analyzed survival rates and marginal bone loss (MBL) of implants based on multiple variables including first year pathologic condition, location of placed implant, age, and sex in both treatments. Patient reported oral measurements (PROMs) regarding functional/esthetic improvement after IC-RPD or IOD treatments and prosthetic complications were also statistically analyzed. After a median observation period of 47.1 months (up to 147 months), we observed 97.3% implant survival rates for IC-RPD and 70.4% for IOD (p < 0.001). Among variables, first year pathologic condition (p < 0.001) and sex (p = 0.027) influenced implant survival rates. The MBL of implants for IC-RPD and IOD groups at the final check-up were 1.12 ± 1.19 mm and 3.31 ± 1.71 mm, respectively (p < 0.001). In both groups, patients with peri-implantitis (p < 0.001) and patients older than 65 years (p = 0.029) showed significantly higher implant MBL regardless of treatment modality. Functional and esthetic satisfaction were significantly improved (p < 0.001) after both treatments. The IOD group showed more frequent prosthetic complications compared to the IC-RPD group. Within the limitations of a retrospective study, we concluded that RPD with few splinted implant crowns is a feasible alternative treatment modality for maxillary edentulous patients with anatomical limitations.

Elucidation of a Novel Role of YebC in Surface Polysaccharides Regulation of Escherichia coli bipA-Deletion.

The BipA (BPI-inducible protein A) protein is ubiquitously conserved in various bacterial species and belongs to the translational GTPase family. Interestingly, the function of Escherichia coli BipA is not essential for cell growth under normal growth conditions. However, cultivation of bipA-deleted cells at 20°C leads to cold-sensitive growth defect and several phenotypic changes in ribosome assembly, capsule production, and motility, suggesting its global regulatory roles. Previously, our genomic library screening revealed that the overexpressed ribosomal protein (r-protein) L20 partially suppressed cold-sensitive growth defect by resolving the ribosomal abnormality in bipA-deleted cells at low temperature. Here, we explored another genomic library clone containing yebC, which encodes a predicted transcriptional factor that is not directly associated with ribosome biogenesis. Interestingly, overexpression of yebC in bipA-deleted cells diminished capsule synthesis and partially restored lipopolysaccharide (LPS) core maturation at a low temperature without resolving defects in ribosome assembly or motility, indicating that YebC may be specifically involved in the regulation of exopolysaccharide and LPS core synthesis. In this study, we collectively investigated the impacts of bipA-deletion on E. coli capsule, LPS, biofilm formation, and motility and revealed novel roles of YebC in extracellular polysaccharide production and LPS core synthesis at low temperature using this mutant strain. Furthermore, our findings suggest that ribosomal defects as well as increased capsule synthesis, and changes in LPS composition may contribute independently to the cold-sensitivity of bipA-deleted cells, implying multiple regulatory roles of BipA.

Overexpressed L20 Rescues 50S Ribosomal Subunit Assembly Defects of bipA-Deletion in Escherichia coli.

The BipA (BPI-inducible protein A) protein is highly conserved in a large variety of bacteria and belongs to the translational GTPases, based on sequential and structural similarities. Despite its conservation in bacteria, bipA is not essential for cell growth under normal growth conditions. However, at 20°C, deletion of bipA causes not only severe growth defects but also several phenotypic changes such as capsule production, motility, and ribosome assembly, indicating that it has global regulatory properties. Our recent studies revealed that BipA is a novel ribosome-associating GTPase, whose expression is cold-shock-inducible and involved in the incorporation of the ribosomal protein (r-protein) L6. However, the precise mechanism of BipA in 50S ribosomal subunit assembly is not completely understood. In this study, to demonstrate the role of BipA in the 50S ribosomal subunit and possibly to find an interplaying partner(s), a genomic library was constructed and suppressor screening was conducted. Through screening, we found a suppressor gene, rplT, encoding r-protein L20, which is assembled at the early stage of ribosome assembly and negatively regulates its own expression at the translational level. We demonstrated that the exogenous expression of rplT restored the growth of bipA-deleted strain at low temperature by partially recovering the defects in ribosomal RNA processing and ribosome assembly. Our findings suggest that the function of BipA is pivotal for 50S ribosomal subunit biogenesis at a low temperature and imply that BipA and L20 may exert coordinated actions for proper ribosome assembly under cold-shock conditions.

A Mixture of Extracts of Kochia scoparia and Rosa multiflora with PPAR α/γ Dual Agonistic Effects Prevents Photoaging in Hairless Mice.

Activation of peroxisome proliferator-activated receptors (PPAR) α/γ is known to inhibit the increases in matrix metalloproteinase (MMP) and reactive oxygen species (ROS) induced by ultraviolet light (UV). Extracts of natural herbs, such as Kochia scoparia and Rosa multiflora, have a PPAR α/γ dual agonistic effect. Therefore, we investigated whether and how they have an antiaging effect on photoaging skin. Eighteen-week-old hairless mice were irradiated with UVA 14 J/cm² and UVB 40 mJ/cm² three times a week for 8 weeks. A mixture of extracts of Kochia scoparia and Rosa multiflora (KR) was topically applied on the dorsal skin of photoaging mice twice a day for 8 weeks. Tesaglitazar, a known PPAR α/γ agonist, and vehicle (propylene glycol:ethanol = 7:3, v/v) were applied as positive and negative controls, respectively. Dermal effects (including dermal thickness, collagen density, dermal expression of procollagen 1 and collagenase 13) and epidermal effects (including skin barrier function, epidermal proliferation, epidermal differentiation, and epidermal cytokines) were measured and compared. In photoaging murine skin, KR resulted in a significant recovery of dermal thickness as well as dermal fibroblasts, although it did not change dermal collagen density. KR increased the expression of dermal transforming growth factor (TGF)-ß. The dermal effects of KR were explained by an increase in procollagen 1 expression, induced by TGF-ß, and a decrease in MMP-13 expression. KR did not affect basal transepidermal water loss (TEWL) or stratum corneum (SC) integrity, but did decrease SC hydration. It also did not affect epidermal proliferation or epidermal differentiation. KR decreased the expression of epidermal interleukin (IL)-1α. Collectively, KR showed possible utility as a therapeutic agent for photoaging skin, with few epidermal side effects such as epidermal hyperplasia or poor differentiation.

Expression of antimicrobial peptides such as LL-37 and hBD-2 in nonlesional skin of atopic individuals.

Recurrent skin infection is one of the major complications of atopic dermatitis and can be partly explained by decreased expression of antimicrobial peptides such as human beta-defensin-2 and cathelicidin (LL-37). In the human epidermis, human beta-defensin-2 is packed in the lamellar body and LL-37 is co-localized with intercellular lipid lamellae of the stratum corneum; together, these antimicrobial peptides constitute the primary defense system. IL-1alpha, a potent inducer of LL-37 and human beta-defensin-2, is also secreted from the disrupted epidermis for barrier homeostasis. In this study, we investigated whether expression of human beta-defensin-2 and LL-37 is constitutively decreased in the skin of atopic individuals. Nonlesional foreskins from atopic (n=7) and nonatopic (n=7) individuals were analyzed. The expression of LL-37, human beta-defensin-2 and IL-1alpha was analyzed using immunohistochemical staining, Western blot, and real-time polymerase chain reaction. Lamellar body density and secretion were evaluated by electron microscope. Quantitative analysis showed that the expression of each parameter was not significantly different between groups. Thus, basal expression of LL-37 and human beta-defensin-2 was not changed in atopic individuals. These results indicate that the expression of antimicrobial peptides at baseline was not different between nonlesional skin of atopic individuals and normal skin of nonatopic individuals.

Topical calcineurin inhibitors compromise stratum corneum integrity, epidermal permeability and antimicrobial barrier function.

BACKGROUND: Topical calcineurin inhibitors (TCIs) such as pimecrolimus and tacrolimus have recently been used for dermatologic diseases including atopic dermatitis instead of topical glucocorticoids, because they display comparable efficacy, but less-frequent side effects. Although even short-term topical glucocorticoid compromise epidermal permeability barrier homeostasis, the effects of TCI on barrier function have not yet been reported. However, viral infections such as eczema herpeticum and molluscum contagiosum, which could indicate an impaired skin barrier, continue to occur with TCI use in atopic dermatitis. OBJECTIVES: We determined here whether TCIs disrupt epidermal permeability barrier and antimicrobial function, and whether these effects can be prevented. METHODS AND RESULTS: In normal humans, topical pimecrolimus and tacrolimus applied twice-daily for 5 days, delay barrier recovery without an increase in basal transepidermal water loss was observed. Co-application of physiologic lipid mixture (PLM) containing an equimolar ratio of ceramides, cholesterol and free fatty acids normalized barrier homeostasis in the face of topical TCIs. In hairless mice, 4 days of TCI treatment also disrupted barrier function significantly. TCIs-treated epidermis showed the decrease of epidermal lipid content, lamellar body number and secretion, and lipid synthesis-related enzymes such as 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, serine-palmitoyl transferase and fatty acid synthase, implying decreased lipid synthesis. TCIs also suppressed expression of IL-1alpha and antimicrobial peptides, CRAMP and mouse beta-defensin 3. However, these TCI-induced abnormalities can be overridden by topical replacement with PLM. CONCLUSIONS: Our results demonstrate that TCIs induce negative effects on the skin barrier including permeability and antimicrobial functions, which are mediated by decreasing epidermal lipid synthesis, lamellar body secretion and antimicrobial peptides expression through suppression of cytokine such as IL-1alpha, therefore co-treatment with PLM would be helpful to overcome these negative effects.

Biopositive effects of low-dose UVB on epidermis: coordinate upregulation of antimicrobial peptides and permeability barrier reinforcement.

Whereas high-dose ultraviolet B (UVB) is detrimental to the epidermal permeability barrier, suberythemal doses of UVB are used to treat atopic dermatitis (AD), which is characterized by defective permeability barrier and antimicrobial function. As epidermal permeability barrier and antimicrobial peptide (AMP) expression are coregulated and interdependent functions, we hypothesized that suberythemal doses of UVB exposure could regulate AMP expression in parallel with permeability barrier function. Hairless mice were exposed to 40 mJ cm(-2) UVB (about 1/2 minimal erythema dose) daily for 1 or 3 days. Twenty-four hours after the last exposure, epidermal barrier function was assessed and skin specimens were taken for western blotting, immunohistochemistry, and quantitative reverse transcription-PCR for mouse beta-defensin (mBD)-2, mBD3 and cathelin-related antimicrobial peptide (CRAMP). mRNA levels of the vitamin D receptor (VDR), 1alpha-hydroxylase and key epidermal lipid synthetic enzymes were also quantified. After 3 days of UVB exposure, acceleration of barrier recovery and augmentation in expression of epidermal differentiation markers (for example, involucrin and filaggrin) occurred in parallel with increased mBD2, mBD3, and CRAMP expression at both the mRNA and protein level. VDR, 1alpha-hydroxylase, and the major epidermal lipid synthetic enzymes were also upregulated. When an inhibitor of 1alpha, 25 dihydroxyvitamin D(3) formation, ketoconazole, was applied immediately after UVB exposure, the cutaneous vitamin D system was inhibited, which in turn blocked epidermal lipid synthesis, AMP expression, and permeability barrier homeostasis, suggesting that the beneficial effect of low-dose UVB depends, at least in part, on activation of the cutaneous vitamin D system. Our results provide new insights into the mechanisms whereby low-dose UVB comprises effective therapy for AD.