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To analyze the tissue morphology around implant-supported prostheses by digital technology and to evaluate the effect of prosthetic contours on the changes in tissues following the free gingiva graft procedure.A total of 53 implants in 32 patients receiving free gingiva grafts were selected. These had previously presented insufficient keratinized mucosa width (KMW). At the follow-up visits (mean: 16.66 ± 9.97 months), the implant position and tissue condition were documented with an oral scanner. Vertical soft tissue thickness (VT), measured from the implant-abutment connection to the marginal tissues, and horizontal soft tissue thickness (HT), at the level of the platform, were calculated. The VT, HT, and emergence angle (EA) of prostheses were assessed by 3Shape analyzing software. The final KMW was measured by clinical assessment. Marginal bone loss (MBL) was calculated in the follow-up bitewing radiographs.The mean VT in the study was 2.65 ± 0.75 mm at the mid-buccal sites, 3.74 ± 1.22 mm at the mesial, 3.16 ± 1.08 mm at the distal, and 2.53 ± 0.92 at the mid-lingual aspects. The mid-buccal HT was 1.45 ± 0.53 mm while the mid-lingual was 1.05 ± 0.43 mm (p = 0.008). Interestingly, prostheses with mid-buccal EAFree gingival grafting is a predictable treatment approach to augmenting soft tissue 3-dimensionally. Prostheses with EA

The literature identified variations in socket seal surgery, each with limitations. This case series aimed to observe the outcome of using autologous dental root (ADR) for socket sealing on socket preservation (SP). A total of 9 patients with 15 extraction sockets were documented. After flapless extraction, the xenograft or alloplastic grafts were placed in the sockets. ADRs were prepared extraorally and applied to seal the socket entrance. All SP sites healed uneventfully. Cone-beam computed tomography (CBCT) scan was performed after 4–6 months of healing to evaluate ridge dimensions. The preserved alveolar ridge profiles were verified on CBCT scans and during implant surgery. Implants were placed successfully with a reduced need for guided bone regeneration. Histological biopsy specimens were examined in three cases. The histological examination demonstrated vital bone formation and osseointegration of graft particles. All patients completed the final restorations and were monitored for 15.56 ± 9.08 months after functional loading. The favorable clinical outcomes support the use of ADR for SP procedures. It was not only accepted to patients but also easy to perform with low complication rates. The ADR technique is thus a feasible method for socket seal surgery.

It is widely accepted that cellular processes are controlled by protein phosphorylation and has become increasingly clear that protein degradation, localization and conformation as well as protein-protein interaction are the examples of subsequent cellular events modulated by protein phosphorylation. Enamel matrix proteins belong to members of the secretory calcium binding phosphoprotein (SCPP) family clustered on chromosome 4q21, and most of the SCPP phosphoproteins have at least one S-X-E motifs (S; serine, X; any amino acid, E; glutamic acid). It has been reported that mutations in C4orf26 gene, located on chromosome 4q21, are associated with autosomal recessive type of Amelogenesis Imperfecta (AI), a hereditary condition that affects enamel formation/mineralization. The enamel phenotype observed in patients with C4orf26 mutations is hypomineralized and partially hypoplastic, indicating that C4orf26 protein may function at both secretory and maturation stages of amelogenesis. The previous in vitro study showed that the synthetic phosphorylated peptide based on C4orf26 protein sequence accelerates hydroxyapatite nucleation. Here we show the molecular cloning of Gm1045, mouse homologue of C4orf26, which has 2 splicing isoforms. Immunohistochemical analysis demonstrated that the immunolocalization of Gm1045 is mainly observed in enamel matrix in vivo. Our report is the first to show that FAM20C, the Golgi casein kinase, phosphorylates C4orf26 and Gm1045 in cell cultures. The extracellular localization of C4orf26/Gm1045 was regulated by FAM20C kinase activity. Thus, our data point out the biological importance of enamel matrix-kinase control of SCPP phosphoproteins and may have a broad impact on the regulation of amelogenesis and AI.

Current rat alveolar ridge preservation models have not been well standardized. In this study, we proposed decoronation-induced infected alveolar socket model of rat. The bilateral maxillary first molars (M1) of twenty-four rats were decoronized or extracted. After 2, 6, 10, and 14 weeks, bone and soft tissue changes at M1 and periodontal conditions of maxillary second (M2) and third molars (M3) were evaluated by micro-computed tomography and histological analysis. Additional eighteen rats with standardized size defects were grafted with Bio-Oss Collagen to compare with unmanipulated contralateral side. Decoronation preserved greater bone and soft tissue dimensions at M1, provided larger three-dimensional (3D) bone contour volume, but also promoted periodontal breakdown of M2 Histological results showed intense inflammatory cell infiltrations and severe bone resorption within M1 socket and at mesial aspect of M2. The critical dimensions to accommodate largest standardized defect at M1 were 2.2–2.3 mm at vertical bone height and 2.8–3.2 mm at alveolar crestal width. Bio-Oss Collagen could not fully preserve buccal or palatal bone height but could be beneficial in preserving ridge width in large alveolar defects. Collectively, if periodontally-involved alveolar bone defect is preferred, we suggest extracting M1 roots 6 weeks after decoronation to allow periodontitis to occur at M2. If standardized critical dimension defect is preferred, we suggest extracting M1 roots 2 weeks after decoronation, and creating defect in the middle of M1 site with size no larger than 2.7 mm diameter to its full depth.

Excessive gingival display is often an aesthetic concern for patients and a challenge for clinicians. Multiple causes are possible and focused treatment options should be considered. The aim of this article is to emphasise the importance of interdisciplinary treatment for patients presenting with excessive gingival display. A 49-year-old female patient who suffered from prominent teeth and a gummy smile was diagnosed with skeletal and dental Class II relationships and a hypermobile upper lip. Aesthetically pleasing outcomes and stable occlusal function were achieved as a result of orthodontic treatment and a surgical lip repositioning procedure. The outcome was maintained after a two-year follow–up.

Background Excessive gingival display is an aesthetic concern for patients and a challenge for clinicians. An accurate diagnosis is essential in order to provide effective treatment. Aim The aim of the present article is to fill a gap in the current literature by providing clinicians with an overview and a stepwise approach to assessing gingival excess, as well as reviewing possible treatment. Method Published articles related to smile aesthetics and excessive gingival display over the last 40 years were identified and analysed through an electronic search. Results An examination should be initiated extra-orally to distinguish contributing skeletal factors. The relationships of four aesthetic parameters (LIPG): lip length (L), incisal lines (I), tooth proportions (P), and gingival lines (G), should be examined individually to determine other possible causes of gingival excess. Contributing factors may include a short or hypermobile upper lip, dentoalveolar extrusion, or altered passive eruption. Further investigation to identify patients who might benefit from orthognathic surgery, orthodontic treatment alone, lip repositioning or botulinum injection, or aesthetic crown lengthening procedures, should be conducted to reconfirm a diagnosis and provide the optimal treatment. Conclusion Clinicians can have a dental blueprint for anterior tooth reconstruction and are able to provide comprehensive treatment in co-operation with other disciplines. By understanding the principles of an aesthetic smile line, the aetiology of an excessive gingival display and treatment concepts, clinicians may provide alternative treatments to avoid extensive surgical procedures while achieving acceptable outcomes.

BACKGROUND/PURPOSE Crestal bone stability, implant rigidity and occlusal loading are issues with small-diameter implants. This article demonstrates the use of two small-diameter implants replacing a missing wide edentulous site and discusses factors that may affect bone changes. METHODS Patients who wanted to restore an edentulous space measuring from 12 to 14 mm wide in the posterior region were offered an alternative treatment option, using two narrow or regular-diameter implants instead of one wide implant. In the study, the crestal bone stability of 12 implants in 6 edentulous sites was assessed by cone beam CTs and periapical radiographs in follow-up visits for up to 4 years. RESULTS The bone level of all the implants was stable at buccal, lingual, mesial and distal sites, with mean values

Data on the shrinkage of free gingival grafts (FGGs) vary. Most studies have analyzed grafts in nonmolar sites because of measurement limitations and have addressed the changes in grafts and keratinized mucosa width (KMW) only in the early healing phase.The purpose of this retrospective clinical study was to assess the dimensional changes of an FGG in the posterior regions and their influencing factors, with the aim of obtaining sufficient and stable KMW after restoration.A total of 77 implants in 40 participants who had undergone an FGG surgery were recruited. Graft sizes during surgery and the surface areas of keratinized mucosa at the follow-up visit after restorations were compared by digital analysis and verified by clinical measurements and photographs. The association between shrinkage and the graft sizes, implant location, and sex and age of the participants was evaluated. The influence of the shrinkage of FGG on the KMW after restoration was analyzed by multivariable linear regression with generalized estimating equation (GEE) models.The mean ±standard deviation shrinkage of FGG around implants in the posterior regions was 24.76 ±14.77%, and the mean ±standard deviation KMW was 4.16 ±1.77 mm at the follow-up visit. Larger grafts had a statistically higher shrinkage ratio (P.001). No statistically significant difference was found regarding the effect of implant location, sex, and age on the shrinkage of FGG and final KMW (P.05). The mean ±standard deviation follow-up period after restoration was 12.45 ±7.73 months CONCLUSIONS: Free gingival grafting was found to be a predictable treatment approach for augmentation of KMW around implants in the posterior region after the fabrication of prostheses as long as grafts of sufficient size were placed. Stable outcomes were shown in the study participants in the follow-up period of up to 3 years.

Background/Purpose: The objectives of this retrospective study are to analyze post-surgical gingival thickness after connective tissue grafting in the Asian population and to assess its tissue stability for up to approximately 3.5 years. Methods: A total of 111 grafted teeth and 57 nearby nongrafted teeth in 28 Asian patients who had undergone connective tissue grafting surgery were selected. Gingival thickness was measured by transgingival probing. The mean gingival thickness of the grafted teeth was compared with adjacent nongrafted teeth in the same individuals. The mean gingival thickness of the grafted teeth in different tooth types and at various time intervals were statistically analyzed. Results: The average gingival thickness following connective tissue grafting is 1.99 ± 0.62 mm compared to 0.96 ± 0.40 mm with nongrafted teeth (P

Introduction Tooth agenesis is a type of dental anomaly in which individuals are missing teeth due to developmental failure as a result of genetic or environmental factors. With approximately one fourth of the population missing ≥1 third molar, tooth agenesis is considered a common dental anomaly. However, the severity of tooth agenesis can range from a missing single tooth to multiple teeth. When suffering from severe tooth agenesis, the patient's health and social relationships are often affected. Case presentation The patient in this report congenitally lost 11 teeth and suffered from compromised esthetics and impaired chewing function. In such a severe tooth agenesis case, interdisciplinary treatments involving orthodontics, periodontics, and prosthodontics were engaged to reconstruct the ideal biology, function, and esthetics for the patient. With an interdisciplinary approach, the periodontist played an important role in the rehabilitation of the edentulous regions with implants in combination with various hard and soft tissue augmentation procedures. In addition, the patient with severe tooth agenesis presented with additional dental anomalies. The periodontist, therefore, had to collaborate with other specialists to provide early detection and intervention to avoid future complications, such as the management of infraoccluded ankylosed deciduous molars and aberrant frenum. The patient at the end of treatment had a good occlusion with improved function and esthetics. Conclusion This case report describes the interdisciplinary treatment approach used and points out the role of periodontists in the treatment of a patient with severe tooth agenesis.

In this study, we fabricated gelatin/nano-hydroxyapatite/metformin scaffold (GHMS) and compared its effectiveness in bone regeneration with extraction-only, Sinbone, and Bio-Oss Collagen® groups in a critical size rat alveolar bone defect model. GHMS was synthesized by co-precipitating calcium hydroxide and orthophosphoric acid within gelatin solution, incorporating metformin, and cross-linked by microbial transglutaminase. The morphology, characterization, and biocompatibility of scaffold were examined. The in vitro effects of GHMS on osteogenic gene and protein expressions were evaluated. In vivo bone formation was assessed in a critical size rat alveolar bone defect model with micro-computed tomography and histological examination by comparing GHMS with extraction-only, Sinbone, and Bio-Oss Collagen®. The synthesized GHMS had a highly interconnected porous structure with a mean pore size of 81.85 ± 13.8 µm. GHMS exhibited good biocompatibility; promoted ALPL, RUNX2, SP7, BGLAP, SPARC and Col1a1 gene expressions; and upregulated the synthesis of osteogenic proteins, including osteonectin, osteocalcin, and collagen type I. In critical size rat alveolar bone defects, GHMS showed superior bone regeneration compared to extraction-only, Sinbone, and Bio-Oss Collagen® groups as manifested by greater alveolar ridge preservation, while more bone formation with a lower percentage of connective tissue and residual scaffold at the defect sites grafted with GHMS in histological staining. The GHMS presented in this study may be used as a potential bone substitute to regenerate alveolar bone. The good biocompatibility, relatively fast degradation, interconnected pores allowing vascularization, and higher bioactivity properties of the components of the GHMS (gelatin, nHA, and metformin) may contribute to direct osteogenesis.

Summary Nutrients and temperature are the major elements in maintaining stable endosymbiotic relationships. The mechanisms and response of cultured Symbiodinium cells in the absence of nitrogen, and at various temperatures are still unclear. The present study investigated the influence of different temperatures and nitrogen-deprivation on free-living Symbiodinium cultures. The physiological responses of free-living Symbiodinium cells cultured at different temperatures during nitrogen deprivation under a 12:12 h light:dark were measured. Symbiodinium cell growth was significantly lower in response to lower temperatures. Transmission electron micrographs (TEMs) revealed the formation of lipid droplets induced by nitrogen deprivation under different temperatures. The results of this study will increase our understanding of adaptive responses occurring in Symbiodinium under environmental stress.

Anterior implant restoration is one of the most challenging restorative procedures, especially for sites with vertical and/or horizontal hard and soft tissue deformities. Orthodontic extrusion before implant placement may be the best means of overcoming vertical deficiencies. This article describes a modification to the standard technique, involving staged orthodontic extrusion and buccal root torque. The main advantage of this modification is that it encourages bone and soft tissue development, thereby allowing the patient to receive immediate or early implant treatment. A clinical procedure is presented to illustrate the modified technique that resulted in an esthetically pleasing and stable 5-year outcome.

Tooth agenesis is a type of dental anomaly in which individuals are missing teeth due to developmental failure as a result of genetic or environmental factors. With approximately one fourth of the population missing ≥1 third molar, tooth agenesis is considered a common dental anomaly. However, the severity of tooth agenesis can range from a missing single tooth to multiple teeth. When suffering from severe tooth agenesis, the patient's health and social relationships are often affected.The patient in this report congenitally lost 11 teeth and suffered from compromised esthetics and impaired chewing function. In such a severe tooth agenesis case, interdisciplinary treatments involving orthodontics, periodontics, and prosthodontics were engaged to reconstruct the ideal biology, function, and esthetics for the patient. With an interdisciplinary approach, the periodontist played an important role in the rehabilitation of the edentulous regions with implants in combination with various hard and soft tissue augmentation procedures. In addition, the patient with severe tooth agenesis presented with additional dental anomalies. The periodontist, therefore, had to collaborate with other specialists to provide early detection and intervention to avoid future complications, such as the management of infraoccluded ankylosed deciduous molars and aberrant frenum. The patient at the end of treatment had a good occlusion with improved function and esthetics.This case report describes the interdisciplinary treatment approach used and points out the role of periodontists in the treatment of a patient with severe tooth agenesis.

Background Royal jelly (RJ), the exclusive food for the larva of queen honeybee, is regarded as the novel supplement to promote human health. The function of RJ may be attributed to its major and unique fatty acid, 10-hydroxy-2-decenoic acid (10-HDA). The current study investigated the anti-inflammory function of 10-HDA on human colon cancer cells, WiDr, as well as its effect on the growth of pathogenic bacterium. Methods The pro-inflammatory cytokines, receptor antagonist cytokine (IL-1ra) and nuclear factor-kappa B (NF-κB) in WiDr cells was analyzed by Enzyme-linked immunosorbent assay (ELISA) or western blot. The growth inhibition of 10-HDA on bacterium was evaluated by determination of minimal inhibitory concentrations (MIC) and minimal bactericide concentrations (MBC). Results The production of pro-inflammatory cytokines, Interleukin (IL)-8, IL-1β and tumor necrosis factor-alpha (TNF-α) in WiDr cells was modulated by 10-HDA. IL-8 were dramatically declined by 10-HDA at 3 mM, while IL-1β and TNF-α were significantly decreased. 10-HDA increased IL-1ra in a dose manner. NF-κB pathway is primarily in response to prototypical pro-inflammatory cytokines, and NF-κB was reduced after 10-HDA treatment. 10-HDA acted as potent bactericide against animal- or human-specific pathogens, including Staphylococcus aureus, Streptococcus alactolyticus, Staphylococcus intermedius B, Staphylococcus xylosus, Salmonella cholearasuis, Vibro parahaemolyticus and Escherichia coli (hemolytic). Conclusions The current study showed that in vitro 10-HDA from RJ exhibited anti-inflammatory activity in WiDr cells, as well as anti-bacterial activity against animal pathogens. 10-HDA showed its potential as anti-imflammtory agent and bactericide to benefit human gastrointestinal tract.

This study first investigates the formation of Pt silicides on Si 1− y C y ( y = 0.024) epilayers in the presence of an interfacial oxide layer. The presence of C atoms is found to retard the growth kinetic of PtSi but significantly improve the thermal stability of PtSi thin films. Experimental results also indicate that an interfacial oxide layer present at the initial Pt/Si 1− y C y interface should have no negative impact on the subsequent Pt silicidation in terms of process integration. We also propose a mechanism to discuss the relationship between microstructures, electrical property, and thermal stability of Pt silicides in terms of C solubility in PtSi. In this mechanism, C atoms accumulated at the PtSi grain boundaries may act as diffusion barriers, which effectively inhibit the grain growth and agglomeration of PtSi and thus widen the low-resistivity process window of PtSi. More importantly, it is possible to gain the benefits of excellent thermal stability of PtSi silicides and enhanced tensile strain in Si 1− y C y epilayers simultaneously if the thermal budget is well controlled during the silicidation process.

The carbon-incorporation behavior in phosphorous-doped Si 1− y C y /Si ( y 1 ∼ 0.018, y 2 ∼ 0.024) epilayers grown by reduced pressure chemical vapor deposition (RPCVD) has been investigated as a function of annealing temperatures. An abnormal interstitial carbon (C i ) re-incorporation was observed in the initial stage of thermal annealing, introducing an additional tensile strain into the Si 1− y C y epilayers. At higher temperature but below β-SiC precipitation threshold, almost complete strain relaxation was found. These strain transitions can be attributed to the competitive behavior between C i re-incorporation and phosphorus deactivation to kick out the substitutional carbon (C sub ) atoms during the post-annealing process. This work demonstrated that Si 1− y C y epilayers grown by RPCVD could keep both enhanced carbon incorporation and nonequilibrium phosphorus activation if the thermal budget is well controlled.

Mutations in the Family with sequence similarity (FAM) 20 gene family are associated with mineralized tissue phenotypes in humans. Among these genes, FAM20A mutations are associated with Amelogenesis Imperfecta (AI) with gingival hyperplasia and nephrocalcinosis, while FAM20C mutations cause Raine syndrome, exhibiting bone and craniofacial/dental abnormalities. Although it has been demonstrated that Raine syndrome associated-FAM20C mutants prevented FAM20C kinase activity and secretion, overexpression of the catalytically inactive D478A FAM20C mutant was detected in both cell extracts and the media. This suggests that FAM20C secretion doesn’t require its kinase activity, and that another molecule(s) may control the secretion. In this study, we found that extracellular FAM20C localization was increased when wild-type (WT), but not AI-forms of FAM20A was co-transfected. On the other hand, extracellular FAM20C was absent in the conditioned media of mouse embryonic fibroblasts (MEFs) derived from Fam20a knock-out (KO) mouse, while it was detected in the media from WT MEFs. We also showed that cells with the conditioned media of Fam20a WT MEFs mineralized, but those with the conditioned media of KO MEFs failed to mineralize in vitro. Our data thus demonstrate that FAM20A controls FAM20C localization that may assist in the extracellular function of FAM20C in mineralized tissues.

Accumulation of oil bodies was successfully induced in a microalga, Chlorella sp., cultured in a nitrogen-limited medium. The oil bodies were initially assembled as many small entities (mostly 0.1-1 μm), and lately found as a major irregular compartment (>3 μm) occupying more than half of the cell space. Approximately, two thirds of oil bodies isolated from Chlorella cells were broken and formed a transparent oil layer on top of the milky compact layer of the remaining stable oil bodies after being washed with 0.1% triton X-100. The stable oil bodies mainly comprised triacylglycerols as examined by thin layer chromatography analysis and confirmed by both Nile red and BODIPY stainings. Integrity of these stable oil bodies was maintained via electronegative repulsion and steric hindrance possibly provided by their surface proteins. Immunological cross-recognition revealed that a major protein of 29 kDa, tentatively identified as caleosin, was exclusively present in Chlorella oil bodies. Mass spectrometric analysis showed that the putative caleosin possessed a trypic fragment of 13 residues matching to that of a hypothetical caleosin in Picea sitchensis. With the aid of a degenerate primer designed according to the tryptic peptide, a complete cDNA fragment encoding this putative caleosin was obtained by PCR. Phylogenetic tree analysis supports that Chlorella caleosin is the most primitive caleosin found in oil bodies to date.

The use of protein fusion tag technology simplifies and facilitates purification of recombinant proteins. In this article, we have found that the starch-binding domain derived from Rhizopus oryzae glucoamylase (RoSBD), a member of carbohydrate-binding module family 21 (CBM21) with raw starch-binding activity, is favorable to be applied as an affinity tag for fusion protein engineering and purification in Escherichia coli and Pichia pastoris systems. To determine suitable spatial arrangement of RoSBD as a fusion handle, enhanced green fluorescent protein (eGFP) was fused to either the N- or C-terminus of the SBD, expressed by E. coli, and purified for yield assessment and functional analysis. Binding assays showed that the ligand-binding capacity was fully retained when the RoSBD was engineered at either the N-terminal or the C-terminal end. Similar results have been obtained with the RoSBD-conjugated phytase secreted by P. pastoris. The effective adsorption onto raw starch and low cost of starch make RoSBD practically applicable in terms of development of a new affinity fusion tag for recombinant protein engineering in an economic manner.

Symbiodinium is a dinoflagellate that plays an important role in the physiology of the symbiotic relationships of Cnidarians such as corals and sea anemones. However, it is very difficult to cultivate free-living dinoflagellates after being isolated from the host, as they are very sensitive to environmental changes. How these symbiont cells are supported by the host tissue is still unclear. This study investigated the characteristics of Symbiodinium cells, particularly with respect to the morphological variability and distinct protein profiles of both cultured and endosymbiotic Symbiodinium which were freshly isolated from Exaiptasia pulchella. The response of the cellular morphology of freshly isolated Symbiodinium cells kept under a 12 h L:12 h D cycle to different temperatures was measured. Cellular proliferation was investigated by measuring the growth pattern of Symbiodinium cells, the results of which indicated that the growth was significantly reduced in response to the extreme temperatures. Proteomic analysis of freshly isolated Symbiodinium cells revealed twelve novel proteins that putatively included transcription translation factors, photosystem proteins and proteins associated with energy and lipid metabolism, as well as defense response. The results of this study will bring more understandings to the mechanisms governing the endosymbiotic relationship between the cnidarians and dinoflagellates.

Natto has recently attracted attention throughout the world due to its healthy benefits. In 1987 a potent fibrinolytic enzyme in the extract of natto was discovered and named as ‘nattokinase’. Many biotechnology companies produce nattokinase via the fermentation of Bacillus subtilis natto. Various factors in the fermentation process may affect the production of nattokinase. This work reports the application of Taguchi parameter design to identifying the importance of those factors and then to obtain the optimal condition of fermentation to achieve the maximum yield of enzyme. Seven controllable factors and one noise factor were involved in the experiment. Based on the analysis of experimental data, both the volume of medium and the fermentation time were considered to have a significant effect on nattokinase activity value. Their nominal values should be set as 80mL and 36h, respectively, to maximise the yield of nattokinase.

Natto has attracted worldwide attention because of its health benefits and long history in Japanese food. It has been found that a potent fibrinolytic enzyme named nattokinase, which is extracted from natto, is able to prevent atherosclerosis. The production of nattokinase may be influenced by various factors such as temperature, shaking speed, volume of medium, fermentation time and so forth. Three-step response surface methodology was applied to obtain the optimal operation conditions of the fermentation process in order to maximize the nattokinase yield. The three major steps are described as follows. First, the important factors for fermentation were identified by L₈ orthogonal array experiment. The chosen factors were temperature (37 or 45C), shaking speed (110 or 150 rpm), volume of medium (80 or 120 mL), Brix of wheat bran extract (1.5 or 3°), Brix of soy meal extract (1 or 2°), glucose concentration (0.6 or 1.2%) and fermentation time (24 or 36 h). Second, a regression equation was established between the response (i.e., the enzyme activity) and the two statistically significant factors (i.e., the volume of medium and fermentation time). Third, the optimal solutions for the volume of medium and fermentation time were obtained based on the response surface of the regression equation. According to the response surface analysis, the optimal operation conditions for the fermentation process should be 80 mL and 37.0817 h for the volume of medium and the fermentation time, respectively, which resulted in 459.11 FU[solidus]mL as the predicted enzyme activity.

Oil bodies of sesame seeds comprise a triacylglycerol matrix, which is surrounded by a monolayer of phospholipids embedded with unique proteins, mainly structural proteins termed oleosins. Artificial oil bodies were successfully reconstituted with various compositions of triacylglycerols, phospholipids, and oil-body proteins. The sizes of reconstituted oil bodies displayed a normal distribution with an average size proportional to the ratio of triacylglycerols to oil-body proteins. Both thermostability and structural stability of reconstituted oil bodies decreased as their sizes increased, and vice versa. Proteinase K digestion indicated that oleosins anchored both native and reconstituted oil bodies via their central hydrophobic domains. The stability of reconstituted oil bodies, as well as the purified ones from sesame seeds, could be substantially enhanced after their surface proteins were cross-linked by glutaraldehyde or genipin.

Royalisin is a 5.5-kDa antibacterial peptide isolated from the royal jelly of the honeybee (Apis mellifera). The antimicrobial activity of royalisin against fungi, Gram-positive and Gram-negative bacteria has been revealed. Compared with another insect antibacterial peptide, there is an extra stretch of 11 amino acid residues at the C-terminus of royalisin. In this study, a recombinant shortened form of royalisin named as royalisin-D, was constructed without the 11 amino acid residues at the C-terminal of royalisin and linked to the C-terminal of oleosin by an inteinS fragment. The recombinant protein was overexpressed in Escherichia coli, purified by artificial oil body system and subsequently released through self-splicing of inteinS induced by the changes of temperature. The antibacterial activity of royalisin-D was compared with royalisin via minimal inhibitory concentration (MIC) assay, minimal bactericidal concentration (MBC) assay, microbial adhesion to solvents (MATS) methods, and cell membrane permeability. Furthermore, the recombinant royalisin and royalisin-D have also been treated with the reducing agent of disulfide bonds, dithiothreitol (DTT), to investigate the importance of the intra-disulfide bond in royalisin. In our results, royalisin-D exhibited similar antimicrobial activity to royalisin. Royalisin and royalisin D lost their antimicrobial activities when the intra-disulfide bonds were reduced by DDT. The intra-disulfide bond plays a more important role than the extra stretch of 11 amino acid residues at the C-terminus of royalisin in terms of the antimicrobial properties of the native royalisin.

The stability of cnidarian-dinoflagellate (genus Symbiodinium spp.) endosymbioses depends on the regulation of nutrient transport between Symbiodinium populations and their hosts. Previously, we successfully induced the production of lipid droplets in the free-living cultured Symbiodinium (clade B) under the nitrogen-deprivation condition for 5 days. Therefore, the present study aimed at understanding the disruption of the endosymbiotic relationship between the cnidarians and dinoflagellates by nitrogen deprivation using Aiptasia pulchella as an example. Transmission electron micrographs revealed the formation of lipid droplets induced by nitrogen deprivation, and the lipid analyses further showed that polyunsaturated fatty acids were drastically enriched in Symbiodinium after 30 days of nitrogen deprivation, although these were unaffected after 5 days of nitrogen starvation. The present study also suggested that the host provided nitrogen to the symbiotic cells during short-term environmental stress. However, the relationship started to deteriorate after 30 days. These findings provide a more detailed understanding of the mechanisms of the symbiotic relationship between the symbiotic dinoflagellates in terms of the nitrogen source, which might provide more information for the explanation of the regulatory mechanism underlying endosymbiotic associations.

Intracellular lipid droplets (LDs) have been proposed to play a key role in the mutualistic endosymbiosis between reef-building corals and the dinoflagellate endosymbiont Symbiodinium spp. This study investigates and identifies LD proteins in Symbiodinium from Euphyllia glabrescens. Discontinuous Percoll gradient centrifugation was used to separate Symbiodinium cells from E. glabrescens tentacles. Furthermore, staining with a fluorescent probe, Nile red, indicated that lipids accumulated in that freshly isolated Symbiodinium cells and lipid analyses further showed polyunsaturated fatty acids (PUFA) was abundant. The stable LDs were purified from endosymbiotic Symbiodinium cells. The structural integrity of the Symbiodinium LDs was maintained via electronegative repulsion and steric hindrance possibly provided by their surface proteins. Protein extracts from the purified LDs revealed a major protein band with a molecular weight of 20 kDa, which was termed Symbiodinium lipid droplet protein (SLDP). Interestingly, immunological cross-recognition analysis revealed that SLDP was detected strongly by the anti-sesame and anti-cycad caleosin antibodies. It was suggested that the stable Symbiodinium LDs were sheltered by this unique structural protein and was suggested that SLDP might be homologous to caleosin to a certain extent.

Oil bodies formed in Auxenochlorella protothecoides induced during limited nutrition had a coating of caleosin. The total lipid content obtained from A. protothecoides in unstressed cultures (first week) was ~210 mg/g compared to the 231 mg/g obtained in the third week (nutrient limited) and 290 mg/g obtained in the fourth week (nutrient limited). The proportion of total saturated fatty acids increased from 28 to 46 %, whereas that of total polyunsaturated fatty acids decreased from 52 to 35 %. The expression levels of the 28 kDa caleosin protein in A. protothecoides rose to a maximum up to 4 weeks; immunolocalization studies showed that caleosin was predominantly associated with the membranes of oil bodies.

Background Escherichia coli heat-labile enterotoxin (LT) with different mutant forms has been used as adjuvant for vaccines due to its ability to enhance immune response to specific antigen in vivo. We hypothesis that LTS61K or LTS61K mixed with dust mite allergen, Der p, (LTS61K/Der p) can modulate dendritic cells (DCs) s' functions thus alleviate allergen-induced airway inflammation. Methods Two protocols (ie, preventive and therapeutic protocol) were designed to evaluate the effects of LTS61K in Der p sensitized and challenged mouse model of asthma. Results Both intranasal inoculations with LTS61K or LTS61K/Der p decreased allergen-induced airway inflammation and alleviated systemic TH2-type immune response. In addition, bronchoalveolar lavage (BAL) fluids and sera from LTS61K/Der p treated mice have higher concentrations of Der p-specific IgA than those of other groups. In the in vitro study, bone marrow-derived dendritic cells (BMDCs) and DC cell line, DC2.4 cells stimulated with LTS61K/Der p both secreted pro-inflammation cytokines IL-6 and TNF-a. In contrast, after LTS61K treatment, only BMDCs decreased production of IL-6 and TNF-a as well as decreased maturation. Furthermore, we found that pre-treatment BMDC with LTS61K inhibited Der p-induced NF-kB translocation which might explain the delayed maturation and decreased productions of IL-6 and TNF-a in LTS61K pre-treated BMDCs. Intratracheally adoptive transferred with LTS61K- or LTS61K/Der p-primed DC2.4 cells or BMDCS into Der p-sensitized mice decreased inflammatory cells infiltration and TH2-type chemokines in BAL fluids and alleviated airway inflammation. Conclusions Our results show that LTS61K may influence DCs maturation and its cytokine production. On the other hands, LTS61K/Der p may induce more Der p-specific IgA production to decrease allergic TH2 cytokine responses and alleviate airway inflammation in murine model of asthma. These finding suggested that LTS61K may have clinical application as an immune-modulator effect on the diseases of allergy and asthma.

Background Rhizopus oryzae glucoamylase (Ro GA) consists of three domains: an amino (N)-terminal raw starch-binding domain (SBD), a glycosylated linker domain, and a carboxy (C)-terminal catalytic domain. The 36-amino-acid linker region (residues 132–167) connects the two functional domains, but its structural and functional roles are unclear. Results To characterize the linker sequences of Ro GA and its involvement in protein expression, a number of Ro GA variants containing deletions and mutations were constructed and expressed in Saccharomyces cerevisiae. Deletion analyses demonstrate that the linker region, especially within residues 161 to 167, is required for protein expression. In addition, site-directed mutagenesis and deglycosylation studies reveal that the linker region of Ro GA contains both N- and O-linked carbohydrate moieties, and the N-linked oligosaccharides play a major role in the formation of active enzyme. Although the linker segment itself appears to have no ordered secondary structural conformation, the flexible region indeed contributes to the stabilization of functional N- and C-terminal domains. Conclusion Our data provide direct evidence that the length, composition, and glycosylation of the interdomain linker play a central role in the structure and function of Ro GA.