Design of an artificial transcriptional system for production of high levels of recombinant proteins in tobacco (Nicotiana benthamiana).

Plants have recently received much attention as a means of producing recombinant proteins because they are easy to grow at a low cost and at a large scale. Although many plant protein expression systems have been developed, there remains a need for improved systems that deliver high yields of recombinant proteins. Transcription of the recombinant gene is a key step in increasing the yield of recombinant proteins. However, revealed strong promoters, terminators, and transcription factors that have been identified do not necessarily lead to high level production of recombinant proteins. Thus, in this study, a robust expression system was designed to produce high levels of recombinant protein consisting of a novel hybrid promoter, FM'M-UD, coupled with an artificial terminator, 3PRt. FM'M-UD contained fragments from three viral promoters (the promoters of Mirabilis mosaic caulimovirus (MMV) full-length transcript, the MMV subgenomic transcript, and figwort mosaic virus subgenomic transcript) and two types of cis-acting elements (four GAL4 binding sites and two zinc finger binding sites). The artificial terminator, 3PRt, consisted of the PINII and 35S terminators plus RB7, a matrix attachment region. The FM'M-UD promoter increased protein levels of reporters GFP, RBD : SD1 (part of S protein from SARS-CoV-2), and human interleukin-6 (hIL6) by 4-6-fold, 2-fold, and 6-fold, respectively, relative to those of the same reporters driven by the CaMV 35S promoter. Furthermore, when the FM'M-UD/3PRt expression cassette was expressed together with GAL4/TAC3d2, an artificial transcription factor that bound the GAL4 binding sites in FM'M-UD, levels of hIL6 increased by 10.7-fold, relative to those obtained from the CaMV 35S promoter plus the RD29B terminator. Thus, this novel expression system led to the production of a large amount of recombinant protein in plants.

Effects on Goat Meat Extracts on α-Glucosidase Inhibitory Activity, Expression of Bcl-2-Associated X (BAX), p53, and p21 in Cell Line and Expression of Atrogin-1, Muscle Atrophy F-Box (MAFbx), Muscle RING-Finger Protein-1 (MuRF-1), and Myosin Heavy Chain-7 (MYH-7) in C2C12 Myoblsts.

This study examined the α-glucosidase inhibitory, and apoptosis- and anti-muscular-related factors of goat meat extracts from forelegs, hind legs, loin, and ribs. The goat meat extracts were evaluated for their α-glucosidase inhibitory activity. The gene and protein expression levels of Bcl-2-associated X (bax), p53, and p21 were examined by reverse transcription polymerase chain reaction (RT-PCR) and immunoblotting in AGS and HT-29 cells. The expression levels of Atrogin-1 and MHC1b were examined by RT-PCR in C2C12 myoblasts, and the expression levels of Atrogin-1, muscle atrophy F-box (MAFbx), muscle RING-finger protein-1 (MuRF-1), and myosin heavy chain-7 were investigated by immunoblotting. α-Glucosidase inhibitory activity was higher in ethanol extract than in hydrous and hot water extracts. BAX and p53 expression levels were higher (p<0.05) in AGS cells treated with goat meat extract than those of cells treated with no goat meat extract. In HT-29 cells, the protein expression levels of BAX, p53, and p21 were higher (p<0.05) in the cells treated with goat meat extract than those of cells not treated with goat meat extract. In dexamethasone-treated C2C12 cells, goat meat extract treatment lower (p<0.05) the expression of Atrogin-1 and lower (p<0.05) the expression of MAFbx and MuRF-1. The results of the present study indicate that goat meat extracts have α-glucosidase inhibitory activity in vitro. In addition, apoptosis was induced in AGS cells and HT-29 cells treated with goat meat extract, and anti-muscular atrophy activity was also observed in C2C12 cells treated with goat meat extract.

Arabidopsis ABCG27 plays an essential role in flower and leaf development by modulating abscisic acid content.

Abscisic acid (ABA) is a phytohormone that mediates stress responses and regulates plant development. Several ATP-binding cassette (ABC) transporters in the G subfamily of ABC (ABCG) proteins have been reported to transport ABA. We investigated whether there are any other ABCG proteins that mediate plant developmental processes regulated by ABA in Arabidopsis (Arabidopsis thaliana). The ABCG27 gene was upregulated in response to exogenous ABA treatment. The abcg27 knockout mutant exhibited two developmental defects: epinastic leaves and abnormally long pistils, which reduced fertility and silique length. ABCG27 expression was induced threefold when flower buds were exposed to exogenous ABA, and the promoter of ABCG27 had two ABA-responsive elements. ABA content in the pistil and true leaves were increased in the abcg27 knockout mutant. Detached abcg27 pistils exposed to exogenous ABA grew longer than those of the wild-type control. ABCG27 fused to GFP localized to the plasma membrane when expressed in Arabidopsis mesophyll protoplasts. A transcriptome analysis of the pistils and true leaves of the wild type and abcg27 knockout mutant revealed that the expression of organ development-related genes changed in the knockout mutant. In particular, the expression of trans-acting small interference (ta-si) RNA processing enzyme genes, which regulate flower and leaf development, was low in the knockout mutant. Together, these results suggest that ABCG27 most likely function as an ABA transporter at the plasma membrane, modulating ABA levels and thereby regulating the development of the pistils and leaves under normal, non-stressed conditions.

ABA Transport Assay in Plant Single-Cell System.

Compare with long history of the abscisic acid (ABA) study, our knowledge of ABA transporters is quite recent. This is due to at least one reason: ABA is a weak acid, and thus, it exists in either protonated form or in anionic form depending on the surrounding pH relative to its pKa value. Because the protonated form of ABA can permeate the cell membrane, it would foreclose a specific uptake transporter of ABA. Notwithstanding this theoretical base, ABA transporters belonging to different protein families have been reported a decade ago, steadily. A critical point of the identification of novel ABA transporters is to prove their transport activity. To do this, heterologous expression system is considered first as a facility of transport activity analysis. However, it is difficult to overexpress membrane proteins in their functional state in heterologous system. They have the tendency to aggregate and produce inclusion bodies caused by mistargeting. Thus, in this chapter, I describe the method of ABA transport assay based on single-cell system originated from plant tissues.

Production of active Exendin-4 in Nicotiana benthamiana and its application in treatment of type-2 diabetics.

GLP-1 (Glucagon-like peptide-1) is a peptide that stimulates insulin secretion from the ß-cell for glycemic control of the plasma blood glucose level. Its mimetic exenatide (synthetic Exendin-4) with a longer half-life of approximately 3.3-4 h is widely used in clinical application to treat diabetes. Currently, exenatide is chemically synthesized. In this study, we report that the GLP-1 analogue recombinant Exendin-4 (Exdn-4) can be produced at a high level in Nicotiana benthamiana, with an estimated yield of 50.0 µg/g fresh biomass. For high-level expression, we generated a recombinant gene, B:GB1:ddCBD1m:8xHis : Exendin-4 (BGC : Exdn-4), for the production of Exendin-4 using various domains such as the BiP signal peptide, the GB1 domain (B1 domain of streptococcal G protein), a double cellulose binding domain 1 (CBD1), and 8 His residues (8xHis) to the N-terminus of Exendin-4. GB1 was used to increase the expression, whereas double CBD1 and 8xHis were included as affinity tags for easy purification using MCC beads and Ni2+-NTA resin, respectively. BGC : Exdn-4 was purified by single-step purification to near homogeneity using both Ni2+-NTA resin and microcrystalline cellulose (MCC) beads. Moreover, Exdn-4 without any extra residues was produced from BGC : Exdn-4 bound onto MCC beads by treating with enterokinase. Plant-produced Exdn-4 (Exendin-4) was as effective as chemically synthesized Exendin-4 in glucose-induced insulin secretion (GIIS) from mouse MIN6m9 cells a pancreatic beta cell line.

High Prevalence of Listeria monocytogenes in Smoked Duck: Antibiotic and Heat Resistance, Virulence, and Genetics of the Isolates.

This study aimed at determining the genetic and virulence characteristics of the Listeria monocytogenes from smoked ducks. L. monocytogenes was isolated by plating, and the isolated colonies were identified by PCR. All the obtained seven L. monocytogenes isolates possessed the virulence genes (inlA, inlB, plcB, and hlyA) and a 385 bp actA amplicon. The L. monocytogenes isolates (SMFM2018 SD 1-1, SMFM 2018 SD 4-1, SMFM 2018 SD 4-2, SMFM 2018 SD 5-2, SMFM 2018 SD 5-3, SMFM 2018 SD 6-2, and SMFM 2018 SD 7-1) were inoculated in tryptic soy broth (TSB) containing 0.6% yeast extract at 60°C, followed by cell counting on tryptic soy agar (TSA) containing 0.6% yeast extract at 0, 2, 5, 8, and 10 min. We identified five heat resistant isolates compared to the standard strain (L. monocytogenes ATCC13932), among which three exhibited the serotype 1/2b and D-values of 5.41, 6.48, and 6.71, respectively at 60°C. The optical densities of the cultures were regulated to a 0.5 McFarland standard to assess resistance against nine antibiotics after an incubation at 30°C for 24 h. All isolates were penicillin G resistant, possessing the virulence genes (inlA, inlB, plcB, and hlyA) and the 385-bp actA amplicon, moreover, three isolates showed clindamycin resistance. In conclusion, this study allowed us to characterize L. monocytogenes isolates from smoked ducks, exhibiting clindamycin and penicillin G resistance, along with the 385-bp actA amplicon, representing higher invasion efficiency than the 268-bp actA, and the higher heat resistance serotype 1/2b.

Lactobacillus fermentum SMFM2017-NK4 Isolated from Kimchi Can Prevent Obesity by Inhibiting Fat Accumulation.

This study evaluated the anti-obesity effects of lactic acid bacteria. Thirty-one lactic acid bacteria were examined in vitro for their ability to inhibit α-glucosidase activity, lipase activity, and 3T3-L1 cell differentiation. Four selected lactic acid bacteria were administered to obese C57BL/6J mice models for 8 weeks. The degree of improvement in obesity was determined by weight gain and serum biochemical analysis. The expression levels of genes (Fas and Cpt-2) related to obesity in the liver were analyzed by quantitative reverse transcription (qRT)-PCR. In addition, antioxidant protein levels (SOD-2, CAT, and GPx-1) in the liver were evaluated. The lactic acid bacteria-treated groups (PPGK1, LFNK3, LPNK2, and LFNK4) showed lower weight increase rate than the control group. The total cholesterol (T-chol), triglyceride (TG), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) levels in the blood serum of the LFNK4 group were the lowest among other groups, compared to the control group. The expression levels of lipid metabolism-related genes (Fas and Cpt-2) in the liver of the LFNK4 group were lower in Fas and higher in Cpt-2 than in the control group. The antioxidant protein expression levels (SOD-2, CAT, and GPx-1) in the liver tissue were also higher in the LFNK4 group. These results indicate that L. fermentum SMFM2017-NK4 has anti-obesity effects.

Development of a Selective Agar for Improving Campylobacter jejuni Detection in Food.

BACKGROUND: Campylobacter jejuni is a major gastroenteritis-causing foodborne pathogen. However, it is difficult to isolate when competing bacteria or cold-damaged cells are present. OBJECTIVE: Herein, a medium (Campylobacter selective agar, CSA) was developed and supplemented with catalase, L-serine, L-cysteine, and quercetin for the selective detection of C. jejuni in food. METHOD: The C. jejuni-detection efficiency in media broth and chicken tenders was evaluated. The pathogen was enumerated on modified charcoal-cefoperazone-deoxycholate agar (mCCDA), CSA supplemented with 4 µM catalase (CSA-C4), 8 µM catalase (CSA-C8), 20 mM L-serine (CSA-S20) or 50 mM L-serine (CSA-S50), and mCCDA supplemented with 0.5 mM L-cysteine (mCCDA-LC0.5), 1 mM L-cysteine (mCCDA-LC1), 40 µM quercetin (mCCDA-Q40) or 320 µM quercetin (mCCDA-Q320). The detection efficiency was then evaluated by counting colonies on the selective agar media. Quantitative assessment was also performed using chicken and duck carcasses. RESULTS: The C. jejuni detection efficiencies were higher (P < 0.05) in the groups CSA-C4 or CSA-C8, and CSA-S20 or CSA-S50, than mCCDA, and the detection efficiencies were maintained even in the presence of Acinetobacter baumannii, a competing bacterium. In the quantitative test, CSA-C8 and CSA-S50 demonstrated higher C. jejuni-detection efficiencies than mCCDA (control). CONCLUSIONS: Therefore, CSA-C8 and CSA-S50 improved the detection efficiency of C. jejuni in poultry products by promoting the recovery of cold-damaged cells. HIGHLIGHTS: When using CSA-C8 or CSA-S50 developed in this study for detection of C. jejuni in food, detection efficiency was higher than mCCDA.

Identification of Pathogenic Variations in Seafood Vibrio parahaemolyticus Isolates by Comparing Genome Sequences.

ABSTRACT: An investigation of the pathogenic characteristics of isolates of Vibrio parahaemolyticus was conducted by identifying the pathogenic tdh gene and then performing adherence and cytotoxicity assays. Genome sequences of the seafood isolates were analyzed using the Illumina HiSeq 2500 platform. The isolated strains were then mapped by comparing the genomes to the reference genome, and variations in the nucleotide sequences and amino acids were identified with the CLC Genomics Workbench program. The tdh gene was identified in four isolates of V. parahaemolyticus, of which three-SMFM201809-CPC7-3, SMFM201809-CF8-2, and SMFM201809-CF8-3-showed high cytotoxicity and differences in cell adhesion. These isolates were selected to identify virulence factors and genomic variations. All three isolates had the same virulence factors, such as adherence, secretion system, and toxin. In addition, amino acid variants were identified in the regions of type IV pilus, T3SS1 and T3SS1 secreted effectors, and thermolabile hemolysin. These results indicate that variations in amino acids found in regions related to adherence and cytotoxicity result in differences in adhesion efficiency and cytotoxicity; therefore, the isolates with these variations may cause more serious foodborne illness compared with other strains.

Synthesis of nitrogen-doped carbon nanodots to destroy bacteria competing with Campylobacter jejuni in enrichment medium, and development of a monoclonal antibody to detect C. jejuni after enrichment.

The objective of this study was to develop a method with improved sensitivity for Campylobacter jejuni detection in foods. Nitrogen-doped carbon nanodots (N-CNDs) were synthesized and added to an enrichment medium (Bolton broth) at a concentration of 10 mg/mL. A light-emitting diode (LED) at a wavelength of 425 nm was used to irradiate the N-CNDs-supplemented enrichment medium to induce an exothermic reaction for 1 h. Additionally, a monoclonal antibody specific to C. jejuni NCTC11168 was developed using hybridoma cells to aid detection. The C. jejuni detection capabilities of N-CNDs-supplemented enrichment medium and the conventional Bolton broth enrichment, were compared using duck samples. C. jejuni in the enrichment was detected with the monoclonal antibody based-indirect enzyme-linked immunosorbent assay (ID-ELISA). The N-CNDs-supplemented enrichment medium showed a better C. jejuni detection capability than the conventional Bolton broth enrichment. Additionally, data from ID-ELISA showed excellent detection efficiency and a shortened detection time in the N-CNDs-supplemented enrichment medium after LED irradiation at 425 nm. These results indicate that 1-h LED irradiation at 425 nm to Bolton broth supplemented with the N-CNDs increased the detection efficiency and shortened the detection time with the monoclonal antibody for C. jejuni in food.

Influence of milk microbiota on Listeria monocytogenes survival during cheese ripening.

This study aimed to compare the three strains of Listeria monocytogenes survival in raw milk cheese and pasteurized milk cheese and to suggest the effect of milk microbiota on survival. L. monocytogenes cell counts decreased in all cheese as ripening time increased, and the survival rate was different for the strains of L. monocytogenes. Furthermore, L. monocytogenes survived longer in raw milk cheese than in pasteurized milk cheese. The difference of bacterial survival in each cheese was independent of Aw or the Lactobacillus spp. populations in cheeses; there was no difference in Aw or Lactobacillus spp. populations in all cheeses. The richness of microbiota in raw milk was little higher than in pasteurized milk, and five phyla (Chloroflexi, Cyanobacteria, Deinococcus-Thermus, Lentisphaerae, and Verrucomicrobia) were present only in raw milk. Also, organic acid-producing bacteria were presented more in pasteurized milk compared with raw milk; thus, the growth of L. monocytogenes was slower in pasteurized milk. In conclusion, differences in the microbial community of milk can affect the growth of L. monocytogenes. Making cheese using raw milk is a risk of L. monocytogenes infection; thus, efforts to prevent growth of L. monocytogenes such as the use of appropriate food additives are required.

Quantitative microbial risk assessment for Clostridium perfringens foodborne illness following consumption of kimchi in South Korea.

The purpose of this study was to conduct a risk assessment for Clostridium perfringens foodborne illness via kimchi consumption in South Korea. Prevalence of C. perfringens in kimchi, kimchi consumption amount and frequency, and distribution conditions (time and temperature) from manufacture to the home were determined. C. perfringens initial contamination level was estimated using Beta distribution [Beta (6, 79)]. Potential C. perfringens cell counts during distribution were predicted using the Weibull model (primary models, R 2 = 0.923-0.953) and a polynomial model [(δ = 1/(0.2385 + (- 0.0307 × Temp) + (0.0011 × Temp2)), R 2 = 0.719]. Average daily consumption data was assessed using Gamma distribution [1.0444, 91.767, RiskShift (0.16895), RiskTruncate (0, 1078)]. The mean risk of C. perfringens-associated foodborne illness following kimchi consumption was found to be 1.21 × 10-17. These results suggest that the risk of C. perfringens foodborne illness from kimchi consumption, under current conditions, can be considered to be very low in S. Korea.

Anti-Inflammatory Effect of a Peptide Derived from the Synbiotics, Fermented Cudrania tricuspidata with Lactobacillus gasseri, on Inflammatory Bowel Disease.

The objective of this study was to evaluate the effects of peptides derived from synbiotics on improving inflammatory bowel disease (IBD). Five-week-old male C57BL/6 mice were administered with dextran sulfate sodium (DSS) via drinking water for seven days to induce IBD (IBD group). The mice in the IBD group were orally administered with PBS (IBD-PBS-positive control), Lactobacillus gasseri 505 (IBD-Pro), fermented powder of CT extract with L. gasseri 505 (IBD-Syn), ß-casein: LSQSKVLPVPQKAVPYPQRDMP (IBD-Pep 1), or α s2-casein: VYQHQKAMKPWIQPKTKVIPYVRYL (IBD-Pep 2) (both peptides are present in the synbiotics) for four more days while inducing IBD. To confirm IBD induction, the weights of the animals and the disease activity index (DAI) scores were evaluated once every two days. Following treatment of probiotics, synbiotics, or peptides for 11 days, the mice were sacrificed. The length of the small and large intestines was measured. The expression of the proinflammatory cytokines IL-1ß, IL-6, TNF-α, and COX-2 in the large intestine was measured. Large intestine tissue was fixed in 10% formalin and stained with hematoxylin and eosin for histopathological analysis. The body weights decreased and DAI scores increased in the IBD group, but the DAI scores were lower in the IBD-Pep 2 group than those in the IBD group treated with PBS, Pro, Syn, or Pep 1. The lengths of the small and large intestines were shorter in the IBD group than in the group without IBD, and the expression levels of the proinflammatory cytokines were lower (p < 0.05) in the IBD-Pep 2 group than those in the IBD-PBS-positive control group. In addition, histopathological analysis showed that IBD was ameliorated in the Pep 2-treated group. These results indicate that Pep 2 derived from α s2-casein was effective in alleviating IBD-associated inflammation. Thus, we showed that these peptides can alleviate inflammation in IBD.

Combined Enrichment and Quantitative Polymerase Chain Reaction to Improve Sensitivity and Reduce Time of Detection of Listeria monocytogenes in Mushrooms.

This study evaluated a combined method for the detection of Listeria monocytogenes in mushrooms, involving enrichment and quantitative real-time polymerase chain reaction (qPCR), to improve sensitivity and reduce detection time. The growth of L. monocytogenes was evaluated in Listeria enrichment broth (LEB) with modified carbon and nitrogen sources, increasing sodium concentrations, and added micronutrients. Primers targeting the L. monocytogenes iap (iap1 and iap2), hlyA (hlyA1-hlyA6), and prfA (prfA1-prfA4) genes were developed and their sensitivity and specificity were evaluated. The greatest increase in L. monocytogenes cell count was observed after 6-h incubation at 30°C in LEB+2 × FAC (LEB plus 20 mL/L ferric ammonium citrate), where cell count increased by 1.4 log CFU (colony-forming unit)/mL, compared with 0.9 log CFU/mL in LEB (p < 0.05). iap2 primers targeting the iap gene showed high specificity and were the most sensitive among those tested, with a detection limit of 2 log CFU/mL in LEB medium, 3.1 log CFU/g in golden needle mushroom, and 3.5 log CFU/g in large oyster mushroom. When applied to detection in golden needle mushrooms, a combination of 3-h incubation in LEB+2 × FAC medium and qPCR analysis with iap2 primers permitted detection of L. monocytogenes, even at an inoculum of 1 log CFU/g. Similarly, in large oyster mushrooms, 10-h enrichment in LEB+2 × FAC medium resulted in a cell count of 3.7 log CFU/g. These results indicate that a combined detection method, using LEB+2 × FAC medium for enrichment followed by qPCR with iap2 primer pair, can reduce enrichment time and improve the sensitivity and specificity of L. monocytogenes detection in mushrooms.

Filling the Gap: Functional Clustering of ABC Proteins for the Investigation of Hormonal Transport in planta.

Plant hormones regulate a myriad of plant processes, from seed germination to reproduction, from complex organ development to microelement uptake. Much has been discovered on the factors regulating the activity of phytohormones, yet there are gaps in knowledge about their metabolism, signaling as well as transport. In this review we analyze the potential of the characterized phytohormonal transporters belonging to the ATP-Binding Cassette family (ABC proteins), thus to identify new candidate orthologs in model plants and species important for human health and food production. Previous attempts with phylogenetic analyses on transporters belonging to the ABC family suggested that sequence homology per se is not a powerful tool for functional characterization. However, we show here that sequence homology might indeed support functional conservation of characterized members of different classes of ABC proteins in several plant species, e.g., in the case of ABC class G transporters of strigolactones and ABC class B transporters of auxinic compounds. Also for the low-affinity, vacuolar abscisic acid (ABA) transporters belonging to the ABCC class we show that localization-, rather than functional-clustering occurs, possibly because of sequence conservation for targeting the tonoplast. The ABC proteins involved in pathogen defense are phylogenetically neighboring despite the different substrate identities, suggesting that sequence conservation might play a role in their activation/induction after pathogen attack. Last but not least, in case of the multiple lipid transporters belong to different ABC classes, we focused on ABC class D proteins, reported to transport/affect the synthesis of hormonal precursors. Based on these results, we propose that phylogenetic approaches followed by transport bioassays and in vivo investigations might accelerate the discovery of new hormonal transport routes and allow the designing of transgenic and genome editing approaches, aimed to improve our knowledge on plant development, plant-microbe symbioses, plant nutrient uptake and plant stress resistance.

Abscisic acid is a substrate of the ABC transporter encoded by the durable wheat disease resistance gene Lr34.

The wheat Lr34res allele, coding for an ATP-binding cassette transporter, confers durable resistance against multiple fungal pathogens. The Lr34sus allele, differing from Lr34res by two critical nucleotide polymorphisms, is found in susceptible wheat cultivars. Lr34res is functionally transferrable as a transgene into all major cereals, including rice, barley, maize, and sorghum. Here, we used transcriptomics, physiology, genetics, and in vitro and in vivo transport assays to study the molecular function of Lr34. We report that Lr34res results in a constitutive induction of transcripts reminiscent of an abscisic acid (ABA)-regulated response in transgenic rice. Lr34-expressing rice was altered in biological processes that are controlled by this phytohormone, including dehydration tolerance, transpiration and seedling growth. In planta seedling and in vitro yeast accumulation assays revealed that both LR34res and LR34sus act as ABA transporters. However, whereas the LR34res protein was detected in planta the LR34sus version was not, suggesting a post-transcriptional regulatory mechanism. Our results identify ABA as a substrate of the LR34 ABC transporter. We conclude that LR34res-mediated ABA redistribution has a major effect on the transcriptional response and physiology of Lr34res-expressing plants and that ABA is a candidate molecule that contributes to Lr34res-mediated disease resistance.

Bone regeneration using three-dimensional hexahedron channel structured BCP block in rabbit calvarial defects.

The purpose of this study is to evaluate the efficacy of bone regeneration and volume maintenance of the three-dimensional (3D) structured biphasic calcium phosphate (BCP) block with porous hexahedron channels in a rabbit calvarial model. In this work, four circular defects (diameter: 8 mm) in calvarium of rabbits were randomly assigned to (1) negative control (control), (2) 3D hexahedron channel structured BCP block, (3) deproteinized bovine bone mineral particle, and (4) deproteinized porcine bone mineral particle. Animals were euthanized at 2 (n = 5) and 8 weeks (n = 5). Outcome measures included micro-computed tomography (CT) and histomorphometrical analysis. Results indicated that in micro-CT, BCP group showed the highest new bone volume with significant difference compared to control (p = 0.008) at 8 weeks. Histomorphometrically, total augmented area of BCP group was the highest with significant difference compared to control (p = 0.008) at 8 weeks. BCP group also maintained total volume of the original defect without collapsing. BCP block with 3D hexahedron channel structure seems to have favorable osteogenic and volume maintaining ability and highly porous structure might attribute to new bone formation. Further studies regarding the optimal internal structure and porosity of the BCP block bone substitute are needed. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 2254-2262, 2019.

3D-printed polycaprolactone scaffold mixed with ß-tricalcium phosphate as a bone regenerative material in rabbit calvarial defects.

Defect-specific bone regeneration using 3-dimensional (3D) printing of block bone has been developed. Polycaprolactone (PCL) is biocompatible polymer that can be used as 3D scaffold. The aim of this study is to assess the biocompatibility and osteogenic efficacy of 3D printed PCL scaffold and to evaluate the effectiveness of ß-tricalcium phosphate (ß-TCP) addition in PCL scaffold. In this work, four circular defects (diameter: 8 mm) in rabbit calvarium were randomly assigned to (1) negative control (control), (2) PCL block (PCL), (3) PCL mixed with 10 wt% ß-TCP (PCL/ß-TCP), and (4) PCL/ß-TCP plus collagen membrane (PCL/ß-TCP + M). Animals were euthanized at 2 (n = 5) and 8 weeks (n = 5). Results indicated that in micro-CT, PCL/ß-TCP + M showed the highest total augmented volume and new bone volume at 8 weeks, but there was no significant difference among four groups. Histomorphometrically, PCL, PCL/ß-TCP, and PCL/ß-TCP + M showed the significantly higher total augmented area compared to the control. PCL/ß-TCP + M showed the highest new bone area but not statistically higher than the control. New bone formation deep inside the scaffold was observed only in ß-TCP added scaffold. PCL showed high biocompatibility with great volume maintenance. Addition of ß-TCP to PCL seemed to increase hydrophilicity and osteoconductivity. Developments in 3D-printed PCL material are expected. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1254-1263, 2019.

Biomimetic characteristics of mussel adhesive protein-loaded collagen membrane in guided bone regeneration of rabbit calvarial defects.

PURPOSE: The aim of the present study was to evaluate the biocompatibility and barrier function of mussel adhesive protein (MAP)-loaded collagen membranes in guided bone regeneration (GBR). METHODS: Eight male New Zealand white rabbits were used. Four circular defects (diameter: 8 mm) were created in the calvarium of each animal. The defects were randomly assigned to 1) a negative control group, 2) a cyanoacrylate (CA)-loaded collagen membrane group (the CA group), 3) a MAP-loaded collagen membrane group (the MAP group), and 4) a group that received a polycaprolactone block with MAP-loaded collagen membrane (the MAP-PCL group). Specimens were harvested at 2 weeks (n=4) and 8 weeks (n=4) postoperatively for observational histology and histometric analysis. RESULTS: In the histologic analysis, MAP was completely absorbed without any byproducts. In contrast, some of the CA adhesive remained, showing an inflammatory reaction, at 8 weeks. In the MAP-PCL group, the MAP-loaded collagen membranes served as a barrier membrane despite their fast degradation in GBR. No significant difference was found in the amount of new bone between the MAP-PCL and MAP groups (1.82±0.86 mm2 and 2.60±0.65 mm2, respectively). CONCLUSIONS: The MAP-loaded collagen membrane functioned efficiently in this rabbit calvarial GBR model, with excellent biocompatibility. Further research is needed to assess clinical applications in defect types that are more challenging for GBR than those used in the current model.

Arabidopsis thaliana Raf22 protein kinase maintains growth capacity during postgerminative growth arrest under stress.

When seeds are exposed to drought and salinity during germination, newly germinated embryos stop growth and enter a quiescent state called postgerminative growth (PGG) arrest. PGG arrest protects embryos from the stress, but it is not known how PGG is restored from the arrest when stress is eased. In this study, we show that under stress- or abscisic acid-induced PGG arrest conditions, Arabidopsis thaliana Raf-type protein kinase 22 (AtRaf22) overexpression accelerated photoautotrophic seedling establishment, whereas atraf22 knockout mutations enhanced the arrest. Furthermore, when the stress intensity was reduced subsequently, AtRaf22 overexpression plants resumed growth and accomplished photoautotrophic transition much faster than the knockout or wild-type plants. These results suggest that AtRaf22 activity is important for maintaining growth capacity during stress-induced PGG arrest, which is most likely critical for competitive growth when the stress subsides and plants resume growth. Such a factor has not been reported before.