Cranial Neural Crest Specific Deletion of Alpl (TNAP) via P0-Cre Causes Abnormal Chondrocyte Maturation and Deficient Cranial Base Growth.

Bone growth plate abnormalities and skull shape defects are seen in hypophosphatasia, a heritable disorder in humans that occurs due to the deficiency of tissue nonspecific alkaline phosphatase (TNAP, Alpl) enzyme activity. The abnormal development of the cranial base growth plates (synchondroses) and abnormal skull shapes have also been demonstrated in global Alpl-/- mice. To distinguish local vs. systemic effects of TNAP on skull development, we utilized P0-Cre to knockout Alpl only in cranial neural crest-derived tissues using Alpl flox mice. Here, we show that Alpl deficiency using P0-Cre in cranial neural crest leads to skull shape defects and the deficient growth of the intersphenoid synchondrosis (ISS). ISS chondrocyte abnormalities included increased proliferation in resting and proliferative zones with decreased apoptosis in hypertrophic zones. ColX expression was increased, which is indicative of premature differentiation in the absence of Alpl. Sox9 expression was increased in both the resting and prehypertrophic zones of mutant mice. The expression of Parathyroid hormone related protein (PTHrP) and Indian hedgehog homolog (IHH) were also increased. Finally, cranial base organ culture revealed that inorganic phosphate (Pi) and pyrophosphate (PPi) have specific effects on cell signaling and phenotype changes in the ISS. Together, these results demonstrate that the TNAP expression downstream of Alpl in growth plate chondrocytes is essential for normal development, and that the mechanism likely involves Sox9, PTHrP, IHH and PPi.

Deletion of the Pyrophosphate Generating Enzyme ENPP1 Rescues Craniofacial Abnormalities in the TNAP-/- Mouse Model of Hypophosphatasia and Reveals FGF23 as a Marker of Phenotype Severity.

Hypophosphatasia is a rare heritable metabolic disorder caused by deficient Tissue Non-specific Alkaline Phosphatase (TNAP) enzyme activity. A principal function of TNAP is to hydrolyze the tissue mineralization inhibitor pyrophosphate. ENPP1 (Ectonucleotide Pyrophosphatase/Phosphodiesterase 1) is a primary enzymatic generator of pyrophosphate and prior results showed that elimination of ENPP1 rescued bone hypomineralization of skull, vertebral and long bones to different extents in TNAP null mice. Current TNAP enzyme replacement therapy alleviates skeletal, motor and cognitive defects but does not eliminate craniosynostosis in pediatric hypophosphatasia patients. To further understand mechanisms underlying craniosynostosis development in hypophosphatasia, here we sought to determine if craniofacial abnormalities including craniosynostosis and skull shape defects would be alleviated in TNAP null mice by genetic ablation of ENPP1. Results show that homozygous deletion of ENPP1 significantly diminishes the incidence of craniosynostosis and that skull shape abnormalities are rescued by hemi- or homozygous deletion of ENPP1 in TNAP null mice. Skull and long bone hypomineralization were also alleviated in TNAP-/-/ENPP1-/- compared to TNAP-/-/ENPP1+/+ mice, though loss of ENPP1 in combination with TNAP had different effects than loss of only TNAP on long bone trabeculae. Investigation of a relatively large cohort of mice revealed that the skeletal phenotypes of TNAP null mice were markedly variable. Because FGF23 circulating levels are known to be increased in ENPP1 null mice and because FGF23 influences bone, we measured serum intact FGF23 levels in the TNAP null mice and found that a subset of TNAP-/-/ENPP1+/+ mice exhibited markedly high serum FGF23. Serum FGF23 levels also correlated to mouse body measurements, the incidence of craniosynostosis, skull shape abnormalities and skull bone density and volume fraction. Together, our results demonstrate that balanced expression of TNAP and ENPP1 enzymes are essential for microstructure and mineralization of both skull and long bones, and for preventing craniosynostosis. The results also show that FGF23 rises in the TNAP-/- model of murine lethal hypophosphatasia. Future studies are required to determine if the rise in FGF23 is a cause, consequence, or marker of disease phenotype severity.

Anabolic actions of parathyroid hormone in a hypophosphatasia mouse model.

Hypophosphatasia, the rare heritable disorder caused by TNAP enzyme mutations, presents wide-ranging severity of bone hypomineralization and skeletal abnormalities. Intermittent PTH (1-34) increased long bone volume in Alpl-/- mice but did not alter the skull phenotype. PTH may have therapeutic value for adults with TNAP deficiency-associated osteoporosis. INTRODUCTION: Hypophosphatasia is the rare heritable disorder caused by mutations in the tissue non-specific alkaline phosphatase (TNAP) enzyme leading to TNAP deficiency. Individuals with hypophosphatasia commonly present with bone hypomineralization and skeletal abnormalities. The purpose of this study was to determine the impact of intermittent PTH on the skeletal phenotype of TNAP-deficient Alpl-/- mice. METHODS: Alpl-/- and Alpl+/+ (wild-type; WT) littermate mice were administered PTH (1-34) (50 µg/kg) or vehicle control from days 4 to 12 and skeletal analyses were performed including gross measurements, micro-CT, histomorphometry, and serum biochemistry. RESULTS: Alpl-/- mice were smaller with shorter tibial length and skull length compared to WT mice. Tibial BV/TV was reduced in Alpl-/- mice and daily PTH (1-34) injections significantly increased BV/TV and BMD but not TMD in both WT and Alpl-/- tibiae. Trabecular spacing was not different between genotypes and was decreased by PTH in both genotypes. Serum P1NP was unchanged while TRAcP5b was significantly lower in Alpl-/- vs. WT mice, with no PTH effect, and no differences in osteoclast numbers. Skull height and width were increased in Alpl-/- vs. WT mice, and PTH increased skull width in WT but not Alpl-/- mice. Frontal skull bones in Alpl-/- mice had decreased BV/TV, BMD, and calvarial thickness vs. WT with no significant PTH effects. Lengths of cranial base bones (basioccipital, basisphenoid, presphenoid) and lengths of synchondroses (growth plates) between the cranial base bones, plus bone of the basioccipitus, were assessed. All parameters were reduced (except lengths of synchondroses, which were increased) in Alpl-/- vs. WT mice with no PTH effect. CONCLUSION: PTH increased long bone volume in the Alpl-/- mice but did not alter the skull phenotype. These data suggest that PTH can have long bone anabolic activity in the absence of TNAP, and that PTH may have therapeutic value for individuals with hypophosphatasia-associated osteoporosis.

Development of Multiplex PCR-based Protocols for Simultaneous Caterpillar Diagnosis of Three Spodoptera and One Mamestra Species (Lepidoptera: Noctuidae).

Since many noctuid moth species are highly destructive crop pests, it is essential to establish proper management strategies, which primarily require accurate and rapid species identification. However, diagnosis of noctuid species in the field, particularly at the larval stage, is very difficult due to their morphological similarity and individual color variation. In particular, caterpillars of Spodoptera exigua (Hübner), Spodoptera litura (Fabricius), Spodoptera frugiperda (Smith), and Mamestra brassicae (L.) (Lepidoptera: Noctuidae) are hard to be identified by morphology and frequently found on the same host crops in the same season, thus requiring a reliable species diagnosis method. To efficiently diagnose these species, we identified species-specific internal transcribed spacer 1 (ITS1) sequences and developed two molecular species diagnosis protocols using ITS1 markers. The first protocol was multiplex conventional PCR in conjunction with subsequent gel electrophoresis for species identification based on amplicon size. The second protocol was based on multiplex real-time PCR using fluorescent dye-labeled primers for single-step diagnosis. Template genomic DNA (gDNA) prepared by the DNA release method was also suitable for both protocols as the template prepared by DNA extraction. The two protocols enabled rapid and robust species diagnosis using a single multiplex PCR step. Depending on laboratory instrumentation, one of the two protocols can be easily adapted for species diagnosis of the four noctuid caterpillars in the field, which is essential for establishing proper management strategies. The multiplex real-time PCR protocol, in particular, will facilitate accurate diagnosis of the four species in a single step regardless of template gDNA quality.

Insecticide resistance in pepper greenhouse populations of Aphis gossypii (Hemiptera: Aphididae) in Korea.

The cotton aphid or melon aphid, Aphis gossypii Glover (Hemiptera: Aphididae), is a polyphagous insect pest with a wide host range. Two distinct genetic clusters were found in A. gossypii populations in Korea. To determine whether the division of the genetic clusters was driven by insecticide selection pressure, the frequencies of insecticide resistance-associated mutations on three representative insecticide target genes [i.e., nicotinic acetylcholine receptor gene (nAChR), voltage-gated sodium channel gene (vgsc), and acetylcholinesterase 1 gene (ace-1)] were predicted in A. gossypii populations with known genetic structures. Most populations revealed heterozygosity-resistant alleles for the nAChR R81T and vgsc M918L mutations, but homozygous-resistant alleles for the ace-1 S431F mutation. However, assessment of the three mutation frequencies revealed no apparent correlation between the genetic structures and the resistance profiles. The regression analysis revealed no correlation between the genetic cluster ratios and resistance allele frequencies (R81T, S431F, and M918L). We used three insecticides that are commonly used in greenhouses: imidacloprid (neonicotinoid), acephate (organophosphate), and esfenvalerate (pyrethroid), to test resistance and susceptibility in A. gossypii populations. The bioassay results revealed that the BS_19 (Busan) and JE_19 (Jeongeup) populations were resistant to imidacloprid and acephate, the HS_19 (Honseong) population was resistant to acephate and esfenvalerate, and susceptible lab strains only exhibited resistance to acephate. The bioassay results were correlated with mutation frequency, but no correlation was detected among genetic clusters. These results suggest that the distinct genetic structure observed in the Korean populations of A. gossypii is not likely influenced by insecticide resistance traits, but rather by other factors.

Species Composition, Abundance, and Seasonal Dynamics of Perilla Seed Bugs (Heteroptera: Lygaeidae) in Weeds and Perilla Fields in Korea.

Perilla seed bugs (Nysius sp.) are considered to be the emerging pests causing nutritional and yield losses in perilla and cereal crops. A survey of perilla seed bugs on weeds and perilla crops was conducted over the course of 2 yr in Korea to determine the species composition, abundance, and seasonal dynamics of perilla seed bugs. Three species of Heteroptera (Nysius plebeius, Nysius hidakai, and Nysius inconspicuus), nymphs of Nysius species, and several parasitoid species were collected from weeds and perilla crops. Nysius hidakai was the most abundant perilla seed bugs. In 2019, adult perilla seed bugs, nymphs of perilla seed bugs, and parasitoid species were more abundant in weed species than in perilla crops. An early peak with a greater number of adult perilla seed bug (N. hidakai) was observed in weeds in 2020. However, an identical peak with a similar number of perilla seed bug (N. hidakai) was found in perilla crops in both years. Peak perilla seed bugs densities were observed in the 4th week of June, 2020 in weeds. Parasitoid species from Aphidiidae (1), Braconidae (11), Eulophidae (7), Figitidae (5), Ichneumonidae (7), Platygastridae (1), and Pteromalidae (5) subfamilies were collected. Perilla seed bugs seem to be a serious and increasingly important pest in several field crop species including perilla crops grown on the southern Korean peninsula. Monitoring and early detection of insect species are vital to predicting seasonal colonization and population build-up of perilla seed bugs on perilla crops from a climate change perspective, and essential for developing appropriate management techniques. Thus, continuous monitoring of perilla seed bugs in alternative weed hosts is needed to protect perilla crops from perilla seed bug infestation.

Development of a LAMP-Based Molecular Species Diagnosis Method for Four Major Agricultural Pests in the Genus Spodoptera (Lepidoptera: Noctuidae).

Molecular-based species identification tools are helpful to identify tiny insect and lepidopteran pests that show morphological similarities in the larval stage and are essential for quarantine as well as agricultural research. Here, we focused on four major Spodoptera pests: S. exigua, S. frugiperda, S. litura, and S. littoralis. S. exigua and S. litura mitochondrial genome sequences were newly identified and species-specific sequence regions were identified in the cytochrome c oxidase subunit II and III regions. Species primers were designed and applied in loop-mediated isothermal amplification (LAMP) and PCR to identify Korean field-collected or overseas samples. The optimal incubation conditions for LAMP were 61 °C for 60 min with four LAMP primers. Additional loop primers increased the amplification efficiency for S. exigua, and the nonspecific amplification for other species. The LAMP assay could detect a wide range of DNA concentrations, with the range 1 ng-1 pg in dependence of four LAMP primers. The DNA-releasing technique, without DNA extraction, in the LAMP assay involved larval or adult tissue sample incubation at 95 °C for 5 min. The entire process takes approximately 70 min. This new molecular diagnostic method is simple and accurate, with application in the field and laboratory and for monitoring and ecological studies.

Investigation of Pathogenic Escherichia coli Contamination of Vegetables Distributed in a Korean Agricultural Wholesale Market.

The purpose of this study was to investigate the monthly contamination rate of pathogenic Escherichia coli, a major cause of food poisoning, in vegetables sold in agricultural wholesale markets, which distribute vegetables from all over the country, in the Incheon Metropolitan City area, South Korea, and to identify a source of the pathogen. In total, 1739 vegetables of 80 types, along with 109 soil, 67 manure, and 33 livestock feces samples, were tested for pathogenic E. coli using polymerase chain reaction, from September 2016 through August 2017. The average annual prevalence rate of vegetables was 5.8%, and the prevalence rate was above 5% from June through October. The highest prevalence rate (15.7%) was recorded in July. Water dropwort showed the highest prevalence rate (28.6%) among the vegetables examined. Pathogenic E. coli was detected in >20 types of the vegetables that were to be consumed without cooking. Among these, the prevalence rates of ponytail radish (n = 21), crown daisy (n = 86), young radish (n = 68), romaine lettuce (n = 133), perilla leaf (n = 103), Korean leek (n = 43), young Chinese cabbage (n = 68), and Chinese cabbage (n = 30) were 9.5%, 8.1%, 7.4%, 6.8%, 4.9%, 4.7%, 4.4%, and 3.3%, respectively. Among the vegetables cooked before consumption, prevalence rates were 28.6%, 27.3%, and 25.0% in wormwood, sweet potato stalk, and edible mountain vegetables (Saussurea sp., etc.), respectively. In soil, manure, and livestock feces, 36.7%, 26.9%, and 90.6% prevalence rates were confirmed, respectively. This study confirmed the pathogenic E. coli contamination of vegetables to be consumed without cooking. Therefore, to produce agricultural products that do not induce food poisoning and are safe for consumption, it is important to develop a process for killing the pathogenic microorganisms and set up a sanitary environment for effectively managing compost. In addition, it is necessary to establish surveillance systems to monitor the production chain.

Development of 50 InDel-based barcode system for genetic identification of tartary buckwheat resources.

Tartary buckwheat (Fagopyrum tataricum Gartn.) is a highly functional crop that is poised to be the target of many future breeding efforts. The reliable ex situ conservation of various genetic resources is essential for the modern breeding of tartary buckwheat varieties. We developed PCR-based co-dominant insertion/deletion (InDel) markers to discriminate tartary buckwheat genetic resources. First, we obtained the whole genome from 26 accessions across a superscaffold-scale reference genome of 569.37 Mb for tartary buckwheat cv. "Daegwan 3-7." Next, 171,926 homogeneous and 53,755 heterogeneous InDels were detected by comparing 26 accessions with the "Daegwan 3-7" reference sequence. Of these, 100 candidate InDels ranging from 5-20 bp in length were chosen for validation, and 50 of them revealed polymorphisms between the 26 accessions and "Daegwan 3-7." The validated InDels were further tested through the assessment of their likelihood to give rise to a single or a few PCR products in 50 other accessions, covering most tartary buckwheat genome types. The major allele frequencies ranged from 0.5616 at the TB42 locus to 0.9863 at the TB48 locus, with the average PIC value of 0.1532 with a range of 0.0267-0.3712. To create a user-friendly system, the homology of the genotypes between and among the accessions were visualized in both one- (1D) and two-dimensional (2D) barcode types by comparing amplicon polymorphisms with the reference variety, "Daegwan 3-7." A phylogenetic tree and population structure of the 76 accessions according to amplicon polymorphisms for the 50 InDel markers corresponded to those using non-synonymous single nucleotide polymorphism variants, indicating that the barcode system based on the 50 InDels was a useful tool to improve the reliability of identification of tartary buckwheat accessions in the germplasm stocks.

Macropore design of tissue engineering scaffolds regulates mesenchymal stem cell differentiation fate.

Craniosynostosis is a debilitating birth defect characterized by the premature fusion of cranial bones resulting from premature loss of stem cells located in suture tissue between growing bones. Mesenchymal stromal cells in long bone and the cranial suture are known to be multipotent cell sources in the appendicular skeleton and cranium, respectively. We are developing biomaterial constructs to maintain stemness of the cranial suture cell population towards an ultimate goal of diminishing craniosynostosis patient morbidity. Recent evidence suggests that physical features of synthetic tissue engineering scaffolds modulate cell and tissue fate. In this study, macroporous tissue engineering scaffolds with well-controlled spherical pores were fabricated by a sugar porogen template method. Cell-scaffold constructs were implanted subcutaneously in mice for up to eight weeks then assayed for mineralization, vascularization, extracellular matrix composition, and gene expression. Pore size differentially regulates cell fate, where sufficiently large pores provide an osteogenic niche adequate for bone formation, while sufficiently small pores (<125 µm in diameter) maintain stemness and prevent differentiation. Cell-scaffold constructs cultured in vitro followed the same pore size-controlled differentiation fate. We therefore attribute the differential cell and tissue fate to scaffold pore geometry. Scaffold pore size regulates mesenchymal cell fate, providing a novel design motif to control tissue regenerative processes and develop mesenchymal stem cell niches in vivo and in vitro through biophysical features.

Development of a simple and accurate molecular tool for Spodoptera frugiperda species identification using LAMP.

BACKGROUND: The fall armyworm, Spodoptera frugiperda is a native species of the Americas. First detected in western and central Africa in early 2016, it has become one of the most serious invasive lepidopteran pests in many African and Asian countries. S. frugiperda has spread very quickly; however, there are no molecular-based, simple and accurate diagnostic tools for identification of this species in the field. Methods to identify invasive S. frugiperda are urgently needed because farmers and agricultural managers have no prior experience with this pest. RESULTS: Based on mitochondrial genome sequence alignment, a S. frugiperda-specific sequence region was identified in the transfer RNA-coding region between NADH dehydrogenase, ND3, and ND5. Using this unique region, species-diagnostic primers were designed and applied in a loop-mediated isothermal amplification (LAMP) assay and a conventional polymerase chain reaction to identify field-collected samples of S. frugiperda. The optimal incubation conditions for the LAMP assay were 61°C for 90 min with four LAMP primers; an additional loop primer increased the amplification efficiency. A response was obtained for a wide range of DNA concentrations in the LAMP assay and the minimum detectable DNA concentration was 10 pg. CONCLUSIONS: We developed a new LAMP-based molecular diagnostic method that it is easy to use and accurate. The LAMP assay was used with a DNA-releasing technique for larval and adult samples, without a DNA extraction step, by incubating the tissue sample at 95°C for 5 min. This method can be applied in intensive field monitoring of S. frugiperda and its ecological studies. © 2021 Society of Chemical Industry.

Tissue Nonspecific Alkaline Phosphatase Function in Bone and Muscle Progenitor Cells: Control of Mitochondrial Respiration and ATP Production.

Tissue nonspecific alkaline phosphatase (TNAP/Alpl) is associated with cell stemness; however, the function of TNAP in mesenchymal progenitor cells remains largely unknown. In this study, we aimed to establish an essential role for TNAP in bone and muscle progenitor cells. We investigated the impact of TNAP deficiency on bone formation, mineralization, and differentiation of bone marrow stromal cells. We also pursued studies of proliferation, mitochondrial function and ATP levels in TNAP deficient bone and muscle progenitor cells. We find that TNAP deficiency decreases trabecular bone volume fraction and trabeculation in addition to decreased mineralization. We also find that Alpl-/- mice (global TNAP knockout mice) exhibit muscle and motor coordination deficiencies similar to those found in individuals with hypophosphatasia (TNAP deficiency). Subsequent studies demonstrate diminished proliferation, with mitochondrial hyperfunction and increased ATP levels in TNAP deficient bone and muscle progenitor cells, plus intracellular expression of TNAP in TNAP+ cranial osteoprogenitors, bone marrow stromal cells, and skeletal muscle progenitor cells. Together, our results indicate that TNAP functions inside bone and muscle progenitor cells to influence mitochondrial respiration and ATP production. Future studies are required to establish mechanisms by which TNAP influences mitochondrial function and determine if modulation of TNAP can alter mitochondrial respiration in vivo.

Development of a Species Diagnostic Molecular Tool for an Invasive Pest, Mythimna loreyi, Using LAMP.

The Mythimna loreyi (Duponchel) is one of the well-known invasive noctuid pests in Africa, Australia, and many Asian countries. However, it is difficult to identify the invasive and morphologically similar species, Mythimna separate, which occur at the cornfield in the larvae stage. Currently, the molecular biology method for diagnosing M. loreyi species is only using the mtCO1 universal primer (LCO1490, HCO2198), which requires a lot of time and effort, such as DNA extraction, PCR, electrophoresis, and sequencing. In this study, the LAMP assay was developed for rapid, simple, effective species identification. By analyzing the mitochondrial genome, the species-specific sequence was found at the coding region of the NADH dehydrogenase subunit 5 gene. Based on this unique sequence, four LAMP primers and two loop primers were designed. The F3 and B3 primers were able to diagnose species-specific, in general, and multiplex PCR and specifically reacted within the inner primers in LAMP assay. The optimal incubation condition of the LAMP assay was 61 °C for 60 min with four LAMP primers, though additional loop primers, BF and LF, did not significantly shorten the amplification time. The broad range of DNA concentration was workable in LAMP assay, in which the minimum detectable DNA concentration was 100 pg. DNA releasing method was applied, which took five minutes of incubation at 95 °C without the DNA extraction process. Only some pieces of tissue of larvae and adult samples were needed to extract DNA. The incidence of invasive pests is gradually diversifying. Therefore, this simple and accurate LAMP assay is possibly applied in the intensive field monitoring for invasive pests and integrated management of Mythimna loreyi.

Genetic background dependent modifiers of craniosynostosis severity.

Craniosynostosis severity varies in patients with identical genetic mutations. To understand causes of this phenotypic variation, we backcrossed the FGFR2+/C342Y mouse model of Crouzon syndrome onto congenic C57BL/6 and BALB/c backgrounds. Coronal suture fusion was observed in C57BL/6 (88% incidence, p < .001 between genotypes) but not in BALB/c FGFR2+/C342Y mutant mice at 3 weeks after birth, establishing that that the two models differ in phenotype severity. To begin identifying pre-existing modifiers of craniosynostosis severity, we compared transcriptome signatures of cranial tissues from C57BL/6 vs. BALB/c FGFR2+/+ mice. We separately analyzed frontal bone with coronal suture tissue from parietal bone with sagittal suture tissues because the coronal suture but not the sagittal suture fuses in FGFR2+/C342Y mice. The craniosynostosis associated Twist and En1 transcription factors were down-regulated, while Runx2 was up-regulated, in C57BL/6 compared to BALB/c tissues, which could predispose to craniosynostosis. Transcriptome analyses under the GO term MAPK cascade revealed that genes associated with calcium ion channels, angiogenesis, protein quality control and cell stress response were central to transcriptome differences associated with genetic background. FGFR2 and HSPA2 protein levels plus ERK1/2 activity were higher in cells isolated from C57BL/6 than BALB/c cranial tissues. Notably, the HSPA2 protein chaperone is central to craniofacial genetic epistasis, and we find that FGFR2 protein is abnormally processed in primary cells from FGFR2+/C342Y but not FGFR2+/+ mice. Therefore, we propose that differences in protein quality control responses may contribute to genetic background influences on craniosynostosis phenotype severity.

Subcritical water extraction of bioactive compounds from Orostachys japonicus A. Berger (Crassulaceae).

Subcritical-water extraction is an ecofriendly method for extracting antioxidant compounds only using water. The Subcritical-water extraction was employed for the extraction of bioactive compounds from Orostachys japonicus known as rock pine by investigating the use of various temperatures (110-260 °C) and extraction times (5-20 min). The Subcritical-water extraction condition at 220 °C for 15 min; the total phenolics content (39.9 ± 4.1 mg/g), flavonoids content (11.4 ± 0.6 mg/g), and antioxidant activities (90.3 ± 2.2%, 96.0 ± 2.9%, and 662.4 ± 17.2 mg/g) of Subcritical-water extract were higher under this condition than for extraction with either methanol or ethanol. Triterpene saponins were observed only in subcritical-water extraction condition at 220 °C for 15 min. Further, some of its phenolic constituents; gallic acid, quercetin, and kaempferol were quantified by high performance liquid chromatography. Subcritical-water extraction is an effective method for extracting valuable bioactive compounds from Orostachys japonicus.

Viral delivery of tissue nonspecific alkaline phosphatase diminishes craniosynostosis in one of two FGFR2C342Y/+ mouse models of Crouzon syndrome.

Craniosynostosis is the premature fusion of cranial bones. The goal of this study was to determine if delivery of recombinant tissue nonspecific alkaline phosphatase (TNAP) could prevent or diminish the severity of craniosynostosis in a C57BL/6 FGFR2C342Y/+ model of neonatal onset craniosynostosis or a BALB/c FGFR2C342Y/+ model of postnatal onset craniosynostosis. Mice were injected with a lentivirus encoding a mineral targeted form of TNAP immediately after birth. Cranial bone fusion as well as cranial bone volume, mineral content and density were assessed by micro CT. Craniofacial shape was measured with calipers. Alkaline phosphatase, alanine amino transferase (ALT) and aspartate amino transferase (AST) activity levels were measured in serum. Neonatal delivery of TNAP diminished craniosynostosis severity from 94% suture obliteration in vehicle treated mice to 67% suture obliteration in treated mice, p<0.02) and the incidence of malocclusion from 82.4% to 34.7% (p<0.03), with no effect on cranial bone in C57BL/6 FGFR2C342Y/+ mice. In contrast, treatment with TNAP increased cranial bone volume (p< 0.01), density (p< 0.01) and mineral content (p< 0.01) as compared to vehicle treated controls, but had no effect on craniosynostosis or malocclusion in BALB/c FGFR2C342Y/+ mice. These results indicate that postnatal recombinant TNAP enzyme therapy diminishes craniosynostosis severity in the C57BL/6 FGFR2C342Y/+ neonatal onset mouse model of Crouzon syndrome, and that effects of exogenous TNAP are genetic background dependent.

Population Genetic Structure of Aphis gossypii Glover (Hemiptera: Aphididae) in Korea.

Aphis gossypii Glover (Hemiptera: Aphididae) is a serious polyphagous agricultural pest worldwide. In the present study, we used eight microsatellite markers to investigate the genetic structure and diversity of A. gossypii populations in Korea. Samples were collected from 37 locations in Korea (18 populations in 2016, 14 populations in 2017, and five populations in 2018) from pepper plants. A. gossypii had low to moderate genetic diversity, and expected heterozygosity (HE) ranged from 0.354 to 0.719. A Mantel test of isolation by distance indicated no relationship between genetic structure and geographic distance among all populations (r2 = 0.0004, p = 0.370), suggesting high gene flow among populations in Korea. Populations of A. gossypii in Korea were divided into two distinct genetic clusters (ΔK = 2). In 2016 and 2017, the genetic clusters changed into opposite genetic structures within one year mostly in northwest and southeast parts of Korea. Possible relevance of study results was discussed. Chemical control, cyclical parthenogenesis, and immigrants from the exterior might have resulted in this low genetic diversity and opposite genetic clusters.

Population genetic structure of Bemisia tabaci MED (Hemiptera: Aleyrodidae) in Korea.

The sweet potato whitefly, Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) is a major agricultural pest that causes economic damages worldwide. In particular, B. tabaci MED (Mediterranean) has resulted in serious economic losses in tomato production of Korea. In this study, 1,145 B. tabaci MED females from 35 tomato greenhouses in different geographic regions were collected from 2016 to 2018 (17 populations in 2016, 13 in 2017, and five in 2018) and analyzed to investigate their population genetic structures using eight microsatellite markers. The average number of alleles per population (NA) ranged from 2.000 to 5.875, the expected heterozygosity (HE) ranged from 0.218 to 0.600, the observed heterozygosity (HO) ranged from 0.061 to 0.580, and the fixation index inbreeding coefficient (FIS) ranged from -0.391 to 0.872 over the three years of the study. Some significant correlation (p < 0.05) was present between genetic differentiations (FST) and geographical distance, and a comparatively high proportion of variation was found among the B. tabaci MED populations. The B. tabaci MED populations were divided into two well-differentiated genetic clusters within different geographic regions. Interestingly, its genetic structures converged into one genetic cluster during just one year. The reasons for this genetic change were speculated to arise from different fitness, insecticide resistance, and insect movement by human activities.

Tissue nonspecific alkaline phosphatase promotes calvarial progenitor cell cycle progression and cytokinesis via Erk1,2.

Bone growth is dependent upon the presence of self-renewing progenitor cell populations. While the contribution of Tissue Nonspecific Alkaline Phosphatase (TNAP) enzyme activity in promoting bone mineralization when expressed in differentiated bone forming cells is well understood, little is known regarding the role of TNAP in bone progenitor cells. We previously found diminished proliferation in the calvarial MC3T3E1 cell line upon suppression of TNAP by shRNA, and in calvarial cells and tissues of TNAP-/- mice. These findings indicate that TNAP promotes cell proliferation. Here we investigate how TNAP mediates this effect. Results show that TNAP is essential for calvarial progenitor cell cycle progression and cytokinesis, and that these effects are mediated by inorganic phosphate and Erk1/2. Levels of active Erk1/2 are significantly diminished in TNAP deficient cranial cells and tissues even in the presence of inorganic phosphate. Moreover, in the absence of TNAP, FGFR2 expression levels are high and FGF2 rescues phospho-Erk1/2 levels and cell cycle abnormalities to a significantly greater extent than inorganic phosphate. Based upon the data we propose a model in which TNAP stimulates Erk1/2 activity via both phosphate dependent and independent mechanisms to promote cell cycle progression and cytokinesis in calvarial bone progenitor cells. Concomitantly, TNAP feeds back to inhibit FGFR2 expression. These results identify a novel mechanism by which TNAP promotes calvarial progenitor cell renewal and indicate that converging pathways exist downstream of FGF signaling and TNAP activity to control craniofacial skeletal development.

Conversion of 6-gingerol to 6-shogaol in ginger (Zingiber officinale) pulp and peel during subcritical water extraction.

Subcritical water extraction is an eco-friendly method for the extraction of less polar compounds without the use of organic solvents. This study determined the extraction conditions that maximize the contents of 6-gingerol and 6-shogaol obtained from ginger pulp and peel. The highest yields of 6-gingerol (0.68 ±â€¯0.08 mg/g), and 6-shogaol (0.39 ±â€¯0.03 mg/g) were obtained from ginger pulp at the extraction conditions of 130 °C/25 min, and 190 °C/15 min. 6-Shogaol content increased with the increasing extraction temperature and extraction time due to the conversion of 6-gingerol to 6-shogaol by thermal cracking. The antioxidant activity of ginger extracts were increased depending on the increasing of 6-shogaol content.