Microbial Community Changes in Silkworms Suspected of Septicemia and Identification of Serratia sp.

Diseases that occur in silkworms include soft rot, hardening disease, digestive diseases, and sepsis. However, research on the causes of bacterial diseases occurring in silkworms and the resulting changes in the microbial community is lacking. Therefore, we examined the morphological characteristics of sepsis and changes in the microbial community between silkworms that exhibit a unique odor and healthy silkworms; thus, we established a relationship between disease-causing microorganisms and sepsis. After producing a 16S rRNA amplicon library for samples showing sepsis, we obtained information on the microbial community present in silkworms using next-generation sequencing. Compared to that in healthy silkworms, in silkworms with sepsis, the abundance of the Firmicutes phylum was significantly reduced, while that of Proteobacteria was increased. Serratia sp. was dominant in silkworms with sepsis. After bacterial isolation, identification, and reinfection through the oral cavity, we confirmed this organism as the disease-causing agent; its mortality rate was 1.8 times higher than that caused by Serratia marcescens. In summary, we identified a new causative bacterium of silkworm sepsis through microbial community analysis and confirmed that the microbial community balance was disrupted by the aberrant proliferation of certain bacteria.

Etiology and clinical characteristics of acute viral hepatitis in South Korea during 2020-2021: a prospective multicenter study.

This prospective, 12-center study investigated the etiology and clinical characteristics of acute viral hepatitis (AVH) during 2020-2021 in South Korea, and the performance of different diagnostic methods for hepatitis E virus (HEV). We enrolled 428 patients with acute hepatitis, of whom 160 (37.4%) were diagnosed with AVH according to predefined serologic criteria. The clinical data and risk factors for AVH were analyzed. For hepatitis E patients, anti-HEV IgM and IgG were tested with two commercial ELISA kits (Abia and Wantai) with HEV-RNA real-time RT-PCR. HAV, HEV, HBV, HCV, Epstein-Barr virus (EBV), cytomegalovirus, and herpes simplex virus accounted for AVH in 78.8% (n = 126), 7.5% (n = 12), 3.1% (n = 5), 1.9% (n = 3), 6.9% (n = 11), 1.2% (n = 2), and 0.6% (n = 1) of 160 patients (median age, 43 years; men, 52.5%; median ALT, 2144 IU/L), respectively. Hospitalization, hemodialysis, and intensive care unit admission were required in 137 (86.7%), 5 (3.2%), and 1 (0.6%) patient, respectively. Two patients developed acute liver failure (1.3%), albeit without mortality or liver transplantation. Ingestion of uncooked clams/oysters and wild boars' blood/bile was reported in 40.5% and 16.7% of patients with HAV and HEV, respectively. The concordance rate between the anti-HEV-IgM results of both ELISA kits was 50%. HEV RNA was detected in only 17% of patients with HEV. The diagnosis of HEV needs clinical consideration due to incomplete HEV diagnostics.

Assessment tools for differential diagnosis of neglect: Focusing on egocentric neglect and allocentric neglect.

BACKGROUND: There are very few studies on the differential diagnosis between egocentric neglect (EN) and allocentric neglect (AN). AIM: To investigate the overall trend of the previously developed assessment tools by conducting a descriptive review of the studies on assessment tools that can perform a differential diagnosis of EN and AN. METHODS: The data were collected by using databases such as Google Scholar, PubMed, and ScienceDirect. The most commonly used search terms were "neglect", "stroke", "egocentric neglect", and "allocentric neglect". RESULTS: A total of seven studies that met the inclusion criteria were selected and analyzed. We were able to confirm the research process, test method, and differential diagnosis criteria of the seven presented assessment tools from four studies on paper-based tests and three studies on computerized tests. The majority of the tests were carried out via the cancellation method using stimuli such as everyday objects or numbers. EN distinguished the left from right based on the test paper, while AN distinguished the left from right based on stimuli. In order to perform differential diagnosis, the difference in the number of left and right responses or non-responses was used based on the EN and AN criteria. CONCLUSION: It was confirmed that all the seven assessment tools can effectively perform differential diagnosis of EN and AN. This study may provide important data that can be used in clinical practice for differential diagnosis and future intervention planning for neglect patients.

Bombyx mori kynurenine 3-monooxygenase gene editing and insect molecular breeding using the clustered regularly interspaced short palindromic repeat/CRISPR associated protein 9 system.

Genome editing by clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR associated protein (Cas)9, a third-generation gene scissors, and molecular breeding at the genome level are attracting considerable attention as future breeding techniques. In the present study, genetic and phenotypic analyses were conducted to examine the molecular breeding of Bombyx mori through CRISPR/Cas9-mediated editing of the kynurenine 3-monooxygenase (KMO) gene. The synthesized guide RNAs (gRNAs) were analyzed using T7 endonuclease I after introduction into the BM-N silkworm cell line. To edit the silkworm gene, K1P gRNA, and Cas9 complexes were microinjected into silkworm embryos. After microinjection, the hatching rate and the incidence of mutation were determined as 18.1% and 60%, respectively. Gene mutation was verified in the heterozygous G0 generation, but no phenotypic change was observed; however, certain embryos and moths produced through sib-mating had significant differences compared to the wild-type. In successive generations, a distinct phenotypic change was also observed by continuous mating. Thus, although there are limitations in the phenotypic expression in breeding through the induction of deletion mutations, as in the present study, the process is believed to yield successful results within a shorter period compared to traditional breeding and is safer than transgenic technology.

Bacillus subtilis strain L1 promotes nitrate reductase activity in Arabidopsis and elicits enhanced growth performance in Arabidopsis, lettuce, and wheat.

Plant growth promoting rhizobacteria (PGPR) are a group of bacteria that promote plants growth in the rhizosphere. PGPRs are involved in various mechanisms that reinforce plant development. In this study, we screened for PGPRs that were effective in early growth of Arabidopsis thaliana when added to the media and one Bacillus subtilis strain L1 (Bs L1) was selected for further study. When Bs L1 was placed near the roots, seedlings showed notably stronger growth than that in the control, particularly in biomass and root hair. Quantitative reverse transcription polymerase chain reaction analysis revealed a high level of expression of the high affinity nitrate transporter gene, NRT2.1 in A. thaliana treated with Bs L1. After considering how Bs L1 could promote plant growth, we focused on nitrate, which is essential to plant growth. The nitrate content was lower in A. thaliana treated with Bs L1. However, examination of the activity of nitrate reductase revealed higher activity in plants treated with PGPR than in the control. Bs L1 had pronounced effects in representative crops (wheat and lettuce). These results suggest that Bs L1 promotes the assimilation and use of nitrate and plant growth.

Risk Factors of Multiple Gastric Polyps according to the Histologic Classification: Prospective Observational Cohort Study.

BACKGROUND/AIMS: The aim of this study was to determine the risk factors of multiple gastric polyps according to the histological classification of gastric polyps. METHODS: Subjects with multiple gastric polyps (at least three) during endoscopy were enrolled prospectively. They were assigned to a fundic gland polyp (FGP) group and hyperplastic polyp (HP) group based on a histological classification of gastric polyps. Helicobacter pylori (H. pylori) was confirmed by its histology. Serum gastrin was measured using the radioimmunoassay method. A questionnaire was taken regarding the intake of proton pump inhibitor and nonsteroidal anti-inflammatory drugs, alcohol, smoking history, and diet. RESULTS: Among the 60 subjects enrolled from 2015 to 2018 at Seoul National University Bungdang Hospital, 47 and 13 subjects were assigned to the FGP and HP groups, respectively. The H. pylori infection rate was 12.8% in the FGP group, which is lower than that in the HP group (69.2%, p<0.001). The gastrin level was higher in the HP group (194.7 pg/dL, range 50.6-387.8 pg/dL) than in the FGP group (57.4 pg/dL, range 24.8-79.0 pg/dL) (p=0.007). Histologically, neutrophil infiltration in the antrum and body of the stomach were higher in the HP group than in the FGP group (p=0.022 and p=0.030, respectively). In contrast, monocyte infiltration in the antrum and body of the stomach were higher in the FGP group than in the HP group (p=0.018 and p<0.001, respectively). CONCLUSIONS: HPs arise from inflammation caused by H. pylori. On the other hand, the FGP was not associated with H. pylori or environmental factors.

Deficiency of AtGFAT1 activity impairs growth, pollen germination and tolerance to tunicamycin in Arabidopsis.

The hexosamine biosynthetic pathway (HBP) plays essential roles in growth and development in plants. However, insight into the biological function of glutamine:fructose-6-phosphate amidotransferase 1 (GFAT1), mediating the first regulatory step of the HBP, remains unclear in plants. Here, we report the molecular characterization of Arabidopsis AtGFAT1 gene. AtGFAT1 was highly expressed in mature pollen grains, but its expression was not detectable in the rest of the organs. Pollen grains bearing the gfat1-2 knockout allele displayed defects in a polar deposition of pectin and callose in the pollen cell wall, leading to no genetic transmission of the gfat1-2 allele through the male gametophyte. AtGFAT1 overexpression increased glucosamine (GlcN) content and enhanced resistance to tunicamycin (Tm) treatment, while RNAi-mediated suppression reduced GlcN content and resistance to Tm treatment. However, the decrease in Tm resistance by RNAi suppression of AtGFAT1 was recovered by a GlcN supplement. The exogenous GlcN supplement also rescued gfat1-2/gaft1-2 mutant plants, which were otherwise not viable. The gfat1-2/gfat1-2 plants stopped growing at the germination stage on GlcN-free medium, but GlcN supplement allowed wild-type growth of gfat1-2/gfat1-2 plants. In addition, reactive oxygen species production, cell death and a decrease in protein N-glycosylation were observed in gfat1-2/gaft1-2 mutant plants grown on GlcN-free medium, whereas these aberrant defects were not detectable on GlcN-sufficient medium. Taken together, these results show that the reduction of protein N-glycosylation was at least partially responsible for many aberrant phenotypes in growth and development as well as the response to Tm treatment caused by AtGFAT1 deficiency in Arabidopsis.

Enhanced anthocyanin accumulation confers increased growth performance in plants under low nitrate and high salt stress conditions owing to active modulation of nitrate metabolism.

Plants require nitrogen (N) for growth and development. However, they are frequently exposed to conditions of nitrogen deficiency. In addition, anthocyanin accumulation is induced under salt stress and nitrate deficiency. To date, most studies have revealed that nitrate deficiency under high sucrose levels induce high levels of anthocyanin accumulation in plants. However, the underlying mechanisms remain unclear. Under nitrate-starved conditions, plant growth rapidly worsens and cells eventually die. In addition, plants are severely affected by salt exposure. Therefore, in this study, we determined whether increased levels of anthocyanin could improve plant growth under salt stress and nitrate-starved conditions. We used PAP1-D/fls1ko and ttg1 plants which have a perturbed anthocyanin biosynthesis pathway to explore the role of anthocyanin in plant adaptation to nitrate-deficient conditions and salt stress. Our results demonstrate that high anthocyanin accumulation in PAP1-D/fls1ko plants confers enhanced tolerance to nitrate-deficient conditions combined with high salinity. PAP1-D/fls1ko plants appeared to use absorbed nitrate efficiently during the nitrate reduction process. In addition, nitrate-related genes such as NRT1.1, NiA1 and NiA2 were upregulated in the PAP1-D/fls1ko plants. On the basis of these findings, it can be concluded that high anthocyanin accumulation helps plants to cope with salt stress under nitrate-deficient conditions via the effective utilization of nitrate metabolism.

Dual role of SND1 facilitates efficient communication between abiotic stress signalling and normal growth in Arabidopsis.

Certain plant cells synthesize secondary cell walls besides primary cell walls. This biosynthesis is strictly controlled by an array of transcription factors. Here, we show that SND1, a regulator of cell-wall biosynthesis, regulates abscisic acid (ABA) biosynthesis to ensure optimal plant growth. In Arabidopsis, the lack of SND1 and its homolog NST1 leads to the deficiency of secondary cell walls, preventing snd1nst1 double mutant seedlings from growing upright. Compared to wild type seedlings, the snd1 knockout mutant seedlings accumulated less anthocyanin and exhibited low tolerance to salt stress. Compared to wild type seedlings, the snd1 knockout seedlings were more sensitive to salt stress. Although SND1 can bind to the promoter of Myb46, we observed that SND1 binds directly to the promoter of the ABI4 gene, thereby reducing ABA levels under normal growth conditions. Thus, plants adjust secondary cell wall thickening and growth via SND1. SND1 has a dual function: it activates the Myb46 pathway, fostering lignin biosynthesis to produce sufficient cell wall components for growth, while maintaining a low ABA concentration, as it inhibits growth. This dual function of SND1 may help plants modulate their growth efficiently.

Paenibacillus pabuli strain P7S promotes plant growth and induces anthocyanin accumulation in Arabidopsis thaliana.

In this study, a novel plant growth-promoting rhizobacteria (PGPR), the bacterial strain Paenibacillus pabuli P7S (PP7S), showed promising plant growth-promoting effects. Furthermore, it induced anthocyanin accumulation in Arabidopsis. When co-cultivated with PP7S, there was a significant increase in anthocyanin content and biomass of Arabidopsis seedlings compared with those of the control. The quantitative reverse transcription-polymerase chain reaction analysis revealed higher expression of many key genes regulating anthocyanin and flavonoid biosynthesis pathways in PP7S-treated seedlings when compared with that of the control. Furthermore, higher expression of pathogen-related genes and microbe-associated molecular pattern genes was also observed in response to PP7S, indicating that the PGPR triggered the induced systemic response (ISR) in A. thaliana. These results suggest that PP7S promotes plant growth in A. thaliana and increases anthocyanin biosynthesis by triggering specific ISRs in plant.

AtMyb56 Regulates Anthocyanin Levels via the Modulation of AtGPT2 Expression in Response to Sucrose in Arabidopsis.

Sucrose is a crucial compound for the growth and development of plants, and the regulation of multiple genes depends on the amount of soluble sugars present. Sucrose acts as a signaling molecule that regulates a proton-sucrose symporter, with its sensor being the sucrose transporter. Flavonoid and anthocyanin biosynthesis are regulated by sucrose, and sucrose signaling can affect flavonoid and anthocyanin accumulation. In the present study, we found a Myb transcription factor affecting accumulation of anthocyanin. AtMyb56 showed an increase in its expression in response to sucrose treatment. Under normal conditions, anthocyanin accumulation was similar between Col-0 (wild type) and atmyb56 mutant seedlings; however, under sucrose treatment, the level of anthocyanin accumulation was lower in the atmyb56 mutant plants than in Col-0 plants. Preliminary microarray analysis led to the investigation of the expression of one candidate gene, AtGPT2, in the atmyb56 mutant. The phosphate translocator, which is a plastidial phosphate antiporter family, catalyzes the import of glucose-6-phosphate (G-6-P) into the chloroplast. AtGPT2 gene expression was altered in atmyb56 seedlings in a sucrose-dependent manner in response to circadian cycle. Furthermore, the lack of AtMyb56 resulted in altered accumulation of maltose in a sucrose-dependent manner. Therefore, the sucrose responsive AtMyb56 regulates AtGPT2 gene expression in a sucrose-dependent manner to modulate maltose and anthocyanin accumulations in response to the circadian cycle.

Evaluation of a Rapid Method for Screening Heat Stress Tolerance Using Three Korean Wheat (Triticum aestivum L.) Cultivars.

Thermotolerance in plants is a topic of concern given the current trends in global warming. Here, we aimed to develop a rapid and reproducible screening method for selection of heat stress-tolerant wheat varieties to expedite the breeding process. We tested the robustness of the screen in three Korean wheat cultivars, "BackJung", "KeumKang", and "ChoKyeong". We showed that 4-day-old seedlings of "KeumKang" had the highest survival rates after a 45 °C treatment for 20 h. Moreover, the ability to retain chlorophyll and antioxidant activity was also highest in "KeumKang". The increase in malondialdehyde content in "ChoKyeong" indicated that this cultivar showed the greatest damage after heat stress. Collectively, our results showed that "KeumKang" is the most heat-tolerant cultivar of the three examined. In conclusion, the most reliable and rapid screening method in our investigation was survival rate examined at lethal temperature.

High accumulation of anthocyanins via the ectopic expression of AtDFR confers significant salt stress tolerance in Brassica napus L.

KEY MESSAGE: The ectopic expression of AtDFR results in increased accumulation of anthocyanins leading to enhanced salinity and drought stress tolerance in B. napus plants. Flavonoids with antioxidant effects confer many additional benefits to plants. Evidence indicates that flavonoids, including anthocyanins, protect tissues against oxidative stress from various abiotic stressors. We determined whether increases in anthocyanins increased abiotic stress tolerance in Brassica napus, because the values of B. napus L. and its cultivation area are increasing worldwide. We overexpressed Arabidopsis dihydroflavonol-4-reductase (DFR) in B. napus. Increased DFR transcript levels for AtDFR-OX B. shoots correlated with higher anthocyanin accumulation. AtDFR-OX Brassica shoots exhibited lower reactive oxygen species (ROS) accumulation than wild-type (WT) shoots under high NaCl and mannitol concentrations. This was corroborated by 3,3-diaminobenzidine staining for ROS scavenging activity in 1,1-diphenyl-2-picryl-hydrazyl assays. Shoots of the AtDFR-OX B. napus lines grown in a high salt medium exhibited enhanced salt tolerance and higher chlorophyll content than similarly grown WT plants. Our observations suggested that the AtDFR gene can be effectively manipulated to modulate salinity and drought stress tolerance by directing to high accumulation of anthocyanins in oilseed plants.

Systematic deletion of the ER lectin chaperone genes reveals their roles in vegetative growth and male gametophyte development in Arabidopsis.

Calnexin (CNX) and calreticulin (CRT) are homologous lectin chaperones in the endoplasmic reticulum (ER) that facilitate glycoprotein folding and retain folding intermediates to prevent their transit via the secretary pathway. The Arabidopsis genome has two CNX (CNX1 and CNX2) and three CRT (CRT1, CRT2 and CRT3) homologs. Despite growing evidence of the biological roles of CNXs and CRTs, little is understood about their function in Arabidopsis growth and development under normal conditions. Here, we report that the deletion of CNX1, but not of CNX2, in the crt1 crt2 crt3 triple mutation background had an adverse effect on pollen viability and pollen tube growth, leading to a significant reduction in fertility. The cnx1 crt1 crt2 crt3 quadruple mutation also conferred severe defects in growth and development, including a shortened primary root, increased root hair length and density, and reduced plant height. Disruption of all five members of the CNX/CRT family was revealed to be lethal. Finally, the abnormal phenotype of the cnx1 crt1 crt2 crt3 quadruple mutants was completely rescued by either the CNX1 or CNX2 cDNA under the control of the CNX1 promoter, suggesting functional redundancy between CNX1 and CNX2. Taken together, these results provide genetic evidence that CNX and CRT play essential and overlapping roles during vegetative growth and male gametophyte development in Arabidopsis.

Drastic anthocyanin increase in response to PAP1 overexpression in fls1 knockout mutant confers enhanced osmotic stress tolerance in Arabidopsis thaliana.

KEY MESSAGE : pap1 - D/fls1ko double mutant plants that produce substantial amounts of anthocyanin show tolerance to abiotic stress. Anthocyanins are flavonoids that are abundant in various plants and have beneficial effects on both plants and humans. Many genes in flavonoid biosynthetic pathways have been identified, including those in the MYB-bHLH-WD40 (MBW) complex. The MYB gene Production of Anthocyanin Pigment 1 (PAP1) plays a particularly important role in anthocyanin accumulation. PAP1 expression in many plant systems strongly increases anthocyanin levels, resulting in a dark purple color in many plant organs. In this study, we generated double mutant plants that harbor fls1ko in the pap1-D background (i.e., pap1-D/fls1ko plants), to examine whether anthocyanins can be further enhanced by blocking flavonol biosynthesis under PAP1 overexpression. We also wanted to examine whether the increased anthocyanin levels contribute to defense against osmotic stresses. The pap1-D/fls1ko mutants accumulated higher anthocyanin levels than pap1-D plants in both control and sucrose-treated conditions. However, flavonoid biosynthesis genes were slightly down-regulated in the pap1-D/fls1ko seedlings as compared to their expression in pap1-D seedlings. We also report the performance of pap1-D/fls1ko seedlings in response to plant osmotic stresses.

Characterization of Arabidopsis thaliana FLAVONOL SYNTHASE 1 (FLS1) -overexpression plants in response to abiotic stress.

Flavonoids are an important group of secondary metabolites that are involved in plant growth and contribute to human health. Many studies have focused on the biosynthesis pathway, biochemical characters, and biological functions of flavonoids. In this report, we showed that overexpression of FLS1 (FLS1-OX) not only altered seed coat color (resulting in a light brown color), but also affected flavonoid accumulation. Whereas fls1-3 mutants accumulated higher anthocyanin levels, FLS1-OX seedlings had lower levels than those of the wild-type. Besides, shoot tissues of FLS1-OX plants exhibited lower flavonol levels than those of the wild-type. However, growth performance and abiotic stress tolerance of FLS1-OX, fls1-3, and wild-type plants were not significantly different. Taken together, FLS1 can be manipulated (i.e., silenced or overexpressed) to redirect the flavonoid biosynthetic pathway toward anthocyanin production without negative effects on plant growth and development.

MYBD employed by HY5 increases anthocyanin accumulation via repression of MYBL2 in Arabidopsis.

Photomorphogenesis is an essential program in plant development. This process is effected by the balanced cooperation of many factors under light and dark conditions. In a previous study, we showed that MYB hypocotyl elongation-related (MYBH) is involved in cell elongation. To expand our understanding of MYBH function, we performed a yeast two-hybrid assay and identified an MYB-like Domain transcription factor (MYBD). In this study, we investigated the function of MYBD, which is an MYBH homolog involved in anthocyanin accumulation. MYBD expression increased in response to light or cytokinin, and MYBD enhanced anthocyanin biosynthesis via repression of MYBL2, which encodes a transcription factor that has a negative effect on this process. In addition, MYBD binding in vivo to the MYBL2 promoter and the lower level of histone H3K9 acetylation at the upstream region of MYBL2 in MYBD over-expressing plants in comparison with wild-type plants imply that MYBD represses MYBL2 expression via an epigenetic mechanism. HY5 directly binds to the MYBD promoter, which indicates that MYBD acts on HY5-downstream in light- or cytokinin-triggered signaling pathways, leading to anthocyanin accumulation. Our results suggest that, although MYBD and MYBH are homologs, they act in opposite ways during plant photomorphogenesis, and these functions should be examined in further studies.

Characterization of Brassica napus Flavonol Synthase Involved in Flavonol Biosynthesis in Brassica napus L.

Recently, Brassica napus has become a very important crop for plant oil production. Flavonols, an uncolored flavonoid subclass, have a high antioxidative effect and are known to have antiproliferative, antiangiogenic, and neuropharmacological properties. In B. napus, some flavonoid structural genes have been identified, such as, BnF3H-1, BnCHS, and BnC4H-1. However, no studies on FLS genes in B. napus have been conducted. Thus, in this study, we cloned and characterized the function of BnFLS gene B. napus. By overexpression of the BnFLS gene, flavonol (kaempferol and quercetin) levels were recovered in the Arabidopsis atfls1-ko mutant. In addition, we found that the higher endogenous flavonol levels of BnFLS-ox in vitro shoots correlated with slightly higher ROS scavenging activities. Thus, our results indicate that the BnFLS gene encodes for a BnFLS enzyme that can be manipulated to specifically increase flavonol accumulation in oilseed plants and other species such as Arabidopsis.

AtMyb7, a subgroup 4 R2R3 Myb, negatively regulates ABA-induced inhibition of seed germination by blocking the expression of the bZIP transcription factor ABI5.

Various Myb proteins have been shown to play crucial roles in plants, including primary and secondary metabolism, determination of cell fate and identity, regulation of development and involvement in responses to biotic and abiotic stresses. The 126 R2R3 Myb proteins (with two Myb repeats) have been found in Arabidopsis; however, the functions of most of these proteins remain to be fully elucidated. In the present study, we characterized the function of AtMyb7 using molecular biological and genetic analyses. We used qRT-PCR to determine the levels of stress-response gene transcripts in wild-type and atmyb7 plants. We showed that Arabidopsis AtMyb7 plays a critical role in seed germination. Under abscisic acid (ABA) and high-salt stress conditions, atmyb7 plants showed a lower germination rate than did wild-type plants. Furthermore, AtMyb7 promoter:GUS seeds exhibited different expression patterns in response to variations in the seed imbibition period. AtMyb7 negatively controls the expression of the gene encoding bZIP transcription factor, ABI5, which is a key transcription factor in ABA signalling and serves as a crucial regulator of germination inhibition in Arabidopsis.