Engineering TALE-linked deaminases to facilitate precision adenine base editing in mitochondrial DNA.

DddA-derived cytosine base editors (DdCBEs) and transcription activator-like effector (TALE)-linked deaminases (TALEDs) catalyze targeted base editing of mitochondrial DNA (mtDNA) in eukaryotic cells, a method useful for modeling of mitochondrial genetic disorders and developing novel therapeutic modalities. Here, we report that A-to-G-editing TALEDs but not C-to-T-editing DdCBEs induce tens of thousands of transcriptome-wide off-target edits in human cells. To avoid these unwanted RNA edits, we engineered the substrate-binding site in TadA8e, the deoxy-adenine deaminase in TALEDs, and created TALED variants with fine-tuned deaminase activity. Our engineered TALED variants not only reduced RNA off-target edits by >99% but also minimized off-target mtDNA mutations and bystander edits at a target site. Unlike wild-type versions, our TALED variants were not cytotoxic and did not cause developmental arrest of mouse embryos. As a result, we obtained mice with pathogenic mtDNA mutations, associated with Leigh syndrome, which showed reduced heart rates.

Evaluation of the Weeds around Capsicum annuum (CA) Cultivation Fields as Potential Habitats of CA-Infecting Viruses.

Capsicum annuum (CA) is grown outdoors across fields in Jeollabuk-do, South Korea. The weeds surrounding these fields were investigated regarding the infection of 11 viruses infecting CA during the year 2014-2018. In the reverse transcription polymerase chain reaction diagnosis, 546 out of 821 CA samples (66.5%) were infected by nine viruses, and 190 out of 918 weed samples (20.7%) were infected by eight viruses. Correlation analysis of the mutual influence of the viruses infecting CA and weeds during these 5 years showed that five viruses had significant positive correlations with the infection in both CA and weeds. Over the study period, the weeds infected by cucumber mosaic virus (CMV) in the previous year were positively correlated with the incidence of CMV infection in CA in the current year, although the correlation was lower for tomato spotted wilt virus (TSWV) compared to CMV. The CMV infection percent was 14.0% in summer annuals, 11.4% in perennials, and 7.8% in winter annuals. However, considering the overwintering period without CA, the infection percent was 5.2% higher in winter annuals and perennials than that in summer annuals, indicating that winter annual and perennial weeds served as the main habitats for insect vectors. The TSWV infection percent in weeds was 10.4% in summer annuals, 6.4% in winter annuals, and 6.2% in perennials. The weeds surrounding CA fields, acting as the intermediate hosts, were found to be the potent sources of infection, influencing the spread and diversity of CA-infecting viruses. The results of this study can contribute to prevent viral infection in agricultural fields.

First Report of Passiflora Latent Virus Infecting Passion Fruit (Passiflora edulis) in South Korea.

Passion fruit (Passiflora edulis) viral diseases caused by papaya leaf curl Guangdong virus, cucumber mosaic virus, East Asian Passiflora virus, and euphorbia leaf curl virus have been reported in South Korea (Joa et al. 2018; Kim et al. 2018). In June 2021, virus-like symptoms, e.g., mosaic pattern, curling, chlorosis, and deformation, were observed on leaves and fruits of greenhouse-grown P. edulis in Iksan, South Korea, with disease incidence greater than 2% (300 plants: 8 symptomatic plants and 292 asymptomatic plants). Total RNA was extracted from a pooled sample of symptomatic leaves of an individual P. edulis plant using the RNeasy Plant Mini Kit (Qiagen, Germany), and a transcriptome library was generated using the TruSeq Stranded Total RNA LT Sample Prep Kit (Illumina, San Diego, CA). Next-Generation Sequencing (NGS) was performed using the Illumina NovaSeq 6000 system (Macrogen Inc., Korea). De novo assembly of the resulting 121,154,740 reads was performed using Trinity (Grabherr et al. 2011). A total of 70,895 contigs was assembled (>200 bp) and annotated against the NCBI viral genome database using BLASTn (ver. 2.12.0). One 827-nt contig was annotated as milk vetch dwarf virus (MVDV), a member of the genus Nanovirus in the family Nanoviridae (Bangladesh isolate, acc. no. LC094159, 96.0% nucleotide identity), and the other 3,639-nt contig corresponded to Passiflora latent virus (PLV), a member of the genus Carlavirus in the family Betaflexiviridae (Israel isolate, acc. no. DQ455582, 90.0% nucleotide identity). For further confirmation, total RNA was isolated from symptomatic leaves of the same P. edulis used for NGS analysis using a viral gene spin DNA/RNA extraction kit (iNtRON Biotechnology, Seongnam, Korea), and reverse transcription polymerase chain reaction (RT-PCR) was performed using specific primers: PLV-F/R (5'-GTGCCCACCGAACATGTTACCTC-3'/5'-CCATGCACTTGGAATGCTTACCC-3') targeting the coat protein region of PLV, MVDV-M-F/R (5'-CTAGTCAGCCATCCAATGGTG-3'/5'-GTGCAGGGTTTGATTGTCTGC-3') targeting the movement protein region, and MVDV-S-F/R (5'-GGATTTTAATACGCGTGGACGATC-3'/5'-AACGGCTATAAGTCACTCCGTAC-3') targeting the coat protein region of MVDV. An expected PCR product of 518 bp corresponding to PLV was amplified, while MVDV was not detected. The amplicon was directly sequenced, and its nucleotide sequence was deposited in GenBank (acc. no. OK274270). A BLASTn analysis showed that the nucleotide sequence of the PCR product shared 93.0% and 96.2% identity with PLV isolates from Israel (MH379331) and Germany (MT723990), respectively. In addition, six passion fruit leaves and two fruit samples with PLV-like symptoms were collected from a total of eight plants grown in the greenhouse in Iksan for RT-PCR analysis, and six samples tested positive for PLV. However, PLV was not detected in one leaf and one fruit among all samples. Mechanical sap inoculation was conducted using extracts of systemic leaves as inoculum on P. edulis and the indicator plants Chenopodium quinoa, Nicotiana benthamiana, N. glutinosa, and N. tabacum. In P. edulis, vein chlorosis and yellowing on systemic leaves were observed 20 days post inoculation (dpi). Necrotic local lesions were observed on inoculated leaves of N. benthamiana and N. glutinosa 15 dpi, and PLV infection was confirmed by RT-PCR assay in symptomatic leaf tissue. This study aimed to determine whether commercially grown passion fruit in the southern part of South Korea could be infected with and potentially spread PLV. Whereas PLV was asymptomatic in persimmon (Diospyros kaki) in South Korea, no pathogenicity testing in passion fruit was reported (Cho et al. 2021). Here, we have shown the natural infection of passion fruit with PLV in South Korea for the first time and associated infection with obvious symptoms. This suggests a need to evaluate potential losses in passion fruit and the selection of healthy propagation material.

Effects of Short-Term Intake of Montmorency Tart Cherry Juice on Sleep Quality after Intermittent Exercise in Elite Female Field Hockey Players: A Randomized Controlled Trial.

The purpose of this study was to investigate the effect of short-term consumption of tart cherry juice on levels of cortisol and melatonin and sleep quality after intermittent exhaustion exercise in female elite field hockey players. A total of 19 field hockey players participated in the present study for 5 days. The individuals were divided into the placebo group (PLA, n = 9) and the tart cherry juice group (TCJ, n = 10), respectively. Actigraphy devices were distributed to analyze sleep quality and participants were required to wear the device while sleeping until the study was completed. Participants consumed tart cherry juice or placebo drinks five times in a total of 48 h while double-blinded after intermittent exhaustion exercise. A significant interaction effect (group × time) between PLA and TCJ groups was not observed in the levels of melatonin and cortisol. The variables of sleep quality showed significant interaction effects with regards to the total time in bed (TTB; p = 0.015), wake after sleep onset (WASO; p = 0.044), and movement index (MI; p = 0.031) variables. As a result, our study confirmed the possibility that the short-term intake of tart cherry juice could not change the levels of melatonin and cortisol in elite female hockey players but could help improve their sleep quality.

The Functional Association of ACQOS/VICTR with Salt Stress Resistance in Arabidopsis thaliana Was Confirmed by CRISPR-Mediated Mutagenesis.

Clustered regularly interspaced palindromic repeat (CRISPR)-mediated mutagenesis has become an important tool in plant research, enabling the characterization of genes via gene knock-out. CRISPR genome editing tools can be applied to generate multi-gene knockout lines. Typically, multiple single-stranded, single guide RNAs (gRNAs) must be expressed in an organism to target multiple genes simultaneously; however, a single gRNA can target multiple genes if the target genes share similar sequences. A gene cluster comprising ACQUIRED OSMOTOLERANCE (ACQOS; AT5G46520) and neighboring nucleotide-binding leucine-rich repeats (NLRs; AT5G46510) is associated with osmotic tolerance. To investigate the role of ACQOS and the tandemly arranged NLR in osmotic tolerance, we introduced small insertion/deletion mutations into two target genes using a single gRNA and obtained transformant plant lines with three different combinations of mutant alleles. We then tested our mutant lines for osmotic tolerance after a salt-stress acclimation period by determining the chlorophyll contents of the mutant seedlings. Our results strongly suggest that ACQOS is directly associated with salt resistance, while the neighboring NLR is not. Here, we confirmed previous findings suggesting the involvement of ACQOS in salt tolerance and demonstrated the usefulness of CRISPR-mediated mutagenesis in validating the functions of genes in a single genetic background.

The efficacy of CRISPR-mediated cytosine base editing with the RPS5a promoter in Arabidopsis thaliana.

CRISPR/Cas9-mediated genome editing is an important and versatile technology in modern biological research. Recent advancements include base-editing CRISPR tools that enable targeted nucleotide substitutions using a fusion protein comprising a nickase variant of Cas9 and a base deaminase. Improvements in base editing efficiencies and inheritable of edited loci need to be made to make CRISPR a viable system in plants. Here, we report efficiency of cytosine base editors (CBEs) in Arabidopsis thaliana by applying the strong endogenous RPS5a promoter to drive the expression of nickase Cas9 and either rAPOBEC1 from rat (BE3) or the PmCDA1 activation-induced cytidine deaminase from sea lamprey (AIDv2). Compared with the strong heterologous CaMV35S promoter of viral origin, the RPS5a promoter improved CBE efficiency by 32% points with the number of T1 plants showing over 50% conversion ratio when the LFY gene was targeted. CBE induced nonsense mutations in LFY via C-to-T conversion, which resulted in loss-of-function lfy phenotypes; defects in LFY function were associated with the targeted base substitutions. Our data suggest that optimal promoter choice for CBE expression may affect base-editing efficiencies in plants. The results provide a strategy to optimize low-efficiency base editors and demonstrate their applicability for functional assays and trait development in crop research.

Cardioprotective Potential of an SGLT2 Inhibitor Against Doxorubicin-Induced Heart Failure.

BACKGROUND AND OBJECTIVES: Recent studies have shown that sodium-glucose co-transporter 2 (SGLT2) inhibitors reduce the risk of heart failure (HF)-associated hospitalization and mortality in patients with diabetes. However, it is not clear whether SGLT2 inhibitors have a cardiovascular benefit in patients without diabetes. We aimed to determine whether empagliflozin (EMPA), an SGLT2 inhibitor, has a protective role in HF without diabetes. METHODS: Cardiomyopathy was induced in C57BL/6J mice using intraperitoneal injection of doxorubicin (Dox). Mice with HF were fed a normal chow diet (NCD) or an NCD containing 0.03% EMPA. Then we analyzed their phenotypes and performed in vitro experiments to reveal underlying mechanisms of the EMPA's effects. RESULTS: Mice fed NCD with EMPA showed improved heart function and reduced fibrosis. In vitro studies showed similar results. Phloridzin, a non-specific SGLT inhibitor, did not show any protective effect against Dox toxicity in H9C2 cells. SGLT2 inhibitor can cause increase in blood ketone levels. Beta hydroxybutyrate (ßOHB), which is well known ketone body associated with SGLT2 inhibitor, showed a protective effect against Dox in H9C2 cells and in Dox-treated mice. These results suggest elevating ßOHB might be a convincing mechanism for the protective effects of SGLT2 inhibitor. CONCLUSIONS: SGLT2 inhibitors have a protective effect in Dox-induced HF in mice. This implied that SGLT2 inhibitor therapy could be a good treatment strategy even in HF patients without diabetes.

Author Correction: Precision genome engineering through adenine base editing in plants.

In Supplementary Fig. 1b originally published with this Brief Communication, the DNA sequence of nickase Cas9 was incorrect; this has now been amended.

Precision genome engineering through adenine base editing in plants.

The recent development of adenine base editors (ABEs) has enabled efficient and precise A-to-G base conversions in higher eukaryotic cells. Here, we show that plant-compatible ABE systems can be successfully applied to protoplasts of Arabidopsis thaliana and Brassica napus through transient transfection, and to individual plants through Agrobacterium-mediated transformation to obtain organisms with desired phenotypes. Targeted, precise A-to-G substitutions generated a single amino acid change in the FT protein or mis-splicing of the PDS3 RNA transcript, and we could thereby obtain transgenic plants with late-flowering and albino phenotypes, respectively. Our results provide 'proof of concept' for in planta ABE applications that can lead to induced neo-functionalization or altered mRNA splicing, opening up new avenues for plant genome engineering and biotechnology.

Eucommia ulmoides leaf (EUL) extract enhances NO production in ox-LDL-treated human endothelial cells.

Eucommia ulmoides leaves (EULs), referred to as Du-zhong, are utilized to lower blood pressure and improve liver and kidney tone, and also have been applied to cardiovascular disease in Korea, China, and Japan. Endothelial dysfunction, which is caused by endothelial nitric oxide synthase (eNOS) uncoupling, is an initial step in atherosclerosis. In this study, we investigated the protective effects of EUL aqueous extract against ox-LDL-induced eNOS uncoupling and its possible mechanisms in human umbilical vein endothelial cells (HUVECs). A EUL component, aucubin, was also applied to ox-LDL-exposed HUVECs. Whereas ox-LDL significantly decreased nitric oxide (NO) levels in HUVECs, EUL extract and aucubin led to significant recovery of NO levels. When treated with ox-LDL in the presence of EUL extracts or aucubin, O2- production was markedly reduced in HUVECs compared to treatment with ox-LDL alone. EUL extract and aucubin also led to recovery of phospho-eNOS Thr495 expression, a critical signaling component in eNOS uncoupling, suggesting that EUL has regulatory effects against eNOS uncoupling and might play preventive/regulatory roles against vascular endothelial dysfunction.

Protective role of quercetin against manganese-induced injury in the liver, kidney, and lung; and hematological parameters in acute and subchronic rat models.

Manganese (Mn) is an important mineral element required in trace amounts for development of the human body, while over- or chronic-exposure can cause serious organ toxicity. The current study was designed to evaluate the protective role of quercetin (Qct) against Mn-induced toxicity in the liver, kidney, lung, and hematological parameters in acute and subchronic rat models. Male Sprague Dawley rats were divided into control, Mn (100 mg/kg for acute model and 15 mg/kg for subchronic model), and Mn + Qct (25 and 50 mg/kg) groups in both acute and subchronic models. Our result revealed that Mn + Qct groups effectively reduced Mn-induced ALT, AST, and creatinine levels. However, Mn + Qct groups had effectively reversed Mn-induced alteration of complete blood count, including red blood cells, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelets, and white blood cells. Meanwhile, the Mn + Qct groups had significantly decreased neutrophil and eosinophil and increased lymphocyte levels relative to the Mn group. Additionally, Mn + Qct groups showed a beneficial effect against Mn-induced macrophages and neutrophils. Our result demonstrated that Mn + Qct groups exhibited protective effects on Mn-induced alteration of GRP78, CHOP, and caspase-3 activities. Furthermore, histopathological observation showed that Mn + Qct groups effectively counteracted Mn-induced morphological change in the liver, kidney, and lung. Moreover, immunohistochemically Mn + Qct groups had significantly attenuated Mn-induced 8-oxo-2'-deoxyguanosine immunoreactivity. Our study suggests that Qct could be a substantially promising organ-protective agent against toxic Mn effects and perhaps against other toxic metal chemicals or drugs.

The influence of physical activity on risk of cardiovascular disease in people who are obese but metabolically healthy.

The metabolic outcomes of metabolically healthy obesity (MHO) remain controversial. The aim of the present study was to determine the effect of physical activity on the cardiovascular disease (CVD) outcomes of MHO. The study included participants who were followed for 10 years and recruited from the Korean Genome and Epidemiology Study (KoGES), a population-based cohort study. Participants with previously recorded CVDs or cancer, or who had received steroids or anticoagulants at baseline were excluded. A total of 8144 participants (3,942 men and 4,202 women) fulfilled inclusion criteria. In a multivariate Cox regression model adjusted for age and sex, MHO participants were not at elevated risk of CVD compared with their metabolically healthy non-obese (MHNO) counterparts (HR, 1.28; 95% CI, 0.96-1.71), although both the non-obese (HR, 1.50; 95% CI, 1.19-1.90) and obese (HR, 1.85; 95% CI, 1.48-2.30) participants with metabolic abnormalities were at elevated risk. However, in the subgroup analysis by physical activity, physically inactive MHO participants had a significantly higher HR for CVD events compared to active MHNO participants (HR, 1.54; 95% CI, 1.03-2.30), while active MHO participants were not at elevated risk (HR, 1.15; 95% CI, 0.70-1.89). Physically inactive MHO participants had significantly increased risk of CVD compared to physically active MHNO participants whereas physically active MHO participants did not.

Curcumin and Curcuma longa L. extract ameliorate lipid accumulation through the regulation of the endoplasmic reticulum redox and ER stress.

For this study, we examined the effects of curcumin against acute and chronic stress, paying specific attention to ROS. We also aimed to clarify the differences between acute and chronic stress conditions. We investigated the effects of curcumin against acute stress (once/1 day CCl4 treatment) and chronic-stress (every other day/4week CCl4 treatment). Compared with acute stress, in which the antioxidant system functioned properly and aspartate transaminase (AST) and ROS production increased, chronic stress increased AST, alanine aminotransferase (ALT), hepatic enzymes, and ROS more significantly, and the antioxidant system became impaired. We also found that ER-originated ROS accumulated in the chronic model, another difference between the two conditions. ER stress was induced consistently, and oxidative intra-ER protein folding status, representatively PDI, was impaired, especially in chronic stress. The PDI-associated client protein hepatic apoB accumulated with the PDI-binding status in chronic stress, and curcumin recovered the altered ER folding status, regulating ER stress and the resultant hepatic dyslipidemia. Throughout this study, curcumin and curcumin-rich Curcuma longa L. extract promoted recovery from CCl4-induced hepatic toxicity in both stress conditions. For both stress-associated hepatic dyslipidemia, curcumin and Curcuma longa L. extract might be recommendable to recover liver activity.

ß-Cell protection and antidiabetic activities of Crassocephalum crepidioides (Asteraceae) Benth. S. Moore extract against alloxan-induced oxidative stress via regulation of apoptosis and reactive oxygen species (ROS).

BACKGROUND: Medicinal plants are becoming more popular in the treatment of various diseases because of the adverse effects of the current therapy, especially antioxidant plant components such as phenols and flavonoids have a protective role against oxidative stress-induced degenerative diseases like diabetes. Thus, the purpose of this study was to investigate ß-cell protection and antidiabetic activities of Crassocephalum crepidioides (Asteraceae) Benth. S. Moore. METHOD: The in-vitro study was conducted by the pancreatic ß-cell culture and α-amylase inhibition technique which includes two methods, namely starch-iodine method and 3,5-dinitrosalicylic acid (DNSA) method. On the other hand, the in-vivo study was performed by oral glucose tolerance test (OGTT) method and alloxan-induced diabetes method by using Wistar albino rat. At the end pancreatic specimens were removed and processed for histopathological study. RESULT: The plant extract showed significant (*p < 0.05, **p < 0.01) effect on hyperglycemia as compared to standard (Gliclazide) in OGTT. The plant extract showed efficient protection activity of pancreatic ß-cell from cell death in INS-1 cell line by significantly reduced (*p < 0.05, **p < 0.01) the levels alloxan-induced apoptosis and intracellular reactive oxygen species (ROS) accumulation. In addition, the plant extract showed a significant (*p < 0.05, **p < 0.01) effect on hyperglycemia by increases in percent of ß-cells present in each islet (45% - 60%) compared to the diabetic group. CONCLUSION: The result showed that C. crepidioides had ß-cell protection and antidiabetic activities in pancreatic ß-cell culture and Wistar albino rat.

Polyphenolic Extract of Euphorbia supina Attenuates Manganese-Induced Neurotoxicity by Enhancing Antioxidant Activity through Regulation of ER Stress and ER Stress-Mediated Apoptosis.

Manganese (Mn) is an important trace element present in human body, which acts as an enzyme co-factor or activator in various metabolic reactions. While essential in trace amounts, excess levels of Mn in human brain can produce neurotoxicity, including idiopathic Parkinson's disease (PD)-like extrapyramidal manganism symptoms. This study aimed to investigate the protective role of polyphenolic extract of Euphorbia supina (PPEES) on Mn-induced neurotoxicity and the underlying mechanism in human neuroblastoma SKNMC cells and Sprague-Dawley (SD) male rat brain. PPEES possessed significant amount of total phenolic and flavonoid contents. PPEES also showed significant antioxidant activity in 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging and reducing power capacity (RPC) assays. Our results showed that Mn treatment significantly reduced cell viability and increased lactate dehydrogenase (LDH) level, which was attenuated by PPEES pretreatment at 100 and 200 µg/mL. Additionally, PPEES pretreatment markedly attenuated Mn-induced antioxidant status alteration by resolving the ROS, MDA and GSH levels and SOD and CAT activities. PPEES pretreatment also significantly attenuated Mn-induced mitochondrial membrane potential (ΔΨm) and apoptosis. Meanwhile, PPEES pretreatment significantly reversed the Mn-induced alteration in the GRP78, GADD34, XBP-1, CHOP, Bcl-2, Bax and caspase-3 activities. Furthermore, administration of PPEES (100 and 200 mg/kg) to Mn exposed rats showed improvement of histopathological alteration in comparison to Mn-treated rats. Moreover, administration of PPEES to Mn exposed rats showed significant reduction of 8-OHdG and Bax immunoreactivity. The results suggest that PPEES treatment reduces Mn-induced oxidative stress and neuronal cell loss in SKNMC cells and in the rat brain. Therefore, PPEES may be considered as potential treat-ment in Mn-intoxicated patients.

Protective effect of Curcuma longa L. extract on CCl4-induced acute hepatic stress.

BACKGROUND: The Curcuma longa L. (CLL) rhizome has long been used to treat patients with hepatic dysfunction. CLL is a member of the ginger family of spices that are widely used in China, India, and Japan, and is a common spice, coloring, flavoring, and traditional medicine. This study was performed to evaluate the hepatoprotective activity of CLL extract and its active component curcumin in an acute carbon tetrachloride (CCl4)-induced liver stress model. METHODS: Acute hepatic stress was induced by a single intraperitoneal injection of CCl4 (0.1 ml/kg body weight) in rats. CLL extract was administered once a day for 3 days at three dose levels (100, 200, and 300 mg/kg/day) and curcumin was administered once a day at the 200 mg/kg/day. We performed alanine transaminase (ALT) and aspartate transaminase (AST). activity analysis and also measured total lipid, triglyceride, and cholesterol levels, and lipid peroxidation. RESULTS: At 100 g CLL, the curcuminoid components curcumin (901.63 ± 5.37 mg/100 g), bis-demethoxycurcumin (108.28 ± 2.89 mg/100 g), and demethoxycurcumin (234.85 ± 1.85 mg/100 g) were quantified through high liquid chromatography analysis. In CCl4-treated rats, serum AST and ALT levels increased 2.1- and 1.2-fold compared with the control. AST but not ALT elevation induced by CCl4 was significantly alleviated in CLL- and curcumin-treated rats. Peroxidation of membrane lipids in the liver was significantly prevented by CLL (100, 200, and 300 mg/kg/day) on tissue lipid peroxidation assay and immunostaining with anti-4HNE antibody. We found that CLL extract and curcumin exhibited significant protection against liver injury by improving hepatic superoxide dismutase (p < 0.05) and glutathione peroxidase activity, and glutathione content in the CCl4-treated group (p < 0.05), leading to a reduced lipid peroxidase level. CONCLUSION: Our data suggested that CLL extract and curcumin protect the liver from acute CCl4-induced injury in a rodent model by suppressing hepatic oxidative stress. Therefore, CLL extract and curcumin are potential therapeutic antioxidant agents against acute hepatotoxicity.

Turmeric extract and its active compound, curcumin, protect against chronic CCl4-induced liver damage by enhancing antioxidation.

BACKGROUND: Curcumin, a major active component of turmeric, has previously been reported to alleviate liver damage. Here, we investigated the mechanism by which turmeric and curcumin protect the liver against carbon tetrachloride (CCl4)-induced injury in rats. We hypothesized that turmeric extract and curcumin protect the liver from CCl4-induced liver injury by reducing oxidative stress, inhibiting lipid peroxidation, and increasing glutathione peroxidase activation. METHODS: Chronic hepatic stress was induced by a single intraperitoneal injection of CCl4 (0.1 ml/kg body weight) into rats. Turmeric extracts and curcumin were administered once a day for 4 weeks at three dose levels (100, 200, and 300 mg/kg/day). We performed ALT and AST also measured of total lipid, triglyceride, cholesterol levels, and lipid peroxidation. RESULT: We found that turmeric extract and curcumin significantly protect against liver injury by decreasing the activities of serum aspartate aminotransferase and alanine aminotransferase and by improving the hepatic glutathione content, leading to a reduced level of lipid peroxidase. CONCLUSIONS: Our data suggest that turmeric extract and curcumin protect the liver from chronic CCl4-induced injury in rats by suppressing hepatic oxidative stress. Therefore, turmeric extract and curcumin are potential therapeutic antioxidant agents for the treatment of hepatic disease.

Green fluorescent protein as a scaffold for high efficiency production of functional bacteriotoxic proteins in Escherichia coli.

The availability of simple, robust, and cost-effective methods for the large-scale production of bacteriotoxic peptides such as antimicrobial peptides (AMPs) is essential for basic and pharmaceutical research. However, the production of bacteriotoxic proteins has been difficult due to a high degree of toxicity in bacteria and proteolytic degradation. In this study, we inserted AMPs into the Green fluorescent protein (GFP) in a loop region and expressed them as insoluble proteins in high yield, circumventing the inherent toxicity of AMP production in Escherichia coli. The AMPs inserted were released by cyanogen bromide and purified by chromatography. We showed that highly potent AMPs such as Protegrin-1, PMAP-36, Buforin-2, and Bactridin-1 are produced in high yields and produced AMPs showed similar activities compared to chemically synthesized AMPs. We increased the yield more than two-fold by inserting three copies of Protegrin-1 in the GFP scaffold. The immunogold electron micrographs showed that the expressed Protegrin-1 in the GFP scaffold forms large and small size aggregates in the core region of the inclusion body and become entirely nonfunctional, therefore not influencing the proliferation of E. coli. Our novel method will be applicable for diverse bacteriotoxic peptides which can be exploited in biomedical and pharmaceutical researches.