Mitigation of ROS-triggered endoplasmic reticulum stress by upregulating Nrf2 retards diabetic nephropathy.

Endoplasmic reticulum stress (ERS) plays a crucial role in the pathogenesis of diabetic nephropathy (DN), and it is often accompanied by an increase in reactive oxygen species (ROS) production. However, the precise relationship between NFE2-related factor-2 (Nrf2), a key regulator of ROS balance, and ERS in DN remains elusive. This study aimed to investigate the impact of Nrf2 on ERS and its therapeutic potential in DN. Herein, ERS-related changes, including increased activating transcription factor-6 (ATF6), glucose-regulated protein 78 (GRP78), and transcription factor C/EBP homologous protein (CHOP) expression, were observed in the renal tissues of streptozotocin-induced DN mice and high glucose cultured human renal proximal tubular (HK-2) cells. Nrf2 knockdown increased the sensitivity of HK-2 cells to ERS under high glucose conditions, underscoring the regulatory role of Nrf2 in ERS modulation. Notably, upregulating Nrf2 in ezetimibe-treated diabetic mice restored ERS markers and ameliorated albuminuria, glomerular hypertrophy, mesangial expansion, and tubulointerstitial fibrosis. Furthermore, the inhibition of ERS in HK-2 cells by the ROS scavenger, N-acetylcysteine, highlights the interplay between ROS and ERS. This study, for the first time, elucidates that the upregulation of Nrf2 may alleviate the negative influence of ROS-mediated ERS, presenting a promising therapeutic avenue for delaying the progression of DN. These findings suggest a potential strategy for targeting Nrf2 and ERS in developing novel therapeutic interventions for DN.

Association of Childhood Adversity With Frailty and the Mediating Role of Unhealthy Lifestyle: A Lifespan Analysis.

OBJECTIVES: Childhood adversity and lifestyle have been associated with frailty in later life, but not much is known about factors that may explain these associations. Therefore, this study aims to investigate the association of childhood adversity with frailty, and the mediating role of unhealthy lifestyle in the association. METHODS: This lifespan analysis included 152,914 adults aged 40-69 years old from the UK Biobank. We measured childhood adversity with five items: physical neglect, emotional neglect, sexual abuse, physical abuse, and emotional abuse through online mental health survey. Frailty was measured by the frailty index; an unhealthy lifestyle score (range: 0-5) was calculated based on unhealthy body mass index, smoking, alcohol consumption, physical inactivity, and unhealthy diet at the baseline survey. Multiple logistic regression and mediation analysis were performed. RESULTS: A total of 10,078 participants (6.6%) were defined as having frailty. Participants with any childhood adversity had higher odds of frailty. For example, in the fully adjusted model, with a one-point increase in cumulative score of childhood adversity, the odds of frailty increased by 38% (odds ratio: 1.38; 95% Confidence Interval: 1.36, 1.40). Unhealthy lifestyle partially mediated the associations of childhood adversity with frailty (mediation proportion: 4.4%-7.0%). The mediation proportions were largest for physical (8.2%) and sexual (8.1%) abuse. CONCLUSIONS: Childhood adversity was positively associated with frailty, and unhealthy lifestyle partially mediated the association. This newly identified pathway highlights the potential of lifestyle intervention strategies among those who experienced childhood adversity (in particular, physical, and sexual abuse) to promote healthy aging.

Acute and chronic toxicity in Sprague-Dawley rats of orally-administered total lignans from Arctii Fructus, a potential therapeutic drug for diabetes.

Arctii Fructus is the dried ripe fruit of Arctium lappa L. (family Asteraceae) and is in the Chinese pharmacopoeia. Previous research showed that the total lignans from Arctii Fructus (TLAF) have pharmacological activities related to diabetes. This study evaluated the acute and chronic (26 weeks) toxicities associated with oral daily administration of TLAF in Sprague-Dawley (SD) rats. An acute-toxicity test showed that TLAF caused 10% mortality at 3,000 mg/kg × 2 (6-h interval), with toxic symptoms, such as dyspnea and tonic convulsions, indicating potential neurotoxicity. A chronic-toxicity study showed no mortality after administration. The no observed adverse-effect level was 1,800 mg/kg (approximately 54 times higher than the human clinical dose) for 26 weeks of TLAF oral administration in SD rats, with toxicity signs of excessive oral and nasal secretions and moist circumferential hair that recovered after TLAF discontinuation. In the toxicokinetic study, the two main components of TLAF, arctigenin plasma level was positively correlated with dose and tended to accumulate after multiple doses. At 1,800 mg/kg, arctiin plasma level increased and tended to accumulate after multiple doses. These results indicated that TLFA has relatively low toxicity and the potential for clinical treatment of diabetes.

Tracing toxic path of antimony: From bioaccumulation to DNA hypomethylation in zebrafish (Danio rerio).

The increasing concentration of Antimony (Sb) in ecological environments has raised serious concerns about its potential biotoxicological impact. This study investigated the toxicokinetics, Global DNA Methylation (GDM), biomarker expression, and Integrated Biological Response (IBR) of Sb at different concentrations in zebrafish. The toxic mechanism of Sb exposure was simulated using molecular dynamics (MD). The results showed that significant differences effect existed (BCFk: liver > ovary > gut > brain) and uptake saturation phenomenon of Sb among zebrafish tissues. Over a 54-day exposure period, the liver emerged as the main target site for Sb-induced GDM, and the restoration was slower than in other tissues during the 54-day recovery period. Moreover, the concentration of Sb had a significant impact on the normally expression of biomarkers, with GSTM1 inhibited and MTF2, MT1, TET3, and p53 showing varying degrees of activation at different Sb concentrations. This could be attributed to Sb3+ potentially occupying the active site or tightly binding to the deep cavity of these genes. The IBR and MD results highlighted DNMT1 as the most sensitive biomarker among those assessed. This heightened sensitivity can be attributed to the stable binding of Sb3+ to DNMT1, resulting in alterations in the conformation of DNMT1's catalytic domain and inhibition of its activity. Consequently, this disruption leads to damage to the integrity of GDM. The study suggests that DNA methylation could serve as a valuable biomarker for assessing the ecotoxicological impact of Sb exposure. It contributes to a better understanding of the toxicity mechanisms in aquatic environments caused potential pollutants.

Transcriptome analysis provides insights into lignin synthesis and MAPK signaling pathway that strengthen the resistance of bitter gourd (Momordica charantia) to Fusarium wilt.

Fusarium wilt is a typical soil-borne disease caused by Fusarium oxysporum f. sp. momordicae (FOM) in bitter gourd. In this study, by comparing sequencing data at multiple time points and considering the difference between resistant (R) and susceptible (S) varieties, differentially expressed genes were screened out. Short time-series expression miner analysis revealed the upregulated expression trend of genes, which were enriched in phenylpropanoid biosynthesis, plant-pathogen interaction, and mitogen-activated protein kinase signaling pathway. Further, observation of the microstructure revealed that the R variety may form tyloses earlier than the S variety to prevent mycelium diffusion from the xylem vessel. After Fusarium wilt infection, the enzymatic activities of superoxide dismutase, peroxidase, phenylalanine ammonia lyase, and catalaseas well as levels of superoxide anion and malondialdehyde were increased in the R variety higher than those in the S variety. This study provides a reference to elucidate the disease resistance mechanism of bitter gourd.

Association of frailty with the incidence risk of cardiovascular disease and type 2 diabetes mellitus in long-term cancer survivors: a prospective cohort study.

BACKGROUND: Comorbidities among cancer survivors remain a serious healthcare burden and require appropriate management. Using two widely used frailty indicators, this study aimed to evaluate whether frailty was associated with the incidence risk of cardiovascular disease (CVD) and type 2 diabetes mellitus (T2DM) among long-term cancer survivors. METHODS: We included 13,388 long-term cancer survivors (diagnosed with cancer over 5 years before enrolment) free of CVD and 6101 long-term cancer survivors free of T2DM, at the time of recruitment (aged 40-69 years), from the UK Biobank. Frailty was assessed by the frailty phenotype (FP_Frailty, range: 0-5) and the frailty index (FI_Frailty, range: 0-1) at baseline. The incident CVD and T2DM were ascertained through linked hospital data and primary care data, respectively. The associations were examined using Cox proportional hazards regression models. RESULTS: Compared with non-frail participants, those with pre-frailty (FP_Frailty [met 1-2 of the components]: hazard ratio [HR]=1.18, 95% confidence interval [CI]: 1.05, 1.32; FI_Frailty [0.10< FI ≤0.21]: HR=1.51, 95% CI: 1.32, 1.74) and frailty (FP_Frailty [met ≥3 of the components]: HR=2.12, 95% CI: 1.73, 2.60; FI_Frailty [FI >0.21]: HR=2.19, 95% CI: 1.85, 2.59) had a significantly higher risk of CVD in the multivariable-adjusted model. A similar association of FI_Frailty with the risk of incident T2DM was observed. We failed to find such an association for FP_Frailty. Notably, the very early stage of frailty (1 for FP_Frailty and 0.1-0.2 for FI_Frailty) was also positively associated with the risk of CVD and T2DM (FI_Frailty only). A series of sensitivity analyses confirmed the robustness of the findings. CONCLUSIONS: Frailty, even in the very early stage, was positively associated with the incidence risk of CVD and T2DM among long-term cancer survivors, although discrepancies existed between frailty indicators. While the validation of these findings is required, they suggest that routine monitoring, prevention, and interventive programs of frailty among cancer survivors may help to prevent late comorbidities and, eventually, improve their quality of life. Especially, interventions are recommended to target those at an early stage of frailty when healthcare resources are limited.

Association of solid fuel use with a risk score capturing dementia risk among middle-aged and older adults: A prospective cohort study.

OBJECTIVES: Whether household air pollution is associated with dementia risk remains unknown. This study examined the associations between solid fuel use for cooking and heating (the main source of household air pollution) and dementia risk. METHODS: This analysis included data on 11,352 participants (aged 45+ years) from the 2011 wave of China Health and Retirement Longitudinal Study, with follow-up to 2018. Dementia risk was assessed by a risk score using the Rotterdam Study Basic Dementia Risk Model (BDRM), which was subsequently standardized for analysis. Household fuel types of cooking and heating were categorized as solid (e.g., coal and crop residue) and clean (e.g., central heating and solar). Multivariable analyses were performed using generalized estimating equations. Moreover, we examined the joint associations of solid fuel use for cooking and heating with the BDRM score. RESULTS: After adjusting for potential confounders, we found an independent and significant association of solid (vs. clean) fuel use for cooking and heating with a higher BDRM score (e.g., ß = 0.17 for solid fuel for cooking; 95% confidence interval [CI]: 0.15-0.19). Participants who used solid (vs. clean) fuel for both cooking and heating had the highest BDRM score (ß = 0.32; 95% CI: 0.29-0.36). Subgroup analysis suggested stronger associations in participants living in rural areas. CONCLUSIONS: Solid fuel use for cooking and heating was independently associated with increased dementia risk in Chinese middle-aged and older adults, particularly among those living in rural areas. Our findings call for more efforts to facilitate universal access to clean energy for dementia prevention.

Catalytic Reactions Directed by a Structurally Well-Defined Aminomethyl Cyclopalladated Complex.

The introduction of N-containing moieties into feedstock molecules to build nitrogenated functional molecules has always been widely studied by the organic chemistry community. Progress in this field paves new roads to the synthesis of N-containing molecules, which are of significant importance in biological activities and play vital roles in pharmaceuticals and functional materials. Remarkable progress has been achieved in the field of transition metal-catalyzed C-N bond-forming reactions, typified by alkene hydroamination and the aza-Wacker reaction. However, the poisoning effect of electron-donating amine substrates on late transition metal catalysts presents a key impediment to these reactions, thus limiting the scope of amine substrates to electron-deficient amide derivatives. To address this problem, our group developed a palladium-aminomethyl complex with a three-membered palladacycle structure that allowed for the incorporation of electron-rich amine building blocks via C-C bond instead of C-N bond construction. This Account details the discovery of the well-defined aminomethyl cyclopalladated complex and recapitulates its applications for the catalysis of a series of aminomethylation reactions. We highlight how the understanding of the fundamental structural properties of the defined complex guided us toward tuning the reactivity of nucleophiles to initiate aminomethylation in different modes. Moreover, principles of designing and establishing further cascade reactions are also described.Aminomethyl cyclopalladated complexes can be prepared via the oxidative addition of aminals or N,O-acetals to Pd0 species. Thorough structural investigations by single-crystal X-ray diffraction analysis of the cyclopalladated complex suggest the presence of both aminomethylene-PdII (3-membered-ring) and Pd0-iminium (π-ligated) resonance forms, which indicates that both the palladium center and the methylene site are electrophilic. This is further verified by analysis of charge distribution. Two general types of reactions can be established, differing by the selective affinity of the nucleophiles to the two electrophilic positions, which is relevant to the "hardness suitability" of the nucleophiles with each electrophilic site. Softer nucleophiles such as alkenes prefer to attack the palladium center to initiate the reaction, mainly via migratory insertion into the Pd-C bond on the 3-membered ring with high strain. Through tandem ß-hydride or reductive elimination, the Heck-type aminomethylation of styrenes, the aminomethylalkoxylation of electron-rich olefins, and even the aminomethylamination of allenes, dienes, enynes, and carbenoids with full atom-economy have been realized in line with this reaction mode. In contrast, harder nucleophiles tend to attack the harder electrophilic methylene site, leading to the aminomethylation of electron-deficient dienes. For secondary amines, a "C-N bond metathesis" process would be furnished through a reductive elimination, 1,3-proton transfer, and oxidative addition sequence. More intriguingly, when using appropriate "dinucleophile" substrates such as electron-rich amine-tethered dienes, sequential C-N bond metathesis and intramolecular insertion would occur to furnish Pd-catalyzed annulation reactions, which exhibits both the hard and soft nucleophile reactivities mentioned above. These transformations provide convenient methods for the preparation of N-containing molecules, such as amines, diamines, amino acetals, and multiple types of N-heterocycles.

MdUGT88F1-mediated phloridzin biosynthesis coordinates carbon and nitrogen accumulation in apple.

The high accumulation of phloridzin makes apple (Malus domestica) unique in the plant kingdom, which suggests a vital role of its biosynthesis in physiological processes. In our previous study, silencing MdUGT88F1 (a key UDP-GLUCOSE: PHLORETIN 2'-O-GLUCOSYLTRANSFERASE gene) revealed the importance of phloridzin biosynthesis in apple development and Valsa canker resistance. Here, results from MdUGT88F1-silenced lines showed that phloridzin biosynthesis was indispensable for normal chloroplast development and photosynthetic carbon fixation by maintaining MdGLK1/2 (GOLDEN2-like1/2) expression. Interestingly, increased phloridzin biosynthesis did not affect plant (or chloroplast) development, but reduced nitrogen accumulation, leading to chlorophyll deficiency, light sensitivity, and sugar accumulation in MdUGT88F1-overexpressing apple lines. Further analysis revealed that MdUGT88F1-mediated phloridzin biosynthesis negatively regulated the cytosolic glutamine synthetase1-asparagine synthetase-asparaginase (GS1-AS-ASPG) pathway of ammonium assimilation and limited chlorophyll synthesis in apple shoots. The interference of phloridzin biosynthesis in the GS1-AS-ASPG pathway was also assumed to be associated with its limitation of the carbon skeleton of ammonium assimilation through metabolic competition with the tricarboxylic acid cycle. Taken together, our findings shed light on the role of MdUGT88F1-mediated phloridzin biosynthesis in the coordination between carbon and nitrogen accumulation in apple trees.

Tunable type-II lateral MoSi2N4/WSi2N4 heterostructures for photocatalytic applications.

Combining various two-dimensional crystals has emerged as an exciting way to tailor the properties of lateral heterostructures for new-generation optoelectronic devices. Herein, a seamless lateral heterostructure based on MoSi2N4 and MoSi2N4 monolayers along armchair interfaces is predicted, and its electronic and optical properties are investigated by using first principles calculations. Our calculations indicate that the MoSi2N4/WSi2N4 lateral heterostructures (HSs) possess excellent stability due to the very small lattice mismatch. In contrast to their parent monolayers with wide indirect band gaps, all (MoSi2N4)m(WSi2N4)n lateral HSs are direct gap semiconductors, and their direct gap nature is independent of compositions and strains. The band alignment of (MoSi2N4)m(WSi2N4)16-m lateral HSs undergoes a quasi-type-I to type-II to quasi-type-II to quasi-type-I band transition with an increase in m. (MoSi2N4)8(WSi2N4)8 is a type-II semiconductor, and the band arrangement changes from type-II to quasi-type-I upon applying tensile strain. Compared with pristine materials, the band edges of MoSi2N4/WSi2N4 lateral HSs are more favorable for photocatalytic water splitting. Furthermore, MoSi2N4/WSi2N4 lateral HSs exhibit higher visible light absorption. These results greatly expand the optoelectronic applications of Mxenes in the 2D realm.

Aging metrics incorporating cognitive and physical function capture mortality risk: results from two prospective cohort studies.

BACKGROUND: Aging metrics incorporating cognitive and physical function are not fully understood, hampering their utility in research and clinical practice. This study aimed to determine the proportions of vulnerable persons identified by three existing aging metrics that incorporate cognitive and physical function and the associations of the three metrics with mortality. METHODS: We considered three existing aging metrics including the combined presence of cognitive impairment and physical frailty (CI-PF), the frailty index (FI), and the motoric cognitive risk syndrome (MCR). We operationalized them using data from the China Health and Retirement Longitudinal Study (CHARLS) and the US National Health and Nutrition Examination Survey (NHANES). Logistic regression models or Cox proportional hazards regression models, and receiver operating characteristic curves were used to examine the associations of the three metrics with mortality. RESULTS: In CHARLS, the proportions of vulnerable persons identified by CI-PF, FI, and MCR were 2.2, 16.6, and 19.6%, respectively. Each metric predicted mortality after adjustment for age and sex, with some variations in the strength of the associations (CI-PF, odds ratio (OR) (95% confidence interval (CI)) 2.87 (1.74-4.74); FI, OR (95% CI) 1.94 (1.50-2.50); MCR, OR (95% CI) 1.27 (1.00-1.62)). CI-PF and FI had additional predictive utility beyond age and sex, as demonstrated by integrated discrimination improvement and continuous net reclassification improvement (all P < 0.001). These results were replicated in NHANES. CONCLUSIONS: Despite the inherent differences in the aging metrics incorporating cognitive and physical function, they consistently capture mortality risk. The findings support the incorporation of cognitive and physical function for risk stratification in both Chinese and US persons, but call for caution when applying them in specific study settings.

Does healthy lifestyle attenuate the detrimental effects of urinary polycyclic aromatic hydrocarbons on phenotypic aging? An analysis from NHANES 2001-2010.

Existing evidence has showed that exposure to polycyclic aromatic hydrocarbons (PAHs) increases the risk of many chronic diseases. Given the close connection between aging (a major risk factor) and chronic diseases, however, very few studies have evaluated the association between PAHs and aging. Furthermore, whether modifiable healthy lifestyle could attenuate the detrimental effect of PAHs on aging remains unknown. Therefore, we conducted this study, aiming to: (1) examine the associations of urinary monohydroxy polycyclic aromatic hydrocarbons (OH-PAHs) and lifestyle with Phenotypic Age Acceleration (PhenoAge.Accel), a novel aging measure that captures morbidity and mortality risk; and (2) evaluate the potential interaction effects of OH-PAHs and lifestyle on PhenoAge.Accel. Cross-sectional data of 2,579 participants (aged 20-84 years, n = 1,292 females) from the National Health and Nutrition Examination Survey for years 2001-2010 were analyzed. A lifestyle index was constructed based on five components (drinking, smoking, body mass index, physical activity, and diet), ranging from 0 to 5. We calculated PhenoAge.Accel using algorithms developed previously. General linear regression models were used to examine the associations. We observed strong associations of OH-PAHs and lifestyle with PhenoAge.Accel. For instance, one unit increase in ∑NAP (sum of 1- and 2-hydroxynaphthalene) was associated with 0.37 year (95% confidence interval [CI]: 0.26, 0.48) increase in PhenoAge.Accel. We did not observe statistically significant interaction effects between OH-PAHs and lifestyle on PhenoAge.Accel. After stratified by sex, we observed strong associations as well as statistically significant interactions of OH-PAHs and lifestyle with PhenoAge.Accel among females. In conclusion, both OH-PAHs and lifestyle were independently associated with phenotypic aging and there were statistically significant interactions between OH-PAHs and lifestyle on phenotypic aging among females. The findings highlight the importance of adherence to a healthy lifestyle to attenuate the detrimental effects of exposures to PAHs on phenotypic aging among females.

Turn-on colorimetric detection of hydroquinone based on Au/CuO nanocomposite nanozyme.

CuO nanorods bearing Au nanoparticles (Au/CuO nanocomposites) were prepared by a solution-phase synthesis and exhibited efficient hydroquinone (HQ)-oxidase activity with good specificity. The Au/CuO nanocomposites effectively catalyzed the oxidation of colorless HQ to brown benzoquinone with an absorbance maximum at 376 nm but did not catalyze the conversions of catechol or resorcinol. Kinetic studies indicated that the Au/CuO nanocomposites exhibited a strong affinity for HQ, with a Michaelis-Menten constant of Km = 0.33 mM. Owing to the high catalytic activity and specificity, a strong color was observed at low concentrations of HQ. Quantitative measurement of HQ was performed via colorimetric analysis, which yielded a detection limit of 3 µM with a linear range of 5-200 µM. This colorimetric sensor was successfully applied to an HQ assay of real water samples with satisfactory results.

Chromatographic fingerprint combined with quantitative analysis of multi-components by single-marker for quality control of total lignans from Fructus arctii by high-performance liquid chromatography.

INTRODUCTION: The total lignans from Fructus arctii (TLFA) is a mixture of a series of lignans isolated from dried ripe fruit of Arctium lappa L. We previously reported on the pharmacological activity of TLFA that is related to diabetes. An accurate and practical TLFA quantitative analysis method for utilising it needs to be established. OBJECTIVE: This study aimed to develop an effective quantitative analysis method for assessing the TLFA quality. METHODS: A total of 11 marker components were confirmed by analysing the high-performance liquid chromatography fingerprints of 24 batches of TLFA samples. The samples were prepared from TLFA and structurally identified as lappaol H, lappaol C, arctiin, arctignan D, arctignan E, matairesinol, arctignan G, isolappaol A, lappaol A, arctigenin, and lappaol F. In the quantitative analysis of multi-components by the single-marker (QAMS) method and with arctiin as an internal reference substance, the content of these lignans in TLFA was simultaneously determined according to their relative correction factors with arctiin. RESULTS: There was no significant difference between results measured by the QAMS and traditional external standard methods. Hierarchical cluster and principal component analyses were performed to evaluate 24 TLFA batches based on the contents of 10 marker components. The results revealed that QAMS method combined with chemometric analyses could accurately measure and clearly distinguish the different quality samples of TLFA. CONCLUSION: The QAMS method is a reliable and promising quality control method for TLFA. It can provide a reference for promoting quality control of complex multi-component systems, especially for traditional Chinese medicine.

Repeated neonatal sevoflurane induced neurocognitive impairment through NF-κB-mediated pyroptosis.

BACKGROUND: Exposure to general anesthesia (GA) during the postnatal period is associated with neuroinflammation and long-term neurocognitive impairment in preclinical and clinical settings. Pyroptosis is a novel type of programmed cell death that, along with inflammation, has been found to play an important role in the mechanism of diverse neurological diseases. However, its roles in GA-induced neuroinflammation and neurocognitive impairment in the developing brain have not been investigated. METHODS: Rats at postnatal day 6 or primary hippocampal neurons at 9 days in vitro received 3% sevoflurane for 2 h daily for three consecutive days. A pharmacological inhibitor of nuclear factor (NF)-κB (BAY 11-7082) was administered to suppress NF-κB activation. Histological and biochemical analyses were performed to assess the pyroptosis as well as neuronal and synaptic damage both in vivo and in vitro. In addition, behavioral tests were performed to evaluate neurocognitive ability in rats. RESULTS: Repeated sevoflurane exposure activated NF-κB-mediated pyroptosis and neuroinflammation in the hippocampus in developing rats, damaged the neuronal morphology and synaptic integrity, and induced neurocognitive impairment in rats. BAY 11-7082 treatment suppressed the activation of pyroptosis, attenuated the neuronal and synaptic damage, and ameliorated the neurocognitive impairment induced by repeated sevoflurane administration to developing rats. CONCLUSIONS: Repeated sevoflurane GA may induce neuroinflammation and neurocognitive impairment in developing rats via the activation of NF-κB-mediated pyroptosis. Our findings characterize a novel role of pyroptosis as a potential therapeutic target in neuroinflammation after repeated neonatal GA.

Pyramiding the antimicrobial PR1aCB and AATCB genes in 'Tarocco' blood orange (Citrus sinensis Osbeck) to enhance citrus canker resistance.

Citrus canker, caused by Xanthomonas citri subsp. citri (Xcc), is a major bacterial disease responsible for substantial economic losses in citrus-producing areas. To breed transgenic citrus plants with enhanced resistance to citrus canker, two antimicrobial peptide genes, PR1aCB and AATCB, were incorporated into 'Tarocco' blood orange (Citrus sinensis Osbeck) plants via co-transformation and sequential re-transformation. The presence of PR1aCB and AATCB in double transgenic plants was confirmed by PCR. The expression of PR1aCB and AATCB in double transformants was demonstrated by quantitative real-time PCR. An in vivo disease resistance assay involving the injection of Xcc revealed that the double transformants were more resistant to citrus canker than the single gene transformants and wild-type plants. An analysis of the bacterial population indicated that the enhanced citrus canker resistance of the double transformants was due to inhibited Xcc growth. These results proved that the pyramiding of multiple genes is a more effective strategy for increasing the disease resistance of transgenic citrus plants than single gene transformations.

A model-driven approach towards rational microbial bioprocess optimization.

Due to sustainability concerns, bio-based production capitalizing on microbes as cell factories is in demand to synthesize valuable products. Nevertheless, the nonhomogenous variations of the extracellular environment in bioprocesses often challenge the biomass growth and the bioproduction yield. To enable a more rational bioprocess optimization, we have established a model-driven approach that systematically integrates experiments with modeling, executed from flask to bioreactor scale, and using ferulic acid to vanillin bioconversion as a case study. The impacts of mass transfer and aeration on the biomass growth and bioproduction performances were examined using minimal small-scale experiments. An integrated model coupling the cell factory kinetics with the three-dimensional computational hydrodynamics of bioreactor was developed to better capture the spatiotemporal distributions of bioproduction. Full-factorial predictions were then performed to identify the desired operating conditions. A bioconversion yield of 94% was achieved, which is one of the highest for recombinant Escherichia coli using ferulic acid as the precursor.

A multiplex PCR assay for the detection of five human pathogenic Vibrio species and Plesiomonas.

A multiplex PCR (mPCR) assay was established to detect five pathogenic Vibrio species and Plesiomonas shigelloides. Twelve genes were included: ompW, ctxA, rfbN, and wbfR from V. cholerae; tl, tdh, and trh from V. parahaemolyticus; toxR and vmhA from V. mimicus; toxR from V. fluvialis; vvhA from V. vulnificus; and the 23S rRNA gene from P. shigelloides. The specificity of the mPCR assay was 100% for the detection of 136 strains and the limits of detection (LoD) were 12.5-50 pg/reaction. The assay exhibited higher sensitivity than cultivation methods in the detection of APW cultures of 113 diarrhea samples. In the analysis of 369 suspected Vibrio populations from estuarine water samples, the specificity of the mPCR for V. cholerae and V. parahaemolyticus was 100% for both, while the sensitivities were 100% and 96.1%, respectively. The assay can be applied to screen enrichment cultures and suspected colonies from environmental and clinical samples.

Overexpressing a NPR1-like gene from Citrus paradisi enhanced Huanglongbing resistance in C. sinensis.

KEY MESSAGE: Overexpression of CiNPR4 enhanced resistance of transgenic citrus plants to Huanglongbing by perceiving the salicylic acid and jasmonic acid signals and up-regulating the transcriptional activities of plant-pathogen interaction genes. Developing transgenic citrus plants with enhanced immunity is an efficient strategy to control citrus Huanglongbing (HLB). Here, a nonexpressor of pathogenesis-related gene 1 (NPR1) like gene from HLB-tolerant 'Jackson' grapefruit (Citrus paradisi Macf.), CiNPR4, was introduced into 'Wanjincheng' orange (Citrus sinensis Obseck). CiNPR4 expression was determined in transgenic citrus plants using quantitative real-time PCR analyses. The Candidatus Liberibacter asiaticus (CLas) pathogen of HLB was successfully transmitted to transgenic citrus plants by grafting infected buds. HLB symptoms developed in transgenic and wild-type (WT) plants by 9 months after inoculation. A CLas population analysis showed that 26.9% of transgenic lines exhibited significantly lower CLas titer levels compared with the CLas-infected WT plants at 21 months after inoculation. Lower starch contents and anatomical aberration levels in the phloem were observed in transgenic lines having enhanced resistance compared with CLas-infected WT plants. CiNPR4 overexpression changed the jasmonic acid, but not salicylic acid, level. Additionally, the jasmonic acid and salicylic acid levels increased after CLas infection. Transcriptome analyses revealed that the enhanced resistance of transgenic plants to HLB resulted from the up-regulated transcriptional activities of plant-pathogen interaction-related genes.

Identification of diarrheagenic Escherichia coli by a new multiplex PCR assay and capillary electrophoresis.

Diarrheagenic Escherichia coli (DEC) is a set of the most common pathogens causing diarrhea. DEC strains are classified into five pathotypes based on the possession of different virulence genes: enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC) or Shiga toxin-producing E. coli (STEC), enteroaggregative E. coli (EAEC), enterotoxigenic E. coli (ETEC), and enteroinvasive E. coli (EIEC). The development of an easy-to-use method to detect the specific virulence genes and distinguish the pathotypes is essential for the diagnosis and surveillance of DEC infections. In this study, a multiplex PCR assay (mPCR) specific to nine virulence genes and an internal control was designed for the identification of five DEC pathotypes. A temperature switch PCR (TSP) strategy was used in the PCR amplification. The PCR products were detected by capillary electrophoresis. The limit of detection (LOD) of the 10-plex reaction was 5 × 103 copies/reaction for stx2 and 5 × 102 copies/reaction for the other targets. The mPCR showed very high specificity, and inclusivity and exclusivity were both 100%. When the mPCR assay was used for the detection of 221 cryopreserved diarrhea specimens, DEC colonies were detected from 49 specimens, and the positive rate was 22.2%. The mPCR assay was sensitive and specific, and the amplified product could be analyzed easily. Thus, this method could be used effectively to identify the suspected colonies of DEC in the primary culture of the specimen.