Prokaryotic Expression and Functional Verification of Antimicrobial Peptide LRGG.

The antimicrobial peptide LRGG (LLRLLRRGGRRLLRLL-NH2) was designed and chemically synthesized in a study conducted by Jia et al. Gram-negative bacteria were found to be sensitive to LRGG and exhibited a high therapeutic index. Genetic engineering methods were used to create the prokaryotic fusion expression vector pQE-GFP-LRGG, and the resulting corresponding fusion protein GFP-LRGG was subsequently expressed and purified. The precursor GFP was then removed by TEV proteolysis, and pure LRGG was obtained after another round of purification and endotoxin removal. The prokaryotic-expressed antimicrobial peptide LRGG displays a broad-spectrum antibacterial effect on Gram-negative bacteria, and its minimum inhibitory activity (MIC) against Escherichia coli can reach 2 µg/mL. Compared to the chemically synthesized LRGG, the prokaryotic-expressed LRGG exhibits similar temperature, pH, salt ion, serum stability, and cell selectivity. Furthermore, prokaryotic-expressed LRGG showed excellent therapeutic effects in both the infection model of cell selectivity and no embryotoxicity in a Galleria mellonella infection model. The mechanism by which LRGG causes bacterial death was found to be the disruption of the Gram-negative cell membrane.

Water quality's responses to water energy variability of the Yangtze River.

River energy serves as an indicator of pollutant-carrying capacity (PCC), influencing regional water quality dynamics. In this study, MIKE21 hydrodynamics-water quality models were developed for two scenarios, and grid-by-grid numerical integration of energy was conducted for the Yangtze River's mainstream. Comparison of predicted and measured values at monitoring points revealed a close fit, with average relative errors ranging from 5.17 to 8.37%. The concept of PCC was introduced to assess water flow's ability to transport pollutants during its course, elucidating the relationship between river energy and water quality. A relationship model between Unit Area Energy (UAE) and PCC was fitted (R2 = 0.8184). Temporally, reservoir construction enhanced the smoothness of UAE distribution by 74.47%, attributable to peak shaving and flow regulation. While this flood-drought season energy transfer reduced PCC differences, it concurrently amplified pollutant retention by 40.95%. Spatially, energy distribution fine-tuned PCC values, showcasing binary variation with energy changes and a critical threshold. Peak PCC values for TP, NH3-N, and COD were 2.46, 2.26, and 54.09 t/(km·a), respectively. These insights support local utility regulators and decision-makers in navigating low-carrying capacity, sensitive areas, enhancing targeted water protection measures for increased effectiveness and specificity.

Microplastics in Chinese coastal waters: A mini-review of occurrence characteristics, sources and driving mechanisms.

The oceans are facing global and irreversible pollution from microplastics, and China is not immune. In this mini-review, information on microplastics in four coastal waters of China and the natural and social environment of key basins were compiled. The results showed that microplastics were ubiquitous in the coastal waters, and the abundance and spatial distribution of microplastics varied significantly under different sampling methods. For trawl samples, microplastic abundance ranged from 0.045 to 1170.8 items m-3, among which the coastal waters of the East China Sea were the most polluted. For filtered samples, microplastic abundance ranged from 46 to 63,600 items m-3, and the coastal waters of the Yellow Sea were the most polluted. Meanwhile, human activities in basin were the key factors affecting microplastic pollution in coastal waters. The main terrestrial source in the coastal waters of the South China Sea was express packaging loss, whereas the main source in the other coastal waters was tyres and road markings wear from vehicle trip. The decoupling results of analytic hierarchy process showed that there was spatial heterogeneity in the impact of socio-economic and natural environmental factors in the basin on the distribution of microplastics in coastal waters. Among the five major basins, the impact weights of the latter were 20.00%, 83.34%, 66.66%, 50.00% and 25.00%, respectively. This study provides the first perspective of land-sea linkage to summarize the characteristics, sources and influencing factors of microplastics in Chinese coastal waters, providing ideas for reducing marine microplastic pollution from the source.

Nutrient Deprivation Coupled with High Light Exposure for Bioactive Chrysolaminarin Production in the Marine Microalga Isochrysis zhangjiangensis.

Chrysolaminarin, a kind of water-soluble bioactive ß-glucan produced by certain microalgae, is a potential candidate for food/pharmaceutical applications. This study identified a marine microalga Isochrysis zhangjiangensis, in which chrysolaminarin production was investigated via nutrient (nitrogen, phosphorus, or sulfur) deprivations (-N, -P, or -S conditions) along with an increase in light intensity. A characterization of the antioxidant activities of the chrysolaminarin produced under each condition was also conducted. The results showed that nutrient deprivation caused a significant increase in chrysolaminarin accumulation, though this was accompanied by diminished biomass production and photosynthetic activity. -S was the best strategy to induce chrysolaminarin accumulation. An increase in light intensity from 80 (LL) to 150 (HL) µE·m-2·s-1 further enhanced chrysolaminarin production. Compared with -N, -S caused more suitable stress and reduced carbon allocation toward neutral lipid production, which enabled a higher chrysolaminarin accumulation capacity. The highest chrysolaminarin content and concentration reached 41.7% of dry weight (%DW) and 632.2 mg/L, respectively, under HL-S, with a corresponding productivity of 155.1 mg/L/day achieved, which exceeds most of the photoautotrophic microalgae previously reported. The chrysolaminarin produced under HL-N (Iz-N) had a relatively competitive hydroxyl radical scavenging activity at low concentrations, while the chrysolaminarin produced under HL-S (Iz-S) exhibited an overall better activity, comparable to the commercial yeast ß-glucan, demonstrating I. zhangjiangensis as a promising bioactive chrysolaminarin producer from CO2.

Ultrasonication followed by aqueous two-phase system for extraction, on-site modification and isolation of microalgal starch with reduced digestibility.

Microalgae are new and sustainable sources of starch with higher productivity and flexible production modes than conventional terrestrial crops, but the downstream processes need further development. Here, ultrasonication (with power of 200 W or 300 W and duration of 10, 15, 20, or 25 min) was applied to simultaneously extract and modify starch from a marine microalga Tetraselmis subcordiformis for reducing the digestibility, and an aqueous two-phase system (ATPS) of ethanol/NaH2PO4 was then used to isolate the starches with varied properties. Increasing ultrasonic duration facilitated the partition of starch into the bottom pellet, while enhancing the ultrasonic power was conducive to the allocation in the interphase of the ATPS. The overall starch recovery yield reached 73 âˆ¼ 87 % and showed no significant difference among the ultrasonic conditions tested. The sequential ultrasonication-ATPS process successfully enriched the starch with purities up to 65 % âˆ¼ 88 %, which was among the top levels reported in microalgal starch isolated. Ultrasonication produced more amylose which was mainly fractionated into the interface of the ATPS. The digestibility of the starch was altered under different ultrasonic conditions and varied from different ATPS phases as well, with the one under the ultrasonic power of 200 W for 15 min at the bottom pellet having the highest resistant starch content (RS, 39.7 %). The structural and compositional analysis evidenced that the ultrasonication-ATPS process could exert impacts on the digestibility through altering the surface roughness and fissures of the starch granules, modulating the impurity compositions (protein and lipid) that could interact with starch, and modifying the long- and short-range ordered structures. The developed ultrasonication-ATPS process provided novel insights into the mechanism and strategy for efficient production of functional starch from microalgae with a potential in industrial application.

A hemicyanine-modified upconversion nanoprobe for NIR-excited evaluating superoxide signaling in drug-induced liver injury.

BACKGROUND: Drug-induced liver injury (DILI) is the most important standard for the entrance of clinical drugs into the pharmaceutical market. The elevation of superoxide anion (O2•-) during drug metabolism can mediate apoptosis of hepatocytes and further generation of liver damage. Therefore, developing an effective imaging method for evaluating O2•- levels during DILI is of great importance. However, current reported O2•- fluorescent probes either use short excitation wavelengths or a single intensity detection system, limiting the accurate quantification of O2•- in deep tissue in vivo. RESULTS: We developed a NIR-excited ratiometric nanoprobe (CyD-UCNPs) by assembly of O2•--sensitive hemicyanine dyes (CyD) on the surface of Tm/Er-codoped upconversion nanoparticles (UCNPs) with the assistance of α-cyclodextrin, which exhibited a robust "turn-on" ratiometric sensing signal. In vitro experiments indicated that CyD-UCNPs respond well to O2•- with high selectivity. Furthermore, by taking advantage of the outstanding optical properties produced by the luminescent resonance energy transfer between the UCNPs and CyD upon the excitation of 980 nm, the ratiometric upconversion luminescence signal of CyD-UCNPs was successfully utilized to monitor the fluctuation of O2•- levels under phorbol-12-myristate-13-acetate (PMA)/cisplatin-induced oxidative stress in living cells, liver tissues, and zebrafish. More importantly, endogenous change in O2•- levels in the liver sites of mice during DILI and its prevention with L-carnitine was visualized using CyD-UCNPs. SIGNIFICANCE: This study provides a ratiometric NIR-excited imaging strategy for investigating the correlation between O2•- levels and DILI and its prevention, which is significant for early diagnosis of DILI and preclinical screening of anti-hepatotoxic drugs in vivo.

Evaluating the Usability of mHealth Apps: An Evaluation Model Based on Task Analysis Methods and Eye Movement Data.

Advancements in information technology have facilitated the emergence of mHealth apps as crucial tools for health management and chronic disease prevention. This research work focuses on mHealth apps for the management of diabetes by patients on their own. Given that China has the highest number of diabetes patients in the world, with 141 million people and a prevalence rate of 12.8% (mentioned in the Global Overview of Diabetes), the development of a usability research methodology to assess and validate the user-friendliness of apps is necessary. This study describes a usability evaluation model that combines task analysis methods and eye movement data. A blood glucose recording application was designed to be evaluated. The evaluation was designed based on the model, and the feasibility of the model was demonstrated by comparing the usability of the blood glucose logging application before and after a prototype modification based on the improvement suggestions derived from the evaluation. Tests showed that an improvement plan based on error logs and post-task questionnaires for task analysis improves interaction usability by about 24%, in addition to an improvement plan based on eye movement data analysis for hotspot movement acceleration that improves information access usability by about 15%. The results demonstrate that this study presents a usability evaluation model for mHealth apps that enables the effective evaluation of the usability of mHealth apps.

NIR-excited imaging of drug-induced liver injury using a superoxide-activated ratiometric upconversion luminescence nanoprobe.

Drug-induced liver injury (DILI) poses a significant risk to human health. Increasing evidence indicates that the superoxide anion (O2•-), as the precursor of the other reactive oxygen species, is key in the pathological processes associated with DILI. Nonetheless, understanding of the mechanisms of DILI is difficult due to the lack of an imaging tool for monitoring the fluctuation of O2•- levels during the progression of DILI. Herein, we developed an upconversion nanoprobe (Rbh-UCNs) for in vivo ratiometric tracking of endogenous O2•- in DILI. In this design, the addition of O2•- triggers the luminescent resonance energy transfer between Rbh and UCNs, which significantly enhances absorption centered at 534 nm and translates into a distinct decrease of the UCL emission at 543 nm, while the UCL emission peak at 654 nm and 800 nm are not significantly affected, offering a ratiometric UCL signal for the quantitative detection of O2•-. In addition, Rbh-UCNs could effectively visualize endogenous O2•- in living cells, zebrafish, and liver tissues upon stimulation with PMA or cisplatin. More importantly, tissue imaging of the liver region of mice revealed that the fluctuation of O2•- levels is associated with DILI and the protective effect of L-carnitine against DILI. Altogether, this study provides an available method for a deeper comprehension of the mechanisms underlying DILI and accelerating the development process of hepatoprotective medicines.

A Novel Multi-Effect Photosensitizer for Tumor Destruction via Multimodal Imaging Guided Synergistic Cancer Phototherapy.

Background: How to ingeniously design multi-effect photosensitizers (PSs), including multimodal imaging and multi-channel therapy, is of great significance for highly spatiotemporal controllable precise phototherapy of malignant tumors. Methods: Herein, a novel multifunctional zinc(II) phthalocyanine-based planar micromolecule amphiphile (ZnPc 1) was successfully designed and synthesized, in which N atom with photoinduced electron transfer effect was introduced to enhance the near-infrared absorbance and nonradiative heat generation. After simple self-assembling into nanoparticles (NPs), ZnPc 1 NPs would exhibit enhanced multimodal imaging properties including fluorescence (FL) imaging (FLI) /photoacoustic (PA) imaging (PAI) /infrared (IR) thermal imaging, which was further used to guide the combined photodynamic therapy (PDT) and photothermal therapy (PTT). Results: It was that under the self-guidance of the multimodal imaging, ZnPc 1 NPs could precisely pinpoint the tumor from the vertical and horizontal boundaries achieving highly efficient and accurate treatment of cancer. Conclusion: Accordingly, the integration of FL/PA/IR multimodal imaging and PDT/PTT synergistic therapy pathway into one ZnPc 1 could provide a blueprint for the next generation of phototherapy, which offered a new paradigm for the integration of diagnosis and treatment in tumor and a promising prospect for precise cancer therapy.

NIR-excited imaging and in vivo visualization of ß-galactosidase activity using a pyranonitrile-modified upconversion nanoprobe.

ß-galactosidase (ß-gal) is a diagnostic biomarker of primary ovarian cancers. The development of effective fluorescent probes for investigating the activity of ß-gal will be beneficial to cancer diagnosis. Herein, a near-infrared (NIR) excited ratiometric nanoprobe (DCM-ß-gal-UCNPs) by assembling pyranonitrile dye (DCM-ß-gal) on the surface of upconversion nanophosphors (UCNPs) was designed for the evaluation of ß-gal activity in vivo. Upon the interaction with ß-gal, a marked decrease of upconversion luminescence (UCL) signal in the green channel was observed owing to the luminescence resonance energy transfer from the UCNPs to pyranonitrile chromophore, whereas the NIR UCL emission at 800 nm was almost no influence. Thus, the ß-gal activity could be quantitatively detected by the UCL intensity ratio of UCL543 nm/UCL800 nm with the limit of detection of 3.1 × 10-4 U/mL. Moreover, DCM-ß-gal-UCNPs was effectively applied for monitoring ß-gal fluctuation in living cells and zebrafish by a ratiometric UCL signal excited by 980 nm laser. We envision that nanoprobe DCM-ß-gal-UCNPs might be used as a potential bioimaging tool to disclose more biological information of ß-gal in ß-gal-associated diseases in the future.

How microplastic loads relate to natural conditions and anthropogenic activities in the Yangtze river basin.

As the third largest river in the world, microplastic pollution in the Yangtze River basin is currently attracting worldwide attention. However, fragmented research information is insufficient to reveal the occurrence and driving mechanisms of microplastics throughout the Yangtze River basin. Building on a systematic review of 20 existing publications, this study constructed a dataset including microplastic data from 366 samples in the Yangtze River basin through a data filtering process, and data on natural conditions and anthropogenic activities from 101 basin municipalities. Further, multivariate statistical analysis was utilized to enhance the understanding of the abundance, composition and drivers of microplastics within the basin. Differences in microplastic abundance among the sampling sites were up to 5 orders of magnitude, with the highest abundance value found in the upstream city of Chengdu. The comprehensive diversity index used to describe the composition characteristics of microplastics ranged from 0.31 to 0.68, slightly higher than the national average. Based on a statistical analysis framework, natural conditions and anthropogenic activities were shown to jointly drive the distribution of microplastics, and the dominant driver shifted between the two with spatial variation. In the upstream, anthropogenic activities dominated by GDP (r = 0.85, P < 0.01) were the main positive factor. In the middle and downstream, natural conditions and anthropogenic activities had comparable driving forces as the stability of natural resistance increased, and both were positively correlated with microplastics. Combining the constructed normalized stepwise linear regression model with GIS spatial analysis, the basin-wide application demonstrated that microplastic pollution in the upstream and delta deserved more attention. After coupling the distance factors, microplastic pollution was concentrated in the middle and downstream of the Yangtze River basin, covering important drinking water sources. This study provided important data support for subsequent targeted microplastic reduction and treatment.

Rational Design of Trimetallic Sulfide Electrodes for Alkaline Water Electrolysis with Ampere-Level Current Density.

Electrochemical water splitting is considered an environmentally friendly approach to hydrogen generation. However, it is difficult to achieve high current density and stability. Herein, we design an amorphous/crystalline heterostructure electrode based on trimetallic sulfide over nickel mesh substrate (NiFeMoS/NM), which only needs low overpotentials of 352 mV, 249 mV, and 360 mV to achieve an anodic oxygen evolution reaction (OER) current density of 1 A cm-2 in 1 M KOH, strong alkaline electrolyte (7.6 M KOH), and alkaline-simulated seawater, respectively. More importantly, it also shows superior stability with negligible decay after continuous work for 120 h at 1 A cm-2 in the strong alkaline electrolyte. The excellent OER performance of the as-obtained electrode can be attributed to the strong electronic interactions between different metal atoms, abundant amorphous/crystalline hetero-interfaces, and 3D porous nickel mesh structure. Finally, we coupled NiFeMoS/NM as both the anode and cathode in the anion exchange membrane electrolyzer, which can achieve low cell voltage and high stability at ampere-level current density, demonstrating the great potential of practicability.

Fine-tuned regulation of photosynthetic performance via γ-aminobutyric acid (GABA) supply coupled with high initial cell density culture for economic starch production in microalgae.

Microalgal starch is considered as renewable and sustainable feedstock for biofuels and biorefinery. High cell density culture is favourable for photoautotrophic starch production in microalgae in the aspects of productivity and economy, but it often encounters low starch content or extra stress exposure that limits the production. This study aimed to economically enhance photosynthetic starch production from CO2 fixation in a green microalga Tetraselmis subcordiformis by regulating photosynthetic stress status with a signalling molecule γ-aminobutyric acid (GABA) combined with the application of high initial cell density culture. By increasing initial cell density (ICD) from the normal of 1.1 g L-1 (NICD) to as high as 2.8 g L-1 (HICD), the starch content, yield, and theoretical productivity were improved by 7%, 63%, and 42%, respectively. The addition of GABA under HICD resulted in 14%, 19%, and 26% of further enhancement in starch content, yield, and theoretical productivity, respectively. GABA exhibited distinct regulatory mechanisms on photosynthesis and stress status under HICD relative to NICD. GABA augmented excessive light energy absorption and electron transfer through photosystem II that reinforced the photoinhibition under NICD, while alleviated the stress reversely under HICD, both of which facilitated starch production by enabling a suitable stress status while simultaneously maintaining a sufficient photosynthetic activity. The increase of ICD and/or GABA supply particularly boosted amylopectin accumulation, leading to the changes in starch composition and was more favourable for fermentation-based biofuels production. Preliminary techno-economic analysis showed that the highest net extra benefit of 9.64 $ m-3 culture could be obtained under HICD with 2.5 mM GABA supply where high starch content (62%DW) and yield (2.5 g L-1) were achieved. The combined HICD-GABA regulation was a promising strategy for economic starch production from CO2 by microalgae for sustainable biomanufacturing.

A self-healing carboxymethyl chitosan/oxidized carboxymethyl cellulose hydrogel with fluorescent bioprobes for glucose detection.

Self-healing hydrogel as a soft material with high durability and life-time has been successfully applied in various fields, including electronic skins, wearable electronic devices, and soft sensors. However, it is still a challenge to design a hydrogel with rapid self-healing, biodegradable and biosensing properties. Here, a self-healing carboxymethyl chitosan (CMCS)/oxidized carboxymethyl cellulose (OCMC) hydrogel with fluorescent bioprobes was developed for glucose detection. In this biosensing system, gold nanoclusters (AuNCs) and glucose oxidase (GOx) were encapsulated into the CMCS/OCMC hydrogel matrix as the fluorescent bioprobes. The CMCS/OCMC hydrogel with fluorescent bioprobes exhibited high sensitivity for glucose sensing with a linearly detection range of 100 µM to 5 mM and a detection limit of 0.029 mM, which covered the level of glucose in clinical detection. Furthermore, this hydrogel exhibited good biocompatibility. Finally, In vitro blood fluorescence tests and in vivo fluorescence investigation of the AuNCs-CMCS/OCMC hydrogel in diabetic mice indicated that this biocompatible and self-healing hydrogel based on fluorescent sensing system had potential application in implantable biosensing area for glucose monitoring.

Changes in nutrient transport from the Yangtze River to the East China Sea linked to the Three-Gorges Dam and water transfer project.

River-sea transition plays a key role in global geochemical cycles. The Yangtze River Estuary of China was selected as the research area, and the Section-Segmented Method was applied to determine the nutrient discharge from the Yangtze River to the East China Sea. A 3-D numerical model for the estuary was established and validated against the field investigated data. By numerical experiments the dynamics of hydrology and nutrient from 1950 to 2016 were simulated under four varied schemes. The individual and combined impacts on the nutrient flux induced by the Three-Gorges Dam (TGD) and the South-to-North Water Transfer Project (SNWTP) were explored. The following results were observed: (1) During the Pre-TGD period, the Yangtze River delivered the loads of 1.32 Tg/yr and 0.08 Tg/yr for TN and TP, respectively. July and Feb. were characterized by the highest and lowest monthly flux, respectively. (2) TGD played a significant role in regulating the temporal nutrient deliveries. After the closing of TGD, the discharges of TN and TP in the dry season respectively went up to 0.55 Tg and 0.032 Tg, with a mean increase of 28.3%. (3) SNWTP reduced the nutrient transport at a relatively stable level, and the total loads of 40.66 Gg and 2.4 Gg were reduced per year for TN and TP, respectively. (4) The combined impacts of TGD and SNWTP varied with seasons. October was characterized by the greatest cumulative effects. In dry seasons, the reduction caused by SNWTP was leveled by TGD-induced increase, limiting the flux variation linked to project operations.

Palliative Care Nursing Competence of Chinese Oncology Nurses and Its Related Factors.

In China, the development of palliative care is challenging because of limited available resources and rapidly increasing demands. The nurses' competence is a significant element in providing high-quality palliative care. This cross-sectional study aimed to describe the palliative care competence among oncology nurses and to examine the relationships between it and palliative care knowledge, attitudes, and workplace learning conditions. A total of 220 nurses with more than 6 months of experience and who worked in inpatient wards were invited to participate in this study. Four questionnaires were administered to collect data-the Palliative Care Quiz for Nurses, the Attitudes Toward Palliative Care Scale, the Workplace Learning Conditions scale, and the Palliative Care Nursing Self-competence Scale. The data were analyzed using descriptive statistics and Pearson correlations. The moderate level of competence was reported by 212 participants (response rate, 96.36%). The scores were lower in the aspects of competence such as spiritual care and ethical and legal issues. Competence was positively related to workplace learning conditions and knowledge but not attitudes. The results highlighted the necessity of improving the palliative care competence among oncology nurses. The optimization of learning conditions in the hospital is recommended to be a vital force in strengthening competence.

Translation of a laboratory-validated equine herpesvirus-1 specific real-time PCR assay into an insulated isothermal polymerase chain reaction (iiPCR) assay for point-of-need diagnosis using POCKIT™ nucleic acid analyzer.

Equine herpesvirus myeloencephalopathy (EHM), a major problem for the equine industry in the United States, is caused by equine herpesvirus-1 (EHV-1). In addition, EHV-1 is associated with upper respiratory disease, abortion, and chorioretinal lesions in horses. Here we describe the development and evaluation of an inexpensive, user-friendly insulated isothermal PCR (iiPCR) method targeting open reading 30 (ORF30) to detect both neuropathogenic and non-neuropathogenic strains on the field-deployable POCKIT™ device for point-of-need detection of EHV-1. The analytical sensitivity of the EHV-1 iiPCR assay was 13 genome equivalents per reaction. The assay did not cross react with ten non-target equine viral pathogens. Performance of the EHV-1 iiPCR assay was compared to two previously described real-time PCR (qPCR) assays in two laboratories by using 104 archived clinical samples. All 53 qPCR-positive and 46 of the 51 qPCR-negative samples tested positive and negative, respectively, by the iiPCR. The agreement between the two assays was 95.19% (confidence interval 90.48-99.90%) with a kappa value of 0.90. In conclusion, the newly developed EHV-1 iiPCR assay is robust to provide specificity and sensitivity comparable to qPCR assays for the detection of EHV-1 nucleic acid in clinical specimens.

Evaluation of two singleplex reverse transcription-Insulated isothermal PCR tests and a duplex real-time RT-PCR test for the detection of porcine epidemic diarrhea virus and porcine deltacoronavirus.

Recent outbreaks of porcine epidemic diarrhea virus (PEDV) and porcine deltacoronavirus (PDCoV) in multiple countries have caused significant economic losses and remain a serious challenge to the swine industry. Rapid diagnosis is critical for the implementation of efficient control strategies before and during PEDV and PDCoV outbreaks. Insulated isothermal PCR (iiPCR) on the portable POCKIT™ device is user friendly for on-site pathogen detection. In the present study, a singleplex PEDV RT-iiPCR, a singleplex PDCoV RT-iiPCR, and a duplex PEDV/PDCoV real-time RT-PCR (rRT-PCR) commercial reagents targeting the M gene were compared to an N gene-based PEDV rRT-PCR and an M gene-based PDCoV rRT-PCR that were previously published and used as reference PCRs. All PCR assays were highly specific and did not cross react with other porcine enteric pathogens. Analytical sensitivities of the PEDV RT-iiPCR, PDCoV RT-iiPCR and duplex PEDV/PDCoV rRT-PCR were determined using in vitro transcribed RNA as well as viral RNA extracted from ten-fold serial dilutions of PEDV and PDCoV cell culture isolates. Performance of each PCR assay was further evaluated using 170 clinical samples (86 fecal swabs, 24 feces, 19 intestines, and 41 oral fluids). Compared to the reference PEDV rRT-PCR, the sensitivity, specificity and accuracy of the PEDV RT-iiPCR were 97.73%, 98.78%, and 98.24%, respectively, and those of the duplex PEDV/PDCoV rRT-PCR were 98.86%, 96.34%, and 97.65%, respectively. Compared to the reference PDCoV rRT-PCR, the sensitivity, specificity and accuracy of the PDCoV RT-iiPCR were 100%, 100%, and 100%, respectively, and those of the PEDV/PDCoV duplex rRT-PCR were 96.34%, 100%, and 98.24%, respectively. Overall, all three new PCR assays were comparable to the reference rRT-PCRs for detection of PEDV and/or PDCoV. The PEDV and PDCoV RT-iiPCRs are potentially useful tools for on-site detection and the duplex PEDV/PDCoV rRT-PCR provides a convenient method to simultaneously detect the two viruses and differentiate PEDV from PDCoV.

Rad23 interaction with the proteasome is regulated by phosphorylation of its ubiquitin-like (UbL) domain.

Rad23 was identified as a DNA repair protein, although a role in protein degradation has been described. The protein degradation function of Rad23 contributes to cell cycle progression, stress response, endoplasmic reticulum proteolysis, and DNA repair. Rad23 binds the proteasome through a UbL (ubiquitin-like) domain and contains UBA (ubiquitin-associated) motifs that bind multiubiquitin chains. These domains allow Rad23 to function as a substrate shuttle-factor. This property is shared by structurally similar proteins (Dsk2 and Ddi1) and is conserved among the human and mouse counterparts of Rad23. Despite much effort, the regulation of Rad23 interactions with ubiquitinated substrates and the proteasome is unknown. We report here that Rad23 is extensively phosphorylated in vivo and in vitro. Serine residues in UbL are phosphorylated and influence Rad23 interaction with proteasomes. Replacement of these serine residues with acidic residues, to mimic phosphorylation, reduced proteasome binding. We reported that when UbL is overexpressed, it can compete with Rad23 for proteasome interaction and can inhibit substrate turnover. This effect is not observed with UbL containing acidic substitutions, consistent with results that phosphorylation inhibits interaction with the proteasome. Loss of both Rad23 and Rpn10 caused pleiotropic defects that were suppressed by overexpressing either Rad23 or Rpn10. Rad23 bearing a UbL domain with acidic substitutions failed to suppress rad23Δ rpn10Δ, confirming the importance of regulated Rad23/proteasome binding. Strikingly, threonine 75 in human HR23B also regulates interaction with the proteasome, suggesting that phosphorylation is a conserved mechanism for controlling Rad23/proteasome interaction.

Cisplatin transiently up-regulates hHR23 expression through enhanced translational efficiency in A549 adenocarcinoma cells.

DNA-damaging agents are commonly used as anticancer therapeutics. Unfortunately, such drugs induced DNA damages as well as DNA repair are important in mediating drug resistance to cancer treatments. To evaluate changes in DNA repair proteins that occur in DNA damage agent treatment, we challenged human A549 lung adenocarcinoma cells with cisplatin. hHR23/RAD23, an accessory protein involved in nucleotide-excision repair (NER) at an early lesion-recognition step, was upregulated by cisplatin in a dose- and time-dependent manner. Upregulation of hHR23 expression by low-dose cisplatin was accompanied by an increase in p53, p21, and XPC protein levels. Importantly, knockdown of hHR23B by RNA interference decreased DNA repair activity, cell survival, and induction of p53 and XPC following treatment with cisplatin. Conversely, overexpression of hHR23B enhanced repair activity towards cisplatin-damaged DNA. Inhibition of MEK/ERK and phosphoinositide 3-kinase (PI3K)/AKT signaling pathways attenuated cisplatin-induced hHR23 expression, indicating that these pathways are involved in the process. The increase in hHR23 protein expression mediated by MEK/ERK signaling was due to increased translational efficiency resulting from phosphorylation/activation of the translation-initiating factor eIF-4B. Taken together, these results suggest that cisplatin-induced increases in hHR23 levels are regulated by proliferative signaling pathways and important for DNA repair.