Stress resilience-enhancing drugs preserve tissue structure and function in degenerating retina via phosphodiesterase inhibition.

Chronic, progressive retinal diseases, such as age-related macular degeneration (AMD), diabetic retinopathy, and retinitis pigmentosa, arise from genetic and environmental perturbations of cellular and tissue homeostasis. These disruptions accumulate with repeated exposures to stress over time, leading to progressive visual impairment and, in many cases, legal blindness. Despite decades of research, therapeutic options for the millions of patients suffering from these disorders remain severely limited, especially for treating earlier stages of pathogenesis when the opportunity to preserve the retinal structure and visual function is greatest. To address this urgent, unmet medical need, we employed a systems pharmacology platform for therapeutic development. Through integrative single-cell transcriptomics, proteomics, and phosphoproteomics, we identified universal molecular mechanisms across distinct models of age-related and inherited retinal degenerations, characterized by impaired physiological resilience to stress. Here, we report that selective, targeted pharmacological inhibition of cyclic nucleotide phosphodiesterases (PDEs), which serve as critical regulatory nodes that modulate intracellular second messenger signaling pathways, stabilized the transcriptome, proteome, and phosphoproteome through downstream activation of protective mechanisms coupled with synergistic inhibition of degenerative processes. This therapeutic intervention enhanced resilience to acute and chronic forms of stress in the degenerating retina, thus preserving tissue structure and function across various models of age-related and inherited retinal disease. Taken together, these findings exemplify a systems pharmacology approach to drug discovery and development, revealing a new class of therapeutics with potential clinical utility in the treatment or prevention of the most common causes of blindness.

Restoring retinal polyunsaturated fatty acid balance and retina function by targeting ceramide in AdipoR1-deficient mice.

Mutations in the adiponectin receptor 1 gene (AdipoR1) lead to retinitis pigmentosa and are associated with age-related macular degeneration. This study explores the effects of AdipoR1 gene deficiency in mice, revealing a striking decline in ω3 polyunsaturated fatty acids (PUFA), an increase in ω6 fatty acids, and elevated ceramides in the retina. The AdipoR1 deficiency impairs peroxisome proliferator-activated receptor α signaling, which is crucial for FA metabolism, particularly affecting proteins associated with FA transport and oxidation in the retina and retinal pigmented epithelium. Our lipidomic and proteomic analyses indicate changes that could affect membrane composition and viscosity through altered ω3 PUFA transport and synthesis, suggesting a potential influence of AdipoR1 on these properties. Furthermore, we noted a reduction in the Bardet-Biedl syndrome proteins, which are crucial for forming and maintaining photoreceptor outer segments that are PUFA-enriched ciliary structures. Diminution in Bardet-Biedl syndrome-proteins content combined with our electron microscopic observations raises the possibility that AdipoR1 deficiency might impair ciliary function. Treatment with inhibitors of ceramide synthesis led to substantial elevation of ω3 LC-PUFAs, alleviating photoreceptor degeneration and improving retinal function. These results serve as the proof of concept for a ceramide-targeted strategy to treat retinopathies linked to PUFA deficiency, including age-related macular degeneration.

Retinylidene chromophore hydrolysis from mammalian visual and non-visual opsins.

Rhodopsin (Rho) and cone opsins are essential for detection of light. They respond via photoisomerization, converting their Schiff-base-adducted 11-cis-retinylidene chromophores to the all-trans configuration, eliciting conformational changes to activate opsin signaling. Subsequent Schiff-base hydrolysis releases all-trans-retinal, initiating two important cycles that maintain continuous vision-the Rho photocycle and visual cycle pathway. Schiff-base hydrolysis has been thoroughly studied with photoactivated Rho but not with cone opsins. Using established methodology, we directly measured the formation of Schiff-base between retinal chromophores with mammalian visual and nonvisual opsins of the eye. Next, we determined the rate of light-induced chromophore hydrolysis. We found that retinal hydrolysis from photoactivated cone opsins was markedly faster than from photoactivated Rho. Bovine retinal G protein-coupled receptor (bRGR) displayed rapid hydrolysis of its 11-cis-retinylidene photoproduct to quickly supply 11-cis-retinal and re-bind all-trans-retinal. Hydrolysis within bRGR in native retinal pigment epithelium microsomal membranes was >6-times faster than that of bRGR purified in detergent micelles. N-terminal-targeted antibodies significantly slowed bRGR hydrolysis, while C-terminal antibodies had no effect. Our study highlights the much faster photocycle of cone opsins relative to Rho and the crucial role of RGR in chromophore recycling in daylight. By contrast, in our experimental conditions, bovine peropsin did not form pigment in the presence of all-trans-retinal nor with any mono-cis retinal isomers, leaving uncertain the role of this opsin as a light sensor.

First Report of Sweet Potato Virus E(SPVE) Infecting Sweet Potato in China.

Sweet potato (Ipomoea batatas [L.] Lam.) is a versatile crop, cultivated in the subtropical and tropical areas, as food, fodder, and industrial raw material crop. In China, sweet potato has been used as a health-care food in recent years, as it contains a wide range of nutrients and xenobiotic phytochemicals. However, viral diseases are major constraint for the sweet potato yield and quality, especially the seed production and quality. Over 30 species of viruses infect sweet potato worldwide (Clark et al. 2012). More recently, a few new viruses infected sweet potato were identified, such as sweet potato virus E (SPVE), which was reported in Korea(Jo et al. 2020). In May 2022, a sweet potato sample (JSXZ1) with virus-like symptom, such as mosaic and vein clearing were collected from sweet potato germplasm Xuzhou resource nursery, Jiangsu Province, China (N34˚16', E117˚18') (Fig. S1A). To investigate the virus disease, the sample JSXZ1 showing the typical symptoms of disease was prepared for Small-RNA (sRNA) deep-sequencing. The sRNA library was constructed using TruSeq™ Small RNA Sample Prep Kits (Illumina, San Diego, USA) and sequenced using the Illumine Hiseq 2500 platform by LC-Bop Technologies (Hangzhou) CO., LTD. The sample was sequenced to obtain 26, 358, 439 raw reads and 22, 969, 139 clean reads after quality control trimming and analysis. The Velvet 1.0.5 software was used to de novo assemble the clean reads (18 to 28 nt) into larger contigs, which were then compared with the nucleotide sequences in the National Center for Biotechnology Information (NCBI) database using the BLASTn algorithm. Viruses found in the sample were sweet potato latent virus (SPLV), sweet potato feathery mottle virus (SPFMV), sweet potato chlorotic stunt virus (SPCSV), sweet potato badnavirus A (SPBV-A) and sweet potato badnavirus B (SPBV-B). Surprisingly, besides the viruses listed above, 28 contigs matched sequences of SPVE isolate GS (MH388502). To verify the result, total RNA was extracted from the sample JSXZ1 and from other leave samples (JSXZ2-JSXZ5) that contained SPFMV, SPVC, SPLV, SPVG respectively stored in lab using FastPure Universal Plant Total RNA Isolation Kit (Vazyme Biotech Co., LTD, Nanjing, China). cDNA was synthesized using random primer (hexadeoxyribonucleotide mixture; pd(N)6). The cDNA serves as template in PCR using a newly designed primer pairs based on SPVE p1 gene (SPVE-F: 5'- TCACCAAAAAGAATGCTACAAC-3'/SPVE-R: 5'-GAAATCCTCCCACTCTCCATA-3'). An expected ~500-bp PCR fragment was obtained in JSXZ1, while none of the fragment was obtained from JSXZ2-JSXZ5 (Fig. S1B). The PCR fragment was cloned into pMD18-T vector (Takara Bio Inc., Beijing, China) and plasmid DNA from transformed Escherichia coli DH5α cell (n=3) were commercially sequenced by Sangon Biotech (Shanghai) Co., Ltd. The sequences of the three fragment clones we obtained were 100% identical when compared. A BLASTN analysis of the sequences revealed that they are specific to SPVE and shared 98.62% nucleotide identity to SPVE GS isolate (MH388502) and one sequence was submitted to GenBank (Accession number OQ948331). To determine the occurrence of SPVE in infected sweet potato plants, a total of 37 leaves samples with viral symptom collected from Shandong Province (n=6) and Jiangsu Province (n=31) were indexed by RT-PCR as described before. Only 9 (24.3%) out of 37 from Shandong (n=1) and Jiangsu (n=8) were positive to SPVE respectively. In addition, five additional viruses (SPFMV, SPVC, SPVG, SPLV, SPCSV) were detected among these 37 samples and always in a mixed infection of two or more viruses. To our knowledge, this is the first report of SPVE infecting sweet potato in China. Sweet potato is an important crop in China and other countries (Zhang et al. 2023). China is the largest sweet potato producer all over the world. In addition, as sweet potato is produced through the vegetative propagation mode, thus, more attention should be paid to detection and monitoring of occurrence of SPVE in China.

Evaluation of nematicides for Meloidogyne enterolobii management in sweetpotato.

Sweetpotato is an important crop whose roots are consumed by people worldwide. Meloidogyne enterolobii stands out as a highly deleterious variant among the species of root-knot nematode that causes significant damage in sweetpotato. In the present study, the activity of four nematicides against M. enterolobii was assessed both in vitro and in growth cabinet experiments. After 48 hours of exposure, fluopyram and cyclobutrifluram had a greater negative effect on the motility of M. enterolobii second-stage juveniles (J2s) compared to fluensulfone and hymexazol, with respective median effective concentration (EC50) values of 0.204, 0.423, 22.335 and 216.622 mg L-1. When M. enterolobii eggs were incubated for 72 hours at the highest concentration of each nematicides, the inhibitory hatching effect of cyclobutrifluram (2.5 mg L-1), fluopyram (1.25 mg L-1) and fluensulfone (80 mg L-1) surpassed 85%, whereas hymexazol (640 mg L-1) was only 67%. Similar results were observed in growth cabinet experiments as well. The disease index (DI) and gall index (GI) were significantly decreased by all four nematicides compared to the control. However, the application of hymexazol did not yield a statistically significant difference in the egg masses index compared to the control, a finding which may be attributed to its potentially limited penetrability through the eggshell barrier. Overall, this study has demonstrated that all four nematicides effectively suppress M. enterolobii in sweetpotato, and this is the first report on the nematicidal activity of cyclobutrifluram and hymexazol against M. enterolobii.

Subchronic toxicity study of ferric oxide nanoparticles through intragastric administration: A 94-d, repeated dose study in Sprague Dawley rats.

In this study, the toxicity of ferric oxide nanoparticles (Fe2O3 NPs) administered through gavage to Sprague Dawley (SD) rats for 94 d, consecutively and the recovery after Fe2O3 NPs withdrawal for 30 d were evaluated. The vehicle control group, low-, medium-, and high-dose groups were administered with the vehicle (0.5% sodium carboxymethyl cellulose [CMC-Na]), 125, 250, and 500 mg/kg of Fe2O3 NPs, respectively, administered every morning for 94 d. There was no significant difference in the body weight, food intake, hematological, blood biochemical, and urine indices of SD rats in each administration group and the control group (P > 0.05). There was no significant difference in organ weight, organ indices, and the coefficient of the visceral brain between the SD rats in the different dosage groups and the SD rats in the vehicle control group (P > 0.05). Histopathological observations showed that there was no correlation between the pathological lesions of the organs observed in this study and the dose of Fe2O3 NPs (P > 0.05). The no-observed-adverse-effect level (NOAEL) dose of Fe2O3 NPs was initially determined to be 500 mg/kg administered to SD rats through oral gavage for 94 d, consecutively, followed by recovery after Fe2O3 NPs withdrawal for 30 d.

Evaluation of subchronic toxicity of the compound of diphenhydramine hydrochloride and caffeine after 28 days of repeated oral administration in Sprague-Dawley rats and beagle dogs.

This study was designed to evaluate the subchronic toxicity of the compound of diphenhydramine hydrochloride (DH) and caffeine in Sprague-Dawley (SD) rats and beagle dogs. A total of 180 SD rats (15/sex/group) were randomly divided into the compound low-, medium- and high-dose groups (51, 102, 204 mg/kg), DH group (60 mg/kg), caffeine group (144 mg/kg) and the vehicle control group. Sixty beagle dogs (5/sex/group) were randomly divided into the compound low-, medium- and high-dose groups (male: 14.20, 28.30, 56.60 mg/kg, female: 5.66, 14.20, 28.30 mg/kg), DH group (male: 16.60 mg/kg, female: 8.30 mg/kg), caffeine group (male: 40.00 mg/kg, female: 20.00 mg/kg) and the vehicle control group. Rats and dogs were given continuous oral administration for 28 days following a 28-day recovery period. The adverse effects of the compound on rats and beagle dogs mainly included anorexia and liver function impairment. Most adverse effects induced by administration were reversible. Under the experimental conditions, the no-observed-adverse-effect level (NOAEL) of the compound of DH and caffeine was 51 mg/kg/day for SD rats and 28.30 mg/kg/day (male) and 5.66 mg/kg/day (female) for beagle dogs.

First Report of Meloidogyne enterolobii Infecting Solanum nigrum in China.

Black nightshade (Solanum nigrum) typically grows as a weed species, but it is also widely used as an herb to treat stomach ulcers and dermal infections in many countries (Jabamalairaj et al. 2019). In April 2023, extensive root galls similar to those associated with by root-knot nematodes (RKNs), Meloidogyne spp., were observed on the roots of black nightshade in several commercial fields in Lufeng county (22°55'57.44″N, 115°33'10.31″E), Guangdong Province, China. Upon inspection, there were one to several female RKN in each gall, and egg masses protruding through the root surface. The disease incidence rate was more than 90% in each field using the random sampling method. The nematode population densities in the samples ranged from 279 to 656 eggs and second-stage juveniles (J2s) per gram of fresh roots. Females and egg masses were collected from the roots, and egg masses were incubated in sterile water at 25°C to obtain J2s. Males were not collected in root galling or soil samples. The J2 tail is thin with a broad, bluntly pointed tip, and a clearly defined hyaline tail terminus. Measurements of J2 (n = 20) included: L= 440 ± 30.5 (384 to 500) µm, stylet = 12.3 ± 0.7 (11.3 to 13.7) µm, tail = 51.6 ± 2.4 (47.9 to 57.0) µm. For females (n = 15), vulval slit length = 25.5 ±1.9 (23.6 to 29.1) µm, vulval slit to anus distance = 22.1 ± 3.0 (18.2 to 27.0) µm. Stylet knobs in females are divided longitudinally by a groove so that each knob appears as two. The perineal patterns are round to ovoid, with coarse and smooth striae, moderate to high dorsal arch and mostly lacking distinct lateral lines. Morphological characteristics from J2s and perineal patterns from adult females fit the original description of M. enterolobii (Yang and Eisenback 1983). Furthermore, species identity was explored by sequencing the D2-D3 region of the 28S rRNA gene using primers D2A/D3B (Vrain et al. 1992), and the mtDNA cytochrome c oxidase I (COI) genes using primers JB3/JB5 (Derycke et al. 2005). The sequences for the target genes were 759 bp (GenBank Accession No. OR046056) and 447 bp (GenBank Accession No. OR042802), respectively. The BLAST analysis suggested 98.17~99.78% similarities to other available M. enterolobii sequences in GenBank. Species identity was further confirmed with the species-specific primer pair Me-F/Me-R (Long et al. 2006). An approximately 240 bp PCR product was produced, which was previously reported only for M. enterolobii, whereas no product was obtained from control populations of M. incognita or M. javanica. The pathogenicity test was conducted in a greenhouse at 28°C using seedlings of S. nigrum maintained in pots containing 500 cm3 sterilized soil. Ten replicates were inoculated with 800 eggs and J2s of the original population of M. enterolobii, while another 10 replicates of control plants were not inoculated. After 7 weeks, the inoculated plants exhibited galling symptoms similar to plants observed in the field, and females and egg masses were obtained by dissecting galls. No galling symptoms were observed on control plants. These results confirmed the nematode's pathogenicity. To our knowledge, this is the first record of M. enterolobii parasitizing black nightshade. M. enterolobii stands out as a highly deleterious variant among the species of RKNs owing to its extensive repertoire of host plants, pathogenicity, and proficiency in thriving and multiplying even on crops possessing resistance genes (Sikandar, 2022). In addition to being a medicinal plant, S. nigrum is a widespread weed found in fields throughout China. This report also showed that S. nigrum could play an important role as a reservoir host of M. enterolobii aiding its survival, reproduction, spread, and increasing the potential damage for host crops.

IbINV Positively Regulates Resistance to Black Rot Disease Caused by Ceratocystis fimbriata in Sweet Potato.

Black rot disease, caused by Ceratocystis fimbriata Ellis & Halsted, severely affects both plant growth and post-harvest storage of sweet potatoes. Invertase (INV) enzymes play essential roles in hydrolyzing sucrose into glucose and fructose and participate in the regulation of plant defense responses. However, little is known about the functions of INV in the growth and responses to black rot disease in sweet potato. In this study, we identified and characterized an INV-like gene, named IbINV, from sweet potato. IbINV contained a pectin methylesterase-conserved domain. IbINV transcripts were most abundant in the stem and were significantly induced in response to C. fimbriata, salicylic acid, and jasmonic acid treatments. Overexpressing IbINV in sweet potato (OEV plants) led to vigorous growth and high resistance to black rot disease, while the down-regulation of IbINV by RNA interference (RiV plants) resulted in reduced plant growth and high sensitivity to black rot disease. Furthermore, OEV plants contained a decreased sucrose content and increased hexoses content, which might be responsible for the increased INV activities; not surprisingly, RiV plants showed the opposite effects. Taken together, these results indicate that IbINV positively regulates plant growth and black rot disease resistance in sweet potato, mainly by modulating sugar metabolism.

Endothelium-Specific GTP Cyclohydrolase I Overexpression Restores Endothelial Function in Aged Mice.

This study tested the hypothesis that endothelium-specific GTP cyclohydrolase I (GTPCH I) overexpression (Tg-GCH) restores age-associated endothelial dysfunction in vivo. Aortic GTPCH I expression and serum nitric oxide (NO) release were measured in young and aged mice. Aortic rings from young and aged wild-type (WT) mice and aged Tg-GCH mice were suspended for isometric tension recording. A hind limb ischemia model was used to measure blood flow recovery. Aged mice showed reduced GTPCH I expression in the aorta and decreased NO levels in serum. Compared with aged WT mice, Tg-GCH significantly elevated NO levels in serum in aged Tg-GCH mice, restored the impaired aortic relaxation in response to acetylcholine, and significantly elevated aortic constriction in response to L-NAME. Importantly, aged Tg-GCH mice displayed a significant increase in blood flow recovery compared with aged WT mice. GTPCH I reduction contributes to aging-associated endothelial dysfunction, which can be retarded by Tg-GCH.

Selenium ameliorates aflatoxin B1-induced uterine injury in female mice and necrosis of human endometrial microvascular endothelial cells.

This study aimed to observe the effects of Selenium (Se) on aflatoxin B1 (AFB1)-induced uterine injury in female mice and necrosis of human endometrial microvascular endothelial cells (HEMECs). Fifty female mice were randomly divided into control group; AFB1 group; Se group (0.2 mg/kg each day); AFB1 + Se group; and positive control group. After continuous treatment for 30 days, uterine tissues were harvested, which were used for hematoxylin and eosin (H&E) staining. Necrosis-related proteins including RIPK1, RIPK3, and MLKL were examined in uterine tissues using western blot and immunohistochemistry. HEMECs were treated with different concentrations of AFB1 or Se, which were used for selecting the optimal concentrations. RIPK1, RIPK3 and MLKL expression was detected in HEMECs exposed to AFB1 and/or Se via western blot and immunofluorescence. H&E staining results showed that AFB1 induced uterine injury of female male, which was ameliorated by Se treatment. According to western blot and immunohistochemistry, RIPK1, RIPK3, and MLKL expression was distinctly increased in uterine tissues of AFB1-treated mice, which was decreased by Se treatment. Cell viability of HEMECs was gradually lowered as the concentrations of AFB1and Se increased. A 10-µM AFB1 exposure significantly increased RIPK1, RIPK3, and MLKL expression in HEMECs, which was improved following co-treatment with 5-µM Se. Thus, our findings revealed that Se may ameliorate AFB1-induced uterine injury in female mice and necrosis of HEMECs.

Novel Two-Dimensional MoS2-Ti4+ Nanomaterial for Efficient Enrichment of Phosphopeptides and Large-Scale Identification of Histidine Phosphorylation by Mass Spectrometry.

Due to its key roles in regulating the occurrence and development of cancer, protein histidine phosphorylation has been increasingly recognized as an important form of post-translational modification in recent years. However, large-scale analysis of histidine phosphorylation is much more challenging than that of serine/threonine or tyrosine phosphorylation, mainly because of its acid lability. In this study, MoS2-Ti4+ nanomaterials were synthesized using a solvothermal method and taking advantage of the electrostatic adsorption between MoS2 nanosheets and Ti4+. The MoS2-Ti4+ nanomaterials have the advantage of the combined affinity of Ti4+ and Mo toward phosphorylation under medium acidic conditions (pH = 3), which is crucial for preventing hydrolysis and loss of histidine phosphorylation during enrichment. The feasibility of using the MoS2-Ti4+ nanomaterial for phosphopeptide enrichment was demonstrated using mixtures of ß-casein and bovine serum albumin (BSA). Further evaluation revealed that the MoS2-Ti4+ nanomaterial is capable of enriching synthetic histidine phosphopeptides from 1000 times excess tryptic-digested HeLa cell lysate. Application of the MoS2-Ti4+ nanomaterials for large-scale phosphopeptide enrichment results in the identification of 10 345 serine, threonine, and tyrosine phosphosites and the successful mapping of 159 histidine phosphosites in HeLa cell lysates, therefore indicating great potential for deciphering the vital biological roles of protein (histidine) phosphorylation.

Artificial neural network-based models used for predicting 28- and 90-day mortality of patients with hepatitis B-associated acute-on-chronic liver failure.

BACKGROUND: This study aimed to develop prognostic models for predicting 28- and 90-day mortality rates of hepatitis B virus (HBV)-associated acute-on-chronic liver failure (HBV-ACLF) through artificial neural network (ANN) systems. METHODS: Six hundred and eight-four cases of consecutive HBV-ACLF patients were retrospectively reviewed. Four hundred and twenty-three cases were used for training and constructing ANN models, and the remaining 261 cases were for validating the established models. Predictors associated with mortality were determined by univariate analysis and were then included in ANN models for predicting prognosis of mortality. The receiver operating characteristic curve analysis was used to evaluate the predictive performance of the ANN models in comparison with various current prognostic models. RESULTS: Variables with statistically significant difference or important clinical characteristics were input in the ANN training process, and eight independent risk factors, including age, hepatic encephalopathy, serum sodium, prothrombin activity, γ-glutamyltransferase, hepatitis B e antigen, alkaline phosphatase and total bilirubin, were eventually used to establish ANN models. For 28-day mortality in the training cohort, the model's predictive accuracy (AUR 0.948, 95% CI 0.925-0.970) was significantly higher than that of the Model for End-stage Liver Disease (MELD), MELD-sodium (MELD-Na), Chronic Liver Failure-ACLF (CLIF-ACLF), and Child-Turcotte-Pugh (CTP) (all p < 0.001). In the validation cohorts the predictive accuracy of ANN model (AUR 0.748, 95% CI: 0.673-0.822) was significantly higher than that of MELD (p = 0.0099) and insignificantly higher than that of MELD-Na, CTP and CLIF-ACLF (p > 0.05). For 90-day mortality in the training cohort, the model's predictive accuracy (AUR 0.913, 95% CI 0.887-0.938) was significantly higher than that of MELD, MELD-Na, CTP and CLIF-ACLF (all p < 0.001). In the validation cohorts, the prediction accuracy of the ANN model (AUR 0.754, 95% CI: 0.697-0.812 was significantly higher than that of MELD (p = 0.019) and insignificantly higher than MELD-Na, CTP and CLIF-ACLF (p > 0.05). CONCLUSIONS: The established ANN models can more accurately predict short-term mortality risk in patients with HBV- ACLF. The main content has been postered as an abstract at the AASLD Hepatology Conference (https://doi.org/10.1002/hep.30257).

A novel mass spectrometry method for the absolute quantification of several cytochrome P450 and uridine 5'-diphospho-glucuronosyltransferase enzymes in the human liver.

Cytochrome P450 (CYP450) and 5'-diphosphate glucuronosyltransferases (UGT) are the two major families of drug-metabolizing enzymes in the human liver microsome (HLM). As a result of their frequent abundance fluctuation among populations, the accurate quantification of these enzymes in different individuals is important for designing patient-specific dosage regimens in the framework of precision medicine. The preparation and quantification of internal standards is an essential step for the quantitative analysis of enzymes. However, the commonly employed stable isotope labeling-based strategy (QconCAT) suffers from requiring very expensive isotopic reagents, tedious experimental procedures, and long labeling times. Furthermore, arginine-to-proline conversion during metabolic isotopic labeling compromises the quantification accuracy. Therefore, we present a new strategy that replaces stable isotope-labeled amino acids with lanthanide labeling for the preparation and quantification of QconCAT internal standard peptides, which leads to a threefold reduction in the reagent costs and a fivefold reduction in the time consumed. The absolute amount of trypsin-digested QconCAT peptides can be obtained by lanthanide labeling and inductively coupled plasma-optical emission spectrometry (ICP-OES) analysis with a high quantification accuracy (%RE < 20%). By taking advantage of the highly selective and facile ICP-OES procedure and multiplexed large-scale absolute target protein quantification using biological mass spectrometry, this strategy was successfully used for the absolute quantification of drug-metabolizing enzymes. We obtained good linearity (correlation coefficient > 0.95) over concentrations spanning 2.5 orders of magnitude with improved sensitivity (limit of quantification = 2 fmol) in nine HLM samples, indicating the potential of this method for large-scale absolute target protein quantification in clinical samples. Graphical abstract.

Protective Effect of Probiotics against Esophagogastric Variceal Rebleeding in Patients with Liver Cirrhosis after Endoscopic Therapy.

BACKGROUND Probiotic therapy has been shown to be beneficial against some liver diseases. However, there is still uncertainty regarding the clinical efficacy of probiotics for the treatment of variceal rebleeding. This research explored the efficacy of probiotics in variceal rebleeding. MATERIAL AND METHODS This was a retrospective study of 704 consecutive patients with liver cirrhosis who recovered from esophagogastric variceal bleeding after endoscopic treatment. Patients were subdivided into a probiotics cohort (n=214) and a non-probiotics cohort (n=490) based on the cumulative defined daily dose (cDDD) of probiotics received during follow-up. Propensity score matching was utilized to obtain a relatively balanced cohort of 200 patients per group for the analysis. Patients were monitored for rebleeding during the one-year follow-up. RESULTS Multivariate Cox regression analysis revealed that probiotic therapy (≥28cDDD) was an independent protector against rebleeding (AHR=0.623; 95% CI=0.488-0.795; P<0.001). After propensity score matching, Kaplan-Meier analysis revealed that the rebleeding rate was higher in the non-probiotics cohort (n=200) than in the probiotics cohort (n=200) (56.0% vs. 44.0%, P=0.002). The incidence of rebleeding decreased with increased probiotic dosage (56.0%, 48.5%, 43.3%, and 38.1% in <28 cDDD, 28-60 cDDD, 61-90 cDDD, and >90 cDDD groups, respectively; P=0.011). The median rebleeding interval in the probiotics cohort (n=95) was significantly longer than that in the non-probiotics cohort (n=261) (147.0 vs. 91.0 days; P<0.001). CONCLUSIONS Adjuvant probiotic therapy significantly reduced the incidence of variceal rebleeding and delayed rebleeding after endotherapy in patients with cirrhosis.

The application of exogenous PopW increases the tolerance of Solanum lycopersicum L. to drought stress through multiple mechanisms.

Tomato is a major cultivated vegetable species of great economic importance throughout the world, but its fruit yield is severely impaired by drought stress. PopW, a harpin protein from Ralstonia solanacearum ZJ3721, plays vital roles in various plant defence responses and growth. In this study, we observed that the foliar application of PopW increased tomato drought tolerance. Our results showed that compared with water-treated plants, PopW-treated plants presented a significantly higher recovery rate and leaf relative water content under drought-stress conditions. PopW decreased the malondialdehyde content and relative electrical conductivity by 40.2% and 21%, respectively. Drought disrupts redox homeostasis through the excessive accumulation of reactive oxygen species (ROS). PopW-treated plants displayed an obvious reduction in ROS accumulation due to enhanced activities of the antioxidant enzyme catalase, superoxide dismutase and peroxidase. Moreover, PopW promoted early stomatal closure, thereby minimizing the water loss rate of plants under drought stress. Further investigation revealed that endogenous abscisic acid (ABA) levels and the transcript levels of drought-responsive genes involved in ABA signal transduction pathways increased in response to PopW. These results confirm that PopW increases drought tolerance through multiple mechanisms involving an enhanced water-retention capacity, balanced redox homeostasis, increased osmotic adjustment, reduced membrane damage and decreased stomatal aperture, suggesting that the application of exogenous PopW may be a potential method to enhance tomato drought tolerance.

Two-Dimensional MoS2-Based Zwitterionic Hydrophilic Interaction Liquid Chromatography Material for the Specific Enrichment of Glycopeptides.

Mass spectrometry (MS)-based glycoproteomics research requires highly efficient sample preparation to eliminate interference from non-glycopeptides and to improve the efficiency of glycopeptide detection. In this work, a novel MoS2/Au-NP (gold nanoparticle)-L-cysteine nanocomposite was prepared for glycopeptide enrichment. The two-dimensional (2D) structured MoS2 nanosheets served as a matrix that could provide a large surface area for immobilizing hydrophilic groups (such as L-cysteine) with low steric hindrance between the materials and the glycopeptides. As a result, the novel nanomaterial possessed an excellent ability to capture glycopeptides. Compared to commercial zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) materials, the novel nanomaterials exhibited excellent enrichment performance with ultrahigh selectivity and sensitivity (approximately 10 fmol), high binding capacity (120 mg g-1), high enrichment recovery (more than 93%), satisfying batch-to-batch reproducibility, and good universality for glycopeptide enrichment. In addition, its outstanding specificity and efficiency for glycopeptide enrichment was confirmed by the detection of glycopeptides from an human serum immunoglobulin G (IgG) tryptic digest in quantities as low as a 1:1250 molar ratio of IgG tryptic digest to bovine serum albumin tryptic digest. The novel nanocomposites were further used for the analysis of complex samples, and 1920 glycopeptide backbones from 775 glycoproteins were identified in three replicate analyses of 50 µg of proteins extracted from HeLa cell exosomes. The resulting highly informative mass spectra indicated that this multifunctional nanomaterial-based enrichment method could be used as a promising tool for the in-depth and comprehensive characterization of glycoproteomes in MS-based glycoproteomics.

Development and external validation of a prognostic nomogram for acute decompensation of chronic hepatitis B cirrhosis.

BACKGROUND: Acute decompensation (AD) has been shown to be associated with a high mortality rate for cirrhosis patients. This study aimed to develop a prognostic nomogram to evaluating the individual prognosis for AD of cirrhosis in chronic hepatitis B (CHB). METHODS: The nomogram was developed using data from a retrospective study on 509 patients hospitalized for AD of CHB cirrhosis from October 2008 to February 2014 at the Beijing Ditan Hospital, Capital Medical University. The predictive accuracy, discriminative ability, and clinical net benefit were evaluated by concordance index (C-index), calibration curves, and decision curve analysis (DCA). The results were validated on 620 patients consecutively enrolled from January 2005 to December 2010 at the Renji Hospital, Shanghai Jiao Tong University,. RESULTS: On multivariate analysis of the derivation cohort, independent factors included in the nomogram were age, previous decompensation, bacterial infection, hepatic encephalopathy, and total bilirubin. The calibration curve for the probability of survival showed good agreement between the nomogram and actual observation. The nomogram had a C-index of 0.897, which was statistically higher than the C-index values of CTP (0.793), MELD (0.821), SOFA (0.868), or the Chronic Liver Failure Consortium AD (CLIF-C AD) (0.716) scores (p <  0.001 for all). Using DCA, the nomogram also demonstrated superior net benefits over other score models. The results were confirmed in the validation cohort. CONCLUSIONS: The proposed nomogram enables more-accurate individualized prediction of survival than MELD, CTP, SOFA, or CLIF-C AD scores for AD of CHB cirrhosis patients.

Ectopic expression of a cyanobacterial flavodoxin in creeping bentgrass impacts plant development and confers broad abiotic stress tolerance.

Flavodoxin (Fld) plays a pivotal role in photosynthetic microorganisms as an alternative electron carrier flavoprotein under adverse environmental conditions. Cyanobacterial Fld has been demonstrated to be able to substitute ferredoxin of higher plants in most electron transfer processes under stressful conditions. We have explored the potential of Fld for use in improving plant stress response in creeping bentgrass (Agrostis stolonifera L.). Overexpression of Fld altered plant growth and development. Most significantly, transgenic plants exhibited drastically enhanced performance under oxidative, drought and heat stress as well as nitrogen (N) starvation, which was associated with higher water retention and cell membrane integrity than wild-type controls, modified expression of heat-shock protein genes, production of more reduced thioredoxin, elevated N accumulation and total chlorophyll content as well as up-regulated expression of nitrite reductase and N transporter genes. Further analysis revealed that the expression of other stress-related genes was also impacted in Fld-expressing transgenics. Our data establish a key role of Fld in modulating plant growth and development and plant response to multiple sources of adverse environmental conditions in crop species. This demonstrates the feasibility of manipulating Fld in crop species for genetic engineering of plant stress tolerance.

Nomogram prediction of individual prognosis of patients with hepatocellular carcinoma.

BACKGROUND: The purpose of this study was to develop an effective nomogram capable of estimating the individual survival outcomes of patients with hepatocellular carcinoma (HCC), and compare the predictive accuracy and discriminative ability with other staging systems. METHODS: The nomogram was established based on a retrospective study of 661 patients newly diagnosed with HCC at the Beijing Ditan Hospital (Beijing, China), Capital Medical University, between October 2008 and July 2012. The predictive accuracy and discriminative ability of the previously developed nomogram were assessed by C-index and calibration curves, and were compared to seven current commonly used staging systems. The results were validated, using a bootstrap approach to correct for bias, in a prospective study of 220 patients consecutively enrolled between August 2012 and March 2013. RESULTS: Multivariate analysis of the primary cohort for survival analysis identified the independent factors to be aspartate aminotransferase, É£-glutamyl transpeptidase, white blood cell count, neutrophil-to-lymphocyte ratio, prothrombin activity, α-fetoprotein, tumor number and size, lymph node metastasis, and portal vein involvement, which were all included to build the nomogram. The calibration curve for predicting the probability of survival showed consistency between the nomogram and the actual observation. The C-index of the nomogram was 0.81 (95% confidence interval, 0.79-0.82), which was statistically better than that of the Tumor, Node, Metastasis staging (0.71), Barcelona Clinic Liver Cancer staging (0.77), Okuda (0.62), Japan Integrated Staging (0.73), Cancer of the Liver Italian Program score (0.76), Chinese University Prognostic Index (0.68), and the Groupe d' Etude et de Traitement du Carcinome Hepatocellulaire Prognostic classification (0.65) (p < 0.001 for all). The results were validated in the prospective validation cohort. CONCLUSIONS: The prognostic nomogram resulted in more accurate individualized risk estimates for overall survival in HCC patients.