RING/U-box E3 protein BIR1 interacts with and ubiquitinates barley growth repressor BROAD LEAF1.

Establishment of final leaf size in plants relies on the precise regulation of two interconnected processes, cell division and cell expansion. The barley (Hordeum vulgare) protein BROAD LEAF1 (BLF1) limits cell proliferation and leaf growth in the width direction. However, how the levels of this potent repressor of leaf growth are controlled remains unclear. Here we used a yeast two-hybrid screen to identify the BLF1-INTERACTING RING/U-BOX 1 (BIR1) E3 ubiquitin ligase that interacts with BLF1 and confirmed the interaction of the two proteins in planta. Inhibiting the proteasome caused overaccumulation of a BLF1-eGFP fusion protein when co-expressed with BIR1, and an in vivo ubiquitination assay in bacteria confirmed that BIR1 can mediate ubiquitination of BLF1 protein. Consistent with regulation of endogenous BLF1 in barley by proteasomal degradation, inhibition of the proteasome in BLF1-vYFP-expressing barley plants caused an accumulation of the BLF1 protein. The BIR1 protein co-localized with BLF1 in nuclei and appeared to reduce BLF1 protein levels. Analysis of bir1-1 knock-out mutants suggested the involvement of BIR1 in leaf growth control, although mainly on leaf length. Together, our results suggest that proteasomal degradation, in part mediated by BIR1, helps fine-tune BLF1 protein levels in barley.

Identifying PE2 and PE5 Proteins from Existing Mass Spectrometry Data Using pFind.

The Chromosome-Centric Human Proteome Project (C-HPP) aims to identify all proteins encoded by the human genome. Currently, the human proteome still contains approximately 2000 PE2-PE5 proteins, referring to annotated coding genes that lack sufficient protein-level evidence. During the past 10 years, it has been increasingly difficult to identify PE2-PE5 proteins in C-HPP approaches due to the limited occurrence. Therefore, we proposed that reanalyzing massive MS data sets in repository with newly developed algorithms may increase the occurrence of the peptides of these proteins. In this study, we downloaded 1000 MS data sets via the ProteomeXchange database. Using pFind software, we identified peptides referring to 1788 PE2-PE5 proteins. Among them, 11 PE2 and 16 PE5 proteins were identified with at least 2 peptides, and 12 of them were identified using 2 peptides in a single data set, following the criteria of the HPP guidelines. We found translation evidence for 16 of the 11 PE2 and 16 PE5 proteins in our RNC-seq data, supporting their existence. The properties of the PE2 and PE5 proteins were similar to those of the PE1 proteins. Our approach demonstrated that mining PE2 and PE5 proteins in massive data repository is still worthy, and multidata set peptide identifications may support the presence of PE2 and PE5 proteins or at least prompt additional studies for validation. Extremely high throughput could be a solution to finding more PE2 and PE5 proteins.

Effects of protein levels on production performance, nutritional values, and phase feeding of two-spotted cricket.

Two experiments were conducted to determine the effects of different protein levels on the growth performance, feed efficiency and nutritional values, and phase feeding of the 2-spotted cricket (Gryllus bimaculatus de Geer). In experiment 1, 4 crude protein (CP) diets were formulated to contain 18%, 20%, 22%, or 24% CP, respectively. A sample of 7-day-old 3,600 crickets was equally divided into 24 plastic boxes (150 crickets each) in a completely randomized design with 4 diets and 6 replications. In experiment 2, 2-phase feedings were used. For starting period (days 7-18), crickets in all treatments were fed a diet containing 22% CP. During the growing period (days 19-35), 3 groups of crickets were fed diets containing 18%, 20%, and 22% CP. In the overall period of experiment 1, the crickets fed with 22% CP diet had greater body weight compared to those fed with 18% CP diet. In addition, the crickets fed with 22% CP diet had the lowest feed conversion ratio (FCR). The broken-line model indicated the growth pattern changed on day 18. In experiment 2, the crickets fed with 20% CP diet from days 19 to 35 had greater growth performance and lower FCR than those fed with 18% CP, but not different from those fed with 22% CP. In conclusion, 22% CP can increase growth performance by improving the feed efficiency of crickets. The implementation of 2-phase feedings using 20% CP, during the growing period, could be considered as a cost-effective strategy for sustainable cricket production.

Assessment of Concentration KRT6 Proteins in Tumor and Matching Surgical Margin from Patients with Head and Neck Squamous Cell Carcinoma.

Head and neck squamous cell carcinomas (HNSCCs) are one of the most frequently detected cancers in the world; not all mechanisms related to the expression of keratin in this type of cancer are known. The aim of this study was to evaluate type II cytokeratins (KRT): KRT6A, KRT6B, and KRT6C protein concentrations in 54 tumor and margin samples of head and neck squamous cell carcinoma (HNSCC). Moreover, we examined a possible association between protein concentration and the clinical and demographic variables. Protein concentrations were measured using enzyme-linked immunosorbent assay (ELISA). Significantly higher KRT6A protein concentration was found in HNSCC samples compared to surgical margins. An inverse relationship was observed for KRT6B and KRT6C proteins. We showed an association between the KRT6C protein level and clinical parameters T and N in tumor and margin samples. When analyzing the effect of smoking and drinking on KRT6A, KRT6B, and KRT6C levels, we demonstrated a statistically significant difference between regular or occasional tobacco and alcohol habits and patients who do not have any tobacco and alcohol habits in tumor and margin samples. Moreover, we found an association between KRT6B and KRT6C concentration and proliferative index Ki-67 and HPV status in tumor samples. Our results showed that concentrations of KRT6s were different in the tumor and the margin samples and varied in relation to clinical and demographic parameters. We add information to the current knowledge about the role of KRT6s isoforms in HNSCC. We speculate that variations in the studied isoforms of the KRT6 protein could be due to the presence and development of the tumor and its microenvironment. It is important to note that the analyses were performed in tumor and surgical margins and can provide more accurate information on the function in normal and cancer cells and regulation in response to various factors.

Cellular control of protein levels: A systems biology perspective.

How cells regulate protein levels is a central question of biology. Over the past decades, molecular biology research has provided profound insights into the mechanisms and the molecular machinery governing each step of the gene expression process, from transcription to protein degradation. Recent advances in transcriptomics and proteomics have complemented our understanding of these fundamental cellular processes with a quantitative, systems-level perspective. Multi-omic studies revealed significant quantitative, kinetic and functional differences between the genome, transcriptome and proteome. While protein levels often correlate with mRNA levels, quantitative investigations have demonstrated a substantial impact of translation and protein degradation on protein expression control. In addition, protein-level regulation appears to play a crucial role in buffering protein abundances against undesirable mRNA expression variation. These findings have practical implications for many fields, including gene function prediction and precision medicine.

Optimization of Higher-Energy Collisional Dissociation Fragmentation Energy for Intact Protein-Level Tandem Mass Tag Labeling.

Isobaric chemical tag labeling (e.g., TMT) is a commonly used approach in quantitative proteomics, and quantification is enabled through detection of low-mass reporter ions generated after MS2 fragmentation. Recently, we have introduced and optimized an intact protein-level TMT labeling platform that demonstrated >90% labeling efficiency in complex samples with top-down proteomics. Higher-energy collisional dissociation (HCD) is commonly utilized for isobaric tag-labeled peptide fragmentation because it produces accurate reporter ion intensities and avoids loss of low mass ions. HCD energies have been optimized for isobaric tag labeled-peptides but have not been systematically evaluated for isobaric tag-labeled intact proteins. In this study, we report a systematic evaluation of normalized HCD fragmentation energies (NCEs) on TMT-labeled HeLa cell lysate using top-down proteomics. Our results suggested that reporter ions often result in higher ion intensities at higher NCEs. Optimal fragmentation of intact proteins for identification, however, required relatively lower NCE. We further demonstrated that a stepped NCE scheme with energies from 30% to 50% resulted in optimal quantification and identification of TMT-labeled HeLa proteins. These parameters resulted in an average reporter ion intensity of ∼4E4 and average proteoform spectrum matches (PrSMs) of >1000 per RPLC-MS/MS run with a 1% false discovery rate (FDR) cutoff.

The basic leucine zipper transcription factor OsbZIP83 and the glutaredoxins OsGRX6 and OsGRX9 facilitate rice iron utilization under the control of OsHRZ ubiquitin ligases.

Under low iron availability, plants induce the expression of various genes for iron uptake and translocation. The rice (Oryza sativa) ubiquitin ligases OsHRZ1 and OsHRZ2 cause overall repression of these iron-related genes at the transcript level, but their protein-level regulation is unclear. We conducted a proteome analysis to identify key regulators whose abundance was regulated by OsHRZs at the protein level. In response to iron deficiency or OsHRZ knockdown, many genes showed differential regulation between the transcript and protein levels, including the TGA-type basic leucine zipper transcription factor OsbZIP83. We also identified two glutaredoxins, OsGRX6 and OsGRX9, as OsHRZ-interacting proteins in yeast and plant cells. OsGRX6 also interacted with OsbZIP83. Our in vitro degradation assay suggested that OsbZIP83, OsGRX6 and OsGRX9 proteins are subjected to 26S proteasome- and OsHRZ-dependent degradation. Proteome analysis and our in vitro degradation assay also suggested that OsbZIP83 protein was preferentially degraded under iron-deficient conditions in rice roots. Transgenic rice lines overexpressing OsGRX9 and OsbZIP83 showed improved tolerance to iron deficiency. Expression of iron-related genes was affected in the OsGRX9 and OsGRX6 knockdown lines, suggesting disturbed iron utilization and signaling. OsbZIP83 overexpression lines showed enhanced expression of OsYSL2 and OsNAS3, which are involved in internal iron translocation, in addition to OsGRX9 and genes related to phytoalexin biosynthesis and the salicylic acid pathway. The results suggest that OsbZIP83, OsGRX6 and OsGRX9 facilitate iron utilization downstream of the OsHRZ pathway.

Cross-Ethnic Variant Screening and Burden Analysis of PTPA in Parkinson's Disease.

BACKGROUND: Recently, homozygous variants in PTPA were identified as the disease cause for two pedigrees with early-onset parkinsonism and intellectual disability. Although the initial link between PTPA and parkinsonism has been established, further replication was still necessary. OBJECTIVES: To evaluate the genetic role of PTPA in Parkinson's disease (PD). METHODS: We analyzed rare variants of PTPA in cohorts of Asian and European ancestries (Ncase = 2743, Ncontrol = 8177) with whole-exome sequencing, and further explored the functional effect of the target variant. RESULTS: One patient with early-onset PD from a consanguineous family carried the homozygous variant p.Met329Val, while her parents and elder sister with heterozygous p.Met329Val were healthy. This patient developed minor cognitive decline within 1 year, with a Montreal Cognitive Assessment (MoCA) score dropping from 28 to 25. Functional exploration with overexpression studies suggested that this variant was associated with decreased protein phosphatase 2A (PTPA) protein level by affecting protein stability, but not mRNA expression. CONCLUSIONS: These results have broadened the mutation spectrum of PTPA, and paved the way for further research into the role of PTPA in PD. © 2023 International Parkinson and Movement Disorder Society.

Intervention of Dietary Protein Levels on Muscle Quality, Antioxidation, and Autophagy in the Muscles of Triploid Crucian Carp (Carassius carassius Triploid).

The aim of this study is to investigate the effect of dietary protein levels on flesh quality, oxidative stress, and autophagy status in the muscles of triploid crucian carp (Carassius carassius triploid), and the related molecular mechanisms. Six experimental diets with different protein levels (26%, 29%, 32%, 35%, 38%, 41%) were formulated. A total of 540 fish with an initial weight of 11.79 ± 0.09 g were randomly assigned to 18 cages and six treatments with three replicates of 30 fish each for 8 weeks feeding. It could be found that the whole-body ash content significantly increased in high protein level groups (p < 0.05). The 29% dietary protein level group exhibited the highest muscle moisture, although there was an inconspicuous decrease in the chewiness of the muscles when compared with the other groups. The dietary protein level influenced the content of free amino acids and nucleotides, especially the content of flavor amino acids, which exhibited an increasing tendency along with the increasing protein level, such as alanine and glutamic acid, while the flavor nucleotides showed different fluctuation trends. Moreover, the genes related to muscle development were shown to be influenced by the dietary protein level, especially the expression of MRF4, which was up-regulated with the increasing dietary protein levels. The 29% dietary protein level promoted the majority of analyzed muscle genes expression to the highest level when compared to other dietary levels, except the Myostain, whose expression reached its highest at 38% dietary protein levels. Furthermore, the effect of dietary protein levels on antioxidant signaling pathway genes were also examined. High protein levels would boost the expression of GSTα; GPX1 and GPX4α mRNA expression showed the highest level at the 32% dietary protein group. The increasing dietary protein level decreased both mRNA and protein expressions of Nrf2 by up-regulating Keap1. Autophagy-related gene expression levels reached the peak at 32% dietary protein level, as evidenced by a similar change in protein expression of FoxO1. In summary, muscle nutritional composition, antioxidative pathways, and autophagy levels were affected by the dietary protein levels. A total of 29-32% dietary protein level would be the appropriate level range to improve muscle quality and promote the antioxidant and autophagy capacity of triploid crucian carp muscles.

Effects of different dietary protein levels on intestinal aquaporins in weaned piglets.

This study was conducted to investigate the relationship between changes in intestinal aquaporins (AQPs) in piglets fed diets with different protein levels and nutritional diarrhoea in piglets. Briefly, 96 weaned piglets were randomly divided into four groups fed diets with crude protein (CP) levels of 18%, 20%, 22% and 24%. The small intestines and colons of the weaned piglets were collected, and several experiments were conducted. In the small intestine, AQP4 protein expression was higher in weaned piglets fed the higher-CP diets (22% and 24% CP) than in those fed the 20% CP diet except at 72 h (p < 0.01). At 72 h, the AQP4 protein expression in the small intestine was lower in the 18% group than in the other three groups (p < 0.01). Under 20% CP feeding, AQP2, AQP4 and AQP9 protein expression in the colons of piglets peaked at certain time points. The AQP2 and AQP4 mRNA levels in the colon and the AQP4 and AQP4 mRNA levels in the distal colon were approximately consistent with the protein expression levels. However, the AQP9 mRNA content in the colon was highest in the 18% group, and the AQP2 mRNA content in the distal colon was significantly higher in the 24% group than in the 20% group. AQP2 and AQP4 were expressed mainly around columnar cells in the upper part of the smooth colonic intestinal villi, and AQP9 was expressed mainly on columnar cells and goblet cells in the colonic mucosa. In conclusion, 20% CP is beneficial to the normal expression of AQP4 in the small intestine, AQP2, AQP4 and AQP9 in the colon of weaned piglets, which in turn maintains the balance of intestinal water absorption and secretion in piglets.

Dietary protein requirement for captive juvenile green turtles (Chelonia mydas).

Head-starting programs are extremely important for restoring the population of sea turtles in wild whereas husbandry conditions and feeding regimens of captive turtles are still limited. In the current study, the optimal dietary protein requirement for green turtle (Chelonia mydas) was investigated to support rearing in head-starting programs. Twenty-five-day-old turtles (44.5-46.2 g body weight, n = 45) were randomly distributed into 15 experimental plastic tanks, comprising three treatment replications of 3 turtles each. They were fed fishmeal-based feeds containing different levels of protein (30%, 35%, 40%, 45%, and 50%) for 8 weeks. At the end of feeding trial, growth performance (specific growth rate = 1.86% body weight/day) and feed utilization (protein efficiency ratio = 3.30 g gain/g protein) were highest in turtles fed with 40% protein in feed (p < .05). These nutritional responses were significantly supported by specific activities of fecal digestive enzymes, especially trypsin, chymotrypsin, amylase, and the amylase/trypsin ratio. Also, this dietary level improved the deposition of calcium and phosphorus in carapace, supporting a hard carapace and strong healthy bones. There were no negative effects in general health status of reared turtles, as indicated by hematological parameters. Based on a broken-line analysis between dietary protein levels and specific growth rate, the optimal protein level for green turtles was estimated as 40.6%. Findings from the current study support the use of artificial diets of specific protein levels to rear captive green turtle before release to natural habitats.

Elucidation of pathological mechanism caused by human disease mutation in CaMKIIß.

Recently, we have identified CaMKIIα and CaMKIIß mutations in patients with neurodevelopmental disorders by whole exome sequencing study. Most CaMKII mutants have increased phosphorylation of Thr286/287, which induces autonomous activity of CaMKII, using cell culture experiments. In this study, we explored the pathological mechanism of motor dysfunction observed exclusively in a patient with Pro213Leu mutation in CaMKIIß using a mouse model of the human disease. The homozygous CaMKIIß Pro213Leu knockin mice showed age-dependent motor dysfunction and growth failure from 2 weeks after birth. In the cerebellum, the mutation did not alter the mRNA transcript level, but the CaMKIIß protein level was dramatically decreased. Furthermore, in contrast to previous result from cell culture, Thr287 phosphorylation of CaMKIIß was also reduced. CaMKIIß Pro213Leu knockin mice showed similar motor dysfunction as CaMKIIß knockout mice, newly providing evidence for a loss of function rather than a gain of function. Our disease model mouse showed similar phenotypes of the patient, except for epileptic seizures. We clearly demonstrated that the pathological mechanism is a reduction of mutant CaMKIIß in the brain, and the physiological aspects of mutation were greatly different between in vivo and cell culture.

Whole blood GBP5 protein levels in patients with and without active tuberculosis.

BACKGROUND: The host blood transcriptional levels of several genes, such as guanylate binding protein 5 (GBP5), have been reported as potential biomarkers for active tuberculosis (aTB) diagnosis. The aim of this study was to investigate whole blood GBP5 protein levels in aTB and non-tuberculosis patients. METHODS: An in-house immunoassay for testing GBP5 protein levels in whole blood was developed, and suspected aTB patients were recruited. Whole blood samples were collected and tested at enrolment using interferon-gamma release assay (IGRA) and the GBP5 assay. RESULTS: A total of 470 participants were enrolled, and 232 and 238 patients were finally diagnosed with aTB and non-TB, respectively. The GBP5 protein levels of aTB patients were significantly higher than those of non-tuberculosis patients (p < 0.001), and the area under the ROC curve of the GBP5 assay for aTB diagnosis was 0.76. The reactivity of the GBP5 assay between pulmonary and extrapulmonary tuberculosis patients was comparable (p = 0.661). With the optimal cut-off value, the sensitivity and specificity of the GBP5 assay for diagnosing aTB were 78.02 and 66.81%, respectively, while those of IGRA were 77.59 and 76.47%. The combination of the GBP5 assay and IGRA results in 88.52% accuracy for diagnosing aTB in 63.83% of suspected patients with a positive predictive value of 89.57% and a negative predictive value of 87.59%. CONCLUSIONS: Whole blood GBP5 protein is a valuable biomarker for diagnosing of aTB. This study provides an important idea for realizing the clinical application of whole blood transcriptomics findings by immunological methods.

Interactive effects of high temperature and crude protein levels on growth performance, nitrogen excretion, and fecal characteristics of broilers.

In the present study, we aimed to explore the interactive effects of high temperature (HT) and dietary crude protein (CP) levels on nitrogen (N) excretion, fecal characteristics, and growth performance of broilers. A total of 288 broilers (Arbor Acres) were divided into six groups with eight replicates (six broilers per replicate). Two temperatures (ambient temperature: AT, 23 °C; HT: 28 ~ 32 ~ 28 °C) and three diets (CP: 14.90%, 18.18%, or 21.19%, with equal amounts of essential amino acids) were examined in a 2 × 3 factorial design. The experiment arrangement was from 4 to 6 weeks of age. The results showed that HT led to a significant decrease in the N excretion (P < 0.0001), average daily feed intake (P < 0.0001), and weight gain of broilers (P < 0.0001), while it markedly increased the fecal pH (P = 0.015), fecal moisture (P = 0.0014), uric acid (UA) contents (P = 0.0018), and feed/gain ratio (P < 0.0001). A low CP diet significantly decreased the N excretion (P < 0.001), fecal pH (P = 0.016), fecal moisture (P < 0.0001), and UA contents (P < 0.0001), while it markedly increased the feed/gain ratio (P < 0.001). In conclusion, HT had a negative impact on the fecal characteristics and growth performance of broilers but showed positive effects on N excretion. Moreover, decreased CP levels had a positive effect on the N excretion and fecal characteristics in broilers.

DYNAMICS OF CHANGES OF C-REACTIVE PROTEIN LEVEL IN BLOOD SERUM IN THE DEVELOPMENT AND COURSE OF EXPERIMENTAL PERIODONTITIS AND THEIR CORRECTION BY FLAVONOL.

OBJECTIVE: The aim: To study the value of C-reactive protein in the experimental animals blood serum with bacterial-immune periodontitis and its correction with quercetin. PATIENTS AND METHODS: Materials and methods: Modeling of periodontitis was performed by the following method: after thiopental anesthesia (at a dose of 40 mg / kg intramuscularly) rats were fixed. A subcostal injection of 0.01 ml of egg protein with cultures of Streptococcus hemolytic and Staphylococcus aureus at a dose of 4 CFU was performed in the area of periodontal tissues of the lower incisor as an initiating inflammatory factor. To enhance the immune process, a complete Freund's adjuvant was introduced into the animal's hind limb at the same time. RESULTS: Results: Analysis of the results of the study of the content of C-reactive protein in the blood serum of animals with experimental bacteria and immune periodontitis, receiving injections of quercetin, showed a significant decrease by 1.31 times, compared with animals with this simulated pathology on the 14th day of the experiment without the use of flavonol. When comparing this indicator on the 14th day of development of experimental periodontitis with correction, it was found that it remained slightly higher than the indicators of the intact group of rats. CONCLUSION: Conclusions: The level of C-reactive protein in the blood serum of experimental animals is an important indicator of the immune-inflammatory response, which increases its activation of the inflammatory system. The administration of flavonoid quercetin for 7 days helps to reduce the level of C-reactive protein in the blood serum of animals with experimental bacterial and immune periodontitis.

Mutation location of HCM-causing troponin T mutations defines the degree of myofilament dysfunction in human cardiomyocytes.

BACKGROUND: The clinical outcome of hypertrophic cardiomyopathy patients is not only determined by the disease-causing mutation but influenced by a variety of disease modifiers. Here, we defined the role of the mutation location and the mutant protein dose of the troponin T mutations I79N, R94C and R278C. METHODS AND RESULTS: We determined myofilament function after troponin exchange in permeabilized single human cardiomyocytes as well as in cardiac patient samples harboring the R278C mutation. Notably, we found that a small dose of mutant protein is sufficient for the maximal effect on myofilament Ca2+-sensitivity for the I79N and R94C mutation while the mutation location determines the magnitude of this effect. While incorporation of I79N and R94C increased myofilament Ca2+-sensitivity, incorporation of R278C increased Ca2+-sensitivity at low and intermediate dose, while it decreased Ca2+-sensitivity at high dose. All three cTnT mutants showed reduced thin filament binding affinity, which coincided with a relatively low maximal exchange (50.5 ± 5.2%) of mutant troponin complex in cardiomyocytes. In accordance, 32.2 ± 4.0% mutant R278C was found in two patient samples which showed 50.0 ± 3.7% mutant mRNA. In accordance with studies that showed clinical variability in patients with the exact same mutation, we observed variability on the functional single cell level in patients with the R278C mutation. These differences in myofilament properties could not be explained by differences in the amount of mutant protein. CONCLUSIONS: Using troponin exchange in single human cardiomyocytes, we show that TNNT2 mutation-induced changes in myofilament Ca2+-sensitivity depend on mutation location, while all mutants show reduced thin filament binding affinity. The specific mutation-effect observed for R278C could not be translated to myofilament function of cardiomyocytes from patients, and is most likely explained by other (post)-translational troponin modifications. Overall, our studies illustrate that mutation location underlies variability in myofilament Ca2+-sensitivity, while only the R278C mutation shows a highly dose-dependent effect on myofilament function.

Quantitative proteomic analysis of pear (Pyrus pyrifolia cv. "Hosui") flesh provides novel insights about development and quality characteristics of fruit.

MAIN CONCLUSION: A total of 6763 proteins were identified in the developing pear flesh, which were further screened for differentially expressed proteins related to fruit quality and ATP-binding cassette transporters. To obtain further details on changes in protein levels during fruit ripening and to identify and evaluate changes in various metabolic pathways that affect fruit quality, a proteomic method using tandem mass tags was implemented at three developmental stages in Pyrus pyrifolia cv. "Hosui" that identified 6763 proteins. Subcellular localization and Gene Ontology enrichment analysis revealed major functions of all identified proteins. Kyoto Encyclopedia of Genes and Genomes pathway analysis suggested that all metabolic processes are reflected in the up- and downregulation of differentially expressed proteins during fruit development, which play predominant roles in cell division, cell expansion, and fruit ripening. Among the examined differentially expressed proteins, 160 related to fruit quality, and 14 ATP-binding cassette transporters related to fruit development were identified and analyzed. The quantitative data were validated by parallel reaction monitoring, which confirmed the reliability of the experimental results.

Culture salinity modulates Nrf2 antioxidant signaling pathway and immune response of juvenile Genetically Improved Farmed Tilapia (GIFT) (Oreochromis niloticus) under different dietary protein levels.

This study aimed to evaluate that dietary protein levels and culture salinity levels affect the health status of juvenile genetically improved farmed tilapia (GIFT, Oreochromis niloticus). Graded protein levels of six diets were prepared, ranging from 18.20% to 49.49% (dry basis), and were used in cultured GIFT at two salinity levels (0‰ and 8‰) for 8 weeks. The results suggested that appropriate protein levels reduced pro-inflammatory gene expressions in the intestine including interleukin 1ß (IL-1ß), interleukin 8 (IL-8) and tumour necrosis factor-α (TNF-α) mRNA levels at two salinity levels (P < 0.05). 8‰ salinity significantly decreased the expression levels of IL-1ß, TNF-α and nuclear factor-kappa B (NF-κB) (P < 0.05). The anti-inflammatory factor interleukin 10 (IL-10) was significantly increased by 36.42% protein level (P < 0.05). Regarding antioxidant capacity, appropriate protein levels and 8‰ salinity significantly improved the antioxidant capacity of fish by regulating the activities of intestinal total superoxide dismutase (T-SOD), glutathione peroxidase (GPx), and the levels of glutathione (GSH) and malondialdehyde (MDA). Furthermore, appropriate protein levels and 8‰ salinity also significantly enhanced the antioxidant gene expressions associated with the Nrf2/keap1 signaling pathway by regulating the expression levels of heme oxygenase-1 (HO-1), GPx, catalase (CAT) and superoxide dismutase (SOD). According to GPx activities and the mRNA levels of IL-10, the optimum dietary protein levels for GIFT juveniles were 31.12%-32.18% (0‰) and 34.25-35.38% (8‰) based on second-degree polynomial regression analysis. The present study found that appropriate protein levels and 8‰ culture salinity are critical in maintaining the health of GIFT juveniles by improving antioxidant and immune capacity.

High platelet × C-reactive protein level multiplier is a negative prognostic marker in rectal cancer treated by neoadjuvant chemoradiotherapy.

PURPOSE: The clinical significance of the platelet count × C-reactive protein level multiplier (P-CRP) in patients with locally advanced rectal cancer (LARC) undergoing neoadjuvant chemoradiotherapy followed by curative surgery has not been fully evaluated. METHODS: In this retrospective study, the correlation between the P-CRP and prognosis was evaluated in 135 patients with LARC. We also performed a subgroup analysis limited to patients with pathological TNM stage III [ypN(+)] LARC. RESULTS: The cut-off value of the P-CRP for prognosis was set at 4.11. The high and low P-CRP groups comprised 39 (28.89%) and 96 (71.11%) patients, respectively. Among the investigated clinicopathological factors, the serum carcinoembryonic antigen level and presence of recurrence were significantly associated with the P-CRP value. In the Kaplan-Meier analysis, both overall survival (OS) and disease-free survival (DFS) were shorter in the high P-CRP group (p < 0.0001 and p = 0.0002, respectively; log-rank test). Multivariate analysis using a Cox proportional hazards model showed that a high P-CRP was an independent prognostic factor for OS [hazard ratio (HR) 29.20; 95% confidence interval (CI), 3.42-294.44; p = 0.0024] and DFS (HR 5.89; 95%CI 1.31-22.69; p = 0.023) in patients with LARC. In addition, a high P-CRP predicted poor OS and DFS in patients with pathological TNM stage III [ypN(+)] LARC (p = 0.0001 and p = 0.0012, respectively; log-rank test). CONCLUSIONS: The P-CRP is a promising predictor of survival and recurrence in patients with LARC treated by neoadjuvant chemoradiotherapy followed by curative surgery.

The Role of Keratin-8 and Keratin-18 Polymorphisms and Protein Levels in the Occurrence and Progression of Vocal Leukoplakia.

OBJECTIVE: This study aimed to evaluate the association between the single-nucleotide polymorphism (SNP) and tissue protein level of keratin-8/18 and the occurrence and progression of vocal leukoplakia. METHODS: The case-control study enrolled 158 patients with vocal leukoplakia, 326 patients with laryngeal squamous cell carcinoma (LSCC), and 268 healthy controls, which were tested for genotype analysis with keratin-8 and keratin-18 gene polymorphisms using pyrosequencing. The tissue protein expression levels of keratin-8 and keratin-18 were evaluated using immunohistochemistry. RESULTS: The keratin-8 SNP RS1907671 showed an obvious increased risk for vocal leukoplakia (OR 1.56, p = 0.002), while the other SNPs (RS2035875, RS2035878, RS4300473) were tested as protective factors for vocal leukoplakia and LSCC (OR <1, p < 0.05). In keratin-18 SNP test, both RS2070876 and RS2638526 polymorphisms demonstrated decreased risks for vocal leukoplakia and LSCC (OR <1, p < 0.05). The protein levels of keratin-8 and keratin-18 in vocal leukoplakia group were significantly higher than those of the LSCC group (p < 0.05). CONCLUSIONS: Keratin-8 and keratin-18 polymorphisms and protein levels are associated with the occurrence and progression of vocal leukoplakia.