The effect of PK gene overexpression on content and antioxidant properties of carotenoids in marine microalga Dunaliella parva.

Dunaliella parva can extensively accumulate carotenoids, which is a promising raw material for carotenoids production. Carotenoids have important medicinal value. D. parva is an ideal organism for studying the mechanism of carotenoid synthesis. Our previous study identified a transcription factor DpAP2 which could regulate carotenoid synthesis in D. parva. In addition, DpAP2 could interact with three proteins with different activities (DNA binding transcription factor activity, protein kinase activity, and alpha-D-phosphohexomutase). To investigate the function of PK gene encoding interacting protein of DpAP2 with protein kinase activity in D. parva, PK gene was cloned into vector pBI221-GFP-UbiΩ-CAT and transformed into D. parva in this study. The results showed that overexpression of PK gene enhanced the contents of carotenoids, total sugars, proteins, and antioxidant activities of carotenoid extract such as superoxide radical scavenging activity, reducing power, hydroxyl radical scavenging activity in transgenic D. parva with overexpression of PK gene. This study explored the function of PK gene, and improved the medicinal value of D. parva.

Red blood cell distribution width as a prognostic factor in patients with aplastic anemia treated with cyclosporin A plus androgen or cyclosporine A alone: a retrospective study.

OBJECTIVE: To explore the prognostic value of red blood cell distribution width (RDW) in newly diagnosed aplastic anemia (AA) patients treated with cyclosporine A (CsA) plus androgen or CsA alone. METHODS: We retrospectively analyzed the clinical outcome of 220 patients with AA. According to the baseline level of RDW before treatment, the patients were divided into the high-RDW group (RDW ≥ 15%) and the normal-RDW group (RDW < 15%). RESULTS: The median RDW of non-severe AA (NSAA) and severe AA (SAA) patients was 15.65% and 15.35%, respectively; this were significantly higher than that of very severe AA (VSAA) patients (13.35%). With median follow-up being 46 months, AA patients in the high-RDW group showed better 5-year OS and PFS than the normal-RDW group (93%: 75.3%; 74.3%: 61%). There was a higher ORR in the high-RDW group than the normal-RDW group (68.7%: 52.3%). The ORR of NSAA patients in the high-RDW group was better than that in the normal RDW group (75.8%: 60%). The 5-year OS of SAA/VSAA patients in the high-RDW group was significantly higher than the normal-RDW group (81.8%: 50.8%). CONCLUSION: This is the first documentation on the prognostic value of RDW in AA patients receiving CsA treatment with long-term follow-up, which had shown that high RDW at diagnosis was a better prognostic factor.

Expression and prognostic value of C-reactive protein in adult immune thrombocytopenia (ITP) patients.

The aim of this study was to investigate the effect of C-reactive protein (CRP) on the prognosis of adult patients with Immune thrombocytopenia purpura (ITP). A retrospective study of 628 adult ITP patients, as well as 100 healthy and 100 infected patients, attending the Affiliated Hospital of Xuzhou Medical University from January 2017 to June 2022 was performed. The ITP patients were grouped according to their CRP levels, and the differences in clinical characteristics of each group and the influencing factors of efficacy in newly diagnosed ITP patients were analyzed. CRP levels were significantly higher in the ITP and infected groups compared with healthy controls (P < 0.001), and platelet counts were significantly lower in the ITP group (P < 0.001). Between the CRP normal and elevated group, their age, white blood cell count, neutrophil count, lymphocyte count, red blood cell count, hemoglobin, platelet count, complement C3 and C4, PAIgG, bleeding score, proportion of severe ITP, and proportion of refractory ITP were significantly different (P < 0.05). Patients of severe ITP (P < 0.001), refractory ITP (P = 0.002), and with active bleeding (P < 0.001) had significantly higher CRP levels. Patients with no response after treatment had significantly higher CRP levels than those who achieved CR or R (P < 0.001). Platelet counts (r = - 0.261, P < 0.001) in newly diagnosed ITP patients and treatment outcomes (r = - 0.221, P < 0.001) were negatively correlated with CRP levels, and bleeding score was positively correlated with CRP levels (r = 0.207, P < 0.001). Treatment outcome was positively correlated with decrease in CRP levels (r = 0.313, P = 0.027). A multifactorial regression analysis of the influencing factors of treatment outcomes on newly diagnosed patients found that CRP was an independent risk factor of the prognosis (P = 0.011). In conclusion, CRP can help assess the severity and predict the prognosis of ITP patients.

First report of leaf spot and stem blight on blueberry (Vaccinium corymbosum cv. Bluerain) caused by Calonectria pseudoreteaudii in China.

Blueberry has high nutritional value and is one of the five healthy fruits. In 2018, leaf spots and stem blights were observed on Vaccinium corymbosum cv. Bluerain in Guangzhou, Guangdong Province, China. Up to 80% of the plants were affected. Initial symptoms of affected leaves were red-brown, irregular, small spots, which gradually coalesced and formed larger irregular necrotic patches. The affected stems showed red-brown and irregular large lesions. Diseased tissues were surface sterilized with 75% alcohol for 15 s, followed by 2.5% NaClO for 30 s, and rinsing three times in sterile distilled water, placed on potato dextrose agar (PDA) and incubated at 25 C. Representative strains, ZHKUCC 21-0021 from diseased leaves and ZHKUCC 21-0073 from diseased stems, were selected for further studies. Colonies grew slowly at 25 C on malt extract agar (MEA) (average 5.68 mm/d), producing white aerial mycelium and red-brown color on the underside after 7 days. Macroconidiophores were hyaline, smooth, consisting of a stipe bearing fertile branches, and a stipe extension terminating in a vesicle. Each terminal branch produced 2-4 phialides, 8-13 × 3-6 µm, reniform or doliiform; Stipe extensions were septate, terminating in a narrowly clavate vesicle, 2-6 µm. Macroconidia were hyaline, straight cylindrical, round at both ends, 83-100 × 7-11 µm (average = 94 × 8 µm; n = 50), with 5 septa. These morphological characteristics were similar to the description of Calonectria pseudoreteaudii (Lombard et al., 2010). The partial calmodulin (cmdA), beta-tubulin (ß-tubulin), and translation elongation factor 1-alpha (tef1-α) genes of the two isolates were respectively amplified using primers CAL-228F/CAL-737R (Carbone et al., 1999), EF1-728F/EF2 and T1/CYLTUB1R (Lombard et al., 2015), and sequences were deposited in GenBank (cmdA: MZ516854 and MZ516855; ß-tubulin: MZ516858 and MZ516859; tef1-α: MZ516856 and MZ516857). BLAST analysis of three gene sequences showed 100% similarity to those of C. pseudoreteaudii. In the maximum likelihood (ML) tree of the concatenated sequences of the three genes, the two isolates from this study were clustered with C. pseudoreteaudii with 100% bootstrap support. Five-mm-diameter hyphal plugs of two representative isolates grown on PDA for five days were used in the pathogenicity test. Leaves were inoculated with ZHKUCC 21-0021, and stems were inoculated with ZHKUCC 21-0073 with five replicates. As controls, sterile PDA plugs were used. All inoculated plants were maintained at 25 C . After 7 days, inoculated leaves and stems developed symptoms similar to field samples, whereas the control plants remained asymptomatic. The pathogen was reisolated from inoculated plants and confirmed to be C. pseudoreteaudii by morphological characteristics. Five Calonectria species (C. canadensis, C. colhounii, C. ilicicola, C. kyotensis and C. pyrochroa), have been reported associated with blueberry (Farr and Rossman, 2022; Fei et al, 2017). Calonectria canadensis and C. ilicicola have been reported to cause stem blight and stem rot in Vaccinium spp. in China (Fei et al, 2017 and 2018). Calonectria colhounii has been reported to cause stem blight in V. angustifolium and V. corymbosum in the United States (Sadowsky et al, 2011). However, this is the first report of C. pseudoreteaudii causing leaf spot and stem blight on Vaccinium spp. worldwide. These results will provide a foundation for future research on prevention and control of this disease.

APB-13 improves the adverse outcomes caused by TGEV infection by correcting the intestinal microbial disorders in piglets.

Porcine transmissible gastroenteritis virus (TGEV) is an enteric coronavirus that has caused high morbidity and mortality of piglets worldwide. Previous studies have shown that the TGEV can lead to severe diarrhoea, vomiting and dehydration in 2-week-old piglets and weaned piglets, resulting in a large number of piglet deaths. Antimicrobial peptides have broad-spectrum antimicrobial activity and a strong killing effect on bacteria, especially on the drug-resistant pathogenic bacteria, and it has attracted broad concern. However, there are very few reports on the effect of APB-13 (an antimicrobial peptide) on the intestinal microbes of piglets infected with TGEV. In this study, 16S rRNA gene sequencing was used to compare the microbial phylum and the genus of piglet's enteric microorganism in different experimental groups, and to predict the metabolic function of the microbial flora. At the same time, the apparent digestibility of nutrients, digestive enzyme activity, daily weight gain and survival rate were also measured. TGEV infection could cause the imbalance of intestinal microbes in piglets, and increase of the relative abundance of Proteobacteria, and decrease of the relative abundance of Firmicutes, Bacteroidetes and Actinobacteri. With the addition of APB-13, this problem can be alleviated, which can reduce the relative abundance of Proteobacteria and improve the balance of intestinal microorganisms. At the microbial genus level, after adding APB-13, the relative abundance of Catenibacterium, Enterobacter and Streptococcus in the intestinal tract of piglets infected with TGEV showed significant decrease, while the relative abundance of Lactobacillus and Ruminococcus increased. Finally, we found that APB-13 can significantly increase the activity of digestive enzyme in the intestinal tract of piglet, thereby improving the apparent digestibility of nutrients and the growth performance of piglets. This study demonstrates that APB-13 can alleviate the adverse outcomes caused by TGEV infection by correcting the intestinal microbial disorders.

Expression of porcine interferon-α and its bioactivity analysis in vitro and in vivo.

Interferon α (IFN-α) plays a crucial role in the host's immune response. In this study, the amino acid sequence of porcine interferon α (PoIFN-α) was analyzed. Seven substitutions, S38F, H40Q, F43L, N78D, Y86C, S151A, and R156T, were mutated and obtained by aligning the sequences of PoIFN-α subtypes. The PoIFN-α mutants were designed, expressed, and purified in E. coli. The antiviral activities of these PoIFN-αs were measured in Vero and swine testis cells against vesicular stomatitis virus (VSV). Their inhibitory abilities on pseudorabies virus (PRV) were also examined. Commercial PoIFN-α was used as a control. We found the ideal inducer concentration of isopropyl ß-D-thiogalactoside was 1 mM, and the best time-point for induction was 8 h. The PoIFN-α mutant named PoIFN-α-156s had the highest antiviral activity, which was about 200-fold more than that of PoIFN-α. PoIFN-α-156s could inhibit VSV and PRV replication in a dose-dependent manner in vitro. The half-life of PoIFN-α-156s was longer than that of PoIFN-α in mice, and the effective antiviral action was higher than PoIFN-α. Animal experiments showed that PoIFN-α-156s could decrease the viral load after infection with VSV. Overall, these results suggest that recombinant PoIFN-α-156s has the ability of antivirus, and is feasible for veterinary clinical applications and fundamental research.

Antiviral effects of Bovine antimicrobial peptide against TGEV in vivo and in vitro.

BACKGROUND: In suckling piglets, transmissible gastroenteritis virus (TGEV) causes lethal diarrhea accompanied by high infection and mortality rates, leading to considerable economic losses. This study explored methods of preventing or inhibiting their production. Bovine antimicrobial peptide-13 (APB-13) has antibacterial, antiviral, and immune functions. OBJECTIVES: This study analyzed the efficacy of APB-13 against TGEV through in vivo and in vitro experiments. METHODS: The effects of APB-13 toxicity and virus inhibition rate on swine testicular (ST) cells were detected using 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT). The impact of APB-13 on virus replication was examined through the 50% tissue culture infective dose (TCID50). The mRNA and protein levels were investigated by real-time quantitative polymerase chain reaction and western blot (WB). Tissue sections were used to detect intestinal morphological development. RESULTS: The safe and effective concentration range of APB-13 on ST cells ranged from 0 to 62.5 µg/mL, and the highest viral inhibitory rate of APB-13 was 74.1%. The log10TCID50 of 62.5 µg/mL APB-13 was 3.63 lower than that of the virus control. The mRNA and protein expression at 62.5 µg/mL APB-13 was significantly lower than that of the virus control at 24 hpi. Piglets in the APB-13 group showed significantly lower viral shedding than that in the virus control group, and the pathological tissue sections of the jejunum morphology revealed significant differences between the groups. CONCLUSIONS: APB-13 exhibited good antiviral effects on TGEV in vivo and in vitro.

Identification and characteristics of a novel cecropin from the armyworm, Mythimna separata.

BACKGROUND: The recent emergence of antibiotic-resistant strains of bacteria has increased the need to develop effective alternatives to antibiotics. Antimicrobial peptides have been considered as a promising product with several advantages. RESULTS: In this present study, we identified a novel cecropin from the armyworm, Mythimna separata (armyworm cecropin 1, AC-1) by transcriptome sequencing and multi-sequence alignment analysis. The AC-1 precursor comprised 63 amino acid residues, containing a conserved cleavage site of the signal peptide, Ala23-Pro24, while the mature AC-1 included 39 amino acid residues. Chemically synthesized AC-1 exhibited low hemolytic activity against chicken red blood cells, low cytotoxicity against swine testis cells, and effective antimicrobial activity against Salmonella, Escherichia coli, Klebsiella pneumonia, and Pseudomonas aeruginosa. Its antimicrobial activity against Salmonella remained after incubation for 1 h at 100 °C or in 250 mM NaCl, KCl, or MgCl2 solution, implying good thermal- and salt-resistant stabilities. The bactericidal effect of AC-1 on E. coli gradually increased with increasing AC-1 concentration, resulting in deformation, severe edema, cytolysis, cell membrane damage, and reducing intracellular electron density. Additionally, recombinant AC-1 protein expressed in E. coli was digested by enterokinase protease to obtain AC-1, which showed similar antimicrobial activity against E. coli to chemically synthesized AC-1. CONCLUSIONS: This study identified a novel antimicrobial peptide that may represent a potential alternative to antibiotics.

The Effect of Selenium on CYP450 Isoform Activity and Expression in Pigs.

Selenium is an essential nutrient in diets; however, the effects of selenium on enzyme metabolic activation are not currently clear. Cytochromes P450 (CYP450) are major phase I metabolic enzymes involved in the biotransformation of xenobiotics and endogenous compounds to form electrophilic reactive metabolites. To investigate the effect of selenium on CYP450 isoform activity, the Landrace pigs were divided into three groups: the control group (containing Se 0.15 mg/kg), the Se-deficient group (Se 0.03 mg/kg), and the Se-supply group (Se 0.35 mg/kg). After 1 week of administration, a mixed solution (20 mg/kg of dextromethorphan, phenacetin, chlorzoxazone, and 10 mg/kg of testosterone in a CMC-Na solution) was intravenously injected into all pigs. The mixed solution content and pharmacokinetic parameters were assayed by HPLC and DAS, respectively. To investigate the effect of selenium on CYP450 isoform expression, RNA-Seq analysis, Western boltting, and qPCR were used. Results showed that Se-supply group significantly increased the activity and expression of CYP1A2 and CYP2D25, and decreased CYP3A29. Se-deficient group decreased the activity of CYP1A2, CYP2D25, and CYP2E1. These results demonstrated that selenium content affecting the activity or expression of the CYP450 isoform may lead to a food-drug interaction.

CdZnTeS quantum dots based electrochemiluminescent image immunoanalysis.

In this work, quaternary CdZnTeS quantum dots (QDs) with a particularly strong electrochemiluminescence (ECL) were synthesized as ECL signal labels. The strong ECL signals can be obtained at both cathode and anode with the ECL efficiencies of 19.78% and 1.62%, respectively. The sandwich-structured ECL immunosensors for the detection of alpha-fetoprotein (AFP) and cancer antigen 125 (CA125) were accomplished with direct ECL image analysis. Under optimal conditions, the QDs-based ECL image immunoanalysis possessed good linearity from 0.5 ng/mL to 20 ng/mL for AFP and from 20 U/mL to 500 U/mL for CA125 with the detection limit of 0.1 ng/mL and 6 U/mL, respectively (S/N = 3), and the lower detection limit obtained by photomultiplier tube were 0.1 fg/mL for AFP and 0.03 mU/mL for CA125 with the wide dynamic range from 0.5 fg/mL to 20 ng/mL and from 0.1 mU/mL to 500 U/mL, respectively (S/N = 3). Furthermore, the ECL immunoanalysis was evaluated with commercial enzyme-linked immunosorbent assay in human serum samples. The good results indicated that CdZnTeS QDs-based ECL biosensor has great potential for fast biomedical screening and multi-assays in clinical diagnosis.