Comparative transcriptomic analyses of brain-liver-muscle in channel catfish (Ictalurus punctatus) with differential growth rate.

Growth is an important economically trait for aquatic animals. The popularity of farmed channel catfish (Ictalurus punctatus) in China has recently surged, prompting a need for research into the genetic mechanisms that drive growth and development to expedite the selection of fast-growing variants. In this study, the brain, liver and muscle transcriptomes of channel catfish between fast-growing and slow-growing groups were analyzed using RNA-Seq. Totally, 63, 110 and 86 differentially expressed genes (DEGs) were from brain, liver and muscle tissues. DEGs are primarily involved in growth, development, metabolism and immunity, which are related to the growth regulation of channel catfish, such as growth hormone receptor b (ghrb), fibroblast growth factor receptor 4 (fgfr4), bone morphogenetic protein 1a (bmp1a), insulin-like growth factor 2a (igf2a), collagen, type I, alpha 1a (col1a1a), acyl-CoA synthetase long chain family member 2 (acsl2) and caveolin 1 (cav1). This study advances our knowledge of the genetic mechanisms accounting for differences in growth rate and offers crucial gene resources for future growth-related molecular breeding programs in channel catfish.

Clinical Impact and Risk Factors of Nonsusceptibility to Third-Generation Cephalosporins Among Hospitalized Adults with Monomicrobial Enterobacteriaceae Bacteremia in Southern Taiwan: A Multicenter Study.

BACKGROUND: Reducing the effectiveness of broad-spectrum cephalosporins against Enterobacteriaceae infections has been recognized. This study aimed to investigate risk factors and clinical significance of third-generation cephalosporin nonsusceptibility (3GC-NS) among the cases of monomicrobial Enterobacteriaceae bacteremia (mEB) at regional or district hospitals. METHODS: The study was conducted at three hospitals in southern Taiwan between Jan. 2017 and Oct. 2019. Only the first episode of mEB from each adult (aged ≥20 years) was included. The primary outcome was in-hospital crude mortality. RESULTS: Overall there were 499 episodes of adults with mEB included, and their mean age was 74.5 years. Female predominated, accounting for 53% of all patients. Escherichia coli (62%) and Klebsiella pneumoniae (21%) were two major causative species. The overall mortality rate was 15% (73/499), and patients infected by 3GC-NS isolates (34%, 172/499) had a higher mortality rate than those by 3GC-susceptible isolates (66%, 327/499) (21% vs 11%, P=0.005). By the multivariate analysis, 3GC-NS was the only independent prognostic determinant (adjusted odds ratio [AOR], 1.78; P=0.04). Of note, male (AOR 2.02, P=0.001), nosocomial-acquired bacteremia (AOR 2.77, P<0.001), and usage of nasogastric tube (AOR 2.01, P=0.002) were positively associated with 3GC-NS, but P. mirabilis bacteremia (AOR 0.28, P=0.01) and age (AOR 0.98, P=0.04) negatively with 3GC-NS. CONCLUSION: For adults with Enterobacteriaceae bacteremia, 3GC-NS signifies a significant prognostic impact. Efforts to rapid identification of such antimicrobial resistance profiles should be incorporated into antimicrobial stewardship programs to achieve favorable outcomes.

The damage and tolerance mechanisms of Phaffia rhodozyma mutant strain MK19 grown at 28 °C.

BACKGROUND: Phaffia rhodozyma has many desirable properties for astaxanthin production, including rapid heterotrophic metabolism and high cell densities in fermenter culture. The low optimal temperature range (17-21 °C) for cell growth and astaxanthin synthesis in this species presents an obstacle to efficient industrial-scale astaxanthin production. The inhibition mechanism of cell growth at > 21 °C in P. rhodozyma have not been investigated. RESULTS: MK19, a mutant P. rhodozyma strain grows well at moderate temperatures, its cell growth was also inhibited at 28 °C, but such inhibition was mitigated, and low biomass 6 g/L was obtained after 100 h culture. Transcriptome analysis indicated that low biomass at 28 °C resulted from strong suppression of DNA and RNA synthesis in MK19. Growth inhibition at 28 °C was due to cell membrane damage with a characteristic of low mRNA content of fatty acid (f.a.) pathway transcripts (acc, fas1, fas2), and consequent low f.a. CONTENT: Thinning of cell wall and low mannose content (leading to loss of cell wall integrity) also contributed to reduced cell growth at 28 °C in MK19. Levels of astaxanthin and ergosterol, two end-products of isoprenoid biosynthesis (a shunt pathway of f.a. biosynthesis), reached 2000 µg/g and 7500 µg/g respectively; ~2-fold higher than levels at 21 or 25 °C. Abundance of ergosterol, an important cell membrane component, compensated for lack of f.a., making possible the biomass production of 6 g/L for MK19 at 28 °C. CONCLUSIONS: Inhibition of growth of P. rhodozyma at 28 °C results from blocking of DNA, RNA, f.a., and cell wall biosynthesis. In MK19, abundant ergosterol made possible biomass production 6 g/L at 28 °C. Significant accumulation of astaxanthin and ergosterol indicated an active MVA pathway in MK19 at 28 °C. Strengthening of the MVA pathway can be a feasible metabolic engineering approach for enhancement of astaxanthin synthesis in P. rhodozyma. The present findings provide useful mechanistic insights regarding adaptation of P. rhodozyma to 28 °C, and improved understanding of feasible metabolic engineering techniques for industrial scale astaxanthin production by this economically important yeast species.

Deregulation of phytoene-ß-carotene synthase results in derepression of astaxanthin synthesis at high glucose concentration in Phaffia rhodozyma astaxanthin-overproducing strain MK19.

BACKGROUND: A major obstacle to industrial-scale astaxanthin production by the yeast Phaffia rhodozyma is the strong inhibitory effect of high glucose concentration on astaxanthin synthesis. We investigated, for the first time, the mechanism of the regulatory effect of high glucose (> 100 g/L) at the metabolite and transcription levels. RESULTS: Total carotenoid, ß-carotene, and astaxanthin contents were greatly reduced in wild-type JCM9042 at high (110 g/L) glucose; in particular, ß-carotene content at 24-72 h was only 14-17% of that at low (40 g/L) glucose. The inhibitory effect of high glucose on astaxanthin synthesis appeared to be due mainly to repression of lycopene-to-ß-carotene and ß-carotene-to-astaxanthin steps in the pathway. Expression of carotenogenic genes crtE, pbs, and ast was also strongly inhibited by high glucose; such inhibition was mediated by creA, a global negative regulator of carotenogenic genes which is strongly induced by glucose. In contrast, astaxanthin-overproducing, glucose metabolic derepression mutant strain MK19 displayed de-inhibition of astaxanthin synthesis at 110 g/L glucose; this de-inhibition was due mainly to deregulation of pbs and ast expression, which in turn resulted from low creA expression. Failure of glucose to induce the genes reg1 and hxk2, which maintain CreA activity, also accounts for the fact that astaxanthin synthesis in MK19 was not repressed at high glucose. CONCLUSION: We conclude that astaxanthin synthesis in MK19 at high glucose is enhanced primarily through derepression of carotenogenic genes (particularly pbs), and that this process is mediated by CreA, Reg1, and Hxk2 in the glucose signaling pathway.

From Molecular Mechanism to the Etiology of Sjogren Syndrome.

Sjogren's Syndrome (SS) is a chronic, female overwhelming fundamental issue of an immune system rheumatic sickness that influences the whole body. It is described by lymphocytic invasion of the exocrine viz. salivary and lacrimal glands and by surprising B-cell hyperactivity. Keratoconjunctivitis sicca (dry eye) and Stomatitis sicca (oral dryness) are the primary visual appearances of SS. The primary SS is recognized from secondary SS which happens as a piece of other immune system maladies. The secondary SS exists together particularly with fundamental lupus erythematosus (15- 36%), rheumatoid joint inflammation (20- 32%) and also restricted and progressive systemic sclerosis (11- 24%), less as often as possible with different sclerosis and immune system hepatitis and thyreoiditis. We assess changes in salivary epidermal growth factor (EGF) intensity and estimate the relationship between salivary EGF levels and the seriousness of intraoral symptoms in SS individuals. The outcomes demonstrated that the salivary EGF levels diminished with the movement of SS, and this crumbling in salivation quality and additionally, hyposalivation could imagine a vital constituent in the pathogenesis of refractory intraoral indication in SS suffering patients. A strong relationship between particular alleles of the MHC and SS improvement has been recommended. The primary hereditary examination on SS revealed a relationship amongst SS and HLA-DR3 in SS population. Subsequent reports featured the relationship amongst SS and the HLA-D locus, with a diverse distribution between primary SS and secondary SS. The motivation behind this manuscript is to give a concise survey on the molecular mechanism, effects of infectious agents and genetic factors in the etiology of Sjogren's Syndrome. Such effects are discussed independently.

Metabolic Redesign of Rhodobacter sphaeroides for Lycopene Production.

Lycopene plays an important role as an antioxidative and anticancer agent, and is an increasingly valuable commodity in the global market. Rhodobacter sphaeroides, a carotenogenic and phototrophic bacterium, is an efficient and practical host for carotenoid production. Herein, we explored the potential of metabolically engineered Rb. sphaeroides as a novel platform to produce lycopene. The basal lycopene-producing strain was generated by introducing an exogenous crtI4 from Rhodospirillum rubrum to replace the native crtI3 and deleting crtC in Rb. sphaeroides. Furthermore, knocking out zwf blocked the competitive pentose phosphate pathway and improved the lycopene content by 88%. Finally, the methylerythritol phosphate pathway was reinforced by integration of dxs combined with zwf deletion, which further increased the lycopene content. The final engineered strain produced lycopene to 10.32 mg/g dry cell weight. This study describes a new lycopene producer and provides insight into a photosynthetic bacterium as a host for lycopene biosynthesis.

Catalpol alleviates ovalbumin-induced asthma in mice: Reduced eosinophil infiltration in the lung.

BACKGROUND: Radix Rehmanniae Preparata is a traditional Chinese herbal medicine used to treat asthma, and catalpol is one of the main active ingredients in this herb. In the present study, the effects of catalpol on asthma and the underlying mechanism were explored. METHODS: Mice with ovalbumin (OVA)-induced asthma were given 5 or 10mg/kg catalpol from Day 15 to Day 28 (intraperitoneal injection). Histopathologic changes were detected by Hematoxylin and Eosin staining and Periodic Acid Schiff staining. The levels of IgE, interleukin (IL)-4, IL-5 and eotaxin were measured by ELISA. The numbers of lymphocytes, monocytes, basophils and eosinophils in the bronchoalveolar lavage fluid were determined by Wright-Giemsa staining. The expression and distribution of eotaxin and C-C chemokine receptor 3 (CCR3) were detected by immunohistochemistry and immunofluorescence. The expression of interleukin-5 receptor α (IL-5Rα) was detected by Western blot assay. RESULTS: Catalpol inhibited OVA-induced inflammation and IgE secretion in the lung. OVA-induced type 2 inflammation was suppressed by catalpol as evidenced by decreased levels of IL-4 and IL-5. Moreover, catalpol inhibited the aberrant eosinophil infiltration in the lungs, and also suppressed OVA-induced elevation of eosinophil chemokine eotaxin and its receptor CCR3. In addition, IL-5Rα expression in the bone marrow cells derived from catalpol-treated asthmatic mice was lower than that from the untreated asthmatic mice. CONCLUSION: Our study demonstrated that catalpol attenuated OVA-induced asthma and inhibit the infiltration of inflammatory cells, especially eosinophils, into the lung. This study suggests that catalpol may become a promising drug for the treatment of asthma.

[Effects of MMPs inhibitors on microleakage with wet bonding technique].

PURPOSE: To observe the effects of matrix metalloproteinases(MMPs) inhibitors on microleakage with wet bonding technique. METHODS: Twenty-seven premolars were randomly assigned to 3 groups. Class Ⅴ cavities were prepared in the neck of the teeth and treated with distilled water, 2% chlorhexidine (CHX) or 2% monocycline (MI) for 60 seconds. Resin was used to restore the prepared cavities following application of luting agent. Samples in each group were soaked for 1 h with 10% NaClO and then 2 layers of nail polish were applied, followed by treatment with 50wt% ammoniated silver nitrate (ASN), and developing solution in turn. Each tooth was sectioned longitudinally to 1~2 mm slices and examined with stereo microscope and scanning electron microscope (SEM). The data were analyzed with SPSS 16.0 software package for Wilcoxon rank sum test. RESULTS: There was varied microleakage at the margin. The difference was significant between group CHX and control group, between group MI and control group; However, there was no significant difference between group CHX and group MI. CONCLUSIONS: MMPs inhibitor can reduce microleakage with wet bonding technique.

Characterization of Two GAS1 Genes and Their Effects on Expression and Secretion of Heterologous Protein Xylanase B in Kluyveromyces lactis.

ß-1,3-glucanosyltransferases play essential roles in cell wall biosynthesis in yeast. Kluyveromyces lactis has six putative ß-1,3-glucanosyltransferase genes. KlGAS1-1 and KlGAS1-2 are homologs of Saccharomyces cerevisiae gene GAS1. RT-qPCR indicated the transcription level of KlGAS1-1 was significantly reduced while heterologous protein (thermostable xylanase B) secretion was enhanced during medium optimization. To evaluate if these two events were related, and to improve xylanase B secretion in K. lactis, we constructed KlGAS1-1 and KlGAS1-2 single deletion strains and double deletion strain, respectively. KlGAS1-1 gene deletion resulted in the highest xylanase B activity among the three mutants. Only the double deletion strain showed morphology similar to that of the GAS1 deletion mutant in S. cerevisiae. The two single deletion strains differed in terms of cell wall thickness and xylanase B secretion. Transcription levels of ß-1,3-glucanosyltransferase genes and genes related to protein secretion and transport were assayed. The ß-1,3-glucanosyltransferase genes displayed transcription complementation in the cell wall synthesis process. KlGAS1-1 and KlGAS1-2 affected transcription levels of secretion- and transport-related genes. Differences in protein secretion ratio among the three deletion strains were associated with changes of transcription levels of secretion- and transport-related genes. Our findings indicate that KlGAS1-1 deletion is an effective tool for enhancing industrial-scale heterologous protein secretion in K. lactis.

Overexpression of a bifunctional enzyme, CrtS, enhances astaxanthin synthesis through two pathways in Phaffia rhodozyma.

BACKGROUND: A moderate-temperature, astaxanthin-overproducing mutant strain (termed MK19) of Phaffia rhodozyma was generated in our laboratory. The intracellular astaxanthin content of MK19 was 17-fold higher than that of wild-type. The TLC profile of MK19 showed a band for an unknown carotenoid pigment between those of ß-carotene and astaxanthin. In the present study, we attempted to identify the unknown pigment and to enhance astaxanthin synthesis in MK19 by overexpression of the crtS gene that encodes astaxanthin synthase (CrtS). RESULTS: A crtS-overexpressing strain was constructed without antibiotic marker. A recombinant plasmid with lower copy numbers was shown to be stable in MK19. In the positive recombinant strain (termed CSR19), maximal astaxanthin yield was 33.5% higher than MK19, and the proportion of astaxanthin as a percentage of total carotenoids was 84%. The unknown carotenoid was identified as 3-hydroxy-3',4'-didehydro-ß,Ψ-carotene-4-one (HDCO) by HPLC, mass spectrometry, and NMR spectroscopy. CrtS was found to be a bifunctional enzyme that helped convert HDCO to astaxanthin. Enhancement of crtS transcriptional level increased transcription levels of related genes (crtE, crtYB, crtI) in the astaxanthin synthesis pathway. A scheme of carotenoid biosynthesis in P. rhodozyma involving alternative bicyclic and monocyclic pathways is proposed. CONCLUSIONS: CrtS overexpression leads to up-regulation of synthesis-related genes and increased astaxanthin production. The transformant CSR19 is a stable, secure strain suitable for feed additive production. The present findings help clarify the regulatory mechanisms that underlie metabolic fluxes in P. rhodozyma carotenoid biosynthesis pathways.

Assembly of functional photosystem complexes in Rhodobacter sphaeroides incorporating carotenoids from the spirilloxanthin pathway.

Carotenoids protect the photosynthetic apparatus against harmful radicals arising from the presence of both light and oxygen. They also act as accessory pigments for harvesting solar energy, and are required for stable assembly of many light-harvesting complexes. In the phototrophic bacterium Rhodobacter (Rba.) sphaeroides phytoene desaturase (CrtI) catalyses three sequential desaturations of the colourless carotenoid phytoene, extending the number of conjugated carbon-carbon double bonds, N, from three to nine and producing the yellow carotenoid neurosporene; subsequent modifications produce the yellow/red carotenoids spheroidene/spheroidenone (N=10/11). Genomic crtI replacements were used to swap the native three-step Rba. sphaeroides CrtI for the four-step Pantoea agglomerans enzyme, which re-routed carotenoid biosynthesis and culminated in the production of 2,2'-diketo-spirilloxanthin under semi-aerobic conditions. The new carotenoid pathway was elucidated using a combination of HPLC and mass spectrometry. Premature termination of this new pathway by inactivating crtC or crtD produced strains with lycopene or rhodopin as major carotenoids. All of the spirilloxanthin series carotenoids are accepted by the assembly pathways for LH2 and RC-LH1-PufX complexes. The efficiency of carotenoid-to-bacteriochlorophyll energy transfer for 2,2'-diketo-spirilloxanthin (15 conjugated C = C bonds; N=15) in LH2 complexes is low, at 35%. High energy transfer efficiencies were obtained for neurosporene (N=9; 94%), spheroidene (N=10; 96%) and spheroidenone (N=11; 95%), whereas intermediate values were measured for lycopene (N=11; 64%), rhodopin (N=11; 62%) and spirilloxanthin (N=13; 39%). The variety and stability of these novel Rba. sphaeroides antenna complexes make them useful experimental models for investigating the energy transfer dynamics of carotenoids in bacterial photosynthesis.

Astaxanthin biosynthesis is enhanced by high carotenogenic gene expression and decrease of fatty acids and ergosterol in a Phaffia rhodozyma mutant strain.

An astaxanthin-overproducing (∼1000 µg g(-1)) strain of Phaffia rhodozyma, termed MK19, was established through 1-methyl-3-nitro-1-nitrosoguanidine and Co60 mutagenesis from wild-type JCM9042 (merely 35-67 µg g(-1)). The total fatty acid content of MK19 was much lower than that of the wild type. Possible causes of the astaxanthin increase were studied at the gene expression level. The expression of the carotenogenic genes crtE, crtI, pbs, and ast, which are responsible for astaxanthin biosynthesis from geranylgeranyl pyrophosphate, was highly induced at the mRNA level, leading to excessive astaxanthin accumulation. In contrast, transcription levels of the genes (hmgs, hmgr, idi, mvk, mpd, fps), responsible for the initial steps in the terpenoid pathway, were essentially the same in wild type and MK19. Although fatty acid and total ergosterol content were reduced by 40-70 mg g(-1) and 760.3 µg g(-1) , respectively, in MK19 as compared with the wild type, but the transcription levels of rate-limiting genes in fatty acid and ergosterol pathways such as acc and sqs were similar. Because fatty acids and ergosterol are two branch pathways of astaxanthin biosynthesis in P. rhodozyma, our findings indicate that enhancement of astaxanthin in MK19 results from decreased fatty acid and ergosterol biosynthesis, leading to precursor accumulation, and transfer to the astaxanthin pathway. Strengthening of the mevalonate pathway is suggested as a promising metabolic engineering approach for further astaxanthin enhancement in MK19.

Reduction of fatty acid flux results in enhancement of astaxanthin synthesis in a mutant strain of Phaffia rhodozyma.

A moderate-temperature mutant strain of the yeast Phaffia rhodozyma, termed MK19, was selected by 1-methyl-3-nitro-1-nitrosoguanidine (NTG) and Co60 mutagenesis. MK19 displayed fast cell growth and elevated astaxanthin content at 25 degrees C, whereas optimal temperature for growth and astaxanthin synthesis of wild-type P. rhodozyma was 17-21 degrees C. Optimized astaxanthin yield for MK19 after 4 days culture in shaking flask at 25 degrees C, determined by response surface methodology, was 25.8 mg/l, which was 17-fold higher than that of the wild-type. MK19 was tolerant of high initial concentration of glucose (>100 g/l) in optimized medium. Total fatty acid content of MK19 was much lower than that of the wild-type. Acetyl-CoA is a common precursor of fatty acid and terpenoid biosynthesis, and it is possible that decreased fatty acid synthesis results in transfer of acetyl-CoA to the carotenoid biosynthetic pathway. Our results indicate that astaxanthin content is negatively correlated with fatty acid content in P. rhodozyma. Nutrient analysis showed that MK19 cells are enriched in lysine, vitamin E, and other rare nutrients, and have potential application as fish food without nutritional supplementation. This moderate-temperature mutant strain is a promising candidate for economical industrial-scale production.