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Cryptophytes are ancestral photosynthetic organisms evolved from red algae through secondary endosymbiosis. They have developed alloxanthin-chlorophyll a/c2-binding proteins (ACPs) as light-harvesting complexes (LHCs). The distinctive properties of cryptophytes contribute to efficient oxygenic photosynthesis and underscore the evolutionary relationships of red-lineage plastids. Here we present the cryo-electron microscopy structure of the Photosystem II (PSII)-ACPII supercomplex from the cryptophyte Chroomonas placoidea. The structure includes a PSII dimer and twelve ACPII monomers forming four linear trimers. These trimers structurally resemble red algae LHCs and cryptophyte ACPI trimers that associate with Photosystem I (PSI), suggesting their close evolutionary links. We also determine a Chl a-binding subunit, Psb-γ, essential for stabilizing PSII-ACPII association. Furthermore, computational calculation provides insights into the excitation energy transfer pathways. Our study lays a solid structural foundation for understanding the light-energy capture and transfer in cryptophyte PSII-ACPII, evolutionary variations in PSII-LHCII, and the origin of red-lineage LHCIIs.
Subject(s)
Cryoelectron Microscopy , Cryptophyta , Light-Harvesting Protein Complexes , Photosystem II Protein Complex , Photosystem II Protein Complex/metabolism , Photosystem II Protein Complex/chemistry , Light-Harvesting Protein Complexes/metabolism , Light-Harvesting Protein Complexes/chemistry , Cryptophyta/metabolism , Photosynthesis , Models, Molecular , Energy Transfer , Photosystem I Protein Complex/metabolism , Photosystem I Protein Complex/chemistry , Chlorophyll A/metabolism , Chlorophyll A/chemistryABSTRACT
BACKGROUND AND AIMS: Direct elimination of cccDNA remains a formidable obstacle due to the persistent and stable presence of cccDNA in hepatocyte nuclei. The silencing of cccDNA transcription enduringly is one of alternative strategies in the treatment of hepatitis B. Protein binding to cccDNA plays an important role in its transcriptional regulation; thus, the identification of key factors involved in this process is of great importance. APPROACHES AND RESULTS: In the present study, high mobility group nucleosome binding domain 1 (HMGN1) was screened out based on our biotin-avidin enrichment system. First, chromatin immunoprecipitation and fluorescent in situ hybridization assays confirmed the binding of HMGN1 with cccDNA in the nucleus. Second, functional experiments in HBV-infected cells showed that the promoting effect of HMGN1 on HBV transcription and replication depended on the functional region of the nucleosomal binding domain, while transfection of the HMGN1 mutant showed no influence on HBV compared with the vector. Third, further mechanistic exploration revealed that the silencing of HMGN1 increased the level of phosphorylase CLK2 and promoted H3 phosphorylation causing the reduced accessibility of cccDNA. Moreover, silenced HMGN1 was mimicked in HBV (r) cccDNA mouse model of HBV infection in vivo. The results showed that silencing HMGN1 inhibited HBV replication in vivo. CONCLUSIONS: In summary, our study identified that a host protein can bind to cccDNA and promote its transcription, providing a candidate strategy for anti-HBV targeting to interfere with the transcriptional activity of cccDNA microchromosomes.
Subject(s)
HMGN1 Protein , Hepatitis B , Animals , Mice , Histones/metabolism , Hepatitis B virus/physiology , HMGN1 Protein/genetics , HMGN1 Protein/metabolism , Chromatin , Carrier Proteins/genetics , Phosphorylation , In Situ Hybridization, Fluorescence , Virus Replication/genetics , DNA, Circular/genetics , DNA, Circular/metabolism , Transcription Factors/genetics , Hepatitis B/metabolism , DNA, Viral/geneticsABSTRACT
Bone defect has always been a major clinical challenge because of its great difficulty and long period of treatment. Drynariae Rhizoma is a commonly used medicine in osteology and traumatology of traditional Chinese medicine, and its active ingredients(mainly flavonoids) facilitate osteoblast differentiation of bone marrow mesenchymal stem cells, osteoclast proliferation, vascular-osteogenic coupling, and inhibit osteoclast activity to promote bone mineralization, and repair and reconstruction of bone defect. As a good substitute for bone regeneration drugs, the active constituents of Drynariae Rhizoma can be loaded on scaffold materials of tissue engineering, which greatly improves the bioavailability of the drug. Meanwhile, the sustained-release microspheres also solve some problems such as sudden drug release from the scaffolds, and the composite scaffolds with active ingredient of Drynariae Rhizoma prepared by them have good ossification activity and osteoinduction, with precise bone repair effects, which meet the diverse performance requirements of bone grafts and have a promising clinical application prospect.
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Objective:To study the clinical manifestations and treatment of neonatal testicular torsion (NTT) and to summarize the experience of diagnosis and treatment.Methods:The clinical data of the patients with NTT admitted to the Department of Pediatric Urology in Guangzhou Women and Children's Medical Center from January 2015 to January 2021 were analyzed retrospectively. The data included demographic information, testicular torsion duration, clinical presentation, ultrasonography findings, surgical procedures, pathological results, follow-ups, and prognosis.Results:A total of 17 cases were enrolled in this study, and the median age was 5.2 d, ranging from 1 d to 17 d; the weight ranging from 2 800 g to 4 000 g. 11 cases of left testicular torsion and 6 cases of right testicular torsion. The median time from onset to medical attention was 3.8 d, ranging from 10 h to 15 d. The first manifestations being reported were scrotal swelling, color change, or painless mass. Emergency ultrasound revealed that the blood flow signal in the testis was significantly reduced or disappeared, and heterogeneous mass echoes were detected in the scrotum. Surgical exploration was performed in all 17 cases and confirmed to be testicular torsion. There were 15 cases of extravaginal torsion and 2 cases of intravaginal torsion. There were 6 cases of clockwise torsion and 11 cases of anticlockwise torsion. The average degree of twisted testis was 450.8°, ranging from 270° to 720°. The rate of orchiectomy was 88.2% (15/17). All patients were followed up for a minimum of 1 year postoperatively. None of the patients experienced postoperative bleeding or infection at the scrotal incision site. There were no instances of recurrent testicular torsion, and the contralateral testicles showed no abnormalities. In 2 cases where the affected testicle was preserved, ultrasound reevaluation at 6 months postoperatively revealed atrophy of the affected testicle.Conclusions:NTT is rare in clinics and lacks specificity, with a high rate of testis loss. When the newborns exhibit scrotal swelling, color change, or other abnormalities, testicular torsion cannot be ruled out, and early surgical exploration is required.
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@#In this study, in order to overcome the shortcomings of the current methods used to identify Bifidobacterium animalis, such as long time, complicated operation and low adaptability of experimental environment, specific primer probes were designed based on ERIC-PCR technology to identify and detect B.animalis.Based on the genomic DNA of B.animalis HP-B1124, the ERIC-PCR reaction conditions of B.animalis HP-B1124 were optimized, and the ERIC-PCR fragments were obtained one by one and sequenced.Two pairs of specific primer probes were designed.The accuracy, specificity, limitation and universality of the two pairs of primer probes were evaluated, and the two pairs of specific primer probes were used for testing the products containing B.animalis in the commercially published formula.The two pairs of specific primer probes designed in this study could be used for identified strains of B.animalis more simply, quickly and targeted.This method has optimized the current relatively traditional methods of pure culture and plate counting identification of B.animalis, and has solved the high requirements of SNP genotyping technology and real-time fluorescence quantitative PCR method for experimental equipment and reagents in the identification of B.animalis to a certain extent.It has the characteristics of low cost, high specificity and earn a broad market development prospect.
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1,3-xylan, an important organic carbon in the ocean, is peculiar to marine algae. 1,3-xylanase-secreting bacteria and their extracellular 1,3-xylanases play pivotal roles in the degradation and biomass conversion of 1,3-xylan. However, only a few 1,3-xylanase-secreting bacteria and 1,3-xylanases have been reported. Here, we identified a novel marine bacterium capable of secreting 1,3-xylanases, designated as strain HB14T. Phylogenetic analysis revealed that strain HB14T clustered tightly with known species of the genus Gilvimarinus, showing the highest 16S rRNA gene sequence similarity (97.7%) with the type strain of Gilvimarinus chinensis. Based on phylogenetic, genomic, chemotaxonomic and phenotypic studies, strain HB14T was classified as a representative of a novel species in the genus Gilvimarinus, for which the name Gilvimarinus xylanilyticus sp. nov. was proposed. The type strain is HB14T (=CCTCC AB 2022109T = KCTC 92379T). Four 1,3-xylanases secreted by strain HB14T were identified based on genome and secretome analyses, and the two (Xyn65 and Xyn80) with relatively higher abundance in secretome were successfully expressed in Escherichia coli and biochemically characterized. They showed the highest activity at pH 6.0-7.0 and 40°C and released mainly 1,3-xylobiose and 1,3-xylotriose from 1,3-xylan. These data suggest that strain HB14T acts as a player in marine 1,3-xylan degradation and recycling and that its extracellular 1,3-xylanases may have a good potential in 1,3-xylooligosaccharides preparation.
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Based on 16S rRNA gene analyses, the same bacterial operational taxonomic units (OTUs) are common to both the Arctic and Antarctic oceans, supporting the concept 'everything is everywhere'. However, whether the same OTUs from both poles have identical genomes, i.e. whether 'everything is still everywhere' at the genomic level has not yet been examined systematically. Here, we isolated, sequenced and compared the genomes of 45 culturable marine bacteria belonging to three genera of Salinibacterium, Psychrobacter and Pseudoalteromonas from both polar oceans. The bacterial strains with identical 16S rRNA genes were common to both poles in every genus, and four identical genomes were detected in the genus Salinibacterium from the Arctic region. However, no identical genomes were observed from opposite poles in this study. Our data, therefore, suggest that 'everything is not everywhere' at the genomic level. The divergence time between bacteria is hypothesized to exert a strong impact on the bacterial biogeography at the genomic level. The geographical isolation between poles was observed for recently diverged, highly similar genomes, but not for moderately similar genomes. This study thus improves our understanding of the factors affecting the genomic-level biogeography of marine microorganisms isolated from distant locations.
Subject(s)
Genomics , Pseudoalteromonas , Antarctic Regions , Geography , Phylogeny , Pseudoalteromonas/genetics , RNA, Ribosomal, 16S/geneticsABSTRACT
Objective:To explore the related factors of anterior cruciate ligament rupture with meniscus injury and the effect of arthroscopic surgery.Methods:From July 2014 to May 2018, 98 patients with anterior cruciate ligament rupture admitted to Shengjing Hospital Affiliated to China Medical University were divided into meniscus injury group(67 cases) and without meniscus injury group(31 cases) according to whether they had meniscus injury.The patients with anterior cruciate ligament rupture and meniscal injury were divided into study group(37 cases underwent arthroscopic reconstruction of anterior cruciate ligament and meniscus injury), and 30 cases in control group(only meniscus was repaired under arthroscopy). The factors affecting the anterior cruciate ligament rupture associated with meniscus were analyzed, and the surgical outcomes of the study group and the control group(the cure rate of meniscus injury and the 1-year reoperation rate, IKDC and Lysholm knee function score) were compared.Results:Multivariate logistic regression analysis showed that early course of disease[95% CI(1.444, 41.68), P<0.05], middle course of disease[95% CI(1.682, 52.147), P<0.05], chronic phase[95% CI(3.623, 180.32), P<0.05]), history of recurrent injury[95% CI(2.649, 27.222), P<0.05]) were risk factors of meniscus injury caused by rupture of anterior cruciate ligament.The treatment rate of meniscus injury in the study group[89.19%(33/37)]was higher than that in the control group[66.67%(20/30)], the reoperation rate in the study group[5.41%(2/37)]was lower than that in the control group[26.67%(8/30)], the differences were statistically significant (χ 2=5.084, 5.898, all P<0.05). At 12 months after operation, the scores of IKDC and Lysholm in the study group were (90.25±14.67)points and (88.36±11.25)points, respectively, which were significantly higher than those in the control group[(73.52±10.12)points and (71.47±10.68)points]( t=12.129, 19.309, all P<0.05). Conclusion:The patients with anterior cruciate ligament rupture complicated with meniscus are associated with history and recurrent injury history.Arthroscopic surgery for simultaneous reconstruction of anterior cruciate ligament and meniscus injury can significantly improve knee joint function, improve the cure rate of meniscus injury in this type of patients, and reduce short-term reoperation rate.
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Extracellular enzymes, initiating the degradation of organic macromolecules, are important functional components of marine ecosystems. Measuring in situ seawater extracellular enzyme activity (EEA) can provide fundamental information for understanding the biogeochemical cycling of organic matter in the ocean. Here we investigate the patterns of EEA and the major factors affecting the seawater EEA of Chinese marginal seas. The geographic distribution of EEA along a latitudinal transect was examined and found to be associated with dissolved organic carbon. Compared with offshore waters, inshore waters had higher enzyme activity. All the tested substrates were hydrolyzed at different rates and phosphatase, ß-glucosidase and protease contributed greatly to summed hydrolysis rates. For any particular enzyme activity, the contribution of dissolved to total EEA was strongly heterogenous between stations. Comparisons of hydrolysis rates of the polymers and their corresponding oligomers suggest that molecule size does not necessarily limit the turnover of marine organic matter. In addition, several typical enzyme-producing clades, such as Bacteroidetes, Planctomycetes, Chloroflexi, Roseobacter, Alteromonas, and Pseudoalteromonas, were detected in the in situ environments. These enzyme-producing clades may be responsible for the production of different enzymes. Overall, each enzyme was found to flexibly respond to environmental conditions and were linked to microbial community composition. It is likely that this activity will profoundly affect organic matter cycling in the Chinese marginal seas.
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Objective To investigate the relationship between the level of serum iron-cofactored superoxide dismutase (SodB) antibody encoded by Helicobacter pylori (H.pylori) genome in infected populations of H.pylori and the occurrence of gastric cancer.Methods Serum samples from 114 cases and 104 control were collected.Indirect ELISA was used to detect serum SodB antibody level in case group and control group.The relationship between serum SodB antibody level and GC risk was analyzed by conditional logistic regression.The value of SodB in serological screening of gastric cancer was analyzed and evaluated by the receiver operating characteristic (ROC) curve.Results The level of serum SodB antibody was correlated with the occurrence of gastric cancer in subjects with OR=2.287 (95%CI:1.191~4.391)(P<0.05).The optimal cutoff value of SodB for screening gastric cancer was determined by using receiver operating characteristic (ROC) curve,and it was 0.028 0.The area under the ROC curve for subjects was 0.575 (95 % CI:0.501 ~ 0.649).Conclusion The level of serum H.pylori SodB antibody was associated with the occurrence of gastric cancer.SodB alone was not effective in screening gastric cancer.It might be used in combination with other biomarkers of gastric cancer to improve further screening of gastric cancer.
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<p><b>OBJECTIVE</b>To investigate the effect of outer membrane vesicles derived from Escherichia coli on proliferation, apoptosis and migration of human neuroblastoma SK-N-SH cells in vitro.</p><p><b>METHODS</b>The outer membrane vesicles (OMVs) were obtained from wild-type Escherichia coli with ultracentrifugation method, and the morphology of the OMVs was observed by transmission electron microscopy and the vesicle diameter was determined using MALVERN ZEN3690. Human neuroblastoma SK-N-SH cells were treated with the OMVs at low (100 µg/mL), moderate (500 µg/mL) and high (1000 µg/mL) doses, and 24, 48 and 72 h later, the cell proliferation activity was detected with MTT assay. The expressions of apoptosis-related marker caspase-3 was detected using Western blotting, and TUNEL assay was performed to detect the cell apoptosis. The migration capacity of SK-N-SH cells was evaluated using Transwell migration assay.</p><p><b>RESULTS</b>The isolated OMVs showed a circular or elliptical hollow structure with double-layer membrane and a diameter range of 30-450 nm. Compared with the control cells, SK-N-SH cells treated with the OMVs showed significantly lowered cell proliferation capacity with enhanced expression of caspase-3. Treatment of the cells with the OMVs resulted in increased cell apoptosis and significantly lowered migration capacity (P<0.05).</p><p><b>CONCLUSION</b>The OMVs derived from Escherichia coli can produce cytotoxicity against SK-N-SH cells and might serve as a therapeutic agent for refractory neuroblastoma.</p>
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OBJECTIVE@#To investigate the effect of Piwil2-induced cancer stem-like cell (Piwil2-iCSC)-derived exosomes on the proliferation,migration and invasion of human umbilical cord blood-derived mesenchymal stem cells (hucMSCs).@*METHODS@#Piwil2-iCSC-derived exosomes were isolated by ultracentrifugation and identified using transmission electron microscopy,nanoparticle tracking analysis and Western blotting.Exosome uptake assay was used to identify the pathway that Piwil2-iCSCderived exosomes utilized.HucMSCs were divided into control group,PBS intervention group and exosome intervention group,and CCK-8 assay,wound healing assay,Transwell assay,Western blotting and cell karyotype analysis were used to observe the proliferation,migration,invasion,expression levels of MMP2 and MMP9 proteins,and chromosome structure of hucMSCs.@*RESULTS@#The diameter of Piwil2-iCSC-derived exosomes ranged from 50 nm to 100 nm,and most of them were oval or spherical capsules rich in CD9,CD63 and Piwil2 proteins.Exosomal uptake assay showed that the exosomes executed theirs functions after entering the cells.Compared with the control cells and PBS-treated cells,hucMSCs treated with the exosomes showed significantly increased number of proliferating cells (<0.05) with accelerated healing rate (<0.05 at 24 h;<0.01 at 48 h),increased invasive cells (<0.01),enhanced protein expressions of MMP2(<0.05 PBS group;<0.01 control group) and MMP9(<0.05),but their karyotype still remained 46XY without any abnormalities.@*CONCLUSIONS@#Piwil2-iCSC-derived exosomes can promote the proliferation,migration and invasion but does not cause cancer-like heterogeneity changes in hucMSCs.
Subject(s)
Humans , Argonaute Proteins , Cell Movement , Physiology , Cell Proliferation , Physiology , Exosomes , Physiology , Fetal Blood , Cell Biology , Karyotyping , Mesenchymal Stem Cells , Pathology , Neoplasm Invasiveness , Neoplastic Stem Cells , Umbilical Cord , Wound HealingABSTRACT
@#Objective To explore the effect of electroacupuncture at Baihui (DU20) and Shenting (DU24) acupoints on learning and memory ability in rats with middle cerebral artery occlusion and reperfusion (MCAO/R), and the potential mechanisms. Methods A total of 90 healthy male Sprague-Dawley rats were randomly divided into sham group (n=18) and operation group (n=72). The MCAO/R model was established by suture method in the operation group. Finally, 54 qualified rats of the operation group were randomly divided into ischemia group (n=18), electroacupuncture group (n=18) and non-acupoint group (n=18). The electroacupuncture group received electroacupuncture at Baihui (DU20) and Shenting (DU24) for 14 days. The cerebral infarction volume was measured by magnetic resonance imaging (MRI). The learning and memory ability was tested by Morris water maze. The protein expression of 5-hydroxytryptamine 1A (5-HT1A) receptor was detected by immunohistochemistry. Results There was no significant difference in the cerebral infarction volume among three groups before intervention (F=1.678, P>0.05). Compared with the ischemia group and the non-acupoint group, the cerebral infarction volume signif-icantly reduced (P<0.001);the latency significantly shortened (P<0.001) and the times crossing the platform decreased (P<0.05);the expres-sion of 5-HT1A receptor decreased in the left hippocampus (P<0.05) in the electroacupuncture group after intervention. There was no signifi-cant difference in all the indices between the non-acupoint group and the ischemia group after intervention (P>0.05). Conclusion Electroacu-puncture at Baihui (DU20) and Shenting (DU24) could effectively increase the learning and memory ability of MCAO/R rats, which might relate with inhibiting the expression of 5-HT1A receptor in hippocampus.
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@#Objective To explore the effect of electroacupuncture at Baihui (DU20) and Shenting (DU24) acupoints on learning and memory ability in rats with middle cerebral artery occlusion and reperfusion (MCAO/R), and the potential mechanisms. Methods A total of 90 healthy male Sprague-Dawley rats were randomly divided into sham group (n=18) and operation group (n=72). The MCAO/R model was established by suture method in the operation group. Finally, 54 qualified rats of the operation group were randomly divided into ischemia group (n=18), electroacupuncture group (n=18) and non-acupoint group (n=18). The electroacupuncture group received electroacupuncture at Baihui (DU20) and Shenting (DU24) for 14 days. The cerebral infarction volume was measured by magnetic resonance imaging (MRI). The learning and memory ability was tested by Morris water maze. The protein expression of 5-hydroxytryptamine 1A (5-HT1A) receptor was detected by immunohistochemistry. Results There was no significant difference in the cerebral infarction volume among three groups before intervention (F=1.678, P>0.05). Compared with the ischemia group and the non-acupoint group, the cerebral infarction volume signif-icantly reduced (P<0.001);the latency significantly shortened (P<0.001) and the times crossing the platform decreased (P<0.05);the expres-sion of 5-HT1A receptor decreased in the left hippocampus (P<0.05) in the electroacupuncture group after intervention. There was no signifi-cant difference in all the indices between the non-acupoint group and the ischemia group after intervention (P>0.05). Conclusion Electroacu-puncture at Baihui (DU20) and Shenting (DU24) could effectively increase the learning and memory ability of MCAO/R rats, which might relate with inhibiting the expression of 5-HT1A receptor in hippocampus.
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AIM To study the chemical constituents from the leaves of Castanopsis fordii Hance.METHODS The 80% ethanol extract from C.fordii leaves was isolated and purified by Sephadex LH-20,MCI,silica and semi-preparative column,then the structures of obtained compounds were identified by spectral data.RESULTS Nine compounds were isolated and identified as quercetin (1),quercetin 3-O-α-L-rhamnopyranoside (2),myricetin 3-rhamnoside (3),myricetin-3-O-(2"-O-galloyl)-α-rhamnopyranoside (4),quercetin 3-O-(2"-O-galloyl)-α-rhamnopyranoside (5),kaempferol-3-O-glucorhamnoside (6),3,5,7,3',5'-pentahydroxy-(2R,3R)-flavanonol 3-O-α-L-rhamnopyranoside (7),astilbin (8),isastilbin (9).CONCLUSION Compounds 2,3 are isolated from this plant for the first time,compounds 1,4-9 are first obatined from genus Castanopsis.
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AIM To study the chemical constituents from the leaves of Castanopsis fordii Hance.METHODS The 80% ethanol extract from C.fordii leaves was isolated and purified by Sephadex LH-20,MCI,silica and semi-preparative column,then the structures of obtained compounds were identified by spectral data.RESULTS Nine compounds were isolated and identified as quercetin (1),quercetin 3-O-α-L-rhamnopyranoside (2),myricetin 3-rhamnoside (3),myricetin-3-O-(2"-O-galloyl)-α-rhamnopyranoside (4),quercetin 3-O-(2"-O-galloyl)-α-rhamnopyranoside (5),kaempferol-3-O-glucorhamnoside (6),3,5,7,3',5'-pentahydroxy-(2R,3R)-flavanonol 3-O-α-L-rhamnopyranoside (7),astilbin (8),isastilbin (9).CONCLUSION Compounds 2,3 are isolated from this plant for the first time,compounds 1,4-9 are first obatined from genus Castanopsis.
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Objective To explore the clinical and laboratory features,and gene diagnosis method of Menkes disease (MD).Methods The clinical and laboratory features and gene diagnosis method of 2 infants with MD were reviewed.Results (1) Clinical features:both infants mentioned in this article were male.Their clinical manifestations were both began at 3-4 months age,including peculiar kinky hair,pale skin,pudgy cheeks,inguinal hernia,vessel abnormality,epilepsy and mental retardation.(2) Laboratory features:the ceruloplasmin concentrations significantly reduced to be < 20 mg/L and 47 mg/L,respectively.The magnetic resonance angiogram images of case 1 showed the abnormal tortuosity of his intracranial vessels.The magnetic resonance images of case 2 showed a rapid progress from normal to severe brain atrophy within half a year.(3) Gene diagnosis:the sequencing of ATP7A gene in case 1 showed a nonsense mutation of c.2110 C > T.The pathogenicity of this mutation had not been reported previously at home and abroad.The sequencing of the gene panel without pathogenic mutation was detected in case 2.But the multiplex ligation-dependent probe amplification test showed a gross deletion of ATP7A gene containing 8-12 exons.This mutation had been documented as a pathogenic mutation of MD.Both mothers of 2 patients were heterozygous mutation carriers of normal phenotype.Conclusions MD is a multisystemic disease caused by ATP7A gene mutation resulting in copper metabolism disorder.MD is inherited as an X-linked recessive trait.MD is characterized by kinky hair,connective tissue abnormalities and progressive neurodegeneration.Clinical diagnosis can be made on the basis of clinical features,findings of blood biochemical examination,and radiological findings.Gene sequencing and multiplex ligation dependent probe amplification test are the main technique widely used for genetic diagnosis.
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Although the Escherichia coli expression system is the most commonly used expression system, some proteins are still difficult to be expressed by this system, such as proteins with high thermolability and enzymes that cannot mature by autoprocessing. Therefore, it is necessary to develop alternative expression systems. In this study, a cold-adapted Pseudoalteromonas expression system was developed. A shuttle vector was constructed, and a conjugational transfer system between E. coli and psychrophilic strain Pseudoalteromonas sp. SM20429 was established. Based on the shuttle vector, three reporter vectors were constructed to compare the strength of the cloned promoters at low temperature. The promoter of xylanase gene from Pseudoalteromonas sp. BSi20429 was chosen due to its high activity at 10-15°C. An expression vector pEV containing the chosen promoter, multiple cloning sites and a His tag was constructed for protein expression and purification. With pEV as expression vector and SM20429 as the host, a cold-adapted protease, pseudoalterin, which cannot be maturely expressed in E. coli, was successfully expressed as an active extracellular enzyme when induced by 2% oat spelt xylan at 15°C for 48 h. Recombinant pseudoalterin purified from the culture by Ni affinity chromatography had identical N-terminal sequence, similar molecular mass and substrate specificity as the native pseudoalterin. In addition, another two cold-adapted enzymes were also successfully expressed by this system. Our results indicate that this cold-adapted Pseudoalteromonas expression system will provide an alternative choice for protein expression, especially for the Pseudoalteromonas proteins intractable for the E. coli system.
Subject(s)
Bacterial Proteins/genetics , Cold Temperature , Escherichia coli/genetics , Pseudoalteromonas/metabolism , Bacterial Proteins/metabolism , Genes, Bacterial , Genetic Vectors , Promoter Regions, GeneticABSTRACT
Sea ice is one of the most frigid environments for marine microbes. In contrast to other ocean ecosystems, microbes in permanent sea ice are space confined and subject to many extreme conditions, which change on a seasonal basis. How these microbial communities are regulated to survive the extreme sea ice environment is largely unknown. Here, we show that filamentous phages regulate the host bacterial community to improve survival of the host in permanent Arctic sea ice. We isolated a filamentous phage, f327, from an Arctic sea ice Pseudoalteromonas strain, and we demonstrated that this type of phage is widely distributed in Arctic sea ice. Growth experiments and transcriptome analysis indicated that this phage decreases the host growth rate, cell density and tolerance to NaCl and H2O2, but enhances its motility and chemotaxis. Our results suggest that the presence of the filamentous phage may be beneficial for survival of the host community in sea ice in winter, which is characterized by polar night, nutrient deficiency and high salinity, and that the filamentous phage may help avoid over blooming of the host in sea ice in summer, which is characterized by polar day, rich nutrient availability, intense radiation and high concentration of H2O2. Thus, while they cannot kill the host cells by lysing them, filamentous phages confer properties advantageous to host survival in the Arctic sea ice environment. Our study provides a foremost insight into the ecological role of filamentous phages in the Arctic sea ice ecosystem.
Subject(s)
Bacteriophages/physiology , Ice Cover/microbiology , Pseudoalteromonas/virology , Seawater/microbiology , Arctic Regions , Bacteriophages/genetics , Bacteriophages/isolation & purification , Ecosystem , Hydrogen Peroxide/metabolism , Pseudoalteromonas/growth & development , Pseudoalteromonas/metabolism , Seasons , Sodium Chloride/metabolismABSTRACT
<p><b>OBJECTIVE</b>To investigate the effects of mannan-binding lectin (MBL) on the maturation and cytokine secretion of human dendritic cells (DC) induced by Candida albicans (C. albicans).</p><p><b>METHODS</b>The plastic-adherent mononuclear cells were prepared from the blood of healthy adult volunteers. The human peripheral blood mononuclear cells-derived dendritic cells (MNC-DC) were induced by 5-day-culture in medium supplemented with rhGM-CSF and rhIL-4, and then cultured for 2 days in presence or absence of C. albicans at varying concentration of human MBL ranging from 1 to 20 mg/L. DC's shape and characters were observed under inverted microscopy, the expression of CD83 and CD86 on DC was analyzed by FACS. The levels of TNF-α and IL-6 were detected by ELISA. FACS also was used to investigate the interaction of MBL with immature DC(imDC) and C. albicans. Western blot was used to detect C. albicans-induced IκBα phosphorylation and p65/NF-κB translocation in DC.</p><p><b>RESULTS</b>MBL at higher concentrations (10-20 mg/L) down-regulated the expression of CD83 and CD86 on the monocyte-derived dentritic cells(MoDC) induced by C. albicans, and inhibited the production of TNF-α and IL-6 induced by C. albicans. FACS showed that MBL could not only bind to C. albicans but also bind to imDCs in a Ca2+-dependent manner. Western blot showed that MBL could decrease the phosphorylation of IκBα and the nuclear translocation of p65/ NF-κB.</p><p><b>CONCLUSION</b>MBL may inhibit TNF-α and IL-6 production induced by C. albicans in DC through NF-κB signaling pathways, suggesting that MBL can play some roles in the regulation of C. albicans-induced immune response.</p>