Diagnosis of colorectal cancer based on folate receptor-positive circulating tumor cell analysis: a retrospective cohort study.

BACKGROUND: Early diagnosis and treatment are crucial to improve the prognosis of colorectal cancer (CRC). At present, there is a lack of an accurate CRC screening factor. We conducted folate receptor-positive circulating tumor cell analysis (FR + CTC analysis) in distinguishing CRC from benign colorectal diseases to evaluate the diagnostic efficiency. METHODS: Clinical data of patients admitted to The First Affiliated Hospital of Anhui Medical University from January 2021 to July 2022 were retrospectively collected. Levels of FR + CTC and other indicators were analyzed. Receiver operating characteristic (ROC) analysis was performed to assess the diagnostic performance of these molecular biomarkers. RESULTS: Data of 103 patients with CRC and 54 patients with benign colorectal diseases were collected. FR + CTC levels were observed significantly higher in CRC patients than in patients with benign colorectal diseases (P < 0.001). FR + CTC level was correlated with tumor diameter, differentiation, T-stage, pathological stage, clinical stage, and intravascular tumor thrombus in patients with CRC (P < 0.05). The optimal cutoff value of FR + CTC level for diagnosing CRC patients was 7.66 FU/3 ml, with a sensitivity of 85.4%, a specificity of 74.1%, and an Area Under Curve (AUC) of 0.855 (95% CI 0.77-0.923). In < 50-years old patients with CRC, the diagnostic efficiency of FR + CTC was excellent, with an AUC of 0.936 (95% CI 0.877-0.995). CONCLUSION: FR + CTC counting has excellent diagnostic efficiency in screening of CRC. FR + CTC count can also predict the tumor stage of CRC patients before surgery, and guide the choice of treatment.

TAFRO syndrome with fatigue and abdominal distension as the first symptom: A case report.

TAFRO syndrome is a peculiar and rare type of multi-centric Castleman's disease which contained a series of symptoms such as thrombocytopenia (T), anasarca (A), fever (F), reticulin fibrosis (R), and organomegaly (O). Here we provide a case of TAFRO syndrome with the manifestation of fatigue, abdominal distension, and low fever at primary diagnosis, characterized by multiple lymphadenopathy of superficial mediastinal and retroperitoneal lymph nodes, and it was finally confirmed by lymph node biopsy. The patient recovered speedy after receiving CHOP chemotherapy. In this case report, the patient has a history of dust-exposure and hepatitis B virus infection, which may be potentially related to the disease. In addition, this case suggests the importance of pathological biopsy of complete lymph nodes in diagnostic process.

The network pharmacology study and molecular docking to investigate the potential mechanism of Acoritataninowii Rhizoma against Alzheimer's Disease.

Alzheimer's Disease is considered as an insidious neurodegenerative progressive disease but its pathogenesis has not been elucidated. Acoritataninowii Rhizoma exhibits anti-dementia effects as a traditional Chinese medicine (TCM), which is linked to its anti- Alzheimer's Disease mechanism. In this study, network pharmacology and molecular docking were used to examine the potential of Acoritataninowii Rhizoma for Alzheimer's Disease. In order to construct PPI networks and drug-component-target-disease networks, disease-related genes and proteins were gathered from the database. Gene ontology (GO), pathway enrichment (KEGG), and molecular docking were used to forecast the potential mechanism of Acoritataninowii Rhizoma on Alzheimer's disease. Therefore, 4 active ingredients and 81 target genes were screened from Acoritataninowii Rhizoma, 6765 specific target genes were screened from Alzheimer's Disease, and 61 drug-disease cross genes were validated. GO analysis showed that Acoritataninowii Rhizoma can regulate processes such as the protein serine/threonine kinase associated with MAPK. KeGG pathway analysis showed that the signaling pathways affected by Acoritataninowii Rhizoma were fluid shear stress and atherosclerosis, AGE-RAGE and other pathways. Molecular docking implied that the pharmacological influences of the bioactive constituents of Acoritataninowii Rhizoma (Cycloaartenol and kaempferol) on Alzheimer's Disease may related to ESR1 and AKT1, respectively. AKT1 and ESR1 may be the core target genes of the treatment for Alzheimer's disease. Kaempferol and Cycloartenol might be core bioactive constituents for treatment.

A Soybean Sucrose Non-Fermenting Protein Kinase 1 Gene, GmSNF1, Positively Regulates Plant Response to Salt and Salt-Alkali Stress in Transgenic Plants.

Soybean is one of the most widely grown oilseed crops worldwide. Several unfavorable factors, including salt and salt-alkali stress caused by soil salinization, affect soybean yield and quality. Therefore, exploring the molecular basis of salt tolerance in plants and developing genetic resources for genetic breeding is important. Sucrose non-fermentable protein kinase 1 (SnRK1) belongs to a class of Ser/Thr protein kinases that are evolutionarily highly conserved direct homologs of yeast SNF1 and animal AMPKs and are involved in various abiotic stresses in plants. The GmPKS4 gene was experimentally shown to be involved with salinity tolerance. First, using the yeast two-hybrid technique and bimolecular fluorescence complementation (BiFC) technique, the GmSNF1 protein was shown to interact with the GmPKS4 protein. Second, the GmSNF1 gene responded positively to salt and salt-alkali stress according to qRT-PCR analysis, and the GmSNF1 protein was localized in the nucleus and cytoplasm using subcellular localization assay. The GmSNF1 gene was then heterologously expressed in yeast, and the GmSNF1 gene was tentatively identified as having salt and salt-alkali tolerance function. Finally, the salt-alkali tolerance function of the GmSNF1 gene was demonstrated by transgenic Arabidopsis thaliana, soybean hairy root complex plants overexpressing GmSNF1 and GmSNF1 gene-silenced soybean using VIGS. These results indicated that GmSNF1 might be useful in genetic engineering to improve plant salt and salt-alkali tolerance.

How Nitrogen and Sulfur Doping Modified Material Structure, Transformed Oxidation Pathways, and Improved Degradation Performance in Peroxymonosulfate Activation.

Current research has widely applied heteroatom doping for the promotion of catalyst activity in peroxymonosulfate (PMS) systems; however, the relationship between heteroatom doping and stimulated activation mechanism transformation is not fully understood. Herein, we introduce nitrogen and sulfur doping into a Co@rGO material for PMS activation to degrade tetracycline (TC) and systematically investigate how heteroatom doping transformed the activation mechanism of the original Co@rGO/PMS system. N was homogeneously inserted into the reduced graphene oxide (rGO) matrix of Co@rGO, inducing a significant increase in the degradation efficiency without affecting the activation mechanism transformation. Additionally, S doping converted Co3O4 to Co4S3 in Co@rGO and transformed the cooperative oxidation pathway into a single non-radical pathway with stronger intensity, which led to a higher stability against environmental interferences. Notably, based on density functional theory (DFT) calculations, we demonstrated that Co4S3 had a higher energy barrier for PMS adsorption and cleavage than Co3O4, and therefore, the radical pathway was not easily stimulated by Co4S3. Overall, this study not only illustrated the improvement due to the heteroatom doping of Co@rGO for TC degradation in a PMS system but also bridged the knowledge gap between the catalyst structure and degradation performance through activation mechanism transformation drawn from theoretical and experimental analyses.

Four Variants of SLCO2A1 Identified in Three Chinese Patients with Chronic Enteropathy Associated with the SLCO2A1 Gene.

BACKGROUND: Chronic enteropathy associated with the SLCO2A1 gene (CEAS) is an enteropathy characterized by multiple small intestinal ulcers of nonspecific histology, also known as chronic nonspecific multiple ulcers of the small intestine. The SLCO2A1 gene encodes a prostaglandin transporter (PGT). AIMS: The aim of this study was to investigate the clinical characteristics of ten Chinese patients with intestinal ulcers of unknown origin, screen them for variants of SLCO2A1, and to investigate the expression of PGT in the small intestinal mucosa of patients with CEAS. METHODS: Ten Chinese patients with intestinal ulcers of unknown origin were included in this study. Blood samples were collected for whole-exome sequencing and Sanger sequencing of candidate gene variants. Immunohistochemical staining was used to investigate the expression of PGT. RESULTS: These ten patients were clinically diagnosed with intestinal ulcers of unknown origin based on criteria established according to earlier publications. Three of them were genetically diagnosed as having CEAS and four candidate variants of the SLCO2A1 gene were identified, among which c.941-1G>A, c.178G>A and c.1681C>T were detected in patients with CEAS for the first time. The terminal ileum was involved in all three patients with CEAS in our study, which was different from the results of Japanese patients. The expression of PGT in the vascular endothelial cells of the intestinal mucosa tissues of patients with CEAS was negative or intermediate. CONCLUSION: We summarized the clinical data of ten Chinese patients with intestinal ulcers of unknown origin and identified three novel SLCO2A1 variants from three patients with CEAS. This study improves our understanding of CEAS and broadens the spectrum of SLCO2A1 variants known to cause CEAS.

Research progress in relation of Helicobacter pylori infection with pregnancy-related diseases and adverse pregnancy outcomes. / å¹½é—¨èžºæ†èŒæ„ŸæŸ“ä¸Žå¦Šå¨ ç›¸å ³ç–¾ç— åŠä¸è‰¯å¦Šå¨ ç»“å±€å ³ç³»çš„ç ”ç©¶è¿›å±•.

Helicobacter pylori (Hp) is a Gram-negative microaerobic bacterium, which is parasitic on gastric mucosa and is associated with the pathogenesis of chronic gastritis, gastric ulcer, gastric cancer and other gastric diseases. Meanwhile, the Hp infection is related with pregnancy-related diseases, pregnancy outcomes, and the health status of offspring, such as infertility, premature delivery, abortion, Hp infection of newborn and neural tube defects. Hp infection is also related to hyperemesis of pregnancy, preeclampsia, gestational diabetes mellitus, iron deficiency anemia, idiopathic thrombocytopenic purpura. It has been suggested that early diagnosis and eradication of Hp and standardized the anti-Hp treatment will benefit pregnant women and their offspring.

Structural mechanism for bacterial oxidation of oceanic trimethylamine into trimethylamine N-oxide.

Trimethylamine (TMA) and trimethylamine N-oxide (TMAO) are widespread in the ocean and are important nitrogen source for bacteria. TMA monooxygenase (Tmm), a bacterial flavin-containing monooxygenase (FMO), is found widespread in marine bacteria and is responsible for converting TMA to TMAO. However, the molecular mechanism of TMA oxygenation by Tmm has not been explained. Here, we determined the crystal structures of two reaction intermediates of a marine bacterial Tmm (RnTmm) and elucidated the catalytic mechanism of TMA oxidation by RnTmm. The catalytic process of Tmm consists of a reductive half-reaction and an oxidative half-reaction. In the reductive half-reaction, FAD is reduced and a C4a-hydroperoxyflavin intermediate forms. In the oxidative half-reaction, this intermediate attracts TMA through electronic interactions. After TMA binding, NADP+ bends and interacts with D317, shutting off the entrance to create a protected micro-environment for catalysis and exposing C4a-hydroperoxyflavin to TMA for oxidation. Sequence analysis suggests that the proposed catalytic mechanism is common for bacterial Tmms. These findings reveal the catalytic process of TMA oxidation by marine bacterial Tmm and first show that NADP+ undergoes a conformational change in the oxidative half-reaction of FMOs.

Structural insights into the cold adaptation of the photosynthetic pigment-protein C-phycocyanin from an Arctic cyanobacterium.

The cold adaptation mechanism of phycobiliproteins, the major photosynthetic pigment-proteins in cyanobacteria and red algae, has rarely been studied. Here we reported the biochemical, structural, and molecular dynamics simulation study of the C-phycocyanin from Arctic cyanobacterial strain Pseudanabaena sp. LW0831. We characterized the phycobilisome components of LW0831 and obtained their gene sequences. Compared to the mesophilic counterpart from Arthrospira platensis (Ar-C-PC), LW0831 C-phycocyanin (Ps-C-PC) has a decreased thermostability (∆Tm of -16°C), one of the typical features of cold-adapted enzymes. To uncover its structural basis, we resolved the crystal structure of Ps-C-PC 1 at 2.04Å. Consistent with the decrease in thermostability, comparative structural analyses revealed decreased intra-trimer and inter-trimer interactions in Ps-C-PC 1, compared to Ar-C-PC. However, comparative molecular dynamics simulations indicated that Ps-C-PC 1 shows similar flexibilities to Ar-C-PC for both the (αß)3 trimer and (αß)6 hexamer. Therefore, the optimization mode is clearly different from cold-adapted enzymes, which usually have increased flexibilities. Detailed analyses demonstrated different optimization modes for the α and ß subunits and it was revealed that hydrophobic interactions are key to this difference, though salt bridges, hydrogen bonds, and surface hydrophobicity are also involved. This study is the first report of the structure of cold-adapted phycobiliproteins and provides insights into the cold-adaptation strategies of non-enzyme proteins.

Supramolecular architecture of photosynthetic membrane in red algae in response to nitrogen starvation.

The availability of nitrogen is one of the most important determinants that can limit the growth of photosynthetic organisms including plants and algae; however, direct observations on the supramolecular architecture of photosynthetic membranes in response to nitrogen stress are still lacking. Red algae are an important evolutionary group of algae which contain phycobilisomes (PBSs) on their thylakoid membranes, as do cyanobacteria. PBSs function not only as light-harvesting antennae but also as nitrogen storage. In this report, alterations of the supramolecular architecture of thylakoid membranes from red alga Porphyridium cruentum during nitrogen starvation were characterized. The morphology of the intact thylakoid membrane was observed to be round vesicles. Thylakoid membranes were reduced in content and PBSs were degraded during nitrogen starvation. The size and density of PBSs were both found to be reduced. PBS size decreased by less than one-half after 20days of nitrogen starvation, but their hemispherical morphology was retained. The density of PBSs on thylakoid membranes was more seriously affected as time proceeded. Upon re-addition of nitrogen led to increasing of PBSs on thylakoid membranes. This work reports the first direct observation on alterations in the supramolecular architecture of thylakoid membranes from a photosynthetic organism in response to nitrogen stress.

A Novel Subfamily Esterase with a Homoserine Transacetylase-like Fold but No Transferase Activity.

Microbial esterases play important roles in deep-sea organic carbon degradation and cycling. Although they have similar catalytic triads and oxyanion holes, esterases are hydrolases and homoserine transacetylases (HTAs) are transferases. Because two HTA homologs were identified as acetyl esterases, the HTA family was recently divided into the bona fide acetyltransferase subfamily and the acetyl esterase subfamily. Here, we identified and characterized a novel HTA-like esterase, Est22, from a deep-sea sedimentary metagenomic library. Est22 could efficiently hydrolyze esters with acyl lengths of up to six carbon atoms but had no transacetylase activity, which is different from HTAs and HTA-like acetyl esterases. Phylogenetic analysis also showed that Est22 and its homologs form a separate branch of the HTA family. We solved the structures of Est22 and its L374D mutant and modeled the structure of the L374D mutant with p-nitrophenyl butyrate. Based on structural, mutational, and biochemical analyses, Phe71 and Met176 in the oxyanion hole and Arg294 were revealed to be the key substrate-binding residues. A detailed structural comparison indicated that differences in their catalytic tunnels lead to the different substrate specificities of Est22 and the other two HTA subfamilies. Biochemical and sequence analyses suggested that Est22 homologs may have the same substrate recognition and catalysis mechanisms as Est22. Due to the significant differences in sequences, structures, and substrate specificities between Est22 (and its homologs) and the other two HTA subfamilies, we suggest that Est22 and its homologs represent a new subfamily in the HTA family.IMPORTANCE Microbial esterases play important roles in the turnover of organic carbon in the deep sea. Esterases and HTAs represent two groups of α/ß hydrolases. Esterases catalyze the hydrolysis of simple esters and are widely used in the pharmaceutical and agrochemical industries, while HTAs catalyze the transfer of an acetyl group from acetyl-coenzyme A (CoA) to homoserine and are essential for microbial growth. Here, we report on a novel HTA-like esterase, Est22, from a deep-sea sediment. Because of the significant differences in sequences, structures, and substrate specificities of HTAs and HTA-like acetyl esterases, Est22 and its homologs represent a new subfamily in the HTA family. This study offers new knowledge regarding marine esterases.

Changchengzhania lutea gen. nov., sp. nov., a new member of the family Flavobacteriaceae isolated from Antarctic intertidal sediment.

A Gram-negative, aerobic, yellow pigmented, non-flagellated, non-gliding, rod-shaped bacterial strain, designated SM1355T, was isolated from Antarctic intertidal sediment collected near the Chinese Antarctic Great Wall Station. The strain grew at 4-35 °C and with 0.5-7.0 % (w/v) NaCl. It hydrolysed aesculin but didn't reduce nitrate to nitrite. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain SM1355T formed a distinct phylogenetic lineage within the family Flavobacteriaceae, sharing the highest 16S rRNA gene sequence similarity with Flaviramulus ichthyoenteri (96.3 %) and fairly high sequence similarities (95.0-96.0 %) with over 20 recognized species in eight genera of the family Flavobacteriaceae. The predominant fatty acids were anteiso-C15 : 0, iso-C15 : 0 and iso-C15 : 1 G. The major polar lipids were phosphatidylethanolamine and one unidentified lipid. The genomic DNA G+C content of strain SM1355T was 36.2 mol%. Based on the results of the polyphasic characterization for strain SM1355T, it is identified as the representative of a novel species in a new genus of the family Flavobacteriaceae, for which the name Changchengzhania lutea gen. nov., sp. nov. is proposed. The type strain of Changchengzhania lutea is SM1355T (=JCM 30336T=CCTCC AB 2014246T).

Molecular insight into bacterial cleavage of oceanic dimethylsulfoniopropionate into dimethyl sulfide.

The microbial cleavage of dimethylsulfoniopropionate (DMSP) generates volatile DMS through the action of DMSP lyases and is important in the global sulfur and carbon cycles. When released into the atmosphere from the oceans, DMS is oxidized, forming cloud condensation nuclei that may influence weather and climate. Six different DMSP lyase genes are found in taxonomically diverse microorganisms, and dddQ is among the most abundant in marine metagenomes. Here, we examine the molecular mechanism of DMSP cleavage by the DMSP lyase, DddQ, from Ruegeria lacuscaerulensis ITI_1157. The structures of DddQ bound to an inhibitory molecule 2-(N-morpholino)ethanesulfonic acid and of DddQ inactivated by a Tyr131Ala mutation and bound to DMSP were solved. DddQ adopts a ß-barrel fold structure and contains a Zn(2+) ion and six highly conserved hydrophilic residues (Tyr120, His123, His125, Glu129, Tyr131, and His163) in the active site. Mutational and biochemical analyses indicate that these hydrophilic residues are essential to catalysis. In particular, Tyr131 undergoes a conformational change during catalysis, acting as a base to initiate the ß-elimination reaction in DMSP lysis. Moreover, structural analyses and molecular dynamics simulations indicate that two loops over the substrate-binding pocket of DddQ can alternate between "open" and "closed" states, serving as a gate for DMSP entry. We also propose a molecular mechanism for DMS production through DMSP cleavage. Our study provides important insight into the mechanism involved in the conversion of DMSP into DMS, which should lead to a better understanding of this globally important biogeochemical reaction.

Interdomain hydrophobic interactions modulate the thermostability of microbial esterases from the hormone-sensitive lipase family.

Microbial hormone-sensitive lipases (HSLs) contain a CAP domain and a catalytic domain. However, it remains unclear how the CAP domain interacts with the catalytic domain to maintain the stability of microbial HSLs. Here, we isolated an HSL esterase, E40, from a marine sedimental metagenomic library. E40 exhibited the maximal activity at 45 °C and was quite thermolabile, with a half-life of only 2 min at 40 °C, which may be an adaptation of E40 to the permanently cold sediment environment. The structure of E40 was solved to study its thermolability. Structural analysis showed that E40 lacks the interdomain hydrophobic interactions between loop 1 of the CAP domain and α7 of the catalytic domain compared with its thermostable homologs. Mutational analysis showed that the introduction of hydrophobic residues Trp(202) and Phe(203) in α7 significantly improved E40 stability and that a further introduction of hydrophobic residues in loop 1 made E40 more thermostable because of the formation of interdomain hydrophobic interactions. Altogether, the results indicate that the absence of interdomain hydrophobic interactions between loop 1 and α7 leads to the thermolability of E40. In addition, a comparative analysis of the structures of E40 and other thermolabile and thermostable HSLs suggests that the interdomain hydrophobic interactions between loop 1 and α7 are a key element for the thermostability of microbial HSLs. Therefore, this study not only illustrates the structural element leading to the thermolability of E40 but also reveals a structural determinant for HSL thermostability.

Structural and molecular basis for the novel catalytic mechanism and evolution of DddP, an abundant peptidase-like bacterial Dimethylsulfoniopropionate lyase: a new enzyme from an old fold.

The microbial cleavage of dimethylsulfoniopropionate (DMSP) generates volatile dimethyl sulfide (DMS) and is an important step in global sulfur and carbon cycles. DddP is a DMSP lyase in marine bacteria, and the deduced dddP gene product is abundant in marine metagenomic data sets. However, DddP belongs to the M24 peptidase family according to sequence alignment. Peptidases hydrolyze C-N bonds, but DddP is deduced to cleave C-S bonds. Mechanisms responsible for this striking functional shift are currently unknown. We determined the structures of DMSP lyase RlDddP (the DddP from Ruegeria lacuscaerulensis ITI_1157) bound to inhibitory 2-(N-morpholino) ethanesulfonic acid or PO4 (3-) and of two mutants of RlDddP bound to acrylate. Based on structural, mutational and biochemical analyses, we characterized a new ion-shift catalytic mechanism of RlDddP for DMSP cleavage. Furthermore, we suggested the structural mechanism leading to the loss of peptidase activity and the subsequent development of DMSP lyase activity in DddP. This study sheds light on the catalytic mechanism and the divergent evolution of DddP, leading to a better understanding of marine bacterial DMSP catabolism and global DMS production.

Nonclassical Phosphomolybdates with Different Degrees of Reduction: Syntheses and Structural and Photo/Electrocatalytic Properties.

Four nonclassical phosphomolybdates, formulated as (H2pytty)8[{Mn(H2pytty)(H2O)3}{Sr⊂P6Mo6(V) Mo12(VI)O73}]2·16H2O (1), [{Mn(H3pytty)(H2O)3}2{Sr⊂P6Mo4(V)Mo14(VI)O73}]·18H2O (2), (H3pytp) (H2pytty)2[{Fe(H2O)4}{Sr⊂P6Mo3(V)Mo15(VI)O73}]·5H2O (3), and (H2pytty)2[{Cd(H2O)4}{Cd(H2O)3 (H3pytty)}{Sr⊂P6Mo5(V)Mo13(VI)O73}]·9H2O (4) (pytty = 3-(pyrazin-2-yl)-5-(1H-1,2,4-triazol-3-yl)-1,2,4-triazolyl; pytp = 4'-(4″-pyridyl)-2,4':6',4″-terpyridine) were hydrothermally synthesized and fully characterized. The penta- and hexa-reduced basket clusters represent the highest reduced level of basket-based polyoxometalate so far. In addition, transition metal complexes as bridge units were introduced to basket system for the first time to induce rare two-dimensional inorganic-organic hybrid layer. The results indicate that reduced degrees of the basket clusters of compounds 1-4 can be tuned by altering the molar ratio of organic ligand pytty and Na2MoO4. Compounds 1-4 exhibit bifunctional electrocatalytic behaviors for oxidation of dopamine and reduction of H2O2. Electrocatalytic mechanism, chronoamperometric experiments and electrocatalytic stability are studied in detail. In addition, the compounds show highly efficient catalytic ability for the degradation of typical dyes under UV irradiation.

Pilot-Scale Production and Thermostability Improvement of the M23 Protease Pseudoalterin from the Deep Sea Bacterium Pseudoalteromonas sp. CF6-2.

Pseudoalterin is the most abundant protease secreted by the marine sedimental bacterium Pseudoalteromonas sp. CF6-2 and is a novel cold-adapted metalloprotease of the M23 family. Proteases of the M23 family have high activity towards peptidoglycan and elastin, suggesting their promising biomedical and biotechnological potentials. To lower the fermentive cost and improve the pseudoalterin production of CF6-2, we optimized the fermentation medium by using single factor experiments, added 0.5% sucrose as a carbon source, and lowered the usage of artery powder from 1.2% to 0.6%. In the optimized medium, pseudoalterin production reached 161.15 ± 3.08 U/mL, 61% greater than that before optimization. We further conducted a small-scale fermentation experiment in a 5-L fermenter and a pilot-scale fermentation experiment in a 50-L fermenter. Pseudoalterin production during pilot-scale fermentation reached 103.48 ± 8.64 U/mL, 77% greater than that before the medium was optimized. In addition, through single factor experiments and orthogonal tests, we developed a compound stabilizer for pseudoalterin, using medically safe sugars and polyols. This stabilizer showed a significant protective effect for pseudoalterin against enzymatic thermal denaturation. These results lay a solid foundation for the industrial production of pseudoalterin and the development of its biomedical and biotechnological potentials.

Mechanistic Insight into Trimethylamine N-Oxide Recognition by the Marine Bacterium Ruegeria pomeroyi DSS-3.

UNLABELLED: Trimethylamine N-oxide (TMAO) is an important nitrogen source for marine bacteria. TMAO can also be metabolized by marine bacteria into volatile methylated amines, the precursors of the greenhouse gas nitrous oxide. However, it was not known how TMAO is recognized and imported by bacteria. Ruegeria pomeroyi DSS-3, a marine Roseobacter, has an ATP-binding cassette transporter, TmoXWV, specific for TMAO. TmoX is the substrate-binding protein of the TmoXWV transporter. In this study, the substrate specificity of TmoX of R. pomeroyi DSS-3 was characterized. We further determined the structure of the TmoX/TMAO complex and studied the TMAO-binding mechanism of TmoX by biochemical, structural, and mutational analyses. A Ca(2+) ion chelated by an extended loop in TmoX was shown to be important for maintaining the stability of TmoX. Molecular dynamics simulations indicate that TmoX can alternate between "open" and "closed" states for binding TMAO. In the substrate-binding pocket, four tryptophan residues interact with the quaternary amine of TMAO by cation-π interactions, and Glu131 forms a hydrogen bond with the polar oxygen atom of TMAO. The π-π stacking interactions between the side chains of Phe and Trp are also essential for TMAO binding. Sequence analysis suggests that the TMAO-binding mechanism of TmoX may have universal significance in marine bacteria, especially in the marine Roseobacter clade. This study sheds light on how marine microorganisms utilize TMAO. IMPORTANCE: Trimethylamine N-oxide (TMAO) is an important nitrogen source for marine bacteria. The products of TMAO metabolized by bacteria are part of the precursors of the greenhouse gas nitrous oxide. It is unclear how TMAO is recognized and imported by bacteria. TmoX is the substrate-binding protein of a TMAO-specific transporter. Here, the substrate specificity of TmoX of Ruegeria pomeroyi DSS-3 was characterized. The TMAO-binding mechanism of TmoX was studied by biochemical, structural, and mutational analyses. Moreover, our results suggest that the TMAO-binding mechanism may have universal significance in marine bacteria. This study sheds light on how marine microorganisms utilize TMAO and should lead to a better understanding of marine nitrogen cycling.

Structural insights into the multispecific recognition of dipeptides of deep-sea gram-negative bacterium Pseudoalteromonas sp. strain SM9913.

UNLABELLED: Peptide uptake is important for nutrition supply for marine bacteria. It is also an important step in marine nitrogen cycling. However, how marine bacteria absorb peptides is still not fully understood. DppA is the periplasmic dipeptide binding protein of dipeptide permease (Dpp; an important peptide transporter in bacteria) and exclusively controls the substrate specificity of Dpp. Here, the substrate binding specificity of deep-sea Pseudoalteromonas sp. strain SM9913 DppA (PsDppA) was analyzed for 25 different dipeptides with various properties by using isothermal titration calorimetry measurements. PsDppA showed binding affinities for 8 dipeptides. To explain the multispecific substrate recognition mechanism of PsDppA, we solved the crystal structures of unliganded PsDppA and of PsDppA in complex with 4 different types of dipeptides (Ala-Phe, Met-Leu, Gly-Glu, and Val-Thr). PsDppA alternates between an "open" and a "closed" form during substrate binding. Structural analyses of the 4 PsDppA-substrate complexes combined with mutational assays indicate that PsDppA binds to different substrates through a precise mechanism: dipeptides are bound mainly by the interactions between their backbones and PsDppA, in particular by anchoring their N and C termini through ion-pair interactions; hydrophobic interactions are important in binding hydrophobic dipeptides; and Lys457 is necessary for the binding of dipeptides with a C-terminal glutamic acid or glutamine. Additionally, sequence alignment suggests that the substrate recognition mechanism of PsDppA may be common in Gram-negative bacteria. All together, our results provide structural insights into the multispecific substrate recognition mechanism of marine Gram-negative bacterial DppA, which provides a better understanding of the mechanisms of marine bacterial peptide uptake. IMPORTANCE: Peptide uptake plays a significant role in nutrition supply for marine bacteria. It is also an important step in marine nitrogen cycling. However, how marine bacteria recognize and absorb peptides is still unclear. This study analyzed the substrate binding specificity of deep-sea Pseudoalteromonas sp. strain SM9913 DppA (PsDppA; the dipeptide-binding protein of dipeptide permease) and solved the crystal structures of unliganded PsDppA and PsDppA in complex with 4 different types of dipeptides. The multispecific recognition mechanism of PsDppA for dipeptides is explained based on structural and mutational analyses. We also find that the substrate-binding mechanism of PsDppA may be common in Gram-negative bacteria. This study sheds light on marine Gram-negative bacterial peptide uptake and marine nitrogen cycling.

Characterization of a novel subtilisin-like protease myroicolsin from deep sea bacterium Myroides profundi D25 and molecular insight into its collagenolytic mechanism.

Collagen is an insoluble protein that widely distributes in the extracellular matrix of marine animals. Collagen degradation is an important step in the marine nitrogen cycle. However, the mechanism of marine collagen degradation is still largely unknown. Here, a novel subtilisin-like collagenolytic protease, myroicolsin, which is secreted by the deep sea bacterium Myroides profundi D25, was purified and characterized, and its collagenolytic mechanism was studied. Myroicolsin displays low identity (<30%) to previously characterized subtilisin-like proteases, and it contains a novel domain structure. Protein truncation indicated that the Pro secretion system C-terminal sorting domain in the precursor protein is involved in the cleavage of the N-propeptide, and the linker is required for protein folding during myroicolsin maturation. The C-terminal ß-jelly roll domain did not bind insoluble collagen fiber, suggesting that myroicolsin may degrade collagen without the assistance of a collagen-binding domain. Myroicolsin had broad specificity for various collagens, especially fish-insoluble collagen. The favored residue at the P1 site was basic arginine. Scanning electron microscopy and atomic force microscopy, together with biochemical analyses, confirmed that collagen fiber degradation by myroicolsin begins with the hydrolysis of proteoglycans and telopeptides in collagen fibers and fibrils. Myroicolsin showed strikingly different cleavage patterns between native and denatured collagens. A collagen degradation model of myroicolsin was proposed based on our results. Our study provides molecular insight into the collagen degradation mechanism and structural characterization of a subtilisin-like collagenolytic protease secreted by a deep sea bacterium, shedding light on the degradation mechanism of deep sea sedimentary organic nitrogen.