Analysis of Short-Term Efficacy of Gasless Single-Port Laparoscopic Inguinal Lymphadenectomy Through Vulva Incision for Vulvar Cancer.

Objective: To investigate the feasibility and short-term efficacy of gasless single-port laparoscopic inguinal lymphadenectomy through vulva incision (VEIL-V). Methods: The data of 9 patients diagnosed as vulvar squamous cell carcinoma who underwent single-port laparoscopic inguinal lymph node dissection through vulvectomy incision were retrospectively analyzed. And 13 patients who underwent laparoscopic inguinal lymph node dissection through lower abdominal subcutaneous approach as the control group (VEIL-H). The operation time, blood loss, numbers of unilateral lymph nodes, hospitalization time, and complications between the two groups were compared. Results: The operation time of VEIL-V was 56.11 ± 5.94 min, which were shorter than that of VEIL-H (74.62 ± 5.50 min; P = 0.013). Bleeding amount in the VEIL-H was 29.44 ± 2.56, which was significantly lower than that of the VEIL-H group (43.08 ± 4.14 ml; P = 0.021). In the two groups, the numbers of unilateral lymph nodes harvested were similar. The differences in the postoperative hospital stay, skin, and lymphatic complications were not statistically significant. Conclusion: Compared with VEIL-H, gasless single-port laparoscopic inguinal lymphadenectomy through vulva incision reduces the difficulty of operation with shorter operation time, and less blood loss, which can be a safe and mini-invasive surgical approach.

An in vitro DNA phosphorothioate modification reaction.

Phosphorothioation (PT) involves the replacement of a nonbridging phosphate oxygen on the DNA backbone with sulfur. In bacteria, the procedure is both sequence- and stereo-specific. We reconstituted the PT reaction using purified DndCDE from Salmonella enterica and IscS from Escherichia coli. We determined that the in vitro process of PT was oxygen sensitive. Only one strand on a double-stranded (ds) DNA substrate was modified in the reaction. The modification was dominant between G and A in the GAAC/GTTC conserved sequence. The modification between G and T required the presence of PT between G and A on the opposite strand. Cysteine, S-adenosyl methionine (SAM) and the formation of an iron-sulfur cluster in DndCDE (DndCDE-FeS) were essential for the process. Results from SAM cleavage reactions support the supposition that PT is a radical SAM reaction. Adenosine triphosphate (ATP) promoted the reaction but was not essential. The data and conclusions presented suggest that the PT reaction in bacteria involves three steps. The first step is the binding of DndCDE-FeS to DNA and searching for the modification sequence, possibly with the help of ATP. Cysteine locks DndCDE-FeS to the modification site with an appropriate protein conformation. SAM triggers the radical SAM reaction to complete the oxygen-sulfur swapping.

Phosphorothioated DNA Is Shielded from Oxidative Damage.

DNA is the carrier of genetic information. DNA modifications play a central role in essential physiological processes. Phosphorothioation (PT) modification involves the replacement of an oxygen atom on the DNA backbone with a sulfur atom. PT modification can cause genomic instability in Salmonella enterica under hypochlorous acid stress. This modification restores hydrogen peroxide (H2O2) resistance in the catalase-deficient Escherichia coli Hpx- strain. Here, we report biochemical characterization results for a purified PT modification protein complex (DndCDE) from S. enterica We observed multiplex oligomeric states of DndCDE by using native PAGE. This protein complex bound avidly to PT-modified DNA. DndCDE with an intact iron-sulfur cluster (DndCDE-FeS) possessed H2O2 decomposition activity, with a Vmax of 10.58 ± 0.90 mM min-1 and a half-saturation constant, K0.5S, of 31.03 mM. The Hill coefficient was 2.419 ± 0.59 for this activity. The protein's activity toward H2O2 was observed to be dependent on the intact DndCDE and on the formation of an iron-sulfur (Fe-S) cluster on the DndC subunit. In addition to cysteine residues that mediate the formation of this Fe-S cluster, other cysteine residues play a catalytic role. Finally, catalase activity was also detected in DndCDE from Pseudomonas fluorescens Pf0-1. The data and conclusions presented suggest that DndCDE-FeS is a short-lived catalase. Our experiments also indicate that the complex binds to PT sites, shielding PT DNA from H2O2 damage. This catalase shield might be able to extend from PT sites to the entire bacterial genome.IMPORTANCE DNA phosphorothioation has been reported in many bacteria. These PT-hosting bacteria live in very different environments, such as the human body, soil, or hot springs. The physiological function of DNA PT modification is still elusive. A remarkable property of PT modification is that purified genomic PT DNA is susceptible to oxidative cleavage. Among the oxidants, hypochlorous acid and H2O2 are of physiological relevance for human pathogens since they are generated during the human inflammation response to bacterial infection. However, expression of PT genes in the catalase-deficient E. coli Hpx- strain restores H2O2 resistance. Here, we seek to solve this obvious paradox. We demonstrate that DndCDE-FeS is a short-lived catalase that binds tightly to PT DNA. It is thus possible that by docking to PT sites the catalase activity protects the bacterial genome against H2O2 damage.

2-(1-Pyrenyl) benzimidazole as a ratiometric and "turn-on" fluorescent probe for iron(III) ions in aqueous solution.

A highly selective and sensitive ratiometric and "turn-on" fluorescent probe for Fe(3+), 2-(1-pyrenyl) benzimidazole (L), was synthesized by a one-step process. In emission spectra, the relative intensity ratio of excimer to monomer fluorescence (IE450/IM387) of L increased 510-fold upon the addition of 30 equiv. of Fe(3+) with a detection limit of 0.2 µM (11.2 ppb) in aqueous solution. Meanwhile, the fluorescence excitation spectra of L showed a fluorescent "turn-on" probe for Fe(3+) with 30-fold enhancement in excitation band intensity of excimer.

Two nucleoside receptors from Streptomyces coelicolor: expression of the genes and characterization of the recombinant proteins.

Streptomyces coelicolor is a soil-dwelling bacterium that undergoes an intricate, saprophytic lifecycle. The bacterium takes up exogenous nucleosides for nucleic acid synthesis or use as carbon and energy sources. However, nucleosides must pass through the membrane with the help of transporters. In the present work, the SCO4884 and SCO4885 genes were cloned into pCOLADuet-1 and overexpressed in Escherichia coli BL21. Each protein was monomeric. Using isothermal titration calorimetry, we determined that SCO4884 and SCO4885 are likely nucleoside receptors with affinity for adenosine and pyrimidine nucleosides. On the basis of bioinformatics analysis and the transporter classification system, we speculate that SCO4884-SCO4888 is an ABC-like transporter responsible for the uptake of adenosine and pyrimidine nucleosides.

Analysis of Streptomyces coelicolor membrane proteome using two-dimensional native/native and native/sodium dodecyl sulfate gel electrophoresis.

Analysis of the oligomeric state of a protein may provide insights into its physiological functions. Because membrane proteins are considered to be the workhorses of energy generation and polypeptide and nutrient transportation, in this study we characterized the membrane-associated proteome of Streptomyces coelicolor by two-dimensional (2D) blue native/sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), high-resolution clear native/native PAGE, and native/SDS-PAGE. A total of 77 proteins were identified, and 20 proteins belonging to 15 complexes were characterized. Moreover, the resolution of high-resolution clear native/SDS-PAGE is much higher than that of blue native/SDS-PAGE. OBP (SCO5477) and BldKB (SCO5113) were identified as the main protein spots from the membrane fractions of S. coelicolor M145, suggesting that these two proteins are involved in extracellular peptide transportation. These two transporters exhibited multiple oligomeric states in the native PAGE system, which may suggest their multiple physiological functions in the development of S. coelicolor.

[Mutagenesis of cysteine residues in dptC from Salmonella enteric serovar Cerro 87 and its effects on DNA phosphorothioate modification].

OBJECTIVE: DNA phosphorothioate modification (DNA sulfur modification, a non-bridging oxygen swapped with a sulfur) exists in diverse bacteria. Salmonella enterica serovar Cerro 87 is one of the bacteria that harbor the DNA sulfur modification. The modification is carried out by the products of a four-membered gene cluster, dptBCDE. Transformation of Escherichia coli DH10B with the dptBCDE gene cluster endows the strain with DNA sulfur modification capability. Deletion of dptC abolished the modification. Here, we studied the function of dptC in DNA sulfur modification. METHODS: Six cysteine residues in dptC were mutated individually within the dptBCDE gene cluster. Mutants were then tested for DNA sulfur modification. RESULTS: Among the 6 cysteine mutations (C39, C146, C262, C273, C280, and C283), 5 abolished DNA modification except for C39, suggesting that C146, C262, C273, C280, and C283 are essential for DNA sulfur modification. Sequence alignment shows that these five cysteine residues are conserved among different strains. CONCLUSION: Mutation at anyone of C146, C262, C273, C280 and C283 of dptC abolished DNA modification. Our results shed light on further study of DNA sulfur modification biochemical pathway.

Mutasynthesis of pyrrole spiroketal compound using calcimycin 3-hydroxy anthranilic acid biosynthetic mutant.

The five-membered aromatic nitrogen heterocyclic pyrrole ring is a building block for a wide variety of natural products. Aiming at generating new pyrrole-containing derivatives as well as to identify new candidates that may be of value in designing new anticancer, antiviral, and/or antimicrobial agents, we employed a strategy on pyrrole-containing compound mutasynthesis using the pyrrole-containing calcimycin biosynthetic gene cluster. We blocked the biosynthesis of the calcimycin precursor, 3-hydroxy anthranilic acid, by deletion of calB1-3 and found that two intermediates containing the pyrrole and the spiroketal moiety were accumulated in the culture. We then fed the mutant using the structurally similar compound of 3-hydroxy anthranilic acid. At least four additional new pyrrole spiroketal derivatives were obtained. The structures of the intermediates and the new pyrrole spiroketal derivatives were identified using LC-MS and NMR. One of them shows enhanced antibacterial activity. Our work shows a new way of pyrrole derivative biosynthetic mutasynthesis.

Characterization of the N-methyltransferase CalM involved in calcimycin biosynthesis by Streptomyces chartreusis NRRL 3882.

Calcimycin is a rare divalent cation specific ionophore antibiotic that has many biochemical and pharmaceutical applications. We have recently cloned and sequenced the Streptomyces chartreusis calcimycin biosynthesis gene cluster as well as identified the genes required for the synthesis of the polyketide backbone of calcimycin. Additional modifying or decorating enzymes are required to convert the polyketide backbone into the biologically active calcimycin. Using targeted mutagenesis of Streptomyces we were able to show that calM from the calcimycin biosynthesis gene cluster is required for calcimycin production. Inactivating calM by PCR targeting, caused high level accumulation of N-demethyl calcimycin. CalM in the presence of S-adenosyl-L-methionine converted N-demethyl calcimycin to calcimycin in vitro. The enzyme was determined to have a kinetic parameter of Km 276 µM, kcat 1.26 min(-1) and kcat/Km 76.2 M(-1) s(-1). These results proved that CalM is a N-methyltransferase that is required for calcimycin biosynthesis, and they set the stage for generating much desired novel calcimycin derivatives by rational genetic and chemical engineering.

A novel target of IscS in Escherichia coli: participating in DNA phosphorothioation.

Many bacterial species modify their DNA with the addition of sulfur to phosphate groups, a modification known as DNA phosphorothioation. DndA is known to act as a cysteine desulfurase, catalyzing a key biochemical step in phosphorothioation. However, bioinformatic analysis revealed that 19 out of the 31 known dnd gene clusters, contain only four genes (dndB-E), lacking a key cysteine desulfurase corresponding gene. There are multiple cysteine desulfurase genes in Escherichia coli, but which one of them participates into DNA phosphorothioation is unknown. Here, by employing heterologous expression of the Salmonella enterica dnd gene cluster named dptBCDE in three E. coli mutants, each of which lacked a different cysteine desulfurase gene, we show that IscS is the only cysteine desulfurase that collaborates with dptB-E, resulting in DNA phosphorothioation. Using a bacterial two-hybrid system, protein interactions between IscS and DptC, and IscS and DptE were identified. Our findings revealed IscS as a key participant in DNA phosphorothioation and lay the basis for in-depth analysis of the DNA phosphorothioation biochemical pathway.

Oxazolomycin biosynthesis in Streptomyces albus JA3453 featuring an "acyltransferase-less" type I polyketide synthase that incorporates two distinct extender units.

The oxazolomycins (OZMs) are a growing family of antibiotics produced by several Streptomyces species that show diverse and important antibacterial, antitumor, and anti-human immunodeficiency virus activity. Oxazolomycin A is a peptide-polyketide hybrid compound containing a unique spiro-linked beta-lactone/gamma-lactam, a 5-substituted oxazole ring. The oxazolomycin biosynthetic gene cluster (ozm) was identified from Streptomyces albus JA3453 and localized to 79.5-kb DNA, consisting of 20 open reading frames that encode non-ribosomal peptide synthases, polyketide synthases (PKSs), hybrid non-ribosomal peptide synthase-PKS, trans-acyltransferases (trans-ATs), enzymes for methoxymalonyl-acyl carrier protein (ACP) synthesis, putative resistance genes, and hypothetical regulation genes. In contrast to classical type I polyketide or fatty acid biosynthases, all 10 PKS modules in the gene cluster lack cognate ATs. Instead, discrete ATs OzmM (with tandem domains OzmM-AT1 and OzmM-AT2) and OzmC were equipped to carry out all of the loading functions of both malonyl-CoA and methoxymalonyl-ACP extender units. Strikingly, only OzmM-AT2 is required for OzmM activity for OZM biosynthesis, whereas OzmM-AT1 seemed to be a cryptic AT domain. The above findings, together with previous results using isotope-labeled precursor feeding assays, are assembled for the OZM biosynthesis model to be proposed. The incorporation of both malonyl-CoA (by OzmM-AT2) and methoxymalonyl-ACP (by OzmC) extender units seemed to be unprecedented for this class of trans-AT type I PKSs, which might be fruitfully manipulated to create structurally diverse novel compounds.

Luminescence switching of CdTe quantum dots in presence of p-sulfonatocalix[4]arene to detect pesticides in aqueous solution.

A simple, rapid, and sensitive identification method of fenamithion and acetamiprid is developed using supramolecular nano-sensitizers combining of CdTe quantum dots (QDs) and p-sulfonatocalix[4]arene as additive by fluorescent spectroscopic technique in water. Depending on p-sulfonatocalix[4]arene, the selectivity of CdTe QDs is tuned between fenamithion and acetamiprid. The luminescence of free CdTe QDs is quenched selectively to fenamithion. While in the presence of p-sulfonatocalix[4]arene, it shows that the fluorescence intensity of QDs is enhanced selectively to acetamiprid due to the cooperation of QDs and p-sulfonatocalix[4]arene. Based on the response characteristics of the QDs, a fluorescent method is performed for tuning selective determination of the pesticides. Under optimum conditions described, it is found that the pesticides effect on the luminescence of the CdTe QDs in concentrations dependence are described by a Stern-Volmer-type equation or a Langmuir binding isotherm equation in the range of 0-10(-4)M (fenamithion) and 0-10(-3)M (acetamiprid), with the corresponding detection limits (3sigma) of 1.2 x 10(-8)M (fenamithion) and 3.4 x 10(-8)M (acetamiprid), respectively. The possible mechanism is discussed.

Functional analysis of spfD gene involved in DNA phosphorothioation in Pseudomonas fluorescens Pf0-1.

DNA phosphorothioation is widespread in many bacterial species. By homology analysis of the dnd gene cluster in Pseudomonas fluorescens Pf0-1, a spfBCDE gene cluster involved in DNA phosphorothioation was localized. Disruption of the spfD gene, a dndD homolog, caused the loss of the Dnd phenotype and demonstrated the involvement of spfD in DNA phosphorothioation in P. fluorescens Pf0-1. The ATPase activity of SpfD suggests that SpfD could hydrolyze ATP to provide the energy required in the DNA phosphorothioate modification process.

Alternative method for site-directed mutagenesis of complex polyketide synthase in Streptomyces albus JA3453.

Sequence analysis of oxazolomycin (OZM) biosynthetic gene cluster from Streptomyces albus JA3453 revealed a gene, ozmH, encoding a hybrid polyketide and non-ribosomal peptide enzyme. Tandem ketosynthase (KS) domains (KS 10-1 and KS 10-2) were characterized and they show significant homology with known KSs. Using an alternative method that involves RecA-mediated homologous recombination, the negative selection marker sacB gene, and temperature-sensitive replications, site-directed mutagenesis of the catalytic triad amino acid cysteines were carried out in each of the tandem KS domains to test the function they play in OZM biosynthesis. HPLC-mass spectrometry analysis of the resulting mutant strains showed that KS 10-2 is essential for OZM biosynthesis but KS 10-1 is not indispensable and might serve as a "redundant" domain. These results confirmed the existence of an "extra domain" in complex polyketide synthase.

A novel DNA modification by sulfur: DndA is a NifS-like cysteine desulfurase capable of assembling DndC as an iron-sulfur cluster protein in Streptomyces lividans.

A novel DNA modification system by sulfur (S) in Streptomyces lividans 66 was reported to be encoded by a cluster of five genes designated dndA-E [Zhou, X., He, X., Liang, J., Li, A., Xu, T., Kieser, T., Helmann, J. D., and Deng, Z. (2005) Mol. Microbiol. 57, 1428-1438]. The dndA gene was cloned and the protein product expressed in Escherichia coli, purified to homogeneity, and characterized as a homodimeric protein of ca. 91 kDa. Purified DndA has a yellow color and UV-visible spectra characteristic of a pyridoxal phosphate-containing enzyme and was proven to be a cysteine desulfurase able to catalyze removal of elemental S atoms from l-cysteine to produce l-alanine with substrate specificity similar to that of E. coli IscS. DndC was also purified to homogeneity and found to contain a 4Fe-4S cluster by spectral analysis and have obvious ATP pyrophosphatase activity. DndA could catalyze iron-sulfur cluster assembly by activation of apo-Fe DndC protein prepared by removal of its iron-sulfur cluster using alpha,alpha'-dipyridyl. A mutated DndA, with serine substituted for cysteine at position 327, which was confirmed to have lost its corresponding cysteine desulfurase activity, also lost its ability to reactivate the apo-Fe DndC. The likely involvement of an interaction between DndA and DndC in the biochemical pathway for the unusual site-specific DNA modification in S. lividans 66 is discussed.

arsRBOCT arsenic resistance system encoded by linear plasmid pHZ227 in Streptomyces sp. strain FR-008.

In the arsenic resistance gene cluster from the large linear plasmid pHZ227, two novel genes, arsO (for a putative flavin-binding monooxygenase) and arsT (for a putative thioredoxin reductase), were coactivated and cotranscribed with arsR1-arsB and arsC, respectively. Deletion of the ars gene cluster on pHZ227 in Streptomyces sp. strain FR-008 resulted in sensitivity to arsenic, and heterologous expression of the ars gene cluster in the arsenic-sensitive Streptomyces strains conferred resistance on the new hosts. The pHZ227 ArsB protein showed homology to the yeast arsenite transporter Acr3p. The pHZ227 ArsC appears to be a bacterial thioredoxin-dependent ArsC-type arsenate reductase with four conserved cysteine thioredoxin-requiring motifs.

Utilization of the methoxymalonyl-acyl carrier protein biosynthesis locus for cloning the oxazolomycin biosynthetic gene cluster from Streptomyces albus JA3453.

Oxazolomycin (OZM), a hybrid peptide-polyketide antibiotic, exhibits potent antitumor and antiviral activities. Using degenerate primers to clone genes encoding methoxymalonyl-acyl carrier protein (ACP) biosynthesis as probes, a 135-kb DNA region from Streptomyces albus JA3453 was cloned and found to cover the entire OZM biosynthetic gene cluster. The involvement of the cloned genes in OZM biosynthesis was confirmed by deletion of a 12-kb DNA fragment containing six genes for methoxymalonyl-ACP biosynthesis from the specific region of the chromosome, as well as deletion of the ozmC gene within this region, to generate OZM-nonproducing mutants.

A novel DNA modification by sulphur.

Streptomyces lividans has a novel DNA modification, which sensitises its DNA to degradation during electrophoresis (the Dnd phenotype). The entire gene cluster (dnd) involved in this modification was localized on an 8 kb DNA fragment and was expressed in a S. lividans deletion mutant (dnd) and in several heterologous hosts. Disruption of the dnd locus abolishes the Dnd phenotype, and gain of the dnd locus conferred the Dnd phenotype respectively. Extensive analysis of the dnd gene cluster revealed five open reading frames, whose hypothetic functions suggested an incorporation of sulphur or a sulphur-containing substance into S. lividans genome, yet in an unknown manner. The Dnd phenotype was also discovered to exist in DNA of widespread bacterial species of variable origin and diverse habitat. Similarly organized gene clusters were found in several bacterial genomes representing different genera and in eDNA of marine organisms, suggesting such modification as a widespread phenomenon. A coincidence between the Dnd phenotype and DNA modification by sulphur was demonstrated to occur in several representative bacterial genomes by the in vivo(35)S-labelling experiments.