Clinical Evaluation of Sequential Transdermal Delivery of Vitamin B6, Compound Glycyrrhizin, Metronidazole, and Hyaluronic Acid Using Needle-Free Liquid Jet in Facial Seborrheic Dermatitis.

Facial seborrheic dermatitis (FSD) is a common facial inflammatory dermatitis. Needle-free transdermal jet injection (NTJI) is a non-invasive injection of drug solution by using a high-pressure liquid injection instrument. To explore a safer, more tolerable, and convenient medical way using NTJI in the treatment of FSD, the patients were treated with vitamin B6, glycyrrhizin compound, metronidazole, and hyaluronic acid sequentially using NTJI every 2 weeks, and only those treated for more than three times were included. A VISIA facial imaging system for the evaluation of erythema, superficial lipid level, and roughness of skin surface and a CK analyzer for biophysical parameters, including the stratum corneum hydration, facial surface lipid, and trans-epidermal water loss, were applied. Erythema was significantly reduced after every treatment (weeks 2, 4, and 6; P < 0.05), whereas superficial lipid level was not improved significantly until week 6 (P < 0.05), and roughness of the skin surface was not improved significantly during the whole treatment. The stratum corneum hydration of lesional skin was significantly increased after three times of treatment (P < 0.05). No observable adverse effect, such as marked erythema, blistering, or atrophy, was observed. Sequential transdermal delivery of small molecular weight drugs (vitamin B6, glycyrrhizin compound, metronidazole, and hyaluronic acid) using NTJI is a safe, low-toxicity, and take-home drug-free therapy for the treatment of FSD.

Complete genomic sequence of the Vibrio alginolyticus bacteriophage Vp670 and characterization of the lysis-related genes, cwlQ and holA.

BACKGROUND: Biocontrol of bacterial pathogens by bacteriophages (phages) represents a promising strategy. Vibrio alginolyticus, a gram-negative bacterium, is a notorious pathogen responsible for the loss of economically important farmed marine animals. To date, few V. alginolyticus phages have been successfully isolated, and only three complete genome sequences of them have been released. The limited available phage resources and poor genomic data hamper research on V. alginolyticus phages and their applications for the biocontrol of V. alginolyticus. RESULTS: We isolated a phage, Vp670, against the V. alginolyticus strain E06333 and obtained its full genomic sequence. It contains 43,121 nucleotides with a GC content of 43.4%, and codes for 49 predicted open reading frames. Observation by electron microscope combined with phylogenetic analysis of DNA polymerase indicates that Vp670 belongs to the subfamily Autographivirinae in the family Podoviridae. orf3 (designated holA) and orf8 (designated cwlQ) are predicted to encode a holin (HolA) and an endolysin (CwlQ), respectively. Expression of holA alone or coexpression of holA and cwlQ from within arrested the growth of Escherichia coli and V. alginolyticus while the expression of cwlQ alone had no effect on the growth of them. Further observation by transmission electron microscopy revealed that the expression of holA vanished the outer membrane and caused the release of cellular contents of V. alginolyticus and the coexpression of holA and cwlQ directly burst the cells and caused a more drastic release of cellular contents. Expression of cwlQ alone in V. alginolyticus did not cause cytomorphological changes. CONCLUSIONS: Phage Vp670 is a V. alginolyticus phage belonging to the family of Podoviridae. The genome of Vp670 contains a two-component lysis module, which is comprised of holA and cwlQ. holA is predicted to encode for the holin protein, HolA, and cwlQ is predicted to encode for the endolysin protein, CwlQ. Both holA and cwlQ likely play important roles during the release of phage progeny.

Comparative genomic analysis of six new-found integrative conjugative elements (ICEs) in Vibrio alginolyticus.

BACKGROUND: Vibrio alginolyticus is ubiquitous in marine and estuarine environments. In 2012-2013, SXT/R391-like integrative conjugative elements (ICEs) in environmental V. alginolyticus strains were discovered and found to occur in 8.9 % of 192 V. alginolyticus strains, which suggests that V. alginolyticus may be a natural pool possessing resourceful ICEs. However, complete ICE sequences originating from this bacterium have not been reported, which represents a significant barrier to characterizing the ICEs of this bacterium and exploring their relationships with other ICEs. In the present study, we acquired six ICE sequences from five V. alginolyticus strains and performed a comparative analysis of these ICE genomes. RESULTS: A sequence analysis showed that there were only 14 variable bases dispersed between ICEValE0601 and ICEValHN492. ICEValE0601 and ICEValHN492 were treated as the same ICE. ICEValA056-1, ICEValE0601 and ICEValHN492 integrate into the 5' end of the host's prfC gene, and their Int and Xis share at least 97 % identity with their counterparts from SXT. ICEValE0601 or ICEValHN492 contain 50 of 52 conserved core genes in the SXT/R391 ICEs (not s025 or s026). ICEValA056-2, ICEValHN396 and ICEValHN437 have a different tRNA-ser integration site and a distinct int/xis module; however, the remaining backbone genes are highly similar to their counterparts in SXT/R391 ICEs. DNA sequences inserted into hotspot and variable regions of the ICEs are of various sizes. The variable genes of six ICEs encode a large array of functions to bestow various adaptive abilities upon their hosts, and only ICEValA056-1 contains drug-resistant genes. Many variable genes have orthologous and functionally related genes to those found in SXT/R391 ICEs, such as genes coding for a toxin-antitoxin system, a restriction-modification system, helicases and endonucleases. Six ICEs also contain a large number of unique genes or gene clusters that were not found in other ICEs. Six ICEs harbor more abundant transposase genes compared with other parts of their host genomes. A phylogenetic analysis indicated that transposase genes in these ICEs are highly diverse. CONCLUSIONS: ICEValA056-1, ICEValE0601 and ICEValHN492 are typical members of the SXT/R391 family. ICEValA056-2, ICEValHN396 and ICEValHN437 form a new atypical group belonging to the SXT/R391 family. In addition to the many genes found to be present in other ICEs, six ICEs contain a large number of unique genes or gene clusters that were not found in other ICEs. ICEs may serve as a carrier for transposable genetic elements (TEs) and largely facilitate the dissemination of TEs.

[Effects of antibiotics on the transfer frequency of SXT/R391 element of Vibrio alginolyticus].

Objective: We studied the effects of nalidixic acid, norfloxacin and kanamycin on the transfer frequency of SXT/R391 element ICEValA056-1 in Vibrio alginolyticus. Methods: The circular ICEValA056-1 in V. alginolyticus A056 was detected by PCR. Conjugation experiments were conducted between V. alginolyticus A056 and Escherichia coli VB111 to explore the frequency variation of the integrating conjugative elements transfer after donor strain A056 was cultured in Luria Broth containing nalidixic acid or norfloxacin or kanamycin in different concentrations for 15 min or 30 min. Results: Circular ICEValA056-1 was detected in V. alginolyticus A056, indicating that ICEValA056-1 had the potential to transfer. Treatment with 40 µg/mL nalidixic acid for 30 min increased the transfer frequency of ICEValA056-1 to19.59 folds. Treatment with 50 µg/mL norfloxacin for 15 min increased the transfer frequency of ICEValA056-1 to 31.25 folds. The transfer frequency of ICEValA056-1 had no significant changes under treatment with different concentrations of kanamycin for 30 min. Conclusion: This study indicates that some antibiotics can obviously increase the transfer frequency of ICEValA056-1, and that antibiotics abuse and arbitrarily discharge might intensify dissemination of integrating conjugative elements from V. alginolyticus to other bacteria.

Developing Universal Genetic Tools for Rapid and Efficient Deletion Mutation in Vibrio Species Based on Suicide T-Vectors Carrying a Novel Counterselectable Marker, vmi480.

Despite that Vibrio spp. have a significant impact on the health of humans and aquatic animals, the molecular basis of their pathogenesis is little known, mainly due to the limited genetic tools for the functional research of genes in Vibrio. In some cases, deletion of target DNAs in Vibrio can be achieved through the use of suicide vectors. However, these strategies are time-consuming and lack universality, and the widely used counterselectable gene sacB does not work well in Vibrio cells. In this study, we developed universal genetic tools for rapid and efficient deletion mutations in Vibrio species based on suicide T-Vectors carrying a novel counterselectable marker, vmi480. We explored two uncharacterized genes, vmi480 and vmi470, in a genomic island from Vibrio mimicus VM573 and confirmed that vmi480 and vmi470 constitute a two-component toxin-antitoxin system through deletion and expression of vmi480 and vmi470. The product of vmi480 exhibited strong toxicity to Escherichia coli cells. Based on vmi480 and the PBAD or PTAC promoter system, we constructed two suicide T-vectors, pLP11 and pLP12, and each of these vectors contained a multiple cloning region with two AhdI sites. Both vectors linearized by AhdI digestion could be stored and directly ligated with purified PCR products without a digestion step. By using pLP11 and pLP12 coupled with a highly efficient conjugation system provided by E. coli ß2163, six genes from four representative Vibrio species were easily deleted. By using the counterselective marker vmi480, we obtained 3-12 positive colonies (deletion mutants) among no more than 20 colonies randomly selected on counterselection plates. The strategy does not require the digestion of PCR products and suicide vectors every time, and it avoids large-scale screening colonies on counterselective plates. These results demonstrate that we successfully developed universal genetic tools for rapid and efficient gene deletion in Vibrio species.