Generation of a novel Streptococcus agalactiae ghost vaccine and examination of its immunogenicity against virulent challenge in tilapia.

Streptococcus agalactiae (S. agalactiae) is a gram-positive pathogen that causes a wide range of infections in fish and other animals including humans. Bacterial ghosts (BGs) are nonliving, empty cell envelopes and are well represented as novel vaccine candidates. In this study, we examined the immunogenicity and protective efficacy of S. agalactiae ghosts (SAG) against a virulent challenge in tilapia. Nonliving SAG was generated by a culture with Penicillin and Streptolysin, and then treated with the MIC of sodium hydroxide. The formation of a transmembrane lysis tunnel structure in SAG was visualized by electron microscopy. To investigate the SAG as a vaccine candidate, fish were divided into three groups, A (SAG immunized), B [Formalin-inactivated S. agalactiae (FSA) immunized] and C (phosphate-buffered saline, PBS-immunized control). The IgM antibody responses were significantly stronger in the SAG-immunized group than in FSA-immunized group, which was higher than in the non-immunized control group (P < 0.05). Moreover, phagocytic activity (percent phagocytes, PP) was significantly higher (p < 0.05) in the SAG-immunized group than in FSA-immunized group, which was higher than in the non-immunized control group (P < 0.05). In addition, non-specific immune immunity, such as lysozyme and superoxide dismutase activities, in the SAG-immunized fish showed significantly higher activities than FSA-immunized fish and the control group fish (P < 0.05). Also, fish immunized with SAG and FSA showed significantly higher (p < 0.05) gene expression of IL-1ß, TNF-α, IFN-γ and TGF-ß in the head kidney and spleen than fish treated with PBS during the whole observed period. In addition, fish immunized with SAG showed significantly higher gene expression of L-1ß, TNF-α, and TGF-ß in the spleen than in the FSA-immunized fish. Although there was no significant (P > 0.05) difference of survival rate (SR) or relative percent survival (RPS) between SAG and FSA immunized groups, they were all significantly more protected against the S. agalactiae challenge (SR: 86.67%, RPS: 76.395) and (SR: 80.00%, RPS: 67.50%) respectively, compared to the PBS-treated group (SR: 33.33%). These results suggest that immunization with SAG induces immune responses and provides protection against a virulent S. agalactiae challenge.

Capture of SO3 isomers in the oxidation of sulfur monoxide with molecular oxygen.

When mixing SO with O2 in N2, Ne, or Ar, an end-on complex OS-OO forms in the gas phase and can subsequently be trapped at cryogenic temperatures (2.8-15.0 K). Upon infrared light irradiation, OS-OO converts to SO3 and SO2 + O with the concomitant formation of a rare 1,2,3-dioxathiirane 2-oxide, i.e., cyclic OS([double bond, length as m-dash]O)O. Unexpectedly, the ring-closure of 16OS-18O18O yields a ca. 2 : 1 mixture of cyclic 18OS([double bond, length as m-dash]16O)18O and 16OS([double bond, length as m-dash]18O)18O. The characterization of OS-OO and OS([double bond, length as m-dash]O)O with IR and UV/Vis spectroscopy is supported by high-level ab initio computations.

Photoinduced Sulfur-Nitrogen Bond Rotation and Thermal Nitrogen Inversion in Heterocumulene OSNSO.

An exotic ternary S, N, O heterocumulene OSNSO in syn-syn (A) and syn-anti (B) conformations has been generated in the gas phase through flash vacuum pyrolysis of CF3S(O)NSO at 700 K. Upon visible light irradiation (570 ± 20 or 532 nm), both A and B, isolated in cryogenic matrices (N2, Ne, Ar, and Kr, <30 K), convert to a higher-energy anti-anti conformer (C). The reverse conformational transformation occurs either through S═N bond rotation (C to A and B) under visible light irradiation (400 ± 20 nm) at 2.8 K or through thermal nitrogen inversion (C to A) in the temperature range of 20-30 K, for which an exceptionally low activation barrier of 1.18 ± 0.07 kcal mol-1 has been experimentally determined.

Magnetically Bistable Nitrenes: Matrix Isolation of Furoylnitrenes in Both Singlet and Triplet States and Triplet 3-Furylnitrene.

Two simple acylnitrenes, 2-furoylnitrene (2) and 3-furoylnitrene (6), were generated through 266 nm laser photolysis of the corresponding azides. Both are magnetically bistable in cryogenic matrices, as evidenced by the direct observation of the closed-shell singlet state with IR spectroscopy in solid Ne, Ar, Kr, Xe, and N2 matrices (3-40 K) and the triplet state in toluene (10 K) with EPR spectroscopy (32: |D/hc| = 1.48 cm-1 and |E/hc| = 0.029 cm-1; 36: |D/hc| = 1.39 cm-1 and |E/hc|c = 0.039 cm-1). Subsequent visible-light and UV laser irradiations led to the formation of furyl isocyanates (3 and 7) and ring-opening product 3-cyanoacrolein (9-E and 9-Z), respectively, in which the elusive 3-furylnitrene (38) was also identified by IR and EPR spectroscopy (|D/hc| = 1.12 cm-1 and |E/hc| = 0.005 cm-1).

Genome size and identification of abundant repetitive sequences in Vallisneria spinulosa.

Vallisneria spinulosa is a freshwater aquatic plant of ecological and economic importance. However, there is limited cytogenetic and genomics information on Vallisneria. In this study, we measured the nuclear DNA content of Vallisneria spinulosa by flow cytometry, performed a de novo assembly, and annotated repetitive sequences by using a combination of next-generation sequencing (NGS) and bioinformatics tools. The genome size of Vallisneria spinulosa is approximately 3,595 Mbp, in which nearly 60% of the genome consists of repetitive sequences. The majority of the repetitive sequences are LTR-retrotransposons comprising 43% of the genome. Although the amount of sequencing data used in this study was not sufficient for a whole-genome assembly, it could generate an overview of representative elements in the genome. These results will lay a new foundation for further studies on various species that belong to the Vallisneria genus.

Fast Heavy-Atom Tunneling in Trifluoroacetyl Nitrene.

Chemical reactions involving quantum mechanical tunneling (QMT) increasingly attract the attention of scientists. In contrast to the hydrogen-tunneling as frequently observed in chemistry and biology, tunneling solely by heavy atoms is rare. Herein, we report heavy-atom tunneling in trifluoroacetyl nitrene, CF3 C(O)N. The carbonyl nitrene CF3 C(O)N in the triplet ground state was generated in cryogenic matrices by laser (193 or 266 nm) photolysis of CF3 C(O)N3 and characterized by IR and EPR spectroscopy. In contrast to the theoretically predicted activation barriers (>10 kcal mol-1 ), CF3 C(O)N undergoes rapid rearrangement into CF3 NCO with half-life times of less than 10 min and unprecedentedly large 14 N/15 N kinetic isotope effects (1.18-1.33) in solid Ar, Ne, and N2 matrices even at 2.8 K. The tunneling disappearance of CF3 C(O)N becomes much slower in the chemically active toluene and in 2-methyltetrahydrofuran at 5 K.

Parent Thioketene S-Oxide H2 CCSO: Gas-Phase Generation, Structure, and Bonding Analysis.

The parent thioketene S-oxide H2 CCSO has been generated in the gas phase through flash vacuum pyrolysis (ca. 1000 K) of vinyl sulfoxide H2 CC(Br)-S(O)CF3 via the intermediacy of a novel vinyl sulfinyl radical H2 C=C(Br)-SO (syn and anti conformers). Upon irradiation at 266 nm, H2 CCSO decomposes into HCCH/SO and H2 CS/CO in cryogenic Ar matrix. Whereas, visible-light irradiations result in syn↔anti conformational interconversion in H2 C=C(Br)-SO. The molecular structures of H2 CCSO and isomers are computationally studied at the CCSD(T)-F12/VTZ-F12 level of theory, and the bonding properties of H2 CCSO are analyzed with the EDA-NOCV method at the M06-2X/TZ2P level.

PTH/SDF-1α cotherapy promotes proliferation, migration and osteogenic differentiation of human periodontal ligament stem cells.

OBJECTIVES: Stromal cell-derived factor-1α (SDF-1α) plays an important role in tissue regeneration in various tissues including the periodontium. A potential limitation for its use derives from its sensitivity to cleavage by dipeptidyl peptidase-IV (DPP-IV). Parathyroid hormone (PTH) reduces enzymatic activity of DPP-IV and is suggested to be a promising agent for periodontal tissue repair. The purpose of this study was to provide insight into how SDF-1α and intermittent PTH treatment might affect proliferation, migration and osteogenic differentiation of human periodontal ligament stem cells (PDLSCs) in vitro. MATERIALS AND METHODS: PDLSCs were isolated by the limiting dilution method. Surface markers were quantified by flow cytometry. Cell-counting kit-8 (CCK8), cell migration assay, alkaline phosphatase (ALP) activity assay, alizarin red staining and RT-PCR were used to determine viability, migration and osteogenic differentiation of PDLSCs. RESULTS: PDLSCs were positive for CD44, CD73, CD90, CD105, CD166 and STRO-1 and negative for CD14, CD34 and CD45. PTH/SDF-1α cotherapy significantly promoted cell proliferation, chemotactic capability, ALP activity and mineral deposition (P<.05). Gene expression level of bone sialoprotein (BSP), runt-related transcription factor 2 (Runx2) and osteocalcin (OCN) were all up-regulated (P<.05). CONCLUSIONS: PTH/SDF-1α cotherapy promoted proliferation, migration and osteogenic differentiation of PDLSCs in vitro. Cotherapy seemed to have potential to promote periodontal tissue regeneration by facilitating chemotaxis of PDLSCs to the injured site, followed by promoting proliferation and osteogenic differentiation of these cells.

Retinoic acid triggers meiosis initiation via stra8-dependent pathway in Southern catfish, Silurus meridionalis.

Existing studies demonstrated that retinoic acid (RA) regulates meiotic initiation via stra8-independent pathway in teleosts which lack stra8 in their genomes. However, stra8 was recently identified from several fish species including Southern catfish (Silurus meridionalis). To explore the existence of stra8-dependent pathway in RA mediated meiotic initiation in fishes, in the present study, the genes encoding RA synthase aldh1a2 and catabolic enzyme cyp26a1 and cyp26b1 were cloned from the Southern catfish. By immunohistochemistry, Aldh1a2 signal was observed in gonads of both sexes during the meiotic initiation period. By real-time PCR, differentially expressed gene was observed for cyp26a1, but not for cyp26b1, in gonads during the meiotic initiation. Administration of exogenous RA or inhibition of endogenous RA degradation by either KET (RA catabolic enzyme inhibitor) or cyp26a1 knockdown using CRISPR/Cas9 induced advanced meiotic initiation in the ovaries as demonstrated by increased Stra8/stra8 expression and appearance of oocytes. In contrast, treatment with RA synthase inhibitor DEAB resulted in delayed meiotic initiation and Stra8/stra8 expression in the ovaries, which was rescued by exogenous RA administration. These results indicated that (1) RA triggers the onset of meiosis via stra8-dependent pathway in stra8 existing teleosts, as it does in tetrapods; (2) exogenous RA can rescue the endogenous RA deficiency; (3) Cyp26a1, instead of Cyp26b1, is the key catabolic enzyme involved in meiosis initiation in teleosts. Taken together, RA might trigger meiotic initiation via stra8-dependent and -independent pathway in different teleosts.

Retinoic acid homeostasis through aldh1a2 and cyp26a1 mediates meiotic entry in Nile tilapia (Oreochromis niloticus).

Meiosis is a process unique to the differentiation of germ cells. Retinoic acid (RA) is the key factor controlling the sex-specific timing of meiotic initiation in tetrapods; however, the role of RA in meiotic initiation in teleosts has remained unclear. In this study, the genes encoding RA synthase aldh1a2, and catabolic enzyme cyp26a1 were isolated from Nile tilapia (Oreochromis niloticus), a species without stra8. The expression of aldh1a2 was up-regulated and expression of cyp26a1 was down-regulated before the meiotic initiation in ovaries and in testes. Treatment with RA synthase inhibitor or disruption of Aldh1a2 by CRISPR/Cas9 resulted in delayed meiotic initiation, with simultaneous down-regulation of cyp26a1 and up-regulation of sycp3. By contrast, treatment with an inhibitor of RA catabolic enzyme and disruption of cyp26a1 resulted in earlier meiotic initiation, with increased expression of aldh1a2 and sycp3. Additionally, treatment of XY fish with estrogen (E2) and XX fish with fadrozole led to sex reversal and reversion of meiotic initiation. These results indicate that RA is indispensable for meiotic initiation in teleosts via a stra8 independent signaling pathway where both aldh1a2 and cyp26a1 are critical. In contrast to mammals, E2 is a major regulator of sex determination and meiotic initiation in teleosts.

Complete feminization of catfish by feeding Limnodilus, an annelid worm collected in contaminated streams.

BACKGROUND: Feminization of animals derived from areas polluted by endocrine disrupting chemicals (EDCs) has been observed in all classes of vertebrates. However, feminization of artificially reared offspring by feeding of specific living organisms has never been reported. METHODS: Different food (including Limnodilus spp collected from the wild) and time treatment were applied to southern catfish. In addition, EDCs in Limnodilus spp., an annelid worm collected from wild contaminated small streams, was detected by LC-MS (Liquid chromatography-mass spectrometry). Serum estradiol-17ß and vitellogenin (VTG) levels and gonadal Sf1, Dmrt1, Foxl2, Cyp19a1a expression levels in the catfish were measured through Estradiol/VTG EIA Kit and real-time PCR. RESULTS: Here we report that feeding of Limnodilus spp. resulted in complete feminization of southern catfish, which has a 1:1 sex ratio in wild conditions. Furthermore, HPLC analysis showed that the extraction of Limnodilus spp. contained EDCs, including bisphenol A (BPA), diethylstilbestrol (DES), 4-tert-octylphenol (4-t-OP) and 4-nonylphenol (4-NP), which were further confirmed by LC-MS. Feeding southern catfish using commercial diets sprayed with EDCs cocktail also resulted in 100% female, whereas the control fish displayed approximate 1:1 sex ratio. Limnodilus spp. fed fish displayed similar serum estradiol-17ß and VTG levels and gonadal Sf1, Dmrt1, Foxl2, Cyp19a1a expression levels to those of female control. CONCLUSION: These results demonstrated that EDCs in Limnodilus spp. cause southern catfish feminization by affecting aromatase expression and endogenous estrogen level. This is the first report showing that feeding of any living organism resulted in complete feminization of a vertebrate.

Ultrabroadband polarization splitter based on three-core photonic crystal fibers.

An ultrabroadband polarization splitter based on three-core photonic crystal fiber (PCF) is proposed. Two fluorine-doped cores and an elliptical modulation core are introduced to achieve an excellent performance and an ultrawide bandwidth. Numerical results demonstrate that the polarization splitter based on three-core PCF has an extinction ratio as low as -20 dB bandwidth as great as 400 nm covering almost all communication bands (O, E, S, C, and L bands). Its Gaussian-like mode-field distributions and suitable effective mode areas make it highly compatible with the standard single-mode fibers. Due to using a uniform size of circular air holes and only one elliptical central air hole, the difficulty of fabrication can be decreased to some extent.

Microarray identification of conserved microRNAs in Pinellia pedatisecta.

Large numbers of microRNAs (miRNAs) reportedly play important roles in plant development. However, none has been reported in Pinellia pedatisecta, an important aroid medicinal plant that possesses the only pedate leaf blades and the largest tubers and inflorescences among all Pinellia species. To detect the miRNAs from P. pedatisecta, an in situ synthesized custom miRNA microarray was employed, following the verification for the presence of the miRNAs through reverse transcription polymerase chain reaction (RT-PCR) and the quantitative RT-PCR (qRT-PCR). A total of 99 miRNAs belonging to 22 miRNA families were identified. The RT-PCR was applied to 14 miRNAs detected to validate the microarray results. The qRT-PCR that targeted seven miRNAs showed different expression levels of miRNAs in different tissues. The current research is the first to report on the miRNAs in P. pedatisecta and will enable further investigation of their roles in P. pedatisecta development.

Microarray-based identification of conserved microRNAs from Pinellia ternata.

A large number of microRNAs (miRNAs) reportedly play important roles in plant development; however, scarcely any of these have been found in Pinellia ternata, a herbaceous plant with special physiologic characteristics and important medicinal value. To detect P. ternata miRNAs, an in situ synthesized custom microarray of plant miRNAs was employed, following verification of the presence of the miRNAs through reverse transcription polymerase chain reaction (RT-PCR) and quantitative RT-PCR (qRT-PCR) in the current study. A total of 54 miRNAs belonging to 23 miRNA families were identified. RT-PCR applied to the eight miRNAs validated the microarray results. qRT-PCR that targeted eleven miRNAs showed the presence of miRNAs in different tissues with different expression levels, especially, miRNA319 expression level in the tubers is nearly 10 times higher than that in the stalks and leaves. This is the first report on the miRNAs in P. ternata, which will enable further investigation of their roles in P. ternata.