BCR-UNet: Bi-directional ConvLSTM residual U-Net for retinal blood vessel segmentation.

Background: High precision segmentation of retinal blood vessels from retinal images is a significant step for doctors to diagnose many diseases such as glaucoma and cardiovascular diseases. However, at the peripheral region of vessels, previous U-Net-based segmentation methods failed to significantly preserve the low-contrast tiny vessels. Methods: For solving this challenge, we propose a novel network model called Bi-directional ConvLSTM Residual U-Net (BCR-UNet), which takes full advantage of U-Net, Dropblock, Residual convolution and Bi-directional ConvLSTM (BConvLSTM). In this proposed BCR-UNet model, we propose a novel Structured Dropout Residual Block (SDRB) instead of using the original U-Net convolutional block, to construct our network skeleton for improving the robustness of the network. Furthermore, to improve the discriminative ability of the network and preserve more original semantic information of tiny vessels, we adopt BConvLSTM to integrate the feature maps captured from the first residual block and the last up-convolutional layer in a nonlinear manner. Results and discussion: We conduct experiments on four public retinal blood vessel datasets, and the results show that the proposed BCR-UNet can preserve more tiny blood vessels at the low-contrast peripheral regions, even outperforming previous state-of-the-art methods.

Hypochlorous acid initiated lipid chlorination at air-water interface.

There has been a surge of interest in interfacial hypochlorous acid (HOCl) chemistry for indoor air quality and public health. Here we combined nanoelectrospray mass spectrometry (nESI-MS) and acoustic levitation (AL) techniques to study the chlorination chemistry of three model lipids (DPPE, POPG, DOPG) mediated by HOCl at the air-water interface of levitated water droplet. For DPPE with no CC double bonds, HOCl was insensitive to the alkane chains, and showed considerable delay directing to head amino groups compared to that in aqueous environment. Chlorination chemistry, for POPG and DOPG with CC double bonds, preferentially reacted with double bonds of one chain. The mechanism was discussed in light of these observations, and it is concluded that the increased hydrophilicity of the chlorinated chain disturbed the lipid packing and attracted it toward the water phase. In addition, the reaction rate constant and reactive uptake coefficient suggested that the chlorination of lipids exposed to HOCl at the air-water interface is likely to occur rapidly. These results gain the knowledge of HOCl mediated lipid interface reaction at the molecule level, and would better understand the adverse health effects associated with elevated indoor pollutants.

Water Microdroplets Allow Spontaneously Abiotic Production of Peptides.

The chemistry of abiotic synthesis of peptides in the context of their prebiotic origins is a continuing challenge that arises from thermodynamic and kinetic constraints in aqueous media. Here we reported a strategy of microdroplets' mass spectrometry for peptide bonds formed from pure amino acids or a mixture in the presence of phosphoric acids in aqueous microdroplets. In contrast to bulk experiments, the condensation reactions proceed spontaneously under ambient conditions. The microdroplet gave a negative free-energy change (ΔG ∼ -1.1 kcal/mol), and product yields of ∼75% were obtained at the scale of a few milliseconds. Experiments in which nebulization gas pressure and external charge were varied established dependence of peptide production on the droplet size that has a high surface-to-volume ratio. It is concluded that the condensation reactions occurred at or near the air-water interfaces of microdroplets. This aqueous microdroplets approach also provides a route for chemistry synthesis in the prebiotic era.

Development of a multiplex polymerase chain reaction assay for the parallel detection of harmful algal bloom-forming species distributed along the Chinese coast.

Harmful algal blooms (HABs) have adverse effects on the marine ecological environment, public health, and marine economy. Thus, methods for the accurate and rapid identification of harmful algal species are urgently needed for the effective monitoring of the occurrence of HABS. A method for the parallel detection of harmful algal species must be established because various HAB-forming algal species coexist in the marine environment. This work developed a multiplex PCR (mPCR) method that can simultaneously detect six common HAB-forming microalgal species distributed along the coast of China: Karlodinium veneficum (Kv), Chattonella marina (Cm), Skeletonema spp., Scrippsiella trochoidea (St), Karenia mikimotoi (Km), and Prorocentrum donghaiense (Pd). Specific mPCR primers were designed from the internal transcribed spacer rDNA or large subunit rDNA gene of the target algal species. The mPCR conditions were optimized. Each mPCR primer was subjected to a cross-reactivity test with other microalgae to confirm the specificity of the developed mPCR system. The results of the system stability test indicated that the background concentration of DNA tested did not affect the performance of the established mPCR system. The results of the sensitivity test showed that the detection limit of the proposed mPCR system for Kv, Cm, Km, and Pd was 0.6 ng µL-1 and that for Skeletonema spp. and St was 0.06 ng µL-1. Additional mPCR analysis with spiked field samples revealed that the detection limit of the mPCR system for Km, Pd, and Kv was 60 cells, whereas that for Cm, Skeletonema spp., and St was 6 cells. The convenience and accuracy of the established mPCR assay were further validated through tests with field samples. The proposed mPCR assay is characterized by parallel analysis, strong specificity, and stability and can be used to supplement morphology-based detection methods for algal species.

Comparison of loop-mediated isothermal amplification with hyperbranched rolling circle amplification as a simple detection method for Heterosigma akashiwo.

The fish-killing alga Heterosigma akashiwo is a globally distributed, toxic, and bloom-forming raphidophyte that has caused great losses to the fishing industry in many coastal countries. Therefore, rapid and sensitive detection methods should be developed to present timely warning of harmful algal blooms. In this study, hyperbranched rolling circle amplification (HRCA) was established for the detection of H. akashiwo and compared with loop-mediated isothermal amplification (LAMP) in terms of specificity and sensitivity. The partial D1-D2 sequence of the large subunit (LSU) of rDNA of H. akashiwo was used to design a specific padlock probe for HRCA and two pairs of specific primers for LAMP. The parameters for HRCA were optimized. Cross-reactivity tests showed that the specificity of the developed HRCA for H. akashiwo was greater than that of LAMP in this study. The sensitivities of HRCA and LAMP were comparable and were 10-fold higher than that of regular PCR. These methods also yielded a detection limit of 20 fg/µL for the recombinant plasmid containing the target LSU D1-D2 and 1 cell for target species. The test with the simulated field samples indicated that the developed HRCA obtained a detection limit of 5 cells mL-1, which was lower than the warning cell density (100 cells mL-1) of H. akashiwo. The visual detection of positive HRCA could be achieved via coloration reaction with the addition of fluorescent SYBR Green I dye to the amplification products. The developed HRCA was also efficient for field samples with target cell densities ranging from 10 cells mL-1 to 1000 cells mL-1. Therefore, the proposed HRCA detection protocols are possibly applicable to the field monitoring of H. akashiwo.

Physiological and molecular responses of Prorocentrum donghaiense to dissolved inorganic phosphorus limitation.

Prorocentrum donghaiense is an important dinoflagellate as it frequently forms harmful algal blooms that cause serious damage to marine ecosystems and fisheries in the coast of East China Sea. Previous studies showed that phosphorus acquisition (especially inorganic phosphorus) was the limiting factor for P. donghaiense growth. However, the responsive mechanism of this microalga under dissolved inorganic phosphorus (DIP) limitation is poorly understood. In this work, the physiological parameters and differentially expressed genes in P. donghaiense response to DIP limitation were comparatively analyzed. DIP-depleted P. donghaiense displayed decreased growth rate, enlarged cell size, decreased cellular phosphorus content, and high AP activities. A forward suppression subtractive hybridization (SSH) library representing differentially upregulated genes in P. donghaiense under DIP-depleted conditions was constructed, and 134 ESTs were finally identified, with a significant identity (E values<1×10-4) to the deposited genes (proteins) in the corresponding databases. Five representative genes, namely, NAD-dependent deacetylase, phosphoglycolate phosphatase, heat shock protein (HSP) 90, rhodopsin, and HSP40 were investigated through real-time quantitative PCR to verify the effectiveness of the established SSH library. Results showed that all the selected genes were differentially expressed and thus indicated that the established SSH library generally represented differentially expressed genes. These genes were classified into 11 categories according to their gene ontology annotations of biological processes. The members involved in functional responses such as cell defense/homeostasis, phosphorus metabolism, and cellular cycles were specially discussed. This study is the first to perform a global analysis of differentially expressed functional genes in P. donghaiense under DIP-depleted condition. It provided new insights into the molecular adaptive mechanisms of dinoflagellate in response to phosphorous limitation and elucidating the formation mechanism of algal blooms.

Application of hyper-branched rolling circle amplification (HRCA) and HRCA-based strip test for the detection of Chattonella marina.

Harmful algal blooms (HABs) are global threats to marine ecosystems, fisheries, and human health. Therefore, developing effective and accurate methods for identifying causative algae and monitoring seawater quality is urgent. However, traditional, microscopy-based methods are complex, inaccurate, and time-consuming. Here, we present a novel method for effective and sensitive detection of Chattonella marina using hyper-branched rolling circle amplification (HRCA) and HRCA-based strip test (HBST). The large subunit (LSU) ribosomal DNA (rDNA) D1-D2 region of C. marina was firstly sequenced to design a species-specific padlock probe (PLP). The HRCA reaction with two amplification primers and further HBST for C. marina was established. The optimized reaction conditions for HRCA were PLP concentration, 20 pM; ligation temperature, 65 °C; ligation time, 60 min; amplification temperature, 61 °C; and amplification time, 60 min. The developed HBST detection procedure involved HRCA reaction, test strip preparation, hybridization, coloration, and judgment of hybridization by the naked eye. Specificity and sensitivity of the established methods were validated. Moreover, the results showed that the established detection methods were specific and sensitive to C. marina. The detection limits of HRCA and HBST assays were 10 copies and 1 copy µL-1 of plasmid with LSU rDNA of C. marina, which are of two and three respective magnitude orders higher than conventional PCR. Finally, the protocols were applied to the simulated field samples and the results showed that the developed HBST assay had higher detection sensitivity than HRCA and PCR. In conclusion, the methods presented in this study are promising for sensitive, intuitive, and specific detection of C. marina in field monitoring natural samples and may provide a good detection model for other harmful algae in the future.

Identification and characterization of a ferritin gene involved in the immune defense response of scallop Chlamys farreri.

Scallop Chlamys farreri is an important aquaculture species in northern China. However, its mass mortality caused by several pathogens can result in great economic loss and negative impacts to the sustainable development of the scallop industry. Thus, improving the overall understanding of immune response mechanisms involved in host-pathogen interactions is necessary. Ferritins are conserved molecules in organisms that are involved in diverse biological processes, such as mediating host-pathogen responses. In this study, we report a novel ferritin gene from C. farreri (denoted as CfFER). The full length of CfFER is 848 bp and contains a 5'-UTR of 113 bp, a 3'-UTR of 219 bp, and a complete open reading frame (ORF) of 516 bp. The ORF encodes a polypeptide of 171 amino acid residues with a molecular weight of approximately 19.95 kDa and an isoelectric point of 5.07. The CfFER protein exhibited typical ferritin structures, namely, a ferroxidase diiron center, a ferrihydrite nucleation center, and an iron-binding response signature. Phylogenetic analysis revealed that CfFER was closely related to other mollusk ferritin proteins. Expression of CfFER in different tissues was analyzed by quantitative real-time PCR, and results showed that CfFER was ubiquitously expressed in all examined tissues. The highest and lowest expression levels of CfFER were measured in the muscle and hemocyte, respectively. The relative mRNA expression of CfFER in response to bacterial (Vibrio anguillarum) and viral (acute viral necrobiotic virus) challenges sharply increased by ca. 5-fold about12 h post-infection (hpi) and then normalized at 48 hpi. Western blot analysis with polyclonal antibodies generated from the recombinant product of CfFER also demonstrated the presence of ferritin protein in hemocytes. These findings strongly suggest that CfFER is involved in the immune response of C. farreri and protection against pathogen challenge.

Molecular characterization and immune response expression of the QM gene from the scallop Chlamys farreri.

The scallop Chlamys farreri is an important aquaculture species in northern China. However, the sustainable development of the scallop industry is currently threatened by several pathogens that cause mass mortality of this mollusk. Therefore, a complete understanding of the immune response mechanisms involved in host-virus interactions is necessary. This study reports a novel QM gene from C. farreri. This gene was first identified as a putative tumor suppressor gene from human and then confirmed to participate in several functions, including immune response. The QM gene from C. farreri (CfQM) was identified by suppression subtractive hybridization, and its full-length (763 bp) cDNA was obtained through rapid amplification of cDNA ends. The cDNA of CfQM contained a short 5'-UTR of 22 bp and a 3'-UTR of 84 bp. Its ORF comprised 657 nucleotides that encode 218 amino acids with a molecular weight of approximately 28.3 kDa and an isoelectric point of 10.06. The deduced amino acid sequence of CfQM contained a series of conserved functional motifs that belong to the QM family. Phylogenetic analysis revealed that CfQM was closely related to other mollusk QM proteins, and altogether they form a mollusk QM protein subfamily that displays evolutionary conservation from yeast to human. The tissue-specific expression and transcriptional regulation of CfQM were investigated by quantitative real-time PCR in response to bacterial (Vibrio anguillarum) and viral (acute viral necrobiotic virus) challenges. The transcript level of CfQM was high in all of the examined tissues in a constitutive manner. The highest and lowest expression levels of CfQM were measured in the hepatopancreas and hemocyte, respectively. Upon bacterial and viral challenges, the relative mRNA expression of CfQM sharply increased at 6 h post-infection (hpi) and then normalized at 48 hpi. These findings suggest that CfQM can respond to and protect against pathogen challenge. To the best of our knowledge, this study is the first report of the QM gene from scallop. The results presented herein provided new insights into the molecular basis of host-pathogen interactions in C. farreri.

CdTe quantum dots enhance feasibility of EvaGreen-based real-time PCR with decent amplification fidelity.

Quantitative real-time PCR (qPCR), as an important quantitative technique for nucleic acids, has been widely used in many fields including clinical diagnosis, molecular biology, and cancer research. However, non-specific amplification products are still a frequent problem in qPCR. In this study, we investigated the effects of QDs on real-time amplification based on either SYBR Green I or EvaGreen. It was found that QDs could raise the amplification sensitivity and thus enhance the efficiency using SYBR Green I detection system. In the case of EvaGreen detection systems, addition of QDs also led to a better correlation coefficient than without QDs. EvaGreen-based system gave sharper peaks for melting curves than SYBR Green I. The experiments indicated that the polymerase activity could be partially blocked by QDs at the pre-PCR temperatures, resulting in the improvement of PCR specificity. These results indicated that CdTe QDs could be used as a descent qPCR enhancer. Good amplification fidelity in QDs-facilitated qPCR was also a plus that has not been reported elsewhere.

QSAR study and the hydrolysis activity prediction of three alkaline lipases from different lipase-producing microorganisms.

The hydrolysis activities of three alkaline lipases, L-A1, L-A2 and L-A3 secreted by different lipase-producing microorganisms isolated from the Bay of Bohai, P. R. China were characterized with 16 kinds of esters. It was found that all the lipases have the ability to catalyze the hydrolysis of the glycerides, methyl esters, ethyl esters, especially for triglycerides, which shows that they have broad substrate spectra, and this property is very important for them to be used in detergent industry. Three QSAR models were built for L-A1, L-A2 and L-A3 respectively with GFA using Discovery studio 2.1. The models equations 1, 2 and 3 can explain 95.80%, 97.45% and 97.09% of the variances (R(2)(adj)) respectively while they could predict 95.44%, 89.61% and 93.41% of the variances (R(2)(cv)) respectively. With these models the hydrolysis activities of these lipases to mixed esters were predicted and the result showed that the predicted values are in good agreement with the measured values, which indicates that this method can be used as a simple tool to predict the lipase activities for single or mixed esters.

[Expressions of LEAFY homologous genes in different organs and stages of Ginkgo biloba].

Expressions of Ginlfy and GinNdly gene were studied by northern blotting in different organs and stages of Ginkgo Biloba. Ginlfy gene was expressed in different organs such as root, stem, leaf of juvenile tree, male tree and female tree, and in different stages of male flower bud and female flower bud. It was inferred that Ginlfy gene could be expressed constitutionally. GinNdly gene was only expressed in leaf of juvenile tree, male tree and female tree and in different stages of male flower bud and female flower bud, while GinNdly gene was not expressed in the other organs. Therefore it was thought that GinNdly gene could be expressed differentially and be a close relation to development of flower.