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Strobilanthes bantonensis Lindau belongs to the family Acanthaceae. It is an antiviral herb that can be used to prevent Influenza virus infections in the border areas between China and Vietnam. Loc...

The complete nucleotide sequence of the Astragalus membranaceus (Fisch.) Bunge var. membranaceus chloroplast genome was reported and characterized in this study. The chloroplast genome is a circular molecule of 123623 bp that belongs to the inverted repeat-lacking clade (IRLC). It comprises 110 genes, including 76 protein-coding genes, four unique rRNAs and 30 tRNAs. Similar to the plastomes of A. membranaceus (Fisch.) Bunge var. mongholicus (Bunge) P. K. Hsiao and other closely related species, rpl22 and rps16 are absent. The phylogenetic analysis of 67 proteins from 29 chloroplast genomes belonging to IRLC provided strong support for the non-monophyly of Galegeae. This genome has provided a wealth of information for distinguishing varieties of A. membranaceus.

Stemona sessilifolia (Miq.) Miq., commonly known as Baibu, is one of the most popular herbal medicines in Asia. In the Chinese Pharmacopoeia, Baibu has multiple authentic sources and there are many similar herbs sold as Baibu in herbal medicine markets. The existence of counterfeits of Baibu brings challenges to its identification. To assist in its accurate identification, we sequenced and analyzed the complete chloroplast genome of S. sessilifolia using next-generation sequencing technology. The genome was found to be 154,037 bp in length, possessing a typical quadripartite structure consisting of a pair of inverted repeats (IRs: 27,090 bp) separated by a large single copy (LSC: 81,949 bp) and a small single copy (SSC: 17,908 bp). A total of 112 unique genes were identified, including 80 protein-coding, 28 transfer RNA and four ribosomal RNA genes. In addition, 45 tandem, 27 forward, 23 palindromic and 104 simple sequence repeats were detected in the genome by repeated analysis. Compared with its counterfeits (Asparagus officinalis and Carludovica palmata) we found that IR expansion and SSC contraction events of S. sessilifolia resulted in two copies of the rpl22 gene in the IR regions and a partial duplication of the ndhF gene in the SSC region. An approximately 3-kb-long inversion was also identified in the LSC region, leading to the petA and cemA genes being presented in the complementary strand of the chloroplast DNA molecule. Comparative analysis revealed some highly variable regions, including trnF-GAA_ndhJ, atpB_rbcL, rps15_ycf1, trnG-UCC_trnR-UCU, ndhF_rpl32, accD_psaI, rps2_rpoC2, trnS-GCU_trnG-UCC, trnT-UGU_trnL-UAA and rps16_trnQ-UUG. Finally, gene loss events were investigated in the context of phylogenetic relationships. In summary, the complete plastome of S. sessilifolia will provide valuable information for the distinction between Baibu and its counterfeits and assist in elucidating the evolution of S. sessilifolia.

Ban-Lan-Gen, the root tissues derived from several morphologically indistinguishable plant species, have been used widely in traditional Chinese medicines for numerous years. The identification of reliable markers to distinguish various source plant species is critical for the effective and safe use of products containing Ban-Lan-Gen. Here, we analyzed and characterized the complete chloroplast (cp) genome sequence of Strobilanthes cusia (Nees) Kuntze to identify high-resolution markers for the species determination of Southern Ban-Lan-Gen. Total DNA was extracted and subjected to next-generation sequencing. The cp genome was then assembled, and the gaps were filled using PCR amplification and Sanger sequencing. Genome annotation was conducted using CpGAVAS web server. The genome was 144,133 bp in length, presenting a typical quadripartite structure of large (LSC; 91,666 bp) and small (SSC; 17,328 bp) single-copy regions separated by a pair of inverted repeats (IRs; 17,811 bp). The genome encodes 113 unique genes, including 79 protein-coding, 30 transfer RNA, and 4 ribosomal RNA genes. A total of 20 tandem, 2 forward, and 6 palindromic repeats were detected in the genome. A phylogenetic analysis based on 65 protein-coding genes showed that S. cusia was closely related to Andrographis paniculata and Ruellia breedlovei, which belong to the same family, Acanthaceae. One interesting feature is that the IR regions apparently undergo simultaneous contraction and expansion, resulting in the presence of single copies of rps19, rpl2, rpl23, and ycf2 in the LSC region and the duplication of psbA and trnH genes in the IRs. This study provides the first complete cp genome in the genus Strobilanthes, containing critical information for the classification of various Strobilanthes species in the future. This study also provides the foundation for precisely determining the plant sources of Ban-Lan-Gen.

The complete nucleotide sequence of the Astragalus membranaceus (Fisch.) Bunge var. membranaceus chloroplast genome was reported and characterized in this study. The chloroplast genome is a circular molecule of 123623 bp that belongs to the inverted repeat-lacking clade (IRLC). It comprises 110 genes, including 76 protein-coding genes, four unique rRNAs and 30 tRNAs. Similar to the plastomes of A. membranaceus (Fisch.) Bunge var. mongholicus (Bunge) P. K. Hsiao and other closely related species, rpl22 and rps16 are absent. The phylogenetic analysis of 67 proteins from 29 chloroplast genomes belonging to IRLC provided strong support for the non-monophyly of Galegeae. This genome has provided a wealth of information for distinguishing varieties of A. membranaceus.

Although H5N1 avian influenza has not yet acquired the capacity to readily infect humans, should it do so, this viral pathogen would present an increasing threat to the immunologically naive human population. Subunit vaccines based on the viral glycoprotein hemagglutinin (HA) can provide protective immunity against influenza. Polyanhydride nanoparticles have been shown to enhance efficacy of subunit vaccines, providing the dual advantages of adjuvanticity and sustained delivery resulting in enhanced protein stability and immunogenicity. In this work, a recombinant trimer of H5 (H53 ) was encapsulated and released from polyanhydride nanoparticles. Release kinetics of the encapsulated H53 were found to be dependent on polymer chemistry (i.e., hydrophobicity and molecular weight). Polyanhydride nanoparticles composed of sebacic anhydride and 1,6-bis(p-carboxyphenoxy)hexane (CPH; that degrade into more acidic monomers) released structurally stable HA H53 , while H53 released from formulations composed of CPH and 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) (that are amphiphilic and whose degradation products are less acidic) displayed unfolding of tertiary structure. However, the antigenicity of the H53 based on binding of a H5-specific monoclonal antibody was preserved upon release from all the formulations studied, demonstrating the value of polyanhydride nanoparticles as a viable platform for HA-based influenza vaccines.

Two variants of equine infectious anemia virus (EIAV) that differed in sensitivity to broadly neutralizing antibody were tested in direct competition assays. No differences were observed in the growth curves and relative fitness scores of EIAVs of principal neutralizing domain variants of groups 1 (EIAV PND-1 ) and 5 (EIAV PND-5 ), respectively; however, the neutralization-resistant EIAV PND-5 variant was less infectious in single-round replication assays. Infectious center assays indicated similar rates of cell-to-cell spread, which was approximately 1,000-fold more efficient than cell-free infectivity. These data indicate that efficient cell-to-cell spread can overcome the decreased infectivity that may accompany immune escape and should be considered in studies assessing the relative levels of fitness among lentivirus variants, including HIV-1.

Precisely identifying biological targets and accurately extracting their relatively weak signals from complicated physiological environments represent daunting challenges in biological detection and biomedical diagnosis. Fluorescence techniques have become the method of choice and offer minimally invasive and ultrasensitive detections, thus, providing a wealth of information regarding the biological mechanisms in living systems. Despite fluorescence analysis has advanced remarkably, conventional detections still encounter considerable limitations. This stems from the fact that the fluorescence intensity signal (I) is sensitive and liable to numerous external factors including temperature, light source, medium characteristics, and dye concentration. The interferences exasperatingly undermine the precision of measurements, and frequently render the signal undetectable. For example, fluorescence from single-molecule emitters can be measured on glass substrates under optimum conditions, but single-molecule events in complicated physiological environments such as live cells can hardly be detected because of autofluorescence interference and other factors. Furthermore, traditional intensity (I) and wavelength (λ) measurements do not reveal the interactive nature between the donor and the acceptor. Thus, innovative detection strategies to circumvent these aforementioned limitations of the conventional techniques are critically needed. With the use of photoswitching-induced donor-acceptor-fluorescence double modulations, we present a novel strategy that introduces three additional physical parameters: modulation amplitude (A), phase shift (ΔΦ), and lock-in frequency (ω), and demonstrate that such a strategy can circumvent the limitation of the conventional fluorescence detection techniques. Together, these five physical quantities (I, λ, A, ΔΦ, ω) reveal insightful information regarding molecular interactive strength between the probe and the analyte and enable extracting weak-fluorescence spectra from large interfering noises in complex environments.

This minireview highlights recent advances of research dedicated to photoswitchable fluorescent nanoparticles and their applications. Recently, several strategies have been developed to synthesize nanoparticles with optically switchable emission properties: either fluorescence on/off or dual-alternating-color fluorescence photoswitching. The underlying mechanisms of fluorescence photoswitching enable many different types of photoswitchable fluorescent nanoparticles to change fluorescence colors, thus validating the basis of the initial photoswitching design. Among all possible applications, the usage of photoswitchable fluorescent nanoparticles to empower super-resolution fluorescence imaging and to label biological targets was subsequently reviewed. Finally, we summarize the important areas regarding future research and development on photoswitchable fluorescent nanoparticles.

Although H5N1 avian influenza has not yet acquired the capacity to readily infect humans, should it do so, this viral pathogen would present an increasing threat to the immunologically naïve human population. Subunit vaccines based on the viral glycoprotein hemagglutinin (HA) can provide protective immunity against influenza. Polyanhydride nanoparticles have been shown to enhance efficacy of subunit vaccines, providing the dual advantages of adjuvanticity and sustained delivery resulting in enhanced protein stability and immunogenicity. In this work, a recombinant trimer of H5 (H53 ) was encapsulated and released from polyanhydride nanoparticles. Release kinetics of the encapsulated H53 were found to be dependent on polymer chemistry (i.e., hydrophobicity and molecular weight). Polyanhydride nanoparticles composed of sebacic anhydride and 1,6-bis(p-carboxyphenoxy)hexane (CPH; that degrade into more acidic monomers) released structurally stable HA H53 , while H53 released from formulations composed of CPH and 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) (that are amphiphilic and whose degradation products are less acidic) displayed unfolding of tertiary structure. However, the antigenicity of the H53 based on binding of a H5-specific monoclonal antibody was preserved upon release from all the formulations studied, demonstrating the value of polyanhydride nanoparticles as a viable platform for HA-based influenza vaccines.

Using the equine infectious anemia virus (EIAV) lentivirus model system, we previously demonstrated protective effects of broadly neutralizing immune plasma in young horses (foals) with severe combined immunodeficiency (SCID). However, in vivo selection of a neutralization-resistant envelope variant occurred. Here, we determined the protective effects of purified immunoglobulin with more potent broadly neutralizing activity. Overall, protection correlated with the breadth and potency of neutralizing activity in vitro . Four of five SCID foals were completely protected against homologous challenge, while partial protection occurred following heterologous challenge. These results support the inclusion of broadly neutralizing antibodies in lentivirus control strategies.

Nanoparticles have played important roles in biological detections and cellular imaging, both in vitro and in vivo . One of the driving forces for the use of nanoprobes is that nanoparticles have numerous advantages such as strong signal strength, resistance to photobleaching, and tunable fluorescence emissions. These physical properties make them the method of choice for tracking vesicle transportation, detecting biomolecular interactions, and imaging live cells. The second driving force for the use of nanoparticles is the fact that their fabrication and conjugation protocols can be customized, and thus, fluorescent nanoparticles are being used more and more frequently to replace the traditional organic dyes. This chapter summarizes recent advances in research devoted to nanoprobes that use fluorescence signals for detection. The nanoparticles discussed in this chapter include polymeric nanoparticles, silica-based particles, metal nanoparticles, and quantum dots. Finally, important areas for future research and improvement of various nanoprobes are highlighted.

We have developed a class of spiropyran dyes and their fluorescence colors can be reversibly photoswitched from red color to green, blue, or nearly dark, thus alternating between two colors. Such individual dyes emit either one color or the other, but not both simultaneously. These photoswitchable dyes-enabled nanoparticles, however, emit either one pure color or a combination of both colors because the nanoparticle fluorescence originates from multiple dyes therein. As a result, the nanoparticle shines >30 times brighter than the state-of-the-art organic dyes such as fluorescein. Interestingly, these copolymer nanoparticles exhibit tunable non-specific interactions with live cells and nanoparticles containing properly balanced butyl acrylate and acrylamide monomers render essentially very little non-specific binding to live cells. Decorated with HMGA1 protein, these optically switchable dual-color nanoparticles undergo endocytosis and unambiguously identify themselves from fluorescence interference including autofluorescence, thus enabling a new tool for live cell imaging.

Beyond-diffraction-limit optical imaging of cells will reveal biological mechanisms, cellular structures, and physiological processes in nanometer scale. Harnessing the photoswitching properties of spiropyran fluorophores, we achieved nanoresolution fluorescence imaging using photoactuated unimolecular logical switching attained reconstruction (PULSAR) microscopy. The PULSAR microscope successfully resolved nanostructures and subcellular organelles when the photoswitchable nanoparticles containing spiropyran dyes were used as fluorescent probes.

Optical imaging in live cells has provided a wealth of information regarding the various biological mechanisms, including using genetically coded green-fluorescent protein-conjugated organic dye molecules and, more recently, highly luminescent quantum dots as optical tags for the target biomolecules. Cells are inherently complex, grow constantly and have autofluorescence covering the entire visible spectrum ranging from green to red. At the single quantum-emitter level, it is often difficult to distinguish optical probes from fortuitous fluorophores inside living cells owing to complexity and constant evolvement. We have developed photoswitchable nanoparticles–optical nanoprobes that can be highlighted in either red or green during fluorescence imaging. Such optically addressable nanoprobes offer unambiguous detection of sites of biological interactions, and successfully implementing such optically switchable nanoprobes should greatly reduce the occurence of false-positives in biomedical imaging and unambiguous detections.

Optically switchable dual-color fluorescent nanoparticles that incorporate two classes of dyes into the polymeric chains have been synthesized using an emulsion polymerization method. The nanoparticles consist of an organic photoisomerizable dye, spiropyran, as an optically responsive component and another fluorescent dye, perylene diimide, as a high-energy emitter. Under UV irradiation, the colorless spiropyran undergoes photoisomerization to yield merocyanine, which absorbs at 588 nm and fluoresces strongly at 670 nm. The absorption band of the merocyanine matches well the fluorescence bands of perylene diimide (at 535 and 575 nm), and therefore fluorescence resonance energy transfer (FRET) converts the high-energy green emission of the perylene into low-energy red emission when the merocyanine form is present. Upon exposure to alternating UV ( 420 nm) light, the nanoparticles cycle between red and green fluorescence as the spiro and mero forms of the optically responsive component...

Emulsion polymerization yields 40–400 nm diameter polymer nanoparticles with spiropyran-merocyanine dyes incorporated into their hydrophobic cavities; in contrast to their virtually nonfluorescent character in most environments, the merocyanine forms of the encapsulated dyes are highly fluorescent. Spiro-mero photoisomerization is reversible, allowing the fluorescence to be switched “on” and “off” by alternating UV and visible light. Immobilizing the dye inside hydrophobic pockets of nanoparticles also improves its photostability, rendering it more resistant than the same dyes in solution to fatigue effects arising from photochemical switching. The photophysical characteristics of the encapsulated fluorophores differ dramatically from those of the same species in solution, making nanoparticle-protected hydrophobic fluorophores attractive materials for potential applications such as optical data storage and switching and biological fluorescent labeling. To evaluate the potential for biological tagging, these optically addressable nanoparticles have been delivered into living cells and imaged with a liquid nitrogen cooled CCD.

Kathleen A Ross,1 Hyelee Loyd,2 Wuwei Wu,2 Lucas Huntimer,3 Shaheen Ahmed,4 Anthony Sambol,5 Scott Broderick,6 Zachary Flickinger,2 Krishna Rajan,6 Tatiana Bronich,4 Surya Mallapragada,1 Michael J Wannemuehler,3 Susan Carpenter,2 Balaji Narasimhan1 1Chemical and Biological Engineering, Iowa State University, Ames, IA, USA; 2Animal Science, Iowa State University, Ames, IA, USA; 3Veterinary Microbiology and Preventive Medicine, Iowa State University, Ames, IA, USA; 4Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, NE, USA; 5Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE, USA; 6Materials Science and Engineering, Iowa State University, Ames, IA, USA Abstract: H5N1 avian influenza is a significant global concern with the potential to become the next pandemic threat. Recombinant subunit vaccines are an attractive alternative for pandemic vaccines compared to traditional vaccine technologies. In particular, polyanhydride nanoparticles encapsulating subunit proteins have been shown to enhance humoral and cell-mediated immunity and provide protection upon lethal challenge. In this work, a recombinant H5 hemagglutinin trimer (H53) was produced and encapsulated into polyanhydride nanoparticles. The studies performed indicated that the recombinant H53 antigen was a robust immunogen. Immunizing mice with H53 encapsulated into polyanhydride nanoparticles induced high neutralizing antibody titers and enhanced CD4+ T cell recall responses in mice. Finally, the H53-based polyanhydride nanovaccine induced protective immunity against a low-pathogenic H5N1 viral challenge. Informatics analyses indicated that mice receiving the nanovaccine formulations and subsequently challenged with virus were similar to naïve mice that were not challenged. The current studies provide a basis to further exploit the advantages of polyanhydride nanovaccines in pandemic scenarios. Keywords: polymer, nanoparticle, vaccine, subunit, neutralizing antibody

Fluorescence imaging of biological systems with spatial resolution beyond diffraction limit is of intensive interests. Recently, it has been discovered that by stochastically photoswitching fluorophores on and off, the diffraction limit can be circumvented in far-field optical imaging. Here we present a small photoswitching dye, spiropyran derivatives with the desired characteristics as the excellent photoswitchable molecular fluorophores.[3] Using such a fluorophore, we have achieved photoactuated unimolecular logical switching attained reconstruction (PULSAR) microscopy, with resolution down to 10-40 nm, far beyond the diffraction limit. Spiropyran requires no special treatment to cells and can be readily modified using chemical functional groups for specific targeting.