Exploring the Formation of Chemical Markers in Chaste Honey by Comparative Metabolomics: From Nectar to Mature Honey.

Identification of chemical markers is important to ensure the authenticity of monofloral honey; however, the formation of chemical markers in honey has received little attention. Herein, using comparative metabolomics, we first identified chemical markers in chaste honey and then explored their formation and accumulation from nectar to mature honey. We identified agnuside and p-hydroxybenzoic acid glucosides as chemical markers for chaste honey. Besides, we developed an UHPLC-MS/MS method for quantifying these markers and found that their levels varied significantly across sample sources. We compared the presence of these compounds in chaste nectar and mature honey. The outcomes underscore that these characteristic compounds are not simply delivered from nectar to mature honey, and activities of honeybees (collecting and processing) play a pivotal role in their formation and accumulation. These observations shed light on how mature honey can form its unique qualities with a rich assortment of natural bioactive compounds, potentially supporting health benefits.

Concise Analysis of Single-Stranded DNA of Recombinant Adeno-Associated Virus By Automated Electrophoresis System.

Recombinant adeno-associated virus (rAAV) is a prominent viral vector currently available for human gene therapy. The diameter of the rAAV capsid is ∼25 nm, and a positive or negative single-stranded DNA is packaged within the vector capsid. In this report, we describe a concise method to examine the extracted rAAV genome using an automated electrophoresis system. The rAAV genome, prepared from vector particles through either heat treatment at 95°C for 10 min or the phenol-chloroform extraction method, was analyzed using an automated electrophoresis system under denaturation conditions. The heat treatment protocol demonstrated a comparable yield with the phenol-chloroform extraction protocol, and the quantified amounts of the rAAV genome obtained using the automated electrophoresis system were consistent with those quantitated by quantitative PCR. Additionally, crude rAAV extractions could also be analyzed by the automated electrophoresis system after DNase I treatment. These results indicated that this simple and quick analysis using automated electrophoresis is highly useful for confirming the purity and integrity of the rAAV genome.

Evaluation of next-generation sequencing performance for in vitro detection of viruses in biological products.

Next-Generation Sequencing (NGS) can detect nucleic acid sequences in a massively parallel sequencing. This technology is expected to be widely applied for the detection of viral contamination in biologics. The recently published ICH-Q5A (R2) draft indicates that NGS could be an alternative or supplement to in vitro viral tests. To examine the performance of NGS for the in vitro detection of viruses, adenovirus type 5 (Ad5), a model virus, was inoculated into Vero cells, which are the most popular indicator cells for the detection of adventitious viruses in the in vitro test. Total RNA extracted from the Vero cells infected with Ad5 was serially diluted with that from non-infected Vero cells, and each sample was analyzed using short- or long-read NGSs. The limits of detection of both NGS methods were almost the same and both methods were sensitive enough to detect viral sequences as long as there was at least one copy in one assay. Although the multiplexing in NGS carries the risk of cross-contamination among the samples, which could lead to false positives, this technology has the potential to become a rapid and sensitive method for detecting adventitious agents in biologics.

Chaste honey in long term-storage: Occurrence and accumulation of Maillard reaction products, and safety assessment.

Honey, a natural sweetener that can be stored long-term, is prone to Maillard reactions. Maillard reaction products (MRPs), such as 5-hydroxymethylfurfural (5-HMF), α-dicarbonyl compounds (α-DCs), and advanced glycation end products (AGEs), negatively affect human health. We analyzed MRP accumulation in chaste honey over four years. In the first year, α-DCs were dominant with total contents of 509.7 mg/kg. In the second year, Amadori compounds increased, accounting for the largest percentage. Their formation at the initial stage showed inhibition of the Maillard reaction over time. AGE contents were approximately 1.00 mg/kg over four years, which is negligible compared to other foods. Increased 5-HMF was significantly correlated with storage time (p < 0.01), making it a suitable indicator of honey quality. Due to the lack of MRP risk assessments, we compared our findings with daily intake of MRPs from other foods, and the levels of MRPs in honey over four years are acceptable.

Quantitation of Residual Host Cell DNA in Recombinant Adeno-Associated Virus Using Droplet Digital Polymerase Chain Reaction.

Recombinant adeno-associated virus (rAAV) is a viral vector commonly used in gene therapy. Residual host cell DNA is an impurity that has been associated with the risk of infection and oncogenicity. Thus, it needs to be monitored for quality control. We aimed to develop a droplet digital polymerase chain reaction (ddPCR) method targeting 18S ribosomal RNA (rRNA) genes to quantitate residual host cell DNA. The copy number of the 18S rRNA gene was determined using two sets of primer pairs for 116- and 247-bp amplicons sharing the C-terminus. For conversion of the copy number of the 18S rRNA gene into the mass concentration of genomic DNA, the accurate copy number of 18S rRNA genes in HEK293 genomic DNA was determined by comparison with copy numbers of three reference genes (EIF5B, DCK, and HBB). Results showed that 88.6-97.9% of HEK293 genomic DNA spiked into rAAV preparations was recovered. The ddPCR-based assay was applied to rAAV preparations to quantitate residual host cell DNA as an impurity. Our findings indicate that the assay can be used for the quantitation and size distribution of residual host cell DNA in rAAV products.

Infectivity assessment of porcine endogenous retrovirus using high-throughput sequencing technologies.

Xenogenic cell-based therapeutic products are expected to alleviate the chronic shortage of human donor organs. For example, porcine islet cell products are currently under development for the treatment of human diabetes. As porcine cells possess endogenous retrovirus (PERV), which can replicate in human cells in vitro, the potential transmission of PERV has raised concerns in the case of products that use living pig cells as raw materials. Although several PERV sequences exist in the porcine genome, not all have the ability to infect human cells. Therefore, polymerase chain reaction analysis, which amplifies a portion of the target gene, may not accurately assess the infection risk. Here, we determined porcine genome sequences and evaluated the infectivity of PERVs using high-throughput sequencing technologies. RNA sequencing was performed on both PERV-infected human cells and porcine cells, and reads mapped to PERV sequences were examined. The normalized number of the reads mapped to PERV regions was able to predict the infectivity of PERVs, indicating that it would be useful for evaluation of the PERV infection risk prior to transplantation of porcine products.

A highly sensitive method for the detection of recombinant PERV-A/C env RNA using next generation sequencing technologies.

Several xenogenic cell-based therapeutic products are currently under development around the world for the treatment of human diseases. Porcine islet cell products for treating human diabetes are a typical example. Since porcine cells possess endogenous retrovirus (PERV), which can replicate in human cells in vitro, the potential transmission of PERV has raised concerns in the development of these products. Four subgroups of infectious PERV have been identified, namely PERV-A, -B, -C, and recombinant PERV-A/C. Among them, PERV-A/C shows a high titre and there was a paper reported that an incidence of PERV-A/C viremia was increased in diseased pigs; thus, it would be important to monitor the emergence of PERV-A/C after transplantation of porcine products. In this study, we developed a highly sensitive method for the detection of PERV-A/C using next generation sequencing (NGS) technologies. A model PERV-C spiked with various doses of PERV-A/C were amplified by RT-PCR and the amplicons were analysed by NGS. We found that the NGS analysis allowed the detection of PERV-A/C at the abundance ratios of 1% and 0.1% with true positive rates of 100% and 57%, respectively, indicating that it would be useful for the rapid detection of PERV-A/C emergence after transplantation of porcine products.

Preferential recognition of monomeric CCR5 expressed in cultured cells by the HIV-1 envelope glycoprotein gp120 for the entry of R5 HIV-1.

Bimolecular fluorescence complementation (BiFC) and western blot analysis demonstrated that CCR5 exists as constitutive homo-oligomers, which was further enhanced by its antagonists such as maraviroc (MVC) and TAK-779. Staining by monoclonal antibodies recognizing different epitopes of CCR5 revealed that CCR5 oligomer was structurally different from the monomer. To determine which forms of CCR5 are well recognized by CCR5-using HIV-1 for the entry, BiFC-positive and -negative cell fractions in CD4-positive 293T cells were collected by fluorescent-activated cell sorter, and infected with luciferase-reporter HIV-1 pseudotyped with CCR5-using Envs including R5 and R5X4. R5 and dual-R5 HIV-1 substantially infected BiFC-negative fraction rather than BiFC-positive fraction, indicating the preferential recognition of monomeric CCR5 by R5 and dual-R5 Envs. Although CCR5 antagonists enhanced oligomerization of CCR5, MVC-resistant HIV-1 was found to still recognize both MVC-bound and -unbound forms of monomeric CCR5, suggesting the constrained use of monomeric CCR5 by R5 HIV-1.

Structure and dynamics of the gp120 V3 loop that confers noncompetitive resistance in R5 HIV-1(JR-FL) to maraviroc.

Maraviroc, an (HIV-1) entry inhibitor, binds to CCR5 and efficiently prevents R5 human immunodeficiency virus type 1 (HIV-1) from using CCR5 as a coreceptor for entry into CD4(+) cells. However, HIV-1 can elude maraviroc by using the drug-bound form of CCR5 as a coreceptor. This property is known as noncompetitive resistance. HIV-1(V3-M5) derived from HIV-1(JR-FLan) is a noncompetitive-resistant virus that contains five mutations (I304V/F312W/T314A/E317D/I318V) in the gp120 V3 loop alone. To obtain genetic and structural insights into maraviroc resistance in HIV-1, we performed here mutagenesis and computer-assisted structural study. A series of site-directed mutagenesis experiments demonstrated that combinations of V3 mutations are required for HIV-1(JR-FLan) to replicate in the presence of 1 µM maraviroc, and that a T199K mutation in the C2 region increases viral fitness in combination with V3 mutations. Molecular dynamic (MD) simulations of the gp120 outer domain V3 loop with or without the five mutations showed that the V3 mutations induced (i) changes in V3 configuration on the gp120 outer domain, (ii) reduction of an anti-parallel ß-sheet in the V3 stem region, (iii) reduction in fluctuations of the V3 tip and stem regions, and (iv) a shift of the fluctuation site at the V3 base region. These results suggest that the HIV-1 gp120 V3 mutations that confer maraviroc resistance alter structure and dynamics of the V3 loop on the gp120 outer domain, and enable interactions between gp120 and the drug-bound form of CCR5.

Generation of a human bone marrow-derived mesenchymal stem cell line expressing and secreting high levels of bioactive α-melanocyte-stimulating hormone.

α-Melanocyte-stimulating hormone (α-MSH) functions as a mediator of inflammation and immunity; however, the short half-life and high dose needed limit the comprehensive clinical application of α-MSH. The aim of this study was to generate human bone marrow-derived mesenchymal stem cells (MSCs) that express and secrete high levels of bioactive α-MSH. MSCs were obtained from a normal donor and assessed for proliferation, surface markers, and adipogenic and osteogenic differentiation. A lentivirus-encoding α-MSH was constructed. MSCs were infected with this lentivirus-encoding α-MSH and assessed for stability and the expression and secretion of bioactive α-MSH. The cumulative MSC expansion rates pre- and post-lentivirus infection were not significantly different (P > 0.05). The MSCs remained stable after infection with the lentivirus-encoding α-MSH. The concentration of α-MSH in the supernatants of MSCs infected with the lentivirus-encoding α-MSH was 17.55 ng/ml (P < 0.001), and a melanin assay indicated that bioactive α-MSH was secreted from MSCs infected with the lentivirus-encoding α-MSH, with an optical absorbance at OD(405) of 0.886 (P < 0.001). These results suggested that MSCs were promising cell carriers for the expression and secretion of high levels of bioactive α-MSH.

The human immunodeficiency virus-1 genotype diversity and drug resistance mutations profile of volunteer blood donors from Chinese blood centers.

BACKGROUND: The global human immunodeficiency virus (HIV)-1 epidemic is becoming increasingly diverse and complex. Molecular epidemiologic characteristics were studied for HIV-1-infected blood donors from five Chinese regions to determine genotype diversity and drug resistance mutations (DRMs) profile. STUDY DESIGN AND METHODS: HIV-1 confirmed-reactive serum samples were collected from 172 blood donors from five blood centers during 2007 to 2010. HIV-1 Pol including whole protease and partial reverse transcriptase genes was amplified, sequenced, and analyzed for the subtype determination and drug resistance profile description. RESULT: A total of 113 amplified sequences including 82 from Kunming blood center and 31 from four other blood centers had the following genotype characteristics: G (0.9%), B (2.7%), circulating recombinant form (CRF) 01_AE (32.7%), CRF07_BC (22.1%), and CRF08_BC (41.6%). Female donors represent 45.1% of all cases and 63.9% cases with DRMs. The prevalence of samples with potential low or higher resistance among Chinese blood donors is 4.4%. CONCLUSION: HIV-1 infection in Chinese blood donors is genetically diverse and the subtype distribution reflects that from the high-risk populations. Our results support continuous molecular epidemiologic surveillance for HIV-1 in blood donors as a part of a comprehensive HIV control program.

A combination of polymorphic mutations in V3 loop of HIV-1 gp120 can confer noncompetitive resistance to maraviroc.

Maraviroc binds to the pocket of extracellular loops of the cell surface CCR5 and prevents R5 HIV-1 from using CCR5 as a coreceptor for entry into CD4-positive cells. To evaluate the contribution of the V3 loop structure in gp120 to maraviroc resistance, we isolated maraviroc-resistant variants from the V3 loop library virus (HIV-1(V3Lib)) containing a set of random combinations of 0-10 polymorphic mutations in vitro. HIV-1(V3Lib) at passage 17 could not be suppressed even at 10 µM (>1400-fold resistance), while HIV-1(JR-FL) at passage 17 revealed an 8-fold resistance to maraviroc. HIV-1(V3Lib-P17) contained T199K and T275M plus 5 mutations in the V3 loop, I304V/F312W/T314A/E317D/I318V. The profile of pseudotyped virus containing I304V/F312W/T314A/E317D/I318V in V3 loop alone revealed a typical noncompetitive resistance, although T199K and/or T275M could not confer noncompetitive resistance. This type of library virus is useful for isolation of escape viruses from effective entry inhibitors.

DrTx(1-42), a C-terminally truncated analogue of drosotoxin, is a candidate of analgesic drugs.

Drosotoxin is an engineered tetrodotoxin-resistant (TTX-R) sodium channel-specific blocker with a non-toxic structural core (Zhu et al. Biochem Pharmacol 2010; 80:1296-302). Here, we report the discovery and functional characterization of a carboxyl-terminally truncated analogue of drosotoxin (named DrTx(1-42)) which selectively inhibited dorsal root ganglion (DRG) neuron TTX-R sodium current (I(Na)) with an IC(50) value of 1.74±0.07µM. Consistent with this effect, DrTx(1-42) significantly attenuates inflammatory hyperalgesia of mice in a formalin-induced pain model with stronger potency than indomethacin, a nonsteroidal anti-inflammatory and analgesic drug. Our mutational experiments indicate that the N-turn insertion is an essential functional determinant for the emergence of neurotoxicity from a non-toxic antifungal scaffold.

Drosotoxin, a selective inhibitor of tetrodotoxin-resistant sodium channels.

The design of animal toxins with high target selectivity has long been a goal in protein engineering. Based on evolutionary relationship between the Drosophila antifungal defensin (drosomycin) and scorpion depressant Na(+) channel toxins, we exploited a strategy to create a novel chimeric molecule (named drosotoxin) with high selectivity for channel subtypes, which was achieved by using drosomycin to substitute the structural core of BmKITc, a depressant toxin acting on both insect and mammalian Na(+) channels. Recombinant drosotoxin selectively inhibited tetrodotoxin-resistant (TTX-R) Na(+) channels in rat dorsal root ganglion (DRG) neurons with a 50% inhibitory concentration (IC(50)) of 2.6+/-0.5muM. This chimeric peptide showed no activity on K(+), Ca(2+) and TTX-sensitive (TTX-S) Na(+) channels in rat DRG neurons and Drosophila para/tipE channels at micromolar concentrations. Drosotoxin represents the first chimeric toxin and example of a non-toxic core scaffold with high selectivity on mammalian TTX-R Na(+) channels.

Two recombinant depressant scorpion neurotoxins differentially affecting mammalian sodium channels.

The scorpion depressant toxins are a group of evolutionarily conserved polypeptides targeting sodium channels, which show preferential ability to induce flaccid paralysis in insects, making them attractive candidates for the construction of transgenic plants or viral vectors to control pests. In this study, two new depressant toxin-like peptides (BmKITc and BmKITc2) differing only at position 52 (Lys for Thr) were produced in Escherichia coli. Circular dichroism analysis indicated that these two recombinant peptides display a typical structural feature similar to native scorpion toxins. They both cause a maintained current component at the last phase of inactivation of the insect sodium channel DmNav1/tipE expressed in Xenopus oocytes and interestingly, they do not produce a beta effect despite of their primary structure as beta-toxins. Furthermore, an inhibitory effect with BmKITc but not with BmKITc2 was observed on TTX-R sodium currents in rat DRG neurons. We hypothesize that such differential potency highlights a crucial role of lysine 52 in channel selectivity. Our results therefore indicate that, in spite of the general idea, not all scorpion depressant toxins interact with mammalian and/or insect sodium channels in the same manner.

Positively selected sites of scorpion depressant toxins: possible roles in toxin functional divergence.

Scorpion depressant toxins represent a distinct pharmacological group of sodium channel neurotoxins, identified by their preferential ability in induction of depressant and flaccid paralysis of insects. However, recent observations that some members in this group exhibit anti-mammal activity raise an interesting evolutionary question of whether it is a consequence of adaptive evolution to the early radiation of mammals on earth. By employing the maximum likelihood method, we provided convincing statistical evidence in favor of positive selection driving the evolution of the depressant toxins, and found that two of three positively selected sites are located on the functional surface of the toxins. A complex model of the scorpion depressant toxin LqhIT2 binding to insect sodium channel alpha-subunit (DmNav1) was constructed by structural bioinformatics approaches which highlights a possible direct interaction between these two sites and insect sodium channels. Our work presented here thus suggests that accelerated substitutions in these site residues could offer an evolutionary advantage for these toxins to adapt different channels from diverse origins.

Functional expression of a Drosophila antifungal peptide in Escherichia coli.

Drosomycin is a key effector molecule involved in Drosophila innate immunity against fungal infection. This peptide is composed of 44 residues stabilized by four disulfide bridges. As the first step towards the understanding of the molecular basis for its specific antifungal activity, rapid and efficient production of the wild-type peptide and its mutants is needed. Here, we report a pGEX system for high-level expression of recombinant Drosomycin. The fusion Drosomycin protein with a carrier of Glutathione S-transferase (GST) was initially purified by affinity chromatography followed by Enterokinase cleavage. The digested product was separated by gel filtration and reverse phase HPLC. Mass spectrometry and circular dichroism spectroscopy analysis revealed that the recombinant peptide has identical molecular weight and correct structural conformation to native Drosomycin. Classical inhibition assay showed clear antifungal activity against Neurospora crassa with the IC(50) of 1.0muM. Successful expression of the CSalphabeta-type antifungal peptide in E. coli offers a basis for further studying its functional surface by alanine scanning mutagenesis strategy. Also, our work should be helpful in developing this peptide to an antifungal drug.