Monitoring the Season-Prevalence Relationship of Vairimorpha ceranae in Honey Bees (Apis mellifera) over One Year and the Primary Assessment of Probiotic Treatment in Taichung, Taiwan.

Microsporidiosis, which is caused by the pathogen Vairimorpha ceranae, is a prevalent disease in the honey bee (Apis mellifera) and might lead to significant adult honey bee mortality. In this study, we conducted an annual survey of the mature spore load of V. ceranae in the guts of nurse bees and forager bees in the apiary of National Chung Hsing University (NCHU) in Taiwan. The results indicated that, on average, honey bees hosted approximately 2.13 × 106 mature spore counts (MSCs)/bee in their guts throughout the entire year. The highest number of MSCs was 6.28 × 106 MSCs/bee, which occurred in April 2020, and the lowest number of MSCs was 5.08 × 105 MSCs/bee, which occurred in November 2020. Furthermore, the guts of forager bees had significantly higher (>58%) MSCs than those of nurse bees. To evaluate the potential of the probiotic to treat microsporidiosis, the lactic acid bacterium Leuconostoc mesenteroides TBE-8 was applied to honey bee colonies. A significant reduction (>53%) in MSCs following probiotic treatment was observed, indicating the potential of probiotic treatment for managing microsporidiosis. This research provided information on V. ceranae MSCs in the honey bee gut at NCHU in Taiwan and the MSCs' correlation with the annual season. Furthermore, a potential probiotic treatment for microsporidiosis was assessed for future management.

Radiographical presentations of inadvertent subdural placement of an epidural catheter for successful labor analgesia: A case report.

RATIONALE: Lumbar epidural analgesia is the gold standard for labor pain control. However, misplacement of epidural catheters into the subdural space may inadvertently happen. Unrecognized subdural administration of local anesthetics could result in serious consequences, including high spinal and brainstem blocks. This case report describes a case where subdural epidural catheter placement was recognized early but labor pain was adequately managed by dosage titration of subdural analgesia. PATIENT CONCERNS: This case report describes a 29-year-old primiparous pregnant woman who was admitted to our obstetric unit for labor induction at the gestational age of 38 weeks. An epidural catheter was inserted via the L2-3 intervertebral space using the standard loss of resistance to air technique. DIAGNOSES: The parturient experienced weakness in the lower extremities and numbness in the upper extremities within 15 minutes after administration of 5 mL of 2% v/v lidocaine as a loading dose and systolic blood pressure also dropped by 25%. INTERVENTIONS: The dose regimen (a mixture of 0.1% ropivacaine and 4 µg/mL fentanyl) for patient-controlled analgesia was given with bolus doses of 0.1 mL per demand and lockout intervals of 20 minutes. The analgesic effects were adequately maintained below the T8 dermatome for more than 12 hours without hypotensive episodes or obvious signs of neurological deficits. Computed tomographic myelography was performed by instillation of a nonionic iodinated contrast medium via the epidural catheter on postpartum day 2 for imaging confirmation of catheter placement in the extradural space. LESSONS: Early recognition that epidural catheters for neuraxial analgesia have been inserted into the subdural space is important for the prevention of high spinal blocks. Subdural analgesia could still be achieved by careful clinical assessment and titration of low analgesic doses. This report also presents important and clear serial computed tomographic images of catheter placement in the thoracic-lumbar subdural spaces and the extent of volume spread in the subdural space following administration of contrast medium.

Exploring the Functional Properties of Propolis, Geopropolis, and Cerumen, with a Special Emphasis on Their Antimicrobial Effects.

Bee propolis has been touted as a natural antimicrobial agent with the potential to replace antibiotics. Numerous reports and reviews have highlighted the functionalities and applications of the natural compound. Despite much clamor for the downstream application of propolis, there remain many grounds to cover, especially in the upstream production, and factors affecting the quality of the propolis. Moreover, geopropolis and cerumen, akin to propolis, hold promise for diverse human applications, yet their benefits and intricate manufacturing processes remain subjects of intensive research. Specialized cement bees are pivotal in gathering and transporting plant resins from suitable sources to their nests. Contrary to common belief, these resins are directly applied within the hive, smoothed out by cement bees, and blended with beeswax and trace components to create raw propolis. Beekeepers subsequently harvest and perform the extraction of the raw propolis to form the final propolis extract that is sold on the market. As a result of the production process, intrinsic and extrinsic factors, such as botanical origins, bee species, and the extraction process, have a direct impact on the quality of the final propolis extract. Towards the end of this paper, a section is dedicated to highlighting the antimicrobial potency of propolis extract.

Transcriptome of Nosema ceranae and Upregulated Microsporidia Genes during Its Infection of Western Honey Bee (Apis mellifera).

Nosema ceranae is one of the fungal parasites of Apis mellifera. It causes physical and behavioral effects in honey bees. However, only a few studies have reported on gene expression profiling during A. mellifera infection. In this study, the transcriptome profile of mature spores at each time point of infection (5, 10, and 20 days post-infection, d.p.i.) were investigated. Based on the transcriptome and expression profile analysis, a total of 878, 952, and 981 differentially expressed genes (DEGs) (fold change ≥ 2 or ≤ -2) were identified in N. ceranae spores (NcSp) at 5 d.p.i., 10 d.p.i., and 20 d.p.i., respectively. Moreover, 70 upregulated genes and 340 downregulated genes among common DEGs (so-called common DEGs) and 166 stage-specific genes at each stage of infection were identified. The Gene Ontology (GO) analysis indicated that the DEGs and corresponding common DEGs are involved in the functions of cytosol (GO:0005829), cytoplasm (GO:0005737), and ATP binding (GO:0005524). Furthermore, the pathway analysis found that the DEGs and common DEGs are involved in metabolism, environmental information processing, and organismal systems. Four upregulated common DEGs with higher fold-change values, highly associated with spore proteins and transcription factors, were selected for validation. In addition, the stage-specific genes are highly involved in the mechanism of pre-mRNA splicing according to GO enrichment analysis; thus, three of them showed high expression at each d.p.i. and were also subjected to validation. The relative gene expression levels showed a similar tendency as the transcriptome predictions at different d.p.i., revealing that the gene expression of N. ceranae during infection may be related to the mechanism of gene transcription, protein synthesis, and structural proteins. Our data suggest that the gene expression profiling of N. ceranae at the transcriptomic level could be a reference for the monitoring of nosemosis at the genetic level.

Effect of harvest time span on physicochemical properties, antioxidant, antimicrobial, and anti-inflammatory activities of Meliponinae honey.

BACKGROUND: The maturity of honey has a great impact on its quality and contents. Additionally, stingless bee honey contains high moisture, which allows microorganisms to survive and ferment, contributing to honey's variable flavor and physicochemical properties. Therefore, there is a need for better quality control of the honey process, especially the harvest time of honey. RESULTS: We gathered honey from the nest of stingless bees Heterotrigona itama and Tetrigona binghami over different time periods, i.e. 15, 30, and 45 days. The results show harvest time considerably affects the physicochemical properties, antioxidant activity, and antimicrobial activity of honey. Good antioxidant activity and antimicrobial activity can be found in honey produced from a longer harvest time. Compared with 15-day harvest time, at 30- or 45-day harvest time water, trehalulose, and protein content and total acidity increased, and the content of reducing sugars, fructose and glucose, and pH values, decreased in both types of honey. Moreover, compared with 15-day harvest time, the sum of six organic acids in the 45-day honey of H. itama fluctuated between 2.78 to 4.12 g 100 g-1 and in the 45-day honey of T. binghami increased from 1.66 to 3.61 g 100 g-1 , respectively. CONCLUSION: Honey harvest time had a significant effect on the physicochemical properties, antioxidant activity, and antimicrobial activity of stingless bee honey (H. itama or T. binghami). This study provides a reference for beekeepers to adjust harvest time to obtain honey with suitable physicochemical parameters. © 2022 Society of Chemical Industry.

A potential probiotic Leuconostoc mesenteroides TBE-8 for honey bee.

An isolated bacterium TBE-8, was identified as Leuconostoc mesenteroides according to the sequences of 16S rDNA and the 16S-23S rDNA intergenic spacer region. The probiotic properties of the L. mesenteroides TBE-8 strain were characterized and revealed that TBE-8 could utilize various carbohydrates, exhibited high tolerance to sucrose's osmotic pressure and acidic conditions, and could mitigate the impact of the bee pathogen Paenibacillus larvae. In addition, we found that the TBE-8 broth increased the expression of the nutrition-related genes major royal jelly protein 1 and vitellogenin in bees by approximately 1400- and 20-fold, respectively. The expression of genes encoding two antibacterial peptides, hymenoptaecin and apidaecin, in the bee abdomen was significantly increased by 17- and 7-fold in bees fed with the TBE-8 fermented broth. Furthermore, we fed four-frame bee colonies with 50% sucrose syrup containing TBE-8 and can detect the presence of approximately 2 × 106 16S rDNA copies of TBE-8 in the guts of all bees in 24 h, and the retention of TBE-8 in the bee gut for at least 5 days. These findings indicate that the L. mesenteroides TBE-8 has high potential as a bee probiotic and could enhance the health of bee colonies.

Nutritive Value of 11 Bee Pollen Samples from Major Floral Sources in Taiwan.

Bee pollen is a nutrient-rich food that meets the nutritional requirements of honey bees and supports human health. This study aimed to provide nutritive composition data for 11 popular bee pollen samples (Brassica napus (Bn), Bidens pilosa var. radiata (Bp), Camellia sinensis (Cs), Fraxinus griffithii (Fg), Prunus mume (Pm), Rhus chinensis var. roxburghii (Rc), Bombax ceiba (Bc), Hylocereus costaricensis (Hc), Liquidambar formosana (Lf), Nelumbo nucifera (Nn), and Zea mays (Zm)) in Taiwan for the global bee pollen database. Macronutrients, such as carbohydrates, proteins, and lipids, were analyzed, which revealed that Bp had the highest carbohydrate content of 78.8 g/100 g dry mass, Bc had the highest protein content of 32.2 g/100 g dry mass, and Hc had the highest lipid content of 8.8 g/100 g dry mass. Only the bee pollen Hc completely met the minimum requirements of essential amino acids for bees and humans, and the other bee pollen samples contained at least 1-3 different limiting essential amino acids, i.e., methionine, tryptophan, histidine, valine, and isoleucine. Regarding the fatty acid profile of bee pollen samples, palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2), and linolenic acid (C18:3) were predominant fatty acids that accounted for 66.0-97.4% of total fatty acids. These data serve as an indicator of the nutritional quality and value of the 11 bee pollen samples.

A Potential Fungal Probiotic Aureobasidium melanogenum CK-CsC for the Western Honey Bee, Apis mellifera.

Aureobasidium melanogenum has been used as an animal feed additive for improving thehealth of pets, however, it has not yet been applied in honey bees. Here, a fungal strain CK-CsC isolated from bee bread pollen, was identified as A. melanogenum. Following characterizing CK-CsC fermentation broth, the 4-days fermentation broth (SYM medium or bee pollen) of the CK-CsC was used to feed newly emerged adult honey bees in cages under laboratory-controlled conditions for analysis of survival, gene expression of nutrient and antibacterial peptide, and gut microbiota of honey bees. It was found that the CK-CsC fermentation broth (SYM medium or bee pollen) is nontoxic to honey bees, and can regularly increase nutrient gene expression of honey bees. However, significant mortality of bees was observed after bees were fed on the supernatant liquid of the fermentation broth. Notably, this mortality can be lowered by the simultaneous consumption of bee pollen. The honey bees that were fed bee pollen exhibited more γ-Proteobacteria, Bacteriodetes, and Actinobacteria in their gut flora than did the honey bees fed only crude supernatant liquid extract. These findings indicate that A. melanogenum CK-CsC has high potential as a bee probiotic when it was fermented with bee pollen.

Honey proteomic signatures for the identification of honey adulterated with syrup, producing country, and nectar source using SWATH-MS approach.

Honey is widely consumed by humans, due to its multiple applications as a food constituent and its therapeutic effects. This study reports on the discrimination of honey products from different geographical and botanical sources, as well as honey products containing distinct forms of syrup used in honey adulteration. Sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH-MS)-based proteomic analysis combined with chemometrics was successfully applied in identifying characteristic proteins that can be used as biomarkers of the original source of honey. Honey samples from different producing regions (Tainan, Changhua, and Taichung), countries (Taiwan and Thailand), and distinct botanical sources (longan and litchi) were clearly distinguished by the developed orthogonal projections to latent structures discriminant analysis (OPLS-DA) model with good fitness and prediction ability. Furthermore, we successfully discriminated the adulteration of honey with syrup in different proportions (even with honey content as low as 20%) with this proteomic SWATH-MS platform.

A nationwide survey of intraoperative management for one-lung ventilation in Taiwan: time to accountable for diversity in protective lung ventilation.

BACKGROUND: There is a major paradigm shift for intraoperative mechanical ventilator support by the introduction of lung protective ventilation strategies to reduce postoperative pulmonary complications and improve overall clinical outcomes in non-thoracic surgeries. However, there is currently a lack of standardized practice guideline for lung protection during thoracic surgeries that require one-lung ventilation (OLV). This study aimed to collect the expert opinions of the thoracic anesthesiologists in perioperative care for OLV surgery in Taiwan. METHODS: This prospective cross-sectional study was undertaken in 16 tertiary hospitals in Taiwan from January to February 2019. A structured survey form was distributed across the participating hospitals and the thoracic anesthesiologists were invited to complete the form voluntarily. The survey form consisted of three parts, including the basic information of the institutional anesthesia care standards, ventilatory settings for a proposed patient receiving OLV surgery and expert opinions on OLV. RESULTS: A total of 71 thoracic anesthesiologists responded to the survey. Double-lumen tubes are the most commonly used (93.8%) airway devices for OLV. The most commonly recommended ventilator setting during OLV is a tidal volume of 6-7 ml/kg PBW (67.6%) and a PEEP level of 4-6 cmH2O (73.5%). Dual controlled ventilator modes are used by 44.1% of the anesthesiologists. During OLV, high oxygen fraction (FiO2 > 0.8) is more commonly supplemented to achieve an oxygen saturation higher than 94%. The consensus of anesthesiologists on the indices for lung protection in thoracic surgery is considerably low. Large majority of the anesthesiologists (91.5%) highly recommend that an international clinical practice guideline on the protective lung ventilation strategy for thoracic anesthesia should be established. CONCLUSIONS: This study found that the thoracic anesthesiologists in Taiwan share certain common practices in ventilator support during OLV. However, they are concerned about the lack of fundamental clinical evidences to support the beneficial outcomes of the current lung protective strategies applicable to OLV. Large-scale trials are needed to form an evidence-based clinical practice guideline for thoracic anesthesia.

Potential probiotic of Lactobacillus strains isolated from the intestinal tracts of pigs and feces of dogs with antibacterial activity against multidrug-resistant pathogenic bacteria.

The occurrence of multidrug-resistant pathogenic bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA), multidrug-resistant Acinetobacter baumannii (MDRAB), extended-spectrum ß-lactamase (ESBL) Escherichia coli, and Pseudomonas aeruginosa, has become a serious problem in animals and public. The objective of this study was to identify and isolate lactic acid bacterial (LAB) strains from the intestinal tracts of pigs and feces of dogs and then characterize them as potential probiotics with antimicrobial activity against multidrug-resistant pathogenic bacteria. In a preliminary isolation screening, 45 of 1167 isolated LAB strains were found to have anti-S. aureus ATCC 27,735 activity. Using 16S rDNA and 16S-23S rDNA intergenic spacer region (ISR) sequences, five of these isolates were further identified as Lactobacillus animalis 30a-2, Lactobacillus reuteri 4-12E, Weissella cibaria C34, Lactococcus lactis 5-12H, and Lactococcus lactis 6-3H. Antimicrobial substance assays suggest that the L. lactis 5-12H, L. lactis 6-3H, L. animalis 30a-2, L. reuteri 4-12E, and W. cibaria C34 strains might produce bacteriocins and hydrogen peroxide (H2O2) as antimicrobial substances. The L. animalis 30a-2 and W. cibaria C34 strains were further characterized for probiotic properties and shown to have high acid and bile salt tolerance. Additionally, they have broad antimicrobial spectra, and can significantly repress the growth of all of the tested strains of MRSA isolates, some MDRAB, ESBL E. coli, and P. aeruginosa isolates, along with food-borne pathogenic bacteria such as Bacillus cereus ATCC 11778, Listeria monocytogens ATCC 19111, Salmonella spp., Shigella spp., and Yersinia enterocolitica BCRC 12986. This is the first report of H2O2-producing L. animalis 30a-2 and W. cibaria C34 isolated from the intestinal tracts of pigs and feces of dogs that have good antimicrobial activity against multidrug-resistant and food-borne pathogenic bacteria and have excellent probiotic properties.

PCR-RFLP of mitochondrial DNA reveals two origins of Apis mellifera in Taiwan.

Beekeeping has been a highly valued industry in Taiwan. As a result, many subspecies of Apis mellifera have been introduced to Taiwan since 1911, leading to the hybridization of different subspecies. In order to know the matrilineal origins of Taiwan A. mellifera, a total of 280 samples collected from 33 apiaries throughout the island were examined. Using PCR-RFLP of four mitochondrial gene fragments, i.e., the non-coding region between tRNAleu and cytochrome c oxidase subunit II (intergenic tRNAleu-COII), cytochrome b (Cyt b), large subunit rRNA (Ls rRNA) and cytochrome c oxidase subunit I (COI), we only found two haplotypes exist in 280 samples. Haplotypes ababa and bbbaa account for 87% of these Western bees belonged to the Eastern European (C) lineage and 13% belonged to the Middle East (Z) lineage, respectively, with the latter being totally absent in northern Taiwan. African (A) and Mellifera (M) lineages, officially imported once in 1990s and 1930s respectively, were not detected. The identification of subspecies of A. mellifera and survey of their distribution on the island are expected to facilitate efficient breeding programs and establish a more booming beekeeping industry.

Gene expression changes in honey bees induced by sublethal imidacloprid exposure during the larval stage.

Honey bee larvae exposed to sublethal doses of imidacloprid show behavioural abnormalities as adult insects. Previous studies have demonstrated that this phenomenon originates from abnormal neural development in response to imidacloprid exposure. Here, we further investigated the global gene expression changes in the heads of newly emerged adults and observed that 578 genes showed more than 2-fold changes in gene expression after imidacloprid exposure. This information might aid in understanding the effects of pesticides on the health of pollinators. For example, the genes encoding major royal jelly proteins (MRJPs), a group of multifunctional proteins with significant roles in the sustainable development of bee colonies, were strongly downregulated. These downregulation patterns were further confirmed through analyses using quantitative reverse transcription-polymerase chain reaction on the heads of 6-day-old nurse bees. To our knowledge, this study is the first to demonstrate that sublethal doses of imidacloprid affect mrjp expression and likely weaken bee colonies.

Rational Re-engineering of a Transcriptional Silencing PreQ1 Riboswitch.

Re-engineered riboswitches that no longer respond to cellular metabolites, but that instead can be controlled by synthetic molecules, are potentially useful gene regulatory tools for use in synthetic biology and biotechnology fields. Previously, extensive genetic selection and screening approaches were employed to re-engineer a natural adenine riboswitch to create orthogonal ON-switches, enabling translational control of target gene expression in response to synthetic ligands. Here, we describe how a rational targeted approach was used to re-engineer the PreQ1 riboswitch from Bacillus subtilis into an orthogonal OFF-switch. In this case, the evaluation of just six synthetic compounds with seven riboswitch mutants led to the identification of an orthogonal riboswitch-ligand pairing that effectively repressed the transcription of selected genes in B. subtilis. The streamlining of the re-engineering approach, and its extension to a second class of riboswitches, provides a methodological platform for the creation of new orthogonal regulatory components for biotechnological applications including gene functional analysis and antimicrobial target validation and screening.

Engineered biosynthesis of enduracidin lipoglycopeptide antibiotics using the ramoplanin mannosyltransferase Ram29.

The lipopeptides ramoplanin from Actinoplanes sp. ATCC 33076 and enduracidin produced by Streptomyces fungicidicus are effective antibiotics against a number of drug-resistant Gram-positive pathogens. While these two antibiotics share a similar cyclic peptide structure, comprising 17 amino acids with an N-terminal fatty acid side chain, ramoplanin has a di-mannose moiety that enduracidin lacks. The mannosyl substituents of ramoplanin enhance aqueous solubility, which was important in the development of ramoplanin as a potential treatment for Clostridium difficile infections. In this study we have determined the function of the putative mannosyltransferase encoded by ram29 from the ramoplanin biosynthetic gene cluster. Bioinformatics revealed that Ram29 is an integral membrane protein with a putative DxD motif that is suggested to bind to, and activate, a polyprenyl phosphomannose donor and an extracytoplasmic C-terminal domain that is predicted to bind the ramoplanin aglycone acceptor. The ram29 gene was cloned into the tetracycline inducible plasmid pMS17 and integrated into the genome of the enduracidin producer S. fungicidicus. Induction of ram29 expression in S. fungicidicus resulted in the production of monomannosylated enduracidin derivatives, which are not present in the WT strain. Tandem MS analysis showed that mannosylation occurs on the Hpg11 residue of enduracidin. In addition to confirming the function of Ram29, these findings demonstrate how the less common, membrane-associated, polyprenyl phosphosugar-dependent glycosyltransferases can be used in natural product glycodiversification. Such a strategy may be valuable in future biosynthetic engineering approaches aimed at improving the physico-chemical and biological properties of bioactive secondary metabolites including antibiotics.

Identification of a high-efficiency baculovirus DNA replication origin that functions in insect and mammalian cells.

UNLABELLED: The p143 gene from Autographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV) has been found to increase the expression of luciferase, which is driven by the polyhedrin gene promoter, in a plasmid with virus coinfection. Further study indicated that this is due to the presence of a replication origin (ori) in the coding region of this gene. Transient DNA replication assays showed that a specific fragment of the p143 coding sequence, p143-3, underwent virus-dependent DNA replication in Spodoptera frugiperda IPLB-Sf-21 (Sf-21) cells. Deletion analysis of the p143-3 fragment showed that subfragment p143-3.2a contained the essential sequence of this putative ori. Sequence analysis of this region revealed a unique distribution of imperfect palindromes with high AT contents. No sequence homology or similarity between p143-3.2a and any other known ori was detected, suggesting that it is a novel baculovirus ori. Further study showed that the p143-3.2a ori can replicate more efficiently in infected Sf-21 cells than baculovirus homologous regions (hrs), the major baculovirus ori, or non-hr oris during virus replication. Previously, hr on its own was unable to replicate in mammalian cells, and for mammalian viral oris, viral proteins are generally required for their proper replication in host cells. However, the p143-3.2a ori was, surprisingly, found to function as an efficient ori in mammalian cells without the need for any viral proteins. We conclude that p143 contains a unique sequence that can function as an ori to enhance gene expression in not only insect cells but also mammalian cells. IMPORTANCE: Baculovirus DNA replication relies on both hr and non-hr oris; however, so far very little is known about the latter oris. Here we have identified a new non-hr ori, the p143 ori, which resides in the coding region of p143. By developing a novel DNA replication-enhanced reporter system, we have identified and located the core region required for the p143 ori. This ori contains a large number of imperfect inverted repeats and is the most active ori in the viral genome during virus infection in insect cells. We also found that it is a unique ori that can replicate in mammalian cells without the assistance of baculovirus gene products. The identification of this ori should contribute to a better understanding of baculovirus DNA replication. Also, this ori is very useful in assisting with gene expression in mammalian cells.

Modular riboswitch toolsets for synthetic genetic control in diverse bacterial species.

Ligand-dependent control of gene expression is essential for gene functional analysis, target validation, protein production, and metabolic engineering. However, the expression tools currently available are difficult to transfer between species and exhibit limited mechanistic diversity. Here we demonstrate how the modular architecture of purine riboswitches can be exploited to develop orthogonal and chimeric switches that are transferable across diverse bacterial species, modulating either transcription or translation, to provide tunable activation or repression of target gene expression, in response to synthetic non-natural effector molecules. Our novel riboswitch-ligand pairings are shown to regulate physiologically important genes required for bacterial motility in Escherichia coli and cell morphology in Bacillus subtilis. These findings are relevant for future gene function studies and antimicrobial target validation, while providing new modular and orthogonal regulatory components for deployment in synthetic biology regimes.

Generation of orthogonally selective bacterial riboswitches by targeted mutagenesis and in vivo screening.

Riboswitches are naturally occurring RNA-based genetic switches that control gene expression in response to the binding of small-molecule ligands, typically through modulation of transcription or translation. Their simple mechanism of action and the expanding diversity of riboswitch classes make them attractive targets for the development of novel gene expression tools. The essential first step in realizing this potential is to generate artificial riboswitches that respond to nonnatural, synthetic ligands, thereby avoiding disruption of normal cellular function. Here we describe a strategy for engineering orthogonally selective riboswitches based on natural switches. The approach begins with saturation mutagenesis of the ligand-binding pocket of a naturally occurring riboswitch to generate a library of riboswitch mutants. These mutants are then screened in vivo against a synthetic compound library to identify functional riboswitch-ligand combinations. Promising riboswitch-ligand pairs are then further characterized both in vivo and in vitro. Using this method, a series of artificial riboswitches can be generated that are versatile synthetic biology tools for use in protein production, gene functional analysis, metabolic engineering, and other biotechnological applications.

Bioengineering natural product biosynthetic pathways for therapeutic applications.

With the advent of next-generation DNA sequencing technologies, the number of microbial genome sequences has increased dramatically, revealing a vast array of new biosynthetic gene clusters. Genomics data provide a tremendous opportunity to discover new natural products, and also to guide the bioengineering of new and existing natural product scaffolds for therapeutic applications. Notably, it is apparent that the vast majority of biosynthetic gene clusters are either silent or produce very low quantities of the corresponding natural products. It is imperative therefore to devise methods for activating unproductive biosynthetic pathways to provide the quantities of natural products needed for further development. Moreover, on the basis of our expanding mechanistic and structural knowledge of biosynthetic assembly-line enzymes, new strategies for re-programming biosynthetic pathways have emerged, resulting in focused libraries of modified products with potentially improved biological properties. In this review we will focus on the latest bioengineering approaches that have been utilised to optimise yields and increase the structural diversity of natural product scaffolds for future clinical applications.

Juvenile hormone induction of glutathione S-transferase activity in the larval fat body of the common cutworm, Spodoptera litura (Lepidoptera: Noctuidae).

The effect of pyriproxyfen, a juvenile hormone analog (JHA), on the pupation of S. litura was examined. A topical application of 100 mug JHA/larva on the newly ecdysed (0-day) sixth instar larvae resulted in more than 80% pupation, while most of the 1- or 2-day-old larvae similarly treated developed into supernumerary larvae. Glutathione S-transferse (GST) activity in the fat body of 0-day-old sixth instar larvae was significantly induced within 12 h of JHA (100 microg/larva) treatment. In contrast, no such induction was found when 1- and 2-day-old sixth instar larvae were similarly treated. This induction phenomenon was also observed when 0-day-old sixth instar larvae were treated with the natural JH III. The suppressive effects of alpha-amanitin and cycloheximide suggest that JHA induction of GST activity in these cutworm larvae presumably occurred at the gene transcription level.