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Urbanization and agricultural intensification continue to reshape landscapes, altering the habitat available to wildlife and threatening species of both economic and conservation concern. The honey bee, Apis mellifera, is a pollinator of economic importance to North American agriculture yet managed colonies are burdened by poor health and high annual mortality. Understanding the factors influencing this species is critical for improving colony health and supporting crop production. We used a nationwide cohort of 638 managed Canadian colonies to study the dominant drivers of colony health and overwintering mortality. We found that fall colony weight – a major predictor of overwintering survival – was strongly associated with landscape composition. Among four broadly defined land cover types, we discovered that urban and forested land covers were the least valuable sources of habitat for colonies, as inferred from fall colony weight measurements. Agricultural land appeared to provide habitat quality of slightly greater value, while herbaceous land cover was most strongly positively associated with fall colony weight. Herbaceous land cover also exhibited an associational effect size which was strongly statistically distinguishable from those of urban and forested land. Our research indicates that recent and ongoing land-use changes exacerbate modern apicultural challenges, and suggests variation in nutrition or floral resource availability plays a major role in modulating honey bee health. Our work highlights the need for additional research investigating whether land use change-associated alterations in floral resource availability increase the potential for resource competition between pollinator species.

To gauge the impact of COVID-19 on the Canadian beekeeping sector, we conducted a survey of over 200 beekeepers in the fall of 2020. Our survey results show Canadian beekeepers faced two major challenges: 1) disrupted importation of honey bees (Hymenoptera: Apidae) (queen and bulk bees) that maintain populations; and 2) disrupted arrival of temporary foreign workers (TFWs). Disruptions in the arrival of bees and labor resulted in fewer colonies and less colony management, culminating in higher costs and lower productivity. Using the survey data, we develop a profitability analysis to estimate the impact of these disruptions on colony profit. Our results suggest that a disruption in either foreign worker or bee arrival allows beekeepers to compensate and while colony profits are lower, they remain positive. When both honey bee and foreign workers arrivals are disrupted for a beekeeper, even when the beekeeper experiences less significant colony health and cost impacts, a colony with a single pollination contract is no longer profitable, and a colony with two pollination contracts has significantly reduced profitability. As COVID-19 disruptions from 2020 and into 2021 become more significant to long-term colony health and more costly to a beekeeping operation, economic losses could threaten the industry’s viability as well as the sustainability of pollination-dependent crop sectors across the country. The economic and agricultural impacts from the COVID-19 pandemic have exposed a vulnerability within Canada’s beekeeping industry stemming from its dependency on imported labor and bees. Travel disruptions and border closures pose an ongoing threat to Canadian agriculture and apiculture in 2021 and highlight the need for Canada’s beekeeping industry to strengthen domestic supply chains to minimize future risks.

Neonicotinoid and fungicide exposure has been linked to immunosuppression and increased susceptibility to disease in honeybees (Apis mellifera). European foulbrood, caused by the bacterium Melissococcus plutonius, is a disease of honeybee larvae which causes economic hardship for commercial beekeepers, in particular those whose colonies pollinate blueberries. We report for the first time in Canada, an atypical variant of M. plutonius isolated from a blueberry-pollinating colony. With this isolate, we used an in vitro larval infection system to study the effects of pesticide exposure on the development of European foulbrood disease. Pesticide doses tested were excessive (thiamethoxam and pyrimethanil) or maximal field-relevant (propiconazole and boscalid). We found that chronic exposure to the combination of thiamethoxam and propiconazole significantly decreased the survival of larvae infected with M. plutonius, while larvae chronically exposed to thiamethoxam and/or boscalid or pyrimethanil did not experience significant increases in mortality from M. plutonius infection in vitro. Based on these results, individual, calculated field-realistic residues of thiamethoxam and/or boscalid or pyrimethanil are unlikely to increase mortality from European foulbrood disease in honeybee worker brood, while the effects of field-relevant exposure to thiamethoxam and propiconazole on larval mortality from European foulbrood warrant further study.

The recent decline in honey bee (Hymenoptera: Apidae) colony health worldwide has had a significant impact on the beekeeping industry as well as on pollination-dependent crop sectors in North America and Europe. The pollinator crisis has been attributed to many environmental and anthropological factors including less nutrient rich agricultural monocultures, pesticide exposure, new parasite and pathogen infestations as well as beekeeper management and weather. Canadian beekeepers have indicated that issues with honey bee queens are the most significant factor affecting their colony health. In Canada, beekeepers manage colony losses by relying on the importation of foreign bees, particularly queens from warmer climates, to lead new replacement colonies. Unfortunately, the risks associated with imported queens include the introduction of new and potentially resistant pests and diseases, undesirable genetics including bees with limited adaptations to Canada’s unique climate and bees negatively affected by transportation. Importing a large proportion of our queens each year also creates an unsustainable dependency on foreign bee sources, putting our beekeeping and pollination sectors at an even greater risk in the case of border closures and restrictions. Increasing the domestic supply of queens is one mitigation strategy that could provide Canadian beekeepers, farmers and consumers with a greater level of agricultural stability through locally bred, healthier queens. Our study is the first rigorous analysis of the economic feasibility of Canadian queen production. We present the costs of queen production for three case study operations across Canada over two years as well as the profitability implications. Our results show that for a small to medium sized queen production operation in Canada, producing queen cells and mated queens can be profitable. Using a mated queen market price ranging from $30 to $50, a producer selling mated queens could earn a profit of between $2 and $40 per queen depending on price and the cost structure of his operation. If the producer chose to rear queens for his own operation, the cost savings would also be significant as imported queen prices continue to rise. Our case studies reveal that there is potential for both skilled labour acquisition over time in queen production as well as cost savings from economies of scale. Our queen producers also reduced their production costs by re-using materials year to year. Domestic queen production could be one viable strategy to help address the current pollinator crisis in Canada.

Innate defense regulator-1 (IDR-1) is a synthetic peptide with no antimicrobial activity that enhances microbial infection control while suppressing inflammation. Previously, the effects of IDR-1 were postulated to impact several regulatory pathways including mitogen-activated protein kinase (MAPK) p38 and CCAAT-enhancer-binding protein, but how this was mediated was unknown. Using a combined stable isotope labeling by amino acids in cell culture-proteomics methodology, we identified the cytoplasmic scaffold protein p62 as the molecular target of IDR-1. Direct IDR-1 binding to p62 was confirmed by several biochemical binding experiments, and the p62 ZZ-type zinc finger domain was identified as the IDR-1 binding site. Co-immunoprecipitation analysis of p62 molecular complexes demonstrated that IDR-1 enhanced the tumor necrosis factor alpha-induced p62 receptor-interacting protein 1 (RIP1) complex formation but did not affect tumor necrosis factor alpha-induced p62-protein kinase zeta complex formation. In addition, IDR-1 induced p38 MAPK activity in a p62-dependent manner and increased CCAAT-enhancer-binding protein beta activity, whereas NF-kappaB activity was unaffected. Collectively, these results demonstrate that IDR-1 binding to p62 specifically affects protein-protein interactions and subsequent downstream events. Our results implicate p62 in the molecular mechanisms governing innate immunity and identify p62 as a potential therapeutic target in both infectious and inflammatory diseases.

We show that an innate defense-regulator peptide (IDR-1) was protective in mouse models of infection with important Gram-positive and Gram-negative pathogens, including methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus and Salmonella enterica serovar Typhimurium. When given from 48 h before to 6 h after infection, the peptide was effective by both local and systemic administration. Because protection by IDR-1 was prevented by in vivo depletion of monocytes and macrophages, but not neutrophils or B- and T-lymphocytes, we conclude that monocytes and macrophages are key effector cells. IDR-1 was not directly antimicrobial: gene and protein expression analysis in human and mouse monocytes and macrophages indicated that IDR-1, acting through mitogen-activated protein kinase and other signaling pathways, enhanced the levels of monocyte chemokines while reducing pro-inflammatory cytokine responses. To our knowledge, an innate defense regulator that counters infection by selective modulation of innate immunity without obvious toxicities has not been reported previously.

The extracellular matrix provides structural components that support the development of tissue morphology and the distribution of growth factors that modulate the overall cellular response to those growth factors. The ability to manipulate the presentation of factors in culture systems should provide an additional degree of control in regulating the stimulation of factor-dependent cells for tissue engineering applications. Cellulose binding domain (CBD) fusion protein technology facilitates the binding of bioactive cytokines to cellulose materials, and has permitted the analysis of several aspects of cell stimulation by surface-localized growth factors. We previously reported the synthesis and initial characterization of a fusion protein comprised of a CBD and murine stem cell factor (SCF) (Doheny et al. [1999] Biochem J 339:429-434). A significant advantage of the CBD fusion protein system is that it permits the stimulation of factor-dependent cells with localized growth factor, essentially free of nonfactor-derived interactions between the cell and matrix. In this work, the long-term stability and bioactivity of SCF-CBD fusions adsorbed to microcrystalline cellulose under cell culture conditions is demonstrated. Cellulose-bound SCF-CBD is shown to stimulate receptor polarization in the cell membrane and adherence to the cellulose matrix. In addition, cellulose-surface presentation of the SCF-CBD attenuates c-kit dephosphorylation kinetics, potentially modulating the overall response of the cell to the SCF signal.

In this work, a new derivative of FX was engineered. It comprises a cellulose-binding module (CBM) fused to the N-terminus of the truncated light chain (E2FX) of FX and a hexahistidine tag (H6) fused to the C-terminus of the heavy chain. The sequence LTR at the site of cleavage of the activation peptide from the N-terminus of the heavy chain is changed to IEGR to render the derivative self-activating. However, N-linked glycans on the CBM of the derivative blocked its binding to cellulose and those on the activation peptide slowed its activation. Therefore, the sites of N-linked glycosylation on the CBM and on the activation peptide were eliminated by mutation. The final derivative can be produced in good yield by cultured mammalian cells. It is purified easily with Ni2+-agarose, it is self-activating, and it can be immobilized on cellulose. When immobilized on a column of cellulose beads, the activated derivative retains ∼80% of its initial activity after 30 days of continuous hydrolysis of a fusion protein substrate. Under these conditions of operation, the effective substrate:enzyme ratio is >104. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 79: 724–732, 2002.

A modified Factor X protein was combined with a cellulose-binding domain tag and expressed in insect cell lines. The protein, CBDFX, was expressed and secreted into the medium. Stable, transformed Hi5 and Sf9 insect cell lines were generated and tested for production of secreted CBDFX. The highest Sf9 and Hi5 CBDFX-producing cell lines were scaled up to 2-liter fermentors to evaluate production of this recombinant protein. Secreted protein production levels reached 4 mg/liter for the stable, transformed Hi5 cell line and 18 mg/liter for the stable, transformed Sf9 cell line. The protein was properly processed as determined by amino terminal sequencing and bound well to the cellulose substrate Avicel. In addition the activated recombinant CBDFX a was capable of recognizing and efficiently processing a Factor X cleavage site.

The family 2a carbohydrate-binding module (CBM), Cel5ACBM2a, from the C-terminus of Cel5A from Cellulomonas fimi, and Xyn10ACBM2a, the family 2a CBM from the C-terminus of Xyn10A from C. fimi, were compared as fusion partners for proteins produced in the methylotrophic yeast Pichia pastoris. Gene fusions of murine stem-cell factor (SCF) with both CBMs were expressed in P. pastoris. The secreted SCF-Xyn10ACBM2a polypeptides were highly glycosylated and bound poorly to cellulose. In contrast, fusion of SCF to Cel5ACBM2a, which lacks potential N-linked glycosylation sites, resulted in the production of polypeptides which bound tightly to cellulose. Cloning and expression of these CBM2a in P. pastoris without a fusion partner confirmed that N-linked glycosylation at several sites was responsible for the poor cellulose binding. The nonglycosylated CBMs produced in E. coli had very similar cellulose-binding properties.

Analysis of 12 baby hamster kidney (BHK) clones in exponential growth revealed a linear relationship between cell-specific recombinant activated protein C (APC) production rates and APC mRNA levels. This correlation indicated that mRNA levels limited APC productivity. Two strategies were employed to increase APC mRNA levels and APC productivity. First, sodium butyrate was added to increase mRNA levels by two- to sixfold in five APC-producing clones to obtain up to 2.7-fold increase in APC production rate. The second strategy was to retransfect an APC-producing BHK cell line with a vector containing additional APC cDNA and a mutant DHFR. This mutant DHFR gene allowed the selection of retransfected clones in higher MTX concentrations. Over two-fold higher mRNA levels were obtained in these retransfected clones and the cell-specific APC production rate increased twofold. At the highest level of APC secretion, increases in mRNA levels did not result in higher rates of APC production. Analysis of the intracellular APC content revealed a possible saturation in the secretory pathway at high mRNA levels. The relation between mRNA level and APC secretion rate was also investigated in batch culture. The levels of total cellular RNA, APC mRNA, and beta-actin mRNA were relatively stable while cells were in the exponential growth phase, but rapidly decreased when cells reached the stationary phase. The decline of cell-specific APC mRNA levels correlated with a decline in APC secretion rates, which indicated that the mRNA levels continued to limit the rates beyond the exponential phase and into the declining growth and stationary phases of batch APC production.

A chimaera of stem cell factor (SCF) and a cellulose-binding domain from the xylanase Cex (CBDCex) effectively immobilizes SCF on a cellulose surface. The fusion protein retains both the cytokine properties of SCF and the cellulose-binding characteristics of CBDCex. When adsorbed on cellulose, SCF–CBDCex is up to 7-fold more potent than soluble SCF–CBDCex and than native SCF at stimulating the proliferation of factor-dependent cell lines. When cells are incubated with cellulose-bound SCF–CBDCex, activated receptors and SCF–CBDCex co-localize on the cellulose matrix. The strong binding of SCF–CBDCex to the cellulose surface permits the effective and localized stimulation of target cells; this is potentially significant for long-term perfusion culturing of factor-dependent cells. It also permits the direct analysis of the effects of surface-bound cytokines on target cells.

The methylotrophic yeast Pichia pastoris can be used to express recombinant genes at high levels under the control of the methanol-inducible alcohol oxidase 1 (AOX1) promoter. Accurate regulation of the methanol concentration in P. pastoris cultures is necessary to maintain induction, while preventing accumulation of methanol to cytotoxic levels. We developed an inexpensive methanol sensor that uses a gas-permeable silicone rubber tube immersed in the culture medium and an organic solvent vapor detector. The sensor was used to monitor methanol concentration continuously throughout a fed-batch shake-flask culture of a P. pastoris clone producing the N-lobe of human transferrin. The sensor calibration was stable for the duration of the culture and the output signal accurately reflected the methanol concentration determined off-line by HPLC. A closed-loop control system utilizing this sensor was developed and used to maintain a 0.3% (v/v) methanol concentration in the culture. Use of this system resulted in a fivefold increase in volumetric protein productivity over levels obtained using the conventional fed-batch protocol.

The secretion rate of activated protein C (APC) by BHK cells was increased 35-fold by increasing the cDNA copy number per cell from 50 to 240. In this range, the relation between APC secretion and cDNA copy number was not linear and the rate of APC secretion per cDNA copy increased sevenfold. This apparent cooperative effect of multiple cDNA copies could be related to their integration in tandem. For cDNA copy numbers higher than 240, the APC secreation rate per cDNA and per cell decreased dramatically. The gamma-carboxylation of glutamic acid residues, a posttranslational modification required for APC biological activity, was also investigated. The proportion of APC that was fully gamma-carboxylated decreased as the secretion rate of APC increased. (c) 1995 John Wiley & Sons, Inc.

The dicistrovirus intergenic region internal ribosome entry site (IRES) utilizes a unique mechanism, involving P-site tRNA mimicry, to directly assemble 80S ribosomes and initiate translation at a specific non-AUG codon in the ribosomal A site. A subgroup of dicistrovirus genomes contains an additional stem-loop 5′-adjacent to the IRES and a short open reading frame (ORFx) that overlaps the viral structural polyprotein ORF (ORF2) in the +1 reading frame. Using mass spectrometry and extensive mutagenesis, we show that, besides directing ORF2 translation, the Israeli acute paralysis dicistrovirus IRES also directs ORFx translation. The latter is mediated by a U∶G base pair adjacent to the P-site tRNA-mimicking domain. An ORFx peptide was detected in virus-infected honey bees by multiple reaction monitoring mass spectrometry. Finally, the 5′ stem-loop increases IRES activity and may couple translation of the two major ORFs of the virus. This study reveals a novel viral strategy in which a tRNA-like IRES directs precise, initiator Met-tRNA-independent translation of two overlapping ORFs.

Background: Disease is a major factor driving the evolution of many organisms. In honey bees, selection for social behavioral responses is the primary adaptive process facilitating disease resistance. One such process, hygienic behavior, enables bees to resist multiple diseases, including the damaging parasitic mite Varroa destructor. The genetic elements and biochemical factors that drive the expression of these adaptations are currently unknown. Proteomics provides a tool to identify proteins that control behavioral processes, and these proteins can be used as biomarkers to aid identification of disease tolerant colonies. Results We sampled a large cohort of commercial queen lineages, recording overall mite infestation, hygiene, and the specific hygienic response to V. destructor. We performed proteome-wide correlation analyses in larval integument and adult antennae, identifying several proteins highly predictive of behavior and reduced hive infestation. In the larva, response to wounding was identified as a key adaptive process leading to reduced infestation, and chitin biosynthesis and immune responses appear to represent important disease resistant adaptations. The speed of hygienic behavior may be underpinned by changes in the antenna proteome, and chemosensory and neurological processes could also provide specificity for detection of V. destructor in antennae. Conclusions Our results provide, for the first time, some insight into how complex behavioural adaptations manifest in the proteome of honey bees. The most important biochemical correlations provide clues as to the underlying molecular mechanisms of social and innate immunity of honey bees. Such changes are indicative of potential divergence in processes controlling the hive-worker maturation.

BACKGROUND: Honey bees are complex eusocial insects that provide a critical contribution to human agricultural food production. Their natural migration has selected for traits that increase fitness within geographical areas, but in parallel their domestication has selected for traits that enhance productivity and survival under local conditions. Elucidating the biochemical mechanisms of these local adaptive processes is a key goal of evolutionary biology. Proteomics provides tools unique among the major 'omics disciplines for identifying the mechanisms employed by an organism in adapting to environmental challenges. RESULTS: Through proteome profiling of adult honey bee midgut from geographically dispersed, domesticated populations combined with multiple parallel statistical treatments, the data presented here suggest some of the major cellular processes involved in adapting to different climates. These findings provide insight into the molecular underpinnings that may confer an advantage to honey bee populations. Significantly, the major energy-producing pathways of the mitochondria, the organelle most closely involved in heat production, were consistently higher in bees that had adapted to colder climates. In opposition, up-regulation of protein metabolism capacity, from biosynthesis to degradation, had been selected for in bees from warmer climates. CONCLUSIONS: Overall, our results present a proteomic interpretation of expression polymorphisms between honey bee ecotypes and provide insight into molecular aspects of local adaptation or selection with consequences for honey bee management and breeding. The implications of our findings extend beyond apiculture as they underscore the need to consider the interdependence of animal populations and their agro-ecological context.

Cationic antimicrobial peptides exhibit potent antimicrobial activity against clinically relevant microorganisms including Propionibacterium acnes. Recent studies showed that, in addition to the antimicrobial activity, these peptides can exhibit an anti-inflammatory effect. These properties make cationic peptides attractive drug candidates for the treatment of acne vulgaris, a disease with both bacterial and inflammatory components. This review focuses on the anti-inflammatory activity of cationic antimicrobial peptides and its application for the treatment of acne vulgaris. The anti-inflammatory activity of cationic peptides in acne vulgaris can be explained by their ability to both bind proinflammatory bacterial factors (e.g. lipoteichoic acid), sequestering them from the site of inflammation, and to inhibit the secretion of proinflammatory cytokines (e.g. tumor necrosis factor-alpha, IL-1) by host cells. These anti-inflammatory effects combined with potent antimicrobial activity may translate into a novel therapeutic option for acne vulgaris.

Endogenous antimicrobial peptides are ubiquitous components of animal and plant host defences. These peptides, usually cationic and amphipathic, kill target cells rapidly and are efficacious against antibiotic-resistant and clinically relevant pathogens. A practical challenge in the development of cationic peptides as therapeutics is to meet the production requirements for large quantities of highly purified drug substance at competitive costs. While chemical peptide synthesis can be used to manufacture cationic peptides, we have developed cost-effective methods for recombinant production by expressing fusion proteins comprised of multiple copies of the peptides. The fusion proteins accumulate in Escherichia coli inclusion bodies and constitute over 50% of the total cellular proteins. Active antimicrobial peptides are released by chemical reagents and purified by chromatography, combining both standard and novel approaches. Challenges of industrial-scale manufacturing of therapeutics were considered in the development of this process.

Many recombinant proteins are synthesized as fusion proteins containing affinity tags to aid in the downstream processing. After purification, the affinity tag is often removed by using a site-specific protease such as factor Xa (FXa). However, the use of FXa is limited by its expense and availability from plasma. To develop a recombinant source of FXa, we have expressed two novel forms of FXa using baby hamster kidney (BHK) cells as host and the expression vector pNUT. The chimeric protein FIIFX consisted of the prepropeptide and the Gla domain of prothrombin linked to the activation peptide and protease region of FXa, together with a cellulose-binding domain (CBD(Cex)) as an affinity tag. A second variant consisted of the transferrin signal peptide linked to the second epidermal growth factor-like domain and the catalytic domain of FX and a polyhistidine tag. Both FX variants were secreted into the medium, their affinity tags were functional, and following activation, both retained FXa-specific proteolytic activity. However, the yield of the FIIFX-CBD(Cex) fusion protein was 10-fold higher than that of FX-CBD(Cex) and other forms of recombinant FX reported to date. The FXa derivatives were used to cleave two different fusion proteins, including a biologically inactive alpha-factor-hirudin fusion protein secreted by Saccharomyces cerevisiae. After cleavage, the released hirudin demonstrated biological activity in a thrombin inhibition assay, suggesting that this method may be applicable to the production of toxic or unstable proteins. The availability of novel FX derivatives linked to different affinity tags allows the development of a versatile system for processing fusion proteins in vitro.

Endogenous antimicrobial peptides are ubiquitous components of animal and plant host defences. These peptides, usually cationic and amphipathic, kill target cells rapidly and are efficacious against antibiotic-resistant and clinically relevant pathogens. A practical challenge in the development of cationic peptides as therapeutics is to meet the production requirements for large quantities of highly purified drug substance at competitive costs. While chemical peptide synthesis can be used to manufacture cationic peptides, we have developed cost-effective methods for recombinant production by expressing fusion proteins comprised of multiple copies of the peptides. The fusion proteins accumulate in Escherichia coli inclusion bodies and constitute over 50% of the total cellular proteins. Active antimicrobial peptides are released by chemical reagents and purified by chromatography, combining both standard and novel approaches. Challenges of industrial-scale manufacturing of therapeutics were considered in the development of this process.