Evaluation of heterologous expression in Pichia pastoris of Pine Weevil TRPA1 by GFP and flow cytometry.

BACKGROUND: The wasabi receptor, also known as the Transient Receptor Potential Ankyrin 1 (TRPA1) ion channel, is a potential target for development of repellents for insects, like the pine weevil (Hylobius abietis) feeding on conifer seedlings and causing damage in forestry. Heterologous expression of TRPA1 from pine weevil in the yeast Pichia pastoris can potentially provide protein for structural and functional studies. Here we take advantage of the Green Fluorescent Protein (GFP) tag to examine the various steps of heterologous expression, to get more insight in clone selection, expression and isolation of the intact purified protein. RESULTS: The sequence of HaTRPA1 is reported and GFP-tagged constructs were made of the full-length protein and a truncated version (Δ1-708 HaTRPA1), lacking the N-terminal ankyrin repeat domain. Clones were screened on GFP expression plates, induced in small liquid cultures and in fed-batch cultures, and evaluated by flow cytometry and fluorescence microscopy. The screening on plates successfully identifies low-expression clones, but fails to predict the ranking of the best performing clones in small-scale liquid cultures. The two constructs differ in their cellular localization. Δ1-708 HaTRPA1 is found in a ring at the perimeter of cell, whereas HaTRPA1 is forming highly fluorescent speckles in interior regions of the cell. The pattern is consistent in different clones of the same construct and persists in fed-batch culture. The expression of Δ1-708 HaTRPA1 decreases the viability more than HaTRPA1, and in fed-batch culture it is clear that intact cells first express Δ1-708 HaTRPA1 and then become damaged. Purifications show that both constructs suffer from degradation of the expressed protein, but especially the HaTRPA1 construct. CONCLUSIONS: The GFP tag makes it possible to follow expression by flow cytometry and fluorescence microscopy. Analyses of localization, cell viability and expression show that the former two parameters are specific for each of the two evaluated constructs, whereas the relative expression of the constructs varies with the cultivation method. High expression is not all that matters, so taking damaged cells into account, something that may be linked to protein degradation, is important when picking the most suitable construct, clone, and expression scheme.

Photochemical cyclopropanation in aqueous micellar media - experimental and theoretical studies.

While in nature, reactions occur in water-based confined compartments, for a long time, water has been often regarded as an unsuitable medium for organic reactions. We have, however, found that photochemical cyclopropanation of styrenes with diazo compounds or their precursors can be performed in micellar systems. COSMO-RS studies revealed that the reactivity correlates with the predicted critical micelle concentration (CMC), with higher CMC values delivering higher yields.

Is asthma in children still increasing? 20-year prevalence trends in northern Sweden.

BACKGROUND: In the present study, we describe prevalence trends of asthma and investigate the association with asthma symptoms, use of asthma medication, and asthma severity among 8-year-old children in Norrbotten, Sweden in 1996, 2006, and 2017. METHODS: Within the Obstructive Lung Disease in Northern Sweden (OLIN) studies, three pediatric cohorts were recruited in 1996, 2006, and 2017 respectively. Identical methods were used; all children in first and second grade (median age 8 years) in three municipalities were invited to a parental questionnaire survey, completed by n = 3430 in 1996 (97% participation), n = 2585 in 2006 (96%), and n = 2785 in 2017 (91%). The questionnaire included questions about respiratory symptoms and diagnosis, treatment, and severity of asthma. RESULTS: The prevalence of wheezing was stable during the study, 10.1% in 1996; 10.8% in 2006; and 10.3% in 2017, p = .621, while physician-diagnosed asthma increased: 5.7%, 7.4%, and 12.2%, p < .001. The use of asthma medication in the last 12 months increased: 7.1%, 8.7%, and 11.5%, p < .001. Among children diagnosed with asthma, the prevalence of asthma symptoms, the impact on daily life, and severe asthma decreased, while the use of inhaled corticosteroids increased from 1996 until 2017. CONCLUSION: The prevalence of wheezing was stable among 8-year-old in this area from 1996 to 2017, while the prevalence of physician-diagnosed asthma doubled but without an increase in asthma morbidity. The increase of physician-diagnosed asthma without a coincident increase in asthma morbidity can partly be explained by more and earlier diagnosis among those with mild asthma.

Antibacterial and Hemolytic Activity of Antimicrobial Hydrogels Utilizing Immobilized Antimicrobial Peptides.

Antimicrobial peptides (AMPs) are viewed as potential compounds for the treatment of bacterial infections. Nevertheless, the successful translation of AMPs into clinical applications has been impeded primarily due to their low stability in biological environments and potential toxicological concerns at higher concentrations. The covalent attachment of AMPs to a material's surface has been sought to improve their stability. However, it is still an open question what is required to best perform such an attachment and the role of the support. In this work, six different AMPs were covalently attached to a long-ranged ordered amphiphilic hydrogel, with their antibacterial efficacy evaluated and compared to their performance when free in solution. Among the tested AMPs were four different versions of synthetic end-tagged AMPs where the sequence was altered to change the cationic residue as well as to vary the degree of hydrophobicity. Two previously well-studied AMPs, Piscidin 1 and Omiganan, were also included as comparisons. The antibacterial efficacy against Staphylococcus aureus remained largely consistent between free AMPs and those attached to surfaces. However, the activity pattern against Pseudomonas aeruginosa on hydrogel surfaces displayed a marked contrast to that observed in the solution. Additionally, all the AMPs showed varying degrees of hemolytic activity when in solution. This activity was entirely diminished, and all the AMPs were non-hemolytic when attached to the hydrogels.

Combining VPS34 inhibitors with STING agonists enhances type I interferon signaling and anti-tumor efficacy.

An immunosuppressive tumor microenvironment promotes tumor growth and is one of the main factors limiting the response to cancer immunotherapy. We have previously reported that inhibition of vacuolar protein sorting 34 (VPS34), a crucial lipid kinase in the autophagy/endosomal trafficking pathway, decreases tumor growth in several cancer models, increases infiltration of immune cells and sensitizes tumors to anti-programmed cell death protein 1/programmed cell death 1 ligand 1 therapy by upregulation of C-C motif chemokine 5 (CCL5) and C-X-C motif chemokine 10 (CXCL10) chemokines. The purpose of this study was to investigate the signaling mechanism leading to the VPS34-dependent chemokine increase. NanoString gene expression analysis was applied to tumors from mice treated with the VPS34 inhibitor SB02024 to identify key pathways involved in the anti-tumor response. We showed that VPS34 inhibitors increased the secretion of T-cell-recruitment chemokines in a cyclic GMP-AMP synthase (cGAS)/stimulator of interferon genes protein (STING)-dependent manner in cancer cells. Both pharmacological and small interfering RNA (siRNA)-mediated VPS34 inhibition increased cGAS/STING-mediated expression and secretion of CCL5 and CXCL10. The combination of VPS34 inhibitor and STING agonist further induced cytokine release in both human and murine cancer cells as well as monocytic or dendritic innate immune cells. Finally, the VPS34 inhibitor SB02024 sensitized B16-F10 tumor-bearing mice to STING agonist treatment and significantly improved mice survival. These results show that VPS34 inhibition augments the cGAS/STING pathway, leading to greater tumor control through immune-mediated mechanisms. We propose that pharmacological VPS34 inhibition may synergize with emerging therapies targeting the cGAS/STING pathway.

Silica-embedded Gold Nanoparticles Analyzed by Atom Probe Tomography.

Nanoparticles are utilized in a multitude of applications due to their unique properties. Consequently, characterization of nanoparticles is crucial, and various methods have been employed in these pursuits. One such method is Atom Probe Tomography (APT). However, existing sample preparation techniques for APT generally involve embedding of the nanoparticles in a matrix different from their environment in solutions or at solid-liquid interfaces. In this work, we demonstrate a methodology based on silica embedding and explore how it can be utilized to form a matrix for nanoparticles suitable for APT analysis. Through chemisorption to a surface, gold nanoparticles were densely packed, ensuring a high probability of encountering at least one particle in the APT analyses. The nanoparticle-covered surface was embedded in a silica film, replacing the water and thus making this method suitable for studying nanoparticles in their hydrated state. The nanoparticle's silver content and its distribution, originating from the nanoparticle synthesis, could be identified in the APT analysis. Sodium clusters, possibly originating from the sodium citrate used to stabilize the particles in solution, were observed on the nanoparticle surfaces. This indicates the potential for silica embedding to be used for studying ligands on nanoparticles in their hydrated state.

No remission in 60% of those with childhood-onset asthma - A population-based cohort followed from 8 to 28 years of age.

BACKGROUND: Although remission occur, childhood-onset asthma may persist until adulthood. Since few longitudinal population-based studies have followed a cohort from childhood until adulthood, the knowledge on predictors of persistence of asthma is sparse. AIM: To estimate persistence of asthma from 8 to 28 years and its associated factors. METHODS: Within the OLIN (Obstructive Lung Disease in Northern Sweden) studies, a cohort was recruited in 1996 (age 8y, n = 3430) and followed annually with questionnaires about asthma and risk factors until 19y. Clinical examinations included skin prick tests (at 8, 12 and 19y) and lung function tests (17 and 19y) whereof a subsample performed bronchial hyperreactivity test. We identified n = 248 with asthma at 8y whereof 170 (69%) participated in a follow-up at 28y (73% of possible to invite). RESULTS: Of the 170 participants at 28y, 105 (61.8%) had persistent asthma (women: 49/76, 64.5%; men: 56/94, 59.6%, p = 0.513). Factors collected at recruitment: allergic sensitization (OR7.8, 95%CI 3.0-20.2), severe respiratory infection (OR2.6, 95%CI 1.1-6.3) and higher asthma severity score (OR1.6, 95%CI 1.1-2.4) were associated with asthma at 28y after adjustment for sex, family history of asthma, breastfeeding <3 months and eczema. Replacing allergic sensitization with rhinoconjunctivitis in the model yielded OR3.4 (95%CI 1.5-8.0). Bronchial hyperreactivity at age 17y associated with asthma at 28y (OR9.0, 95%CI 1.7-47.0). CONCLUSIONS: Among children with asthma onset by 8y, 62% still had asthma at age 28 years. Persistent asthma was associated with allergic sensitization, rhinoconjunctivitis, severe respiratory infection, a more severe asthma and bronchial hyperreactivity.

Successful Use of Easyhaler® Dry Powder Inhaler in Patients with Chronic Obstructive Pulmonary Disease; Analysis of Peak Inspiratory Flow from Three Clinical Trials.

INTRODUCTION: There is increasing pressure to use environmentally friendly dry powder inhalers (DPI) instead of pressurized metered-dose inhalers (pMDI). However, correct inhalation technique is needed for effective inhaler therapy, and there is persistent concern whether patients with chronic obstructive pulmonary disease (COPD) can generate sufficient inspiratory effort to use DPIs successfully. The aims of this study were to find clinical predictors for peak inspiratory flow rate (PIF) and to assess whether patients with COPD had difficulties in generating sufficient PIF with a high resistance DPI. METHODS: Pooled data of 246 patients with COPD from previous clinical trials was analyzed to find possible predictors of PIF via the DPI Easyhaler (PIFEH) and to assess the proportion of patients able to achieve an inhalation flow rate of 30 l/min, which is needed to use the Easyhaler successfully. RESULTS: The mean PIF was 56.9 l/min and 99% (243/246) of the study patients achieved a PIF ≥ 30 l/min. A low PIF was associated with female gender and lower forced expiratory volume in 1 s (FEV1), but the association was weak and a statistical model including both only accounted for 18% of the variation seen in PIFEH. CONCLUSIONS: Based on our results, impaired expiratory lung function or patient characteristics do not predict patients' ability to use DPIs in COPD; 99% of the patients generated sufficient PIFEH for successful dose delivery. Considering the targets for sustainability in health care, this should be addressed as DPIs are a potential option for most patients when choosing the right inhaler for the patient. TRIAL REGISTRATION: Two of three included trials were registered under numbers NCT04147572 and NCT01424137. Third trial preceded registration platforms and therefore, was not registered.

Decreasing prevalence of atopic dermatitis in Swedish schoolchildren: three repeated population-based surveys.

BACKGROUND: The prevalence of atopic dermatitis (AD) has increased over several decades and now affects about one-fifth of all children in high-income countries (HICs). While the increase continues in lower-income countries, the prevalence of AD might have reached a plateau in HICs. OBJECTIVES: To investigate trends in the prevalence of AD and atopic comorbidity in schoolchildren in Sweden. METHODS: The study population consisted of three cohorts of children (median age 8 years) in Norrbotten, Sweden, for 1996 (n = 3430), 2006 (n = 2585) and 2017 (n = 2785). An identical questionnaire that included questions from the International Study of Asthma and Allergies in Childhood (ISAAC) protocol was used in all three cohorts. Trends in AD prevalence were estimated, as well as trends in atopic comorbidity. AD prevalence was estimated both according to the ISAAC definition of AD and by adding the reported diagnosis by a physician (D-AD). RESULTS: The prevalence of AD decreased in the last decade, from 22.8% (1996) and 21.3% (2006) to 16.3% (2017; P < 0.001). The prevalence of D-AD was lower, but the same pattern of decrease was seen, from 9.3% (1996) and 9.4% (2006) to 5.7% (2017; P < 0.001). In all three cohorts, AD was more common among girls than boys (18.9% vs. 13.8% in 2017; P < 0.001). Children from the mountain inlands had a higher prevalence of AD than children from coastal cities (22.0% vs. 15.1% in 2017; P < 0.001). In comparing D-AD, there were no significant differences between the sexes or between inland or coastal living. Concomitant asthma increased over the years from 12.2% (1996) to 15.8% (2006) to 23.0% (2017; P < 0.001). Concomitant allergic rhinitis and allergic sensitization increased from 1996 (15.0% and 27.5%) to 2006 (24.7% and 49.5%) but then levelled off until 2017 (21.0% and 46.7%). CONCLUSIONS: The prevalence of AD among schoolchildren in Sweden decreased over the study period, whereas atopic comorbidity among children with AD increased. Although a decrease was seen, AD is still common and the increase in atopic comorbidity among children with AD, especially the increase in asthma, is concerning.

Estimation of the Added Cancer Risk Derived From EVAR and CTA Follow-Up.

OBJECTIVE: The aim of this study was to assess the risk of radiation-induced cancer development in patients that have undergone an infrarenal EVAR, stratifying the relative contributions of the procedure and the preoperative and postoperative CTAs. METHODS AND MATERIALS: The organ-specific absorbed radiation doses from CTA and the EVAR procedure were estimated from the radiation exposures of 95 and 45 male patients, respectively. Lifetime attributable risk (LAR) cancer predictions were calculated for 14 different organs. Life expectancy was assumed from a previous cohort of patients undergoing infra-renal EVAR. RESULTS: The calculated total excess cancer risk was 0.0046, ie, 1 out of 220 patients will develop a neoplasm after being exposed to the ionizing radiation from the preoperative CTA, the EVAR and annual CTA examinations for 15 years. The procedure and the preoperative CTA contributed with 38% of the total excess risk, while the rest was derived from the follow-up. If the entire CTA based follow-up would have been eliminated, an excess risk of 0.0018 (1/560) would remain. CONCLUSIONS: 1 out of 219 patients who have undergone EVAR of an infra-renal AAA have a lifetime risk of developing cancer secondary to the radiation exposures related to the procedure and the CTAs used preoperatively and during follow-up. This risk derives mostly from the yearly postoperative CTAs, underlining the potential benefits of reducing or replacing their use. CLINICAL IMPACT: A simulation-based estimation reinforced the potential deleterious effects of the radiation exposure for patients undergoing Endovascular Aneurysm Repair (EVAR) of Abdominal Aortic Aneurysms (AAA) and subsequently followed by yearly Computer Tomography Angiographies (CTAs). The risk could be as high as 1 out 219 patients developing a neoplasm after 15 years. The largest exposure derives from the follow-up CTAs and efforts to minimize their use as well as the intraoperative radiation are greatly needed. The simulation-based estimations done in this study reinforce potential deleterious effects of the radiation exposure for patients undergoing EVAR of AAA. Efforts should be done to minimize the intraoperative radiation and the number of CTAs used during follow-up.

Prevalence and risk factors for allergic sensitization: 3 cross-sectional studies among schoolchildren from 1996 to 2017.

Background: The prevalence of allergic sensitization and allergic diseases has increased for decades in Northern Europe, but recent studies are lacking. Objective: We sought to study the prevalence trends of allergic sensitization, associated risk factors, and the association with asthma and allergic rhinitis (AR) among children in Northern Sweden. Methods: Three cohorts of children aged 7 to 8 years participated in a skin prick test (SPT) with 10 airborne allergens in 1996, 2006, and 2017, with 2148, 1693, and 1762 participants tested, respectively, representing 87% to 90% of schoolchildren in the catchment communities. Adjusted Poisson regression was used to identify risk factors for allergic sensitization and the association with asthma and AR. Results: The prevalence of any positive SPT response increased from 21% in 1996 to 30% in 2006 and remained at 30% in 2017 (P < .001). Sensitization to cat was the most common for all the years. The risk factor pattern for a positive SPT response was similar in all examinations, with positive and significant associations with a family history of allergy (risk ratio, 1.4-1.5) and negative and significant associations with having a cat at home (risk ratio, 0.7-0.8). The prevalence of physician-diagnosed asthma increased, but the association with allergic sensitization weakened. The opposite trends were found for AR-decreasing prevalence and strengthened association with allergic sensitization. Conclusions: The prevalence of allergic sensitization increased from 1996 to 2006 but plateaued in the next decade, whereas the risk factor pattern remained stable. The diverging trends of associations between allergic sensitization and asthma and AR suggest secular trends in the clinical management of allergic diseases.

First-Principles Prediction of Amorphous Silica Nanoparticle Surface Charge: Effect of Size, pH, and Ionic Strength.

The quantification of surface charge properties of silica nanoparticles is essential for several applications. To determine these properties, many experimental and theoretical methods have been introduced, which are time-consuming and/or challenging to use. In this study, a first-principles approach is developed to determine the surface charge properties of amorphous silica nanoparticles against the nanoparticle size, pH, and ionic strength without relying on experimental data. An amorphous silica nanoparticle of 1.34 nm diameter is simulated by using integrated molecular dynamics and Monte Carlo methods. A detailed analysis of the nanoparticle structure is provided by analyzing the types of silanol groups on the surface. Moreover, a model is developed to estimate the probability distribution of the surface silanol groups based on the nearest neighbor distances and the diameter of the nanoparticle to determine the number of surface silanols on larger nanoparticles. Thereafter, a computational chemistry approach is used to calculate the acid dissociation constants of the corresponding deprotonation reactions. The calculated constants and the point of zero charge value are in excellent agreement with experiments. The surface charge properties of the nanoparticle with various diameters are then estimated by using a mean-field model at different pH and ionic strength values. The results of the developed model are compared to the Poisson-Boltzmann equation as a reference model. The developed model predictions agree well with the reference model for low and mid-electrolyte concentrations (1 and 10 mM) and small nanoparticles (smaller than 100 nm). However, the developed model seems to qualitatively predict the surface charge properties more accurately than the Poisson-Boltzmann model for high electrolyte concentrations.

Occupational physical activity and resting blood pressure in male construction workers.

OBJECTIVE: This study investigated the association between occupational physical activity (OPA) and resting blood pressure in a cohort of Swedish construction workers. METHODS: The final sample included 241,176 male construction workers. Occupations with low OPA were foremen and white-collar workers. The most frequent occupations in the medium OPA group were electricians, pipe workers, and machine operators, and in the high OPA group woodworkers, concrete workers, and painters. RESULTS: Mixed effects models showed higher systolic and lower diastolic blood pressure with higher OPA, but the associations varied depending on the year of participation and participant age as shown by significant interaction terms (OPA*age, OPA*calendar year, age*calendar year). Age-stratified linear regression analyses showed a pattern of slightly higher systolic (1.49, 95% confidence interval: 1.08-1.90 mmHg) and lower diastolic (0.89, 95% confidence interval: 0.65-1.13 mmHg) blood pressure when comparing low with high OPA, but not among the oldest age groups. CONCLUSION: Despite a rather large contrast in OPA, the differences in systolic and diastolic blood pressure according to OPA were small.

Clinical investigation of use of an antimicrobial peptide hydrogel wound dressing on intact skin.

A material with the ability to rapidly eradicate bacteria via a contact-killing mechanism has the benefit of a more localised treatment that is easy to implement when needed to prevent or treat a bacterial infection. Here, we present an antimicrobial material based on covalently attached antimicrobial peptides (AMPs) to a soft amphiphilic hydrogel. This results in a material that exhibits an antimicrobial effect based on contact-killing. In this study, the antimicrobial efficacy of the AMP-hydrogel was investigated by observing the changes in total bioburden on the intact skin of healthy human volunteers when the AMP-hydrogel dressing was placed on the forearm for three hours. The AMP-hydrogel significantly reduced the bioburden on the skin from a mean value of 1200CFU/cm2 for the untreated skin to 23CFU/cm2. Biocompatibility evaluations of the AMP-hydrogel showed no sign of cytotoxicity, acute systemic toxicity, irritation or sensitisation, demonstrating the safety of the AMP-hydrogel as a potential wound dressing. Leachability studies confirmed no release of AMPs and that the antimicrobial effect was localised to the surface of the hydrogels, demonstrating a pure contact-killing mode of action.

In Silico Prediction of Eye Irritation Using Hansen Solubility Parameters and Predicted pKa Values.

An in silico method has been developed that permits the binary differentiation between pure liquids causing serious eye damage or eye irritation, and pure liquids with no need for such classification, according to the UN GHS system. The method is based on the finding that the Hansen Solubility Parameters (HSP) of a liquid are collectively important predictors for eye irritation. Thus, by applying a two-tier approach in which in silico-predicted pKa values (firstly) and a trained model based solely on in silico-predicted HSP data (secondly) were used, we have developed, and validated, a fully in silico approach for predicting the outcome of a Draize test (in terms of UN GHS Cat. 1/Cat. 2A/Cat. 2B or UN GHS No Cat.) with high validation set performance (sensitivity = 0.846, specificity = 0.818, balanced accuracy = 0.832) using SMILES only. The method is applicable to pure non-ionic liquids with molecular weight below 500 g/mol, fewer than six hydrogen bond donors (e.g. nitrogen-hydrogen or oxygen-hydrogen bonds) and fewer than eleven hydrogen bond acceptors (e.g. nitrogen or oxygen atoms). Due to its fully in silico characteristics, this method can be applied to pure liquids that are still at the desktop design stage and not yet in production.

Lyotropic liquid crystal elastomers for drug delivery.

Silicone elastomers like polydimethylsiloxane (PDMS) possess a combination of attractive material and biological properties motivating their widespread use in biomedical applications. Development of elastomers with capacity to deliver active therapeutic substances in the form of drugs is of particular interest to produce medical devices with added functionality. In this work, silicone-based lyotropic liquid crystal elastomers with drug-eluting functionality were developed using PDMS and triblock copolymer (diacrylated Pluronic F127, DA-F127). Various ternary PDMS-DA-F127-H2O compositions were explored and evaluated. Three compositions were found to have specific properties of interest and were further investigated for their nanostructure, mechanical properties, water retention capacity, and morphology. The ability of the elastomers to encapsulate and release polar and nonpolar substances was demonstrated using vancomycin and ibuprofen as model drugs. It was shown that the materials could deliver both types of drugs with a sustained release profile for up to 6 and 5 days for vancomycin and ibuprofen, respectively. This works demonstrates a lyotropic liquid crystal, silicone-based elastomer with tailorable mechanical properties, water retention capacity and ability to host and release polar and nonpolar active substances.

PlyKp104, a Novel Phage Lysin for the Treatment of Klebsiella pneumoniae, Pseudomonas aeruginosa, and Other Gram-Negative ESKAPE Pathogens.

Klebsiella pneumoniae and Pseudomonas aeruginosa are two leading causes of burn and wound infections, pneumonia, urinary tract infections, and more severe invasive diseases, which are often multidrug resistant (MDR) or extensively drug resistant. Due to this, it is critical to discover alternative antimicrobials, such as bacteriophage lysins, against these pathogens. Unfortunately, most lysins that target Gram-negative bacteria require additional modifications or outer membrane permeabilizing agents to be bactericidal. We identified four putative lysins through bioinformatic analysis of Pseudomonas and Klebsiella phage genomes in the NCBI database and then expressed and tested their intrinsic lytic activity in vitro. The most active lysin, PlyKp104, exhibited >5-log killing against K. pneumoniae, P. aeruginosa, and other Gram-negative representatives of the multidrug-resistant ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, K. pneumonia, Acinetobacter baumannii, P. aeruginosa, and Enterobacter species) without further modification. PlyKp104 displayed rapid killing and high activity over a wide pH range and in high concentrations of salt and urea. Additionally, pulmonary surfactants and low concentrations of human serum did not inhibit PlyKp104 activity in vitro. PlyKp104 also significantly reduced drug-resistant K. pneumoniae >2 logs in a murine skin infection model after one treatment of the wound, suggesting that this lysin could be used as a topical antimicrobial against K. pneumoniae and other MDR Gram-negative infections.

Characterization of olfactory sensory neurons in the striped ambrosia beetle Trypodendron lineatum.

Introduction: The striped ambrosia beetle Trypodendron lineatum (Coleoptera, Curculionidae, Scolytinae) is a major forest pest in the Holarctic region. It uses an aggregation pheromone and host and non-host volatiles to locate suitable host trees, primarily stressed or dying conifer trees. The beetles bore into the xylem and inoculate spores of their obligate fungal mutualist Phialophoropsis ferruginea inside their excavated egg galleries, with the fungus serving as the main food source for the developing larvae. Olfactory sensory neuron (OSN) responses to pheromones and host volatiles are poorly understood in T. lineatum and other ambrosia beetles, and nothing is known about potential responses to fungal volatiles. Methods: We screened responses of OSNs present in 170 antennal olfactory sensilla using single sensillum recordings (SSR) and 57 odor stimuli, including pheromones, host and non-host compounds, as well as volatiles produced by P. ferruginea and fungal symbionts of other scolytine beetles. Results and Discussion: Thirteen OSN classes were characterized based on their characteristic response profiles. An OSN class responding to the aggregation pheromone lineatin was clearly the most abundant on the antennae. In addition, four OSN classes responded specifically to volatile compounds originating from the obligate fungal mutualist and three responded to non-host plant volatiles. Our data also show that T. lineatum has OSN classes tuned to pheromones of other bark beetles. Several OSN classes showed similar response profiles to those previously described in the sympatric bark beetle Ips typographus, which may reflect their shared ancestry.