Delivery of endolysin across outer membrane of Gram-negative bacteria using translocation domain of botulinum neurotoxin.

The emergence of multidrug-resistant pathogens has outpaced the development of new antibiotics, leading to renewed interest in endolysins. Endolysins have been investigated as novel biocontrol agents for Gram-positive bacteria. However, their efficacy against Gram-negative species is limited by the barrier presented by their outer membrane, which prevents endolysin access to the peptidoglycan substrate. Here, we used the translocation domain of botulinum neurotoxin to deliver endolysin across the outer membrane of Gram-negative bacteria. The translocation domain selectively interacts with and penetrates membranes composed of anionic lipids, which have been used in nature to deliver various proteins into animal cells. In addition to the botulinum neurotoxin translocation domain, we have fused bacteriophage-derived receptor binding protein to endolysins. This allows the attached protein to efficiently bind to a broad spectrum of Gram-negative bacteria. By attaching these target-binding and translocation machineries to endolysins, we aimed to develop an engineered endolysin with broad-spectrum targeting and enhanced antibacterial activity against Gram-negative species. To validate our strategy, we designed engineered endolysins using two well-known endolysins, T5 and LysPA26, and tested them against 23 strains from six species of Gram-negative bacteria, confirming that our machinery can act broadly. In particular, we observed a 2.32 log reduction in 30 min with only 0.5 µM against an Acinetobacter baumannii isolate. We also used the SpyTag/SpyCatcher system to easily attach target-binding proteins, thereby improving its target-binding ability. Overall, our newly developed endolysin engineering strategy may be a promising approach to control multidrug-resistant Gram-negative bacterial strains.

Design of PD-L1-Targeted Lipid Nanoparticles to Turn on PTEN for Efficient Cancer Therapy.

Lipid nanoparticles (LNPs) exhibit remarkable mRNA delivery efficiency, yet their majority accumulate in the liver or spleen after injection. Tissue-specific mRNA delivery can be achieved through modulating LNP properties, such as tuning PEGylation or varying lipid components systematically. In this paper, a streamlined method is used for incorporating tumor-targeting peptides into the LNPs; the programmed death ligand 1 (PD-L1) binding peptides are conjugated to PEGylated lipids via a copper-free click reaction, and directly incorporated into the LNP composition (Pep LNPs). Notably, Pep LNPs display robust interaction with PD-L1 proteins, which leads to the uptake of LNPs into PD-L1 overexpressing cancer cells both in vitro and in vivo. To evaluate anticancer immunotherapy mediated by restoring tumor suppressor, mRNA encoding phosphatase and tensin homolog (PTEN) is delivered via Pep LNPs to PTEN-deficient triple-negative breast cancers (TNBCs). Pep LNPs loaded with PTEN mRNA specifically promotes autophagy-mediated immunogenic cell death in 4T1 tumors, resulting in effective anticancer immune responses. This study highlights the potential of tumor-targeted LNPs for mRNA-based cancer therapy.

Conversion of Host Cell Receptor into Virus Destructor by Immunodisc to Neutralize Diverse SARS-CoV-2 Variants.

The decreasing efficacy of antiviral drugs due to viral mutations highlights the challenge of developing a single agent targeting multiple strains. Using host cell viral receptors as competitive inhibitors is promising, but their low potency and membrane-bound nature have limited this strategy. In this study, the authors show that angiotensin-converting enzyme 2 (ACE2) in a planar membrane patch can effectively neutralize all tested severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants that emerged during the COVID-19 pandemic. The ACE2-incorporated membrane patch implemented using nanodiscs replicated the spike-mediated membrane fusion process outside the host cell, resulting in virus lysis, extracellular RNA release, and potent antiviral activity. While neutralizing antibodies became ineffective as the SARS-CoV-2 evolved to better penetrate host cells the ACE2-incorporated nanodiscs became more potent, highlighting the advantages of using receptor-incorporated nanodiscs for antiviral purposes. ACE2-incorporated immunodisc, an Fc fusion nanodisc developed in this study, completely protected humanized mice infected with SARS-CoV-2 after prolonged retention in the airways. This study demonstrates that the incorporation of viral receptors into immunodisc transforms the entry gate into a potent virucide for all current and future variants, a concept that can be extended to different viruses.

Development mechanism of resistant population post-exposure to tigecycline in tigecycline-heteroresistant Acinetobacter baumannii strain.

Tigecycline heteroresistance is highly prevalent in Acinetobacter baumannii clinical isolates, reducing the efficacy of tigecycline treatment. This study investigated the population dynamics of A. baumannii with tigecycline heteroresistance to determine the origin of resistance that occurs over time after antibiotic exposure. Tigecycline heteroresistance was imitated by mixing tigecycline-susceptible and -resistant A. baumannii isolates in a 1:10-6 ratio, and confirmed using population analysis profiling. Growth curves and an in-vitro competition assay found no difference in bacterial fitness between tigecycline-resistant and -susceptible populations. The green fluorescent protein (GFP) expression system and flow cytometry were used to monitor the population dynamics of the heteroresistant population, while differentiating the resistant population from the susceptible population. The mimicked tigecycline heteroresistance was confirmed to be reproducible and stable without tigecycline. The GFP-expressing population (i.e. the resistant population) nearly went undetected because it only represented approximately 10-6 of the entire population. However, when the mimicked tigecycline-heteroresistant strain was treated with tigecycline, most subpopulations expressing GFP were detected. The surviving A. baumannii population, upon exposure to tigecycline, exhibited a high minimum inhibitory concentration for tigecycline, equivalent to that of tigecycline-resistant isolates that were used to mimic heteroresistance. These results indicate that the development of resistance in tigecycline-heteroresistant A. baumannii strains, resulting in decreased antibiotic efficacy, may depend on the selection of a pre-existing resistant subpopulation.

Directed Evolution of Soluble α-1,2-Fucosyltransferase Using Kanamycin Resistance Protein as a Phenotypic Reporter for Efficient Production of 2'-Fucosyllactose.

2'-Fucosyllactose (2'-FL), the most abundant fucosylated oligosaccharide in human milk, has multiple beneficial effects on human health. However, its biosynthesis by metabolically engineered Escherichia coli is often hampered owing to the insolubility and instability of α-1,2-fucosyltransferase (the rate-limiting enzyme). In this study, we aimed to enhance 2'-FL production by increasing the expression of soluble α-1,2-fucosyltransferase from Helicobacter pylori (FucT2). Because structural information regarding FucT2 has not been unveiled, we decided to improve the expression of soluble FucT2 in E. coli via directed evolution using a protein solubility biosensor that links protein solubility to antimicrobial resistance. For such a system to be viable, the activity of kanamycin resistance protein (KanR) should be dependent on FucT2 solubility. KanR was fused to the C-terminus of mutant libraries of FucT2, which were generated using a combination of error-prone PCR and DNA shuffling. Notably, one round of the directed evolution process, which consisted of mutant library generation and selection based on kanamycin resistance, resulted in a significant increase in the expression level of soluble FucT2. As a result, a batch fermentation with the ΔL M15 pBCGW strain, expressing the FucT2 mutant (F#1-5) isolated from the first round of the directed evolution process, resulted in the production of 0.31 g/l 2'-FL with a yield of 0.22 g 2'-FL/g lactose, showing 1.72- and 1.51-fold increase in the titer and yield, respectively, compared to those of the control strain. The simple and powerful method developed in this study could be applied to enhance the solubility of other unstable enzymes.

Optimization of protein trans-splicing in an inducible plasmid display system for high-throughput screening and selection of soluble proteins.

Directed evolution is widely used to optimize protein folding and solubility in cells. Although the screening and selection of desired mutants is an essential step in directed evolution, it generally requires laborious optimization and/or specialized equipment. With a view toward designing a more practical procedure, we previously developed an inducible plasmid display system, in which the intein (auto-processing) and Oct-1 DNA-binding (DBD) domains were used as the protein trans-splicing domain and DNA-binding module, respectively. Specifically, the N-terminal (CfaN) and C-terminal (CfaC) domains of intein were fused to the C-terminal end of the His-tag and the N-terminal end of Oct-1 DBD to generate His6-CfaN and CfaC-Oct-1, respectively. For such a system to be viable, the efficiency of protein trans-splicing without the protein of interest (POI) should be maximized, such that the probability of occurrence is solely dependent on the solubility of the POI. To this end, we initially prevented the degradation of l-arabinose (the inducer of the PBAD promoter) by employing an Escherichia coli host strain deficient in the metabolism of l-arabinose. Given that a low expression of His6-CfaN, compared with that of CfaC-Oct-1, was found to be conducive to the generation to a soluble product of the protein trans-splicing event, we designed the expression of His6-CfaN and CfaC-Oct-1 to be inducible from the PBAD and PT7 promoters, respectively. The optimized system thus obtained enabled in vitro selection of the plasmid-protein complex with high yield. We believe that the inducible plasmid display system developed in this study would be applicable to high-throughput screening and/or selection of protein variants with enhanced solubility.

Association between exercise and fear of falling in community-dwelling elderly Koreans: results of a cross-sectional public opinion survey.

OBJECTIVE: To investigate the association between habitual exercise and fear of falling in an older Korean population. DESIGN: Cross-sectional study conducted in a population-based sample of an urban city. SETTING: Urban city in South Korea. PARTICIPANTS: Randomly sampled older Korean people (N=828; aged ≥65y) living in a typical urban city located in South Korea. INTERVENTION: Standardized telephone interview. MAIN OUTCOME MEASURES: Data on exercise habits, history of falls during the previous year, and fear of falling were obtained using a random digit dialing telephone survey method. Multiple linear regression and multiple logistic regression analyses were used to examine the association between exercise habits and the fear of falling. RESULTS: The incidence of falls in the study cohort was 13%, and the prevalence of the fear of falling was 67.4% (47.6% in men and 80.8% in women). About 30% of those that expressed a fear of falling stated that this was the cause of their limited activity. Older men and women who exercised regularly showed a similar level of fear of falling, but they were less likely to experience fear-related activity restriction than nonexercisers. CONCLUSIONS: A regular exercise habit was found to be inversely associated with fear-related activity restriction regardless of fall experience among older Korean men and women.

Longitudinal change of treatment-related upper limb dysfunction and its impact on late dysfunction in breast cancer survivors: a prospective cohort study.

BACKGROUND AND OBJECTIVES: To investigate the prevalence of upper limb dysfunction (ULD) and subtypes after breast cancer surgery and to identify factors associated with late ULD. METHODS: Among 191 enrolled patients, 191 were evaluated at 3 months, 187 at 6 months, and 183 at 12 months after surgery. Pain, shoulder range of motion, muscle strength, and arm circumference were assessed. Based on symptoms and physical examinations, the types of ULD common after breast cancer treatment were diagnosed and categorized. RESULTS: The prevalence of ULD after surgery were 24.6%, 20.9%, and 26.8% at 3, 6, and 12 months, respectively. The most common types of ULD were pectoralis tightness at 3 and 6 months and lymphedema at 12 months. Patients with pectoralis tightness or lymphedema at 3 or 6 months showed a higher prevalence of rotator cuff disease at 12 months compared with those without early pectoralis tightness or lymphedema. CONCLUSIONS: The major post-operative ULD were pectoralis tightness at 3 and 6 months and lymphedema at 12 months. Late ULD such as rotator cuff disease were associated with pectoral tightness or lymphedema at earlier stages. Diagnosis and treatment of ULD should take place as soon as possible after surgery.

Cross-cultural adaptation and clinimetric property of Korean version of the Chronic Pain Coping Inventory-42 in patients with chronic low back pain.

STUDY DESIGN: Validation of a translated, culturally adapted questionnaire. OBJECTIVE: We developed a Korean version of the Chronic Pain Coping Inventory-42 (CPCI-42) by performing a cross-cultural adaptation, and evaluated its reliability and validity. SUMMARY OF BACKGROUND DATA: The CPCI is widely used and validated instruments for measuring coping strategies in chronic pain. However, no validated and culturally adapted version was available in Asian countries. METHODS: We assessed 142 patients with chronic low back pain using the CPCI-42 and measures of physical disability, pain, and quality of life. Results for 93 of the 142 patients exhibited test-retest reliability. The interval time of collecting retest data varied from 2 weeks to 1 month. Criterion validity was evaluated using correlations between the CPCI-42 and the Oswestry Disability Index, the Brief Pain Inventory, and the Short Form 36-item Health Survey (version 2.0). Construct validity was computed using exploratory factor analysis. RESULTS: The Korean version of the CPCI-42 had a high internal consistency (Cronbach's alpha >0.70) with the exception of results for task persistence and relaxation. Illness-focused coping (guarding, resting, asking for assistance) and other-focused coping (seeking social support) were most significantly correlated with Oswestry Disability Index, Brief Pain Inventory, and Short Form 36-item Health Survey, respectively. Outcomes for task persistence were contrary to other subscales in wellness-focused coping. Construct validity by factor analysis produced similar results to the original CPCI subscale. However, several factors showed cross-loading in 8 factor solutions. CONCLUSION: Despite linguistic and cultural differences, the Korean version of the CPCI-42 is overall a meaningful tool, and produces results sufficiently similar to the original CPCI-42.

The effect of back school integrated with core strengthening in patients with chronic low-back pain.

OBJECTIVE: To assess the effect of back school integrated with core-strengthening exercises on back-specific disability and pain-coping strategies and to examine how reactions to pain affect the outcomes of back school in patients with chronic low back pain. DESIGN: A single-center prospective trial was conducted with 142 participants with chronic low-back pain (38 men and 104 women) who completed a back school program at the spine center of a university hospital. The subjects participated in a 4-wk program integrated with core-strengthening exercises. Back-specific disabilities were measured as a primary outcome before and after the program. Secondary outcomes were pain, Chronic Pain Coping Inventory, general health status assessed by the SF-36, and quantitative functional evaluations of factors, such as trunk muscle strength, endurance, and the back performance scale. A subgroup of 28 subjects (12 men and 16 women) of the total sample of 142 subjects was used to analyze the longitudinal association between coping strategies and the primary outcome in a long-term follow-up study. These participants were divided into three groups (much improved, slightly improved, and unimproved) based on changes in back-specific disability scores. RESULTS: Participants improved significantly in terms of back-specific disability, pain, general health, and quantitative functional tests according to the short-term evaluation. They used more relaxation and exercise/stretching techniques as coping strategies. Of the groups participating in the longer-term follow-up (T3), the much-improved group showed significant improvement between T1 (before back school) and T2 (after back school) in scores for relaxation (1.6 +/- 1.0 vs. 2.6 +/- 1.1), task persistence (2.9 +/- 1.2 vs. 3.7 +/- 1.2), and exercise (3.3 +/- 1.1 vs. 5.2 +/- 1.9), but the coping strategies of those in the slightly improved and unimproved groups did not change significantly at T2. CONCLUSIONS: Our back school program may help patients with chronic low back pain reduce back-specific disability and pain and develop wellness-focused coping strategies such as exercise and stretching.

Discrepancies between balance confidence and physical performance among community-dwelling Korean elders: a population-based study.

BACKGROUND: This study investigates discrepancies between the confidence expressed by Korean elders in their balance and their actual physical performances according to sex and age. It also attempts to identify the factors responsible for such gaps or discrepancies. METHODS: The Korean Longitudinal Study on Health and Aging (KLoSHA) was designed as a population-based prospective cohort study on the health and aging of Korean adults aged 65 years and over. We evaluated 1000 of 1118 randomly selected subjects in terms of activities-specific balance confidence (ABC) and performance in regard to balance and gait (POMA). Activities of daily living (ADL) and instrumental activities of daily living (IADL) were assessed to measure disability. Pain-related dysfunctions, depression and cognitive functions were assessed. Next, we assessed the relationship between balance confidence and physical performance and functioning by sex and age. RESULTS: The balance confidence of elderly women was lower than that of elderly men, although the physical performances of men and women under the age of 80 were similar. Women showed increased functional disability related to lower extremity pain and closely associated with balance confidence. However, psychological variables such as depression and cognitive functioning did not affect balance confidence. CONCLUSION: We found a discrepancy between balance confidence and physical performance according to sex and age among Koreans elders. Low balance confidence among women elders was correlated with pain-related dysfunctions other than those reflected in POMA scores. Therefore, enhancing confidence and controlling pain as a means of preventing disability should be emphasized when developing models for maintaining and promoting health in elders.