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Alzheimer's disease (AD) is an age-related progressive neurodegenerative disorder characterized by memory loss, cognitive decline, and behavioral changes, impacting millions of individuals worldwide. Despite significant research into its cellular and molecular mechanisms, no cure has been found to treat AD to date. For over two decades, research aimed at treating AD has focused on targeting amyloid-β (Aβ); however, these strategies have not demonstrated substantial effectiveness. Consequently, research is now expanding towards targeting other hallmarks of the disease, such as tau protein and brain metal ions. Among potential therapeutics against these pathophysiological targets, peptide-based inhibitors are notable for their high selectivity and low toxicity. Despite these advantages, they face obstacles such as a short half-life in vivo and low efficiencies in crossing the blood-brain barrier (BBB). The use of nanoparticles (NPs) to deliver peptide-based inhibitors to the brain offers unique advantages, such as enhanced stability against degradation, improvement in targeted delivery, and reduced potential for immunogenic responses. This review aims to provide a comprehensive overview of emerging peptides tested as treatments for AD against Aβ, tau protein, and brain metal ions and to evaluate NPs as a means to overcome the limitations. These peptide-based inhibitors are promising, as they not only alleviate symptoms but also aim to prevent progressive neuronal loss, and NPs can be highly effective in delivering these inhibitors., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

An ischemic cerebral stroke results from the interruption of blood flow to the brain, triggering rapid and complex cascades of excitotoxicity, oxidative stress, and inflammation. Current reperfusion therapies, including intravenous thrombolysis and mechanical thrombectomy, cause further brain injury due to reperfusion-induced cytotoxicity. To date, novel cytoprotective therapies that could address these challenges have yet to be developed, likely due to the limitations of targeting a single pathologic mechanism. To address these unmet clinical needs, we investigated a synthetic verbenone derivative, SP-8356, as a potential multi-target cytoprotective agent for acute ischemic strokes. In transient middle cerebral artery occlusion (MCAO) rats, SP-8356 significantly reduced brain infarct and edema volumes while improving acute neurological deficits in a dose-dependent manner. Furthermore, SP-8356 improved long-term outcomes, particularly by reducing mortality. These potent cytoprotective effects of SP-8356 were achieved by suppressing the excessive production of free radicals and pro-inflammatory cytokines, reducing the infiltration of inflammatory cells, and mitigating increases in blood-brain barrier permeability. Additional research is needed to determine whether co-administration of SP-8356 can extend the therapeutic time window of reperfusion therapies by mitigating ischemia/reperfusion injury.

Background: Pharyngeal infection is more difficult to diagnose and treat than genital or rectal infection and can act as a reservoir for gonococcal infection. We determined the prevalence of pharyngeal gonorrhea in Korean men with urethritis and analyzed the molecular characteristics and antimicrobial susceptibility of the isolates., Methods: Seventy-two male patients with symptoms of urethritis who visited a urology clinic in Wonju, Korea, between September 2016 and March 2018 were included. Urethral and pharyngeal gonococcal cultures, antimicrobial susceptibility testing, Neisseria gonorrhoeae multi-antigen sequence typing (NG-MAST), and multiplex real-time PCR (mRT-PCR) were performed., Results: Among the 72 patients, 59 tested positive for gonococcus by mRT-PCR. Of these 59 patients, 18 (30.5%) tested positive in both the pharynx and urethra, whereas 41 tested positive only in the urethra. NG-MAST was feasible in 16 out of 18 patients and revealed that 14 patients had the same sequence types in both urethral and pharyngeal specimens, whereas two patients exhibited different sequence types between the urethra and pharynx. Of the 72 patients, 33 tested culture-positive. All patients tested positive only in urethral specimens, except for one patient who tested positive in both. All culture-positive specimens also tested positive by mRT-PCR. All isolates were susceptible to azithromycin and spectinomycin, but resistance rates to ceftriaxone and cefixime were 2.9% and 14.7%, respectively., Conclusions: The prevalence of pharyngeal gonorrhea in Korean men with gonococcal urethritis is as high as 30.5%, highlighting the need for pharyngeal screening in high-risk groups. Ceftriaxone is the recommended treatment for pharyngeal gonorrhea.

Background: The objective of this study lies in identifying the dominant genotype of C. trachomatis isolated in Republic of Korea (ROK) between 2017 and 2018., Methods: A total of 504 clinical cervicovaginal swabs from patients were collected and inoculated on McCoy cell monolayers to isolate Chlamydial agents. C. trachomatis isolates were analyzed by sequencing of its ompA gene., Results: A total of 54 C. trachomatis isolates were obtained. The constructed phylogenetic tree revealed the genotypes of isolates are D/Ep6 (48, 88.89%), D/Ep6-like (5, 9.26%), and Ja (1, 1.85%). The C. trachomatis D/Ep6-like have one single nucleotide polymorphism (SNP) compared to C. trachomatis D/Ep6, leading to E870K amino acid change., Conclusions: Phylogenetic analysis demonstrated globally rare C. trachomatis D/Ep6 was dominant in the ROK from 2017 to 2018. Findings in this study act as a keystone, bridging past and present molecular epidemiology of C. trachomatis in context of ROK.

Inflammation drives many age-related, especially neurological, diseases, and likely mediates age-related proteotoxicity. For example, dementia due to Alzheimer's Disease (AD), cerebral vascular disease, many other neurodegenerative conditions is increasingly among the most devastating burdens on the American (and world) health system and threatens to bankrupt the American health system as the population ages unless effective treatments are developed. Dementia due to either AD or cerebral vascular disease, and plausibly many other neurodegenerative and even psychiatric conditions, is driven by increased age-related inflammation, which in turn appears to mediate Abeta and related proteotoxic processes. The functional significance of inflammation during aging is also supported by the fact that Humira, which is simply an antibody to the pro-inflammatory cytokine TNF-a, is the best-selling drug in the world by revenue. These observations led us to develop parallel high-throughput screens to discover small molecules which inhibit age-related Abeta proteotoxicity in a C. elegans model of AD AND LPS-induced microglial TNF-a. In the initial screen of 2560 compounds (Microsource Spectrum library) to delay Abeta proteotoxicity, the most protective compounds were, in order, phenylbutyrate, methicillin, and quetiapine, which belong to drug classes (HDAC inhibitors, beta lactam antibiotics, and tricyclic antipsychotics, respectably) already robustly implicated as promising to protect in neurodegenerative diseases, especially AD. RNAi and chemical screens indicated that the protective effects of HDAC inhibitors to reduce Abeta proteotoxicity are mediated by inhibition of HDAC2, also implicated in human AD, dependent on the HAT Creb binding protein (Cbp), which is also required for the protective effects of both dietary restriction and the daf-2 mutation (inactivation of IGF-1 signaling) during aging. In addition to methicillin, several other beta lactam antibiotics also delayed Abeta proteotoxicity and reduced microglial TNF-a. In addition to quetiapine, several other tricyclic antipsychotic drugs also delayed age-related Abeta proteotoxicity and increased microglial TNF-a, leading to the synthesis of a novel congener, GM310, which delays Abeta as well as Huntingtin proteotoxicity, inhibits LPS-induced mouse and human microglial and monocyte TNF-a, is highly concentrated in brain after oral delivery with no apparent toxicity, increases lifespan, and produces molecular responses highly similar to those produced by dietary restriction, including induction of Cbp inhibition of inhibitors of Cbp, and genes promoting a shift away from glycolysis and toward metabolism of alternate (e.g., lipid) substrates. GM310, as well as FDA-approved tricyclic congeners, prevented functional impairments and associated increase in TNF-a in a mouse model of stroke. Robust reduction of glycolysis by GM310 was functionally corroborated by flux analysis, and the glycolytic inhibitor 2-DG inhibited microglial TNF-a and other markers of inflammation, delayed Abeta proteotoxicity, and increased lifespan. These results support the value of phenotypic screens to discover drugs to treat age-related, especially neurological and even psychiatric diseases, including AD and stroke, and to clarify novel mechanisms driving neurodegeneration (e.g., increased microglial glycolysis drives neuroinflammation and subsequent neurotoxicity) suggesting novel treatments (selective inhibitors of microglial glycolysis).

Genetically engineered fusion polypeptides have been investigated to introduce unique bio-functionality and improve some therapeutic activity for anti-angiogenesis. We report herein that stimuli-responsive, vascular endothelial growth factor receptor 1 (VEGFR1) targeting fusion polypeptides composed of a VEGFR1 (fms-like tyrosine kinase-1 (Flt1)) antagonist, an anti-Flt1 peptide, and a thermally responsive elastin-based polypeptide (EBP) were rationally designed at the genetic level, biosynthesized, and purified by inverse transition cycling to develop potential anti-angiogenic fusion polypeptides to treat neovascular diseases. A series of hydrophilic EBPs with different block lengths were fused with an anti-Flt1 peptide, forming anti-Flt1-EBPs, and the effect of EBP block length on their physicochemical properties was examined. While the anti-Flt1 peptide decreased phase-transition temperatures of anti-Flt1-EBPs, compared with EBP blocks, anti-Flt1-EBPs were soluble under physiological conditions. The anti-Flt1-EBPs dose dependently inhibited the binding of VEGFR1 against vascular endothelial growth factor (VEGF) as well as tube-like network formation of human umbilical vein endothelial cells under VEGF-triggered angiogenesis in vitro because of the specific binding between anti-Flt1-EBPs and VEGFR1. Furthermore, the anti-Flt1-EBPs suppressed laser-induced choroidal neovascularization in a wet age-related macular degeneration mouse model in vivo. Our results indicate that anti-Flt1-EBPs as VEGFR1-targeting fusion polypeptides have great potential for efficacious anti-angiogenesis to treat retinal-, corneal-, and choroidal neovascularization.

Background: National reference standards for anti-HIV-1 antibody are needed to evaluate the performance and maintain the quality control of anti-HIV-1 antibody assays. The aim of this study was to prepare a mixed-titer performance panel and assess its suitability as a national reference standard for anti-HIV-1 antibody according to stability, collaboration, and other studies., Methods: Nineteen serum samples from different HIV patients were obtained, along with 15 units of fresh frozen plasma samples with negative anti-HIV-1 antibody results. Ten anti-HIV-1 antibody-positive candidate standards and two negative candidate standards were prepared based on the reactivity in the Alinity i HIV Ag/Ab combo assay (Abbott Laboratories, Wiesbaden, Germany). A collaborative study was conducted across eight laboratories using five anti-HIV-1 antibody assays. Real-time and accelerated stability were evaluated to assess the long-term stability., Results: In the collaborative study, results of all five anti-HIV-1 antibody assays were positive for all 10 candidate standards prepared using HIV patient samples. The CV of each assay for every candidate standard was within 10%, except for one assay result. No real-time and accelerated stability change trend was observed at -70°C or -20°C, supporting that the reference standards were maintained in a stable state at -70°C for long-term storage., Conclusions: The overall results suggest that the 12 candidate standards could serve as national reference standards for anti-HIV-1 antibody.

While the coronavirus disease 2019 pandemic is ongoing, monkeypox has been rapidly spreading in non-endemic countries since May 2022. Accurate and rapid laboratory tests are essential for identifying and controlling monkeypox. Korean Society for Laboratory Medicine and the Korea Disease Prevention and Control Agency have proposed guidelines for diagnosing monkeypox in clinical laboratories in Korea. These guidelines cover the type of tests, selection of specimens, collection of specimens, diagnostic methods, interpretation of test results, and biosafety. Molecular tests are recommended as confirmatory tests. Skin lesion specimens are recommended for testing in the symptomatic stage, and the collection of both blood and oropharyngeal swabs is recommended in the presymptomatic or prodromal stage.

Purpose: The Janus tyrosine kinase and signal transducers and activators of transcription (JAK/STAT) pathway is involved in vascular endothelial growth factor (VEGF) expression, but the role of this pathway in diabetic retinopathy (DR) remains unclear. We investigated the role of the JAK/STAT pathway on DR and VEGF expression using a streptozotocin (STZ)-induced DR mouse model., Methods: Cultured ARPE-19 cells were exposed to high-glucose conditions and treated with JAK/STAT inhibitors (JAK inhibitor I [JAKiI], tofacitinib, STAT3 inhibitor [STAT3i]) for 48 h. Reverse-transcription polymerase chain reaction, western blotting, and enzyme-linked immunosorbent assay were used to investigate p-JAK/STAT and VEGF expression. Diabetes was induced by intraperitoneal injection of STZ (50 mg/kg) in C57BL/6 mice for 5 days. DR development was evaluated every 4 weeks. JAK/STAT inhibitors were administered for 8 weeks. Immunofluorescence was used to measure the activation status of the JAK/STAT pathway and VEGF production in the retinal tissue., Results: In ARPE-19 cells exposed to high-glucose conditions, the mRNA and secretory protein levels of VEGF, p-JAK1, p-JAK2, p-STAT3, and p-STAT5 levels were significantly increased. Treatment with JAKiI, tofacitinib, and STAT3i significantly suppressed VEGF to basal levels at both the mRNA and secretory levels in vitro. In STZ-induced mice, retinal vascular leakage, p-JAK1, p-JAK2, p-JAK3, p-STAT3, and VEGF were significantly increased after diabetes induction. Diabetes-induced retinal vascular leakage was significantly reduced by treatment with JAKiI and tofacitinib. Increased p-JAK1 and VEGF in STZ-induced mice were significantly reduced by JAKiI (p < 0.05, p < 0.001) and tofacitinib (p < 0.001, respectively)., Conclusion: JAK1 may be more involved in VEGF production and DR progression in mice than JAK2, JAK3, and STAT3., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The horizon of immunotherapy using CAR-T cells is continuously extending to treat solid tumors beyond the success in the treatment of liquid tumors. Precise in-vitro evaluations of CAR-T cells for their phenotypes, quantity and quality of activation in various tumor microenvironments including different antigen densities, and the resulting effector functions are critical for the successful development of CAR-T therapies and safe translation to clinics. Unfortunately, the development of methods and tools to accommodate these needs have been lagging behind. Here, we developed a novel biomaterial platform, acellular artificial target particles (aaTPs) against CAR-T cells, using magnetic microbeads that are already widely employed in the manufacturing of T cell products. By devising a simple and standardized procedure, we precisely controlled the antigen surface densities presented on the aaTPs for a wide range. By co-incubation of aaTPs with CAR-T cells followed by flow cytometry and cytokine assays, we quantitatively determined the antigen-specific and dose-dependent activation of anti-HER2 CAR-T cells. We also demonstrated that the aaTP can serve as a clean target cell in in-vitro assays to prove the proposed mechanism of action of a next-generation CAR-T product. Overall, the simple, inexpensive, modular and precisely controllable synthetic nature of aaTPs enables the development of clean and standardized in-vitro assays for CAR-T cells, which provides critical advantages over the conventional assays using target cell lines. The design of aaTPs can be extended to include other tumor antigens and relevant surface molecules of physiological target cells. Thus, the aaTP platform has great potential as a standardized tool for the development and evaluation of both conventional and new CAR-T products in the context of approval from regulatory agencies and clinical translation., Competing Interests: Authors RH-S, LC, and FM were employed by the company Refuge Biotechnologies. The authors declare that this study received funding from Refuge Biotechnologies. The funder had the following involvement in the study: the study design, collection, analysis, interpretation of data, and the writing of this article., (Copyright © 2022 Harari-Steinfeld, Abhinav Ayyadevara, Cuevas, Marincola and Roh.)

With the rapid spread of the coronavirus disease (COVID-19), the need for rapid testing and diagnosis and consequently, the demand for mobile laboratories have increased. Despite this need, there are no clear guidelines for the operation, maintenance, or quality control of mobile laboratories. We provide guidelines for the operation, management, and quality control of mobile laboratories, and specifically for the implementation and execution of COVID-19 molecular diagnostic testing. These practical guidelines are primarily based on expert opinions and a laboratory accreditation inspection checklist. The scope of these guidelines includes the facility, preoperative evaluation, PCR testing, internal and external quality control, sample handling, reporting, laboratory personnel, biosafety level, and laboratory safety management. These guidelines are useful for the maintenance and operation of mobile laboratories not only in normal circumstances but also during public health crises and emergencies.

Korean Society for Laboratory Medicine and the Korea Disease Prevention and Control Agency have announced guidelines for diagnosing coronavirus disease (COVID-19) in clinical laboratories in Korea. With the ongoing pandemic, we propose an update of the previous guidelines based on new scientific data. This update includes recommendations for tests that were not included in the previous guidelines, including the rapid molecular test, antigen test, antibody test, and self-collected specimens, and a revision of the previous recommendations. This update will aid clinical laboratories in performing laboratory tests for diagnosing COVID-19.

Background: Adoptive transfer of chimeric antigen receptor (CAR)-engineered T cells combined with checkpoint inhibition may prevent T cell exhaustion and improve clinical outcomes. However, the approach is limited by cumulative costs and toxicities., Methods: To overcome this drawback, we created a CAR-T (RB-340-1) that unites in one product the two modalities: a CRISPR interference-(CRISPRi) circuit prevents programmed cell death protein 1 (PD-1) expression upon antigen-encounter. RB-340-1 is engineered to express an anti-human epidermal growth factor receptor 2 (HER2) CAR single chain variable fragment (scFv), with CD28 and CD3ζ co-stimulatory domains linked to the tobacco etch virus (TEV) protease and a single guide RNA (sgRNA) targeting the PD-1 transcription start site (TSS). A second constructs includes linker for activation of T cells (LAT) fused to nuclease-deactivated spCas9 (dCas9)-Kruppel-associated box (KRAB) via a TEV-cleavable sequence (TCS). Upon antigen encounter, the LAT-dCas9-KRAB (LdCK) complex is cleaved by TEV allowing targeting of dCas9-KRAB to the PD-1 gene TSS., Results: Here, we show that RB-340-1 consistently demonstrated higher production of homeostatic cytokines, enhanced expansion of CAR-T cells in vitro, prolonged in vivo persistence and more efficient suppression of HER2 + FaDu oropharyngeal cancer growth compared to the respective conventional CAR-T cell product., Conclusions: As the first application of CRISPRi toward a clinically relevant product, RB-340-1 with the conditional, non-gene editing and reversible suppression promotes CAR-T cells resilience to checkpoint inhibition, and their persistence and effectiveness against HER2-expressing cancer xenografts., (© 2021. The Author(s).)

The rapid antigen test (RAT) for coronavirus disease (COVID-19) represents a potent diagnostic method in situations of limited molecular testing resources. However, considerable performance variance has been reported with the RAT. We evaluated the clinical performance of Standard Q COVID-19 RAT (SQ-RAT; SD Biosensor, Suwon, Korea), the first RAT approved by the Korean Ministry of Food and Drug Safety. In total, 680 nasopharyngeal swabs previously tested using real-time reverse-transcription PCR (rRT-PCR) were retested using SQ-RAT. The clinical sensitivity of SQ-RAT relative to that of rRT-PCR was 28.7% for all specimens and was 81.4% for specimens with RNA-dependent RNA polymerase gene ( RdRp ) threshold cycle (Ct) values ≤23.37, which is the limit of detection of SQ-RAT. The specificity was 100%. The clinical sensitivity of SQ-RAT for COVID-19 diagnosis was assessed based on the Ct distribution at diagnosis of 33,294 COVID-19 cases in Korea extracted from the laboratory surveillance system of Korean Society for Laboratory Medicine. The clinical sensitivity of SQ-RAT for COVID-19 diagnosis in the Korean population was 41.8%. Considering the molecular testing capacity in Korea, use of the RAT for COVID-19 diagnosis appears to be limited.

Despite the tremendous progress in immunotherapy regimens using T cells, efforts to modulate the functions of T cells are still significantly hampered by the lack of reliable methods to deliver various cargoes into the T cells. This ongoing challenge originates from the intrinsic resistance of T cells in taking up exogenous materials. Here, we strategically aimed to hijack the natural endocytosis of Interleukin-2 (IL2) by the activated T cells for the targeted association and intracellular delivery of cargoes in varying sizes. First, we carefully characterized the fluctuations in the expression levels of IL2 receptor (IL2R) subunits (CD25, CD122, and CD132) during the murine primary T cell cultures over 12 days. We identified the highest fraction of T cells that would express the high-affinity trimeric IL2R on Day 3. By examining the association and uptake efficiencies of IL2 molecules that are biotinylated via either random lysine-targeting chemical reaction (using NHS-PEG4-Biotin) or site-specific enzymatic modification (using Avitag sequence), we demonstrated that the most efficient delivery of cargo can be achieved by C-terminal conjugation. Upon confirmation of successful delivery of a small model cargo, streptavidin, we employed superparamagnetic iron oxide nanoparticles (SPIONs) as bigger model cargoes having core diameters of 50, 100, and 200 nm. We examined the association and intracellular delivery of the IL2-conjugated nanocargoes using flow cytometry, confocal laser scanning microscopy, and transmission electron microscopy. While cargoes of all tested sizes were successfully associated with the IL2R-expressing T cells in comparable efficiencies, the uptake efficiencies were inversely proportional to the sizes of the cargoes. Nevertheless, our current definitive report confirms that nanocargoes with a practical maximum size limit around 100-200 nm can be intracellularly delivered into activated primary T cells using IL2R-mediated endocytosis, which opens a new horizon for engineering and manufacturing of various T cell immunotherapeutics.

Classification of clinical symptoms and diagnostic microbiology are essential to effectively employ antimicrobial therapy for lower respiratory tract infections (LRTIs) in a timely manner. Empirical antibiotic treatment without microbial identification hinders the selective use of narrow-spectrum antibiotics and effective patient treatment. Thus, the development of rapid and accurate diagnostic procedures that can be readily adopted by the clinic is necessary to minimize non-essential or excessive use of antibiotics and accelerate patient recovery from LRTI-induced damage. We developed and validated a multiplex real-time polymerase chain reaction (mRT-PCR) assay with good analytical performance and high specificity to simultaneously detect four bacterial pathogens causing pneumonia: Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, and Moraxella catarrhalis. The analytical performance of mRT-PCR against target pathogens was evaluated by the limit of detection (LOD), specificity, and repeatability. Two hundred and ten clinical specimens from pneumonia patients were processed using an automatic nucleic acid extraction system for the "respiratory bacteria four" (RB4) mRT-PCR assay, and the results were directly compared to references from bacterial culture and/or Sanger sequencing. The RB4 mRT-PCR assay detected all target pathogens from sputum specimens with a coefficient of variation ranging from 0.29 to 1.71 and conservative LOD of DNA corresponding to 5 × 102 copies/reaction. The concordance of the assay with reference-positive specimens was 100%, and additional bacterial infections were detected from reference-negative specimens. Overall, the RB4 mRT-PCR assay showed a more rapid turnaround time and higher performance that those of reference assays. The RB4 mRT-PCR assay is a high-throughput and reliable tool that assists decision-making assessment and outperforms other standard methods. This tool supports patient management by considerably reducing the inappropriate use of antibiotics., Competing Interests: The authors have declared that no competing interests exist.

The detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in upper and lower respiratory specimens and coinfection with other respiratory pathogens in patients with coronavirus disease 2019 (COVID-19) was investigated. Study subjects ( N  = 342) were retrospectively enrolled after being confirmed as SARS-CoV-2 positive, and their nasopharyngeal swab (NPS), oropharyngeal swab (OPS), and sputum specimens were restored for SARS-CoV-2 retesting and respiratory pathogen detection. The majority of the subjects (96.5%, N  = 330) were confirmed as SARS-CoV-2 positive using NPS/OPS specimens. Among the COVID-19 patients ( N  = 342), 7.9% ( N  = 27) and 0.9% ( N  = 3) were coinfected with respiratory viruses and Mycoplasma pneumoniae , respectively, yielding an 8.8% ( N  = 30) overall respiratory pathogen coinfection rate. Of the respiratory virus coinfection cases ( N  = 27), 92.6% ( N  = 25) were coinfected with a single respiratory virus and 7.4% ( N  = 2) with two viruses (metapneumovirus/adenovirus and rhinovirus/bocavirus). No triple coinfections of other respiratory viruses or bacteria with SARS-CoV-2 were detected. Respiratory viruses coinfected in the patients with COVID-19 were as follows: rhinovirus ( N  = 7, 2.1%), respiratory syncytial virus A and B ( N  = 6, 1.8%), non-SARS-CoV-2 coronaviruses (229E, NL63, and OC43, N  = 5, 1.5%), metapneumovirus ( N  = 4, 1.2%), influenza A ( N  = 3, 0.9%), adenovirus ( N  = 3, 0.9%), and bocavirus ( N  = 1, 0.3%). In conclusion, the diagnostic value of utilizing NPS/OPS specimens is excellent, and, as the first report in Korea, coinfection with respiratory pathogens was detected at a rate of 8.8% in patients with COVID-19., Competing Interests: All authors declare no conflicts of interest., (Copyright © 2021 Kyoung Ho Roh et al.)

In response to the ongoing coronavirus disease 2019 (COVID-19) pandemic, an online laboratory surveillance system was established to monitor severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) real-time reverse transcription-PCR (rRT-PCR) testing capacities and results. SARS-CoV-2 rRT-PCR testing data were collected from 97 clinical laboratories, including 84 medical institutions and 13 independent clinical laboratories in Korea. We assessed the testing capacities to utilize SARS-CoV-2 rRT-PCR based on surveillance data obtained from February 7th to June 4th, 2020 and evaluated positive result characteristics according to the reagents used and sample types. A total of 1,890,319 SARS-CoV-2 rRT-PCR testing were performed, 2.3% of which were positive. Strong correlations were observed between the envelope ( E ) gene and RNA-dependent RNA polymerase ( RdRp )/nucleocapsid ( N ) genes threshold cycle (Ct) values for each reagent. No statistically significant differences in gene Ct values were observed between the paired upper and lower respiratory tract samples, except in the N gene for nasopharyngeal swab and sputum samples. Our study showed that clinical laboratories in Korea have rapidly expanded their testing capacities in response to the COVID-19 outbreak, with a peak daily capacity of 34,193 tests. Rapid expansion in testing capacity is a critical component of the national response to the ongoing pandemic.

Pattern-recognition receptors including Toll-like receptors (TLRs) recognize invading pathogens and trigger an immune response in mammals. Here we show that mammalian ste20-like kinase 1/serine/threonine kinase 4 (MST1/STK4) functions as a negative regulator of lipopolysaccharide (LPS)-induced activation of the TLR4-NF-κB signaling pathway associated with inflammation. Myeloid-specific genetic ablation of MST1/STK4 increased the susceptibility of mice to LPS-induced septic shock. Ablation of MST1/STK4 also enhanced NF-κB activation triggered by LPS in bone marrow-derived macrophages (BMDMs), leading to increased production of proinflammatory cytokines by these cells. Furthermore, MST1/STK4 inhibited TRAF6 autoubiquitination as well as TRAF6-mediated downstream signaling induced by LPS. In addition, we found that TRAF6 mediates the LPS-induced activation of MST1/STK4 by catalyzing its ubiquitination, resulting in negative feedback regulation by MST1/STK4 of the LPS-induced pathway leading to cytokine production in macrophages. Together, our findings suggest that MST1/STK4 functions as a negative modulator of the LPS-induced NF-κB signaling pathway during macrophage activation.

CD3ε is expressed on T lymphocytes as a part of the T cell receptor (TCR)-CD3 complex. Together with other CD3 molecules, CD3ε is responsible for the activation of T cells via transducing the event of antigen recognition by the TCR into intracellular signaling cascades. The present study first aims to identify a novel peptide ligand that binds to human CD3ε in a specific manner and to perform an initial evaluation of its biological efficacy on the human T cell line, Jurkat cells. We screened a phage-display peptide library against human CD3ε using a subtractive biopanning process, from which we identified 13 phage clones displaying unique peptide sequences. One dominant phage clone displaying the 7 amino acid sequence of WSLGYTG, which occupied 90% of tested plaques (18 out of 20) after the 5th round of biopanning, demonstrated a superior binding behavior to other clones in the binding assays against recombinant CD3ε on microbeads or Jurkat cells. The synthesized peptide also showed specific binding to Jurkat cells in a dose-dependent manner but not to B cell lymphoma line, 2PK3 cells. Molecular modeling and docking simulation confirmed that the selected peptide ligand in an energetically stable conformation binds to a pocket of CD3ε that is not hidden by either CD3γ or CD3δ. Lastly, magnetic microbeads conjugated with the synthesized peptide ligands showed a weak but specific association with Jurkat cells and induced the calcium flux, a hallmark indication of proximal T cell receptor signaling, which gave rise to an enhancement of IL-2 section and cell proliferation. The novel peptide ligand and its various multivalent forms have a great potential in applications related to T cell biology and T cell immunotherapy.

[This corrects the article on p. 439 in vol. 40.].

Jurkat, an immortalized cell line derived from human leukemic T lymphocytes, has been employed as an excellent surrogate model of human primary T-cells for the advancement of T-cell biology and their applications in medicine. However, presumably due to its T-cell origin, Jurkat cells are very difficult to transfect. Thus, for the genetic modification of Jurkat cells, expensive and time-consuming viral vectors are normally required. Despite many previous efforts, non-viral vectors have not yet overcome the hurdles of low transfection efficiency and/or high toxicity in transfection of Jurkat cells. Here, we report that a simple addition of calcium ions (Ca 2+ ) into culture media at optimal concentrations can enhance the efficiency of the polyplex-mediated transfection using poly(ethylene imine) (PEI) by up to 12-fold when compared to the polyplex-only control. We show that calcium enhances the association between polyplex and Jurkat, which is at least partially responsible for the increase in transmembrane delivery of polyplex and consequential enhancement in expression of transgene. Other cations, Mg 2+ or Na + did not show similar enhancement. Interestingly, addition of Ca 2+ was rather detrimental for the transfection of lipoplex on Jurkat cells. Observation of significant enhancement in the transfection of non-viral vectors with a simple and physiologically relevant reagent like Ca 2+ in the engineering of hard-to-transfect cells such as Jurkat warrants further investigation on similar strategies.

Coronavirus disease 2019 (COVID-19) is a respiratory disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Early detection of COVID-19 and immediate isolation of infected patients from the naive population are important to prevent further pandemic spread of the infection. Real-time reverse transcription (RT)-PCR to detect SARS-CoV-2 RNA is currently the most reliable diagnostic method for confirming COVID-19 worldwide. Guidelines for clinical laboratories on the COVID-19 diagnosis have been recently published by Korean Society for Laboratory Medicine and the Korea Centers for Disease Control and Prevention. However, these formal guidelines do not address common practical laboratory issues related to COVID-19 real-time RT-PCR testing and their solutions. Therefore, this guideline is intended as a practical and technical supplement to the "Guidelines for Laboratory Diagnosis of COVID-19 in Korea".

The outbreak of coronavirus disease 2019 (COVID-19), which began in December 2019, is still ongoing in Korea, with >9,000 confirmed cases as of March 25, 2020. COVID-19 is a severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) infection, and real-time reverse transcription-PCR is currently the most reliable diagnostic method for COVID-19 around the world. Korean Society for Laboratory Medicine and the Korea Centers for Disease Prevention and Control propose guidelines for diagnosing COVID-19 in clinical laboratories in Korea. These guidelines are based on other related domestic and international guidelines, as well as expert opinions and include the selection of test subjects, selection of specimens, diagnostic methods, interpretation of test results, and biosafety., Competing Interests: No potential conflicts of interest relevant to this article were reported., (© The Korean Society for Laboratory Medicine.)

Since carbapenems have been used for the treatment of infections in medical settings, multidrug-resistant Pseudomonas aeruginosa containing resistance for carbapenems has become a major cause of nosocomial infections worldwide. Information on carbapenemase-producing P. aeruginosa isolates at community hospitals, including long-term care facilities and general hospitals, has rarely been reported in South Korea. The aims of this study were to describe the characteristics of seven carbapenemase-producing P. aeruginosa isolates recovered from two long-term care facilities in South Korea. The carbapenemase genes were identified by PCR and sequencing. Strain typing was assessed by pulsed field gel electrophoresis and multilocus sequence typing (MLST) analysis. Isolates with a genomic island and class I integron surrounding bla GES-type were confirmed by the PCR mapping method. Of seven GES-type carbapenemase-producing P. aeruginosa isolates, the bla GES-24 gene was detected in six isolates, and the bla GES-5 gene was detected in one isolate. The epidemiological relatedness of the seven isolates carrying bla GES-24 and bla GES-5 showed >81% similarity. Five isolates carrying bla GES-24 were sequence type 155 (ST155) by MLST, followed by one ST244 isolate carrying bla GES-24 and one ST308 isolate carrying bla GES-5 . bla GES-type genes were embedded in two different class I integrons in a genomic island-15-like region. Our results indicate the possible spread of carbapenemase-producing P. aeruginosa and present a current threat of antimicrobial resistance in community hospitals.

Stress relaxation is an important design parameter of biomaterials that can provide an artificial microenvironment mimicking natural extracellular matrix (ECM). Here, we report a novel hydrogel platform based on sodium alginate (NaAlg) with tunable stress relaxation. We first developed a new synthesis route to introduce alkoxyamine functional groups into the alginate polymer backbone. By mixing the resulting polymer (NaAlg-AA) with aldehyde-containing oxidized alginate (NaAlg-Ald), oxime cross-linked alginate hydrogels were prepared. We demonstrate that highly tunable stress relaxation and mechanical properties can be achieved by systematically varying the composition (concentration, polymer mixing ratios, degree of oxidation of NaAlg-Ald) or environmental factors (pH, temperature, and use of catalyst). Combined with the natural capability of the alginate to be cross-linked by divalent cations, the developed hydrogel formations possess the unique capability of dual cross-linking mechanisms with different gelation kinetics. We demonstrated that this dual cross-linking capability can (i) be utilized for the creation of hydrogels in microbead or microthread geometries and (ii) be useful for biomedical applications that require both the fast encapsulation of cells in hydrogels (fast calcium cross-linking) and the provision of controlled viscoelastic environments to cultured cells for an extended period (durable oxime cross-linking). With biocompatibility confirmed by the culture of a B-cell line encapsulated within the developed hydrogel, this novel hydrogel platform provides a good prospect in various applications where stress relaxation plays a key role in cell-matrix interactions.

Background: Pigment epithelium-derived factor (PEDF)-derived 34-mer peptide (PEDF34, Asp 44 -Asn 77 ) has anti-angiogenic activity but has limitations in clinical application because of an inverted bell-shaped dose-effect relationship and a short half-life. In this study, we attempted to mitigate these problems by mixing PEDF34 with type I collagen., Methods: The anti-angiogenic activity of the PEDF34/atelocollagen mixture was evaluated by HUVEC tube formation assay and in a laser-induced choroidal neovascular (CNV) mouse model. PEDF34 and/or collagen were administrated using intravitreal injections or eye drops. CNV lesion size was quantified using FITC-dextran-perfused retinal whole mounts. Western blot analysis and inhibitor assays were used to define the action mechanisms of PEDF34 and the mixture., Results: Collagen broadened the effective dose range of PEDF34 in the tube formation assay by > 250 times (from 0.2 to 50 nM). In the CNV model, five intravitreal injections of PEDF34 were required for therapeutic effect, whereas the mixture had a significant therapeutic effect following a single injection. Eye drops of the mixture showed significantly stronger CNV-suppressive effects than drops of PEDF34 alone. The anti-angiogenic activity of PEDF34 might be mediated by inhibition of ERK and JNK activation by VEGF, and collagen potentiated these effects., Conclusions: Collagen can serve as a carrier and reservoir of PEDF34. PEDF peptide/collagen mixture is easy to prepare than conventional methods for maintaining the therapeutic effect of PEDF peptide.

The nuclear factor (NF)-κB pathway plays a central role in inflammatory and immune responses, with aberrant activation of NF-κB signaling being implicated in various human disorders. Here, we show that mammalian ste20-like kinase 1 (MST1) is a previously unrecognized component of the tumor necrosis factor α (TNFα) receptor 1 signaling complex (TNF-RSC) and attenuates TNFα-induced NF-κB signaling. Genetic ablation of MST1 in mouse embryonic fibroblasts and bone marrow-derived macrophages potentiated the TNFα-induced increase in IκB kinase (IKK) activity, as well as the expression of NF-κB target genes. TNFα induced the recruitment of MST1 to TNF-RSC and its interaction with HOIP, the catalytic component of the E3 ligase linear ubiquitin assembly complex (LUBAC). Furthermore, MST1 activated in response to TNFα stimulation mediates the phosphorylation of HOIP and thereby inhibited LUBAC-dependent linear ubiquitination of NEMO/IKKγ. Together, our findings suggest that MST1 negatively regulates TNFα-induced NF-κB signaling by targeting LUBAC., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The cultivation of genetically modified (GM) crops has raised many questions regarding their environmental risks, particularly about their ecological impact on non-target organisms, such as their closely-related relative species. Although evaluations of transgene flow from GM crops to their conventional crops has been conducted under large-scale farming system worldwide, in particular in North America and Australia, few studies have been conducted under smallholder farming systems in Asia with diverse crops in co-existence. A two-year field study was conducted to assess the potential environmental risks of gene flow from glufosinate-ammonium resistant (GR) Brassica napus to its conventional relatives, B. napus, B. juncea, and Raphanus sativus under simulated smallholder field conditions in Korea. Herbicide resistance and simple sequence repeat (SSR) markers were used to identify the hybrids. Hybridization frequency of B. napus × GR B. napus was 2.33% at a 2 m distance, which decreased to 0.007% at 75 m. For B. juncea, it was 0.076% at 2 m and decreased to 0.025% at 16 m. No gene flow was observed to R. sativus. The log-logistic model described hybridization frequency with increasing distance from GR B. napus to B. napus and B. juncea and predicted that the effective isolation distances for 0.01% gene flow from GR B. napus to B. napus and B. juncea were 122.5 and 23.7 m, respectively. Results suggest that long-distance gene flow from GR B. napus to B. napus and B. juncea is unlikely, but gene flow can potentially occur between adjacent fields where the smallholder farming systems exist., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Pollen-mediated gene flow (PMGF) from genetically modified (GM) Brassica napus to its wild relatives by wind and insects is a major ecological concern in agricultural ecosystems. This study conducted is to estimate maximum potential gene flow and differentiate between wind- and bee-mediated gene flows from herbicide resistant (HR) B. napus to its closely-related male sterile (MS) relatives, B. napus, B. juncea and Raphanus sativus. Various markers, including pods formation in MS plants, herbicide resistance, and SSR markers, were used to identify the hybrids. Our results revealed the following: 1) maximum potential gene flow (a maximum % of the progeny of pollen recipient confirmed hybrid) to MS B. napus ranged from 32.48 to 0.30% and from 14.69 to 0.26% at 2-128 m from HR B. napus under open and wind pollination conditions, respectively, and to MS B. juncea ranged from 21.95 to 0.24% and from 6.16 to 0.16%, respectively; 2) estimates of honeybee-mediated gene flow decreased with increasing distance from HR B. napus and ranged from 17.78 to 0.03% at 2-128 m for MS B. napus and from 15.33 to 0.08% for MS B. juncea; 3) a small-scale donor plots would strongly favour insect over wind pollination; 4) no gene flow occurred from HR B. napus to MS R. sativus. Our approach and findings are helpful in understanding the relative contribution of wind and bees to gene flow and useful for estimating maximum potential gene flow and managing environmental risks associated with gene flow., (Copyright © 2018. Published by Elsevier B.V.)

B cells play a major role in the adaptive immune response by producing antigen-specific antibodies against pathogens and imparting immunological memory. Following infection or vaccination, antibody-secreting B cells and memory B cells are generated in specialized regions of lymph nodes and spleens, called germinal centers. Here, we report a fully synthetic ex-vivo system that recapitulates the generation of antigen-specific germinal-center (GC) like B cells using material-surface driven polyvalent signaling. This synthetic germinal center (sGC) reaction was effectively induced using biomaterial-based artificial "follicular T helper cells (T FH )" that provided both natural CD40-CD40L ligation as well as crosslinking of CD40 and by mimicking artificial "follicular dendritic cells (FDC)" to provide efficient, polyvalent antigen presentation. The artificial sGC reaction resulted in efficient B cell expansion, immunoglobulin (Ig) class switching, and expression of germinal center phenotypes. Antigen presentation during sGC reaction selectively enhanced the antigen-specific B cell population and induced somatic hyper-mutations for potential affinity maturation. The resulting B cell population consisted primarily of GC-like B cells (centrocytes) as well as some plasma-like B cells expressing CD138. With concurrent cell sorting, we successfully created highly enriched populations of antigen-specific B cells. Adoptive transfer of these GC-like B cells into non-irradiated isogeneic or non-lethally irradiated congenic recipient mice showed successful engraftment and survival of the donor cells for the 4 week test period. We show that this material-surface driven sGC reaction can be successfully applied to not only splenic B cells but also B cells isolated from more therapeutically relevant sources such as peripheral blood mononuclear cells (PBMCs), thus making our current work an exciting prospect in the new era of personalized medicine and custom-immunotherapy., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Combination therapies with phosphoinositide 3-kinase (PI3K) inhibitors and trastuzumab (anti-human epidermal growth factor receptor [HER]2/neu antibody) are effective against HER2+ breast cancer. Isoform-selective PI3K inhibitors elicit anti-tumor immune responses that are distinct from those induced by inhibitors of class I PI3K isoforms (pan-PI3K inhibitors). The present study investigated the therapeutic effect and potential for stimulating anti-tumor immunity of combined therapy with an anti-HER2/neu antibody and pan-PI3K inhibitor (GDC-0941) or a PI3K p110α isoform-selective inhibitor (A66) in mouse models of breast cancer. The anti-neu antibody inhibited tumor growth and enhanced anti-tumor immunity in HER2/neu+ breast cancer TUBO models, whereas GDC-0941 or A66 alone did not. Anti-neu antibody and PI3K inhibitor synergistically promoted anti-tumor immunity by increasing functional T cell production. In the presence of the anti-neu antibody, A66 was more effective than GDC-0941 at increasing the fraction of CD4 + , CD8 + , and IFN-γ + CD8 + T cells in the tumor-infiltrating lymphocyte population. Detection of IFN-γ levels by enzyme-linked immunospot assay showed that the numbers of tumor-specific T cells against neu and non-neu tumor antigens were increased by combined PI3K inhibitor plus anti-neu antibody treatment, with A66 exhibiting more potent effects than GDC-0941. In a TUBO (neu+) and TUBO-P2J (neu-) mixed tumor model representing immunohistochemistry 2+ tumors, A66 suppressed tumor growth and prolonged survival to a greater extent than GDC-0941 when combined with anti-neu antibody. These results demonstrate that a PI3K p110α-isoform-selective inhibitor is an effective adjunct to trastuzumab in the treatment of HER2-positive breast cancer.

Antigen-specific immunity conferred by T lymphocytes is a result of complex molecular interactions at the immunological synapse. A variety of biomimetic approaches have been devised to artificially induce T cell activation either to study the T cell biology or to expand and prime the therapeutic T cell populations. Here we first briefly review the molecular and cellular, structural and phenotypical bases that are involved in T cell activation. The artificial methods for T cell activation are then discussed in two grand categories, the soluble (3D) and the surface-anchored (2D) platforms with their design parameters. With the growing number of successful adoptive T cell therapies, the spurring demands for effective and safe T cell expansion as well as precise control over resulting T cell functions and phenotypes warrant the extensions of engineering parameters in the development of novel methodologies for T cell activation.

LC3-family member proteins play a critical role in autophagy, a cellular process responsible for the degradation of massive cellular components including intracellular pathogens. A variety of molecules involved in the autophagic pathway engage in specific interactions with a unique sequence motif referred to as the LIR (LC3-interacting region) motif. Although identification of conserved structural features of LIR motifs in complex with LC3-family members has established a canonical LIR motif, atypical conformations of LIR motifs have recently been revealed. Here, we determined the three-dimensional crystal structures of LC3B in complex with three different LIR motifs of RavZ from Legionella pneumophila, an intracellular pathogen that can manipulate the host autophagy system. The tandem LIR motifs located in the N-terminal region of RavZ adopt a novel β-sheet conformation and thus provide specific ionic interactions with LC3B in addition to canonical hydrophobic plugged-in interactions. Consequently, these motifs possess higher binding affinity to LC3-family members than canonical LIR motifs, although the tandem repeats can only bind to one LC3 molecule. These findings broaden our understanding of the functional repertoire of LIR motifs in autophagy., (Copyright © 2017 Elsevier Inc. All rights reserved.)

MST1 deficiency causes T and B cell lymphopenia, resulting in combined immunodeficiency. However, MST1-deficient patients also exhibit autoimmune-like symptoms such as hypergammaglobulinemia and autoantibody production. Recent studies have shown that the autoimmune responses observed in MST1-deficient patients were most likely attributable to defective regulatory T (Treg) cells instead of intrinsic signals in MST1-lacking B cells. Nevertheless, it is not determined how MST1 deficiency in T cells breaks B cell tolerance and causes systemic autoimmune-like phenotypes. In this study, we confirmed that Mst1 -/- mice developed hypergammaglobulinemia associated with increased levels of IgG, IgA, and IgE. We also showed that uncontrolled B cell responses were resulted from the IL-4-rich environment created by CD4 + T cells. Defective MST1-FOXO1 signaling down-regulated Treg cells, resulting in the collapse of immune tolerance where the populations of Th2 and T follicular helper cells expanded. In conclusion, we suggest that MST1 acts as a molecular brake to maintain immune tolerance by regulating T cell-mediated B cell activation., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: In contrast to the significant advantages of laparoscopic versus open gastrectomy, robotic gastrectomy has shown little benefit over laparoscopic gastrectomy. This study aimed to compare multi-dimensional aspects of surgical outcomes after open, laparoscopic, and robotic gastrectomy., Methods: Data from 915 gastric cancer patients who underwent gastrectomy by one surgeon between March 2009 and May 2015 were retrospectively reviewed. Perioperative parameters were analyzed for short-term outcomes. Surgical success was defined as the absence of conversion to open surgery, major complications, readmission, positive resection margin, or fewer than 16 retrieved lymph nodes., Results: This study investigated 241 patients undergoing open gastrectomy, 511 patients undergoing laparoscopic gastrectomy, and 173 patients undergoing robotic gastrectomy. For each approach, the respective incidences were as follows: conversion to open surgery (not applicable, 0.4%, and 0%; p = 0.444), in-hospital major complications (5.8, 2.7, and 1.2%; p = 0.020), delayed complications requiring readmission (2.9, 2.0, and 1.2%; p = 0.453), positive resection margin (1.7, 0, and 0%; p = 0.003), and inadequate number of retrieved lymph nodes (0.4, 4.1, and 1.7%; p = 0.010). Compared with open and laparoscopic surgery, robotic gastrectomy had the highest surgical success rate (90, 90.8, and 96.0%). Learning-curve analysis of success using cumulative sum plots showed success with the robotic approach from the start. Multivariate analyses identified age, sex, and gastrectomy extent as significant independent parameters affecting surgical success. Surgical approach was not a contributing factor., Conclusions: Open, laparoscopic, and robotic gastrectomy exhibited different incidences and causes of surgical failure. Robotic gastrectomy produced the best surgical outcomes, although the approach method itself was not an independent factor for success.

Post-transcriptional gene regulation is an important step in eukaryotic gene expression. The last step to govern production of nascent peptides is during the process of mRNA translation. mRNA translation is controlled by many translation initiation factors that are susceptible to post-translational modifications. Here we report that one of the translation initiation factors, eIF4E, is phosphorylated by Mammalian Ste20-like kinase (MST1). Upon phosphorylation, eIF4E weakly interacts with the 5' CAP to inhibit mRNA translation. Simultaneously, active polyribosome is more associated with long noncoding RNAs (lncRNAs). Moreover, the linc00689-derived micropeptide, STORM (Stress- and TNF-α-activated ORF Micropeptide), is triggered by TNF-α-induced and MST1-mediated eIF4E phosphorylation, which exhibits molecular mimicry of SRP19 and, thus, competes for 7SL RNA. Our findings have uncovered a novel function of MST1 in mRNA and lncRNA translation by direct phosphorylation of eIF4E. This novel signaling pathway will provide new platforms for regulation of mRNA translation via post-translational protein modification., (Published by Elsevier B.V.)

Hosts utilize macroautophagy/autophagy to clear invading bacteria; however, bacteria have also developed a specific mechanism to survive by manipulating the host cell autophagy mechanism. One pathogen, Legionella pneumophila, can hinder host cell autophagy by using the specific effector protein RavZ that cleaves phosphatidylethanolamine-conjugated LC3 on the phagophore membrane. However, the detailed molecular mechanisms associated with the function of RavZ have hitherto remained unclear. Here, we report on the biochemical characteristics of the RavZ-LC3 interaction, the solution structure of the 1:2 complex between RavZ and LC3, and crystal structures of RavZ showing different conformations of the active site loop without LC3. Based on our biochemical, structural, and cell-based analyses of RavZ and LC3, both distant flexible N- and C-terminal regions containing LC3-interacting region (LIR) motifs are important for substrate recognition. These results suggest a novel mechanism of RavZ action on the phagophore membrane and lay the groundwork for understanding how bacterial pathogens can survive autophagy.

In this paper, we investigate the optimal consumption and portfolio selection problem with negative wealth constraints for an economic agent who has a quadratic utility function of consumption and receives a constant labor income. Due to the property of the quadratic utility function, we separate our problem into two cases and derive the closed-form solutions for each case. We also illustrate some numerical implications of the optimal consumption and portfolio.

Mesenchymal stem cells (MSCs) have been proven to be therapeutically effective against atopic dermatitis (AD) in preclinical studies. However, the safety and efficacy of MSCs against AD have not yet been investigated in a clinical study. To establish the safety and efficacy of human umbilical cord blood-derived MSCs (hUCB-MSCs) in AD, 34 adult patients with moderate-to-severe AD were enrolled in two phase trials with a follow-up for 1 month and 3 months, respectively. Patients were randomly allocated to receive low dose (2.5 × 10 7 ) or high dose (5.0 × 10 7 ) of hUCB-MSCs subcutaneously. An Eczema Area and Severity Index (EASI) score, Investigator's Global Assessment (IGA) score, Severity Scoring for Atopic Dermatitis (SCORAD) score, adverse effect assessments, and serum biomarker levels were evaluated as end points. A single treatment of hUCB-MSCs resulted in dose-dependent improvements in AD manifestation. Fifty-five percent of patients in high dose hUCB-MSC-treated group showed a 50% reduction in the EASI score. The IGA score and SCORAD score decreased by 33% and 50%, respectively, in high dose-treated group. Particularly, the administration of high dose hUCB-MSCs reduced the pruritus score by 58%. The serum IgE levels and number of blood eosinophils were downregulated by the treatment. No serious adverse events occurred, and none of the patients discontinued the trial due to adverse events. This is the first report to demonstrate a marked improvement of AD features with cell therapeutics. These data suggest that the infusion of hUCB-MSCs might be an effective therapy for patients with moderate-to-severe AD. Stem Cells 2017;35:248-255., (© 2016 AlphaMed Press.)

Key Message: We described identification, expression, subcellular localization, and functions of genes that encode fatty acid desaturase enzymes in Perilla frutescens var. frutescens. Perilla (Perilla frutescens var. frutescens) seeds contain approximately 40 % of oil, of which α-linolenic acid (18:3) comprise more than 60 % in seed oil and 56 % of total fatty acids (FAs) in leaf, respectively. In perilla, endoplasmic reticulum (ER)-localized and chloroplast-localized ω-3 FA desaturase genes (PfrFAD3 and PfrFAD7, respectively) have already been reported, however, microsomal oleate 12-desaturase gene (PfrFAD2) has not yet. Here, four perilla FA desaturase genes, PfrFAD2-1, PfrFAD2-2, PfrFAD3-2 and PfrFAD7-2, were newly identified and characterized using random amplification of complementary DNA ends and sequence data from RNAseq analysis, respectively. According to the data of transcriptome and gene cloning, perilla expresses two PfrFAD2 and PfrFAD3 genes, respectively, coding for proteins that possess three histidine boxes, transmembrane domains, and an ER retrieval motif at its C-terminal, and two chloroplast-localized ω-3 FA desaturase genes, PfrFAD7-1 and PfrFAD7-2. Arabidopsis protoplasts transformed with perilla genes fused to green fluorescence protein gene demonstrated that PfrFAD2-1 and PfrFAD3-2 were localized in the ER, and PfrFAD7-1 and PfrFAD7-2 were localized in the chloroplasts. PfrFAD2 and perilla ω-3 FA desaturases were functional in budding yeast (Saccharomyces cerevisiae) indicated by the presence of 18:2 and 16:2 in yeast harboring the PfrFAD2 gene. 18:2 supplementation of yeast harboring ω-3 FA desaturase gene led to the production of 18:3. Therefore, perilla expresses two functional FAD2 and FAD3 genes, and two chloroplast-localized ω-3 FA desaturase genes, which support an evidence that P. frutescens cultivar is allotetraploid plant.

T cells play a central role in immune-homeostasis; specifically in the induction of antigen-specific adaptive immunity against pathogens and mutated self with immunological memory. The thymus is the unique organ where T cells are generated. In this review, first the complex structures and functions of various thymic microcompartments are briefly discussed to identify critical engineering targets for regeneration of thymic functions in vitro and in vivo. Then the biomimetic regenerative engineering approaches are reviewed in three categories: 1) reconstruction of 3-D thymic architecture, 2) cellular engineering, and 3) biomaterials-based artificial presentation of critical biomolecules. For each engineering approach, remaining challenges and clinical opportunities are also identified and discussed.

Stem cells and other functionally defined therapeutic cells (e.g., T cells) are promising to bring hope of a permanent cure for diseases and disorders that currently cannot be cured by conventional drugs or biological molecules. This paradigm shift in modern medicine of using cells as novel therapeutics can be realized only if suitable manufacturing technologies for large-scale, cost-effective, reproducible production of high-quality cells can be developed. Here we review the state of the art in therapeutic cell manufacturing, including cell purification and isolation, activation and differentiation, genetic modification, expansion, packaging, and preservation. We identify current challenges and discuss opportunities to overcome them such that cell therapies become highly effective, safe, and predictively reproducible while at the same time becoming affordable and widely available.

Further understanding of male human papillomavirus (HPV) infection is necessary to prevent infection in men, as well as transmission to women. In our current study, we investigated patterns of HPV infection and genotype distributions in male genital warts using the Anyplex II HPV28 Detection kit. We reviewed the medical records of 80 male patients who presented to 5 neighborhood clinics in Ulsan, Korea, for the treatment of genital warts between April 2014 and January 2015. All patients underwent HPV genotyping. The prevalence and characteristics of HPV infection were analyzed, and the patterns of HPV infection according to age were assessed. Among the study patients, 13 (16.3%) were negative for HPV infection, 46 (57.3%) were infected with low-risk HPV, and 21 (26.3%) were infected with high-risk HPV. Patients with multiple HPV infection were more likely to have high-risk HPV infection (P = 0.001). The prevalence of HPV infection was much higher in samples obtained by tissue excision due to a definite lesion (P = 0.001). There were no differences in high-risk HPV infection (P = 0.459), multiple HPV infection (P = 0.185), and recurrence at diagnosis (P = 0.178) according to age. HPV-6 and HPV-11 were the most common type overall (39.7% and 13.8%, respectively). HPV-16 and HPV-18 were the most common high-risk infections (both 3.4%). HPV infection is not only commonly encountered in male genital warts, but is also accompanied by high-risk HPV and multiple infections.

Background: Extensively drug-resistant (XDR) Pseudomonas aeruginosa and Acinetobacter baumannii are a threat to hospitalized patients. We evaluated the effects of antimicrobial combinations on XDR P. aeruginosa and A. baumannii isolates., Methods: P. aeruginosa and A. baumannii isolates, which were resistant to all antibiotics except colistin (CL), were collected from eight hospitals in Korea. Genes encoding metallo-β-lactamases (MBLs) and OXA carbapenemases were detected by PCR in eight P. aeruginosa and 30 A. baumannii isolates. In vitro synergy of antimicrobial combinations was tested by using the checkerboard method., Results: Minimum inhibitory concentrations of β-lactams, aminoglycosides, and fluoroquinolones were very high, while that of CL was low for majority of XDR P. aeruginosa and A. baumannii isolates. Antimicrobial combinations including Imipenem (IPM)-CL, ceftazidime (CAZ)-CL, and rifampin (RIF)-CL exerted only additive/indifferent effects on majority of XDR P. aeruginosa isolates. Proportions of XDR A. baumannii isolates that showed synergistic and additive/indifferent inhibition after treatment with antimicrobial combinations used are as follows: IPM-ampicillin-sulbactam (AMS), 17% and 80% isolates, respectively; IPM-rifampin (RIF), 13% and 81% isolates, respectively; IPM-CL, 13% and 87% isolates, respectively; and RIF-COL, 20% and 73% isolates, respectively. Significant proportion (19%) of XDR P. aeruginosa isolates produced MBLs, and majority (82%) of A. baumannii isolates produced either MBLs or OXA-23., Conclusions: Our results suggest that combinations of IPM-AMS, IPM-RIF, IPM-CL, and RIF-CL are more useful than individual drugs for treating 13-20% of XDR A. baumannii infections.

Reactive oxygen species (ROS) have many physiological and pathological effects on diverse cellular events. In particular, excessive ROS causes oxidative stress that leads to cell death. The mammalian STE20-like kinase-1 (MST1), a multifunctional serine-threonine kinase, plays a pivotal role in oxidative stress-induced cellular signaling events. Tumor necrosis factor receptor (TNFR)-associated factor 2 (TRAF2) is also known to be essential for oxidative stress-induced cell death. Here, we showed that H2O2 induced the physical interaction between TRAF2 and MST1, and that this interaction promoted the homodimerization as well as the activation of MST1. Furthermore, TRAF2 was required for MST1 to mediate the H2O2-induced stimulation of c-Jun N-terminal kinase and p38 kinase as well as apoptosis. Taken together, our results suggest that TRAF2 functions as a key activator of MST1 in oxidative stress-induced intracellular signaling processes., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Purpose: The a-disintegrin-and-metalloprotease (ADAM) family proteins are widely expressed in the different layers of the retina throughout development. The effect of ADAM proteins on the epithelial-to-mesenchymal transition (EMT) in proliferative vitreoretinopathy (PVR) or AMD is yet to be elucidated. In this study we used Epstein-Barr virus (EBV)-transformed adult retinal pigment epithelial (ARPE) cells to investigate how sorafenib, a multikinase inhibitor, modulates ADAM proteins to control EMT., Methods: Epithelial to mesenchymal transition and related mechanisms in EBV-infected ARPE cells were determined by RT-PCR, Western blot, invasion assay, ELISA assay, and gene silencing with siRNA., Results: Mesenchymal-like ARPE/EBV cells exhibited considerably increased cellular migration and invasion compared with ARPE cells and produced EMT-related cytokines. Sorafenib significantly inhibited production of TGF-β1, VEGF, IL-6, IL-8, MCP-1, and TNF-α and blocked the activation of migration-related signaling molecules, such as HIF-1α, p-STAT3, MMP2, and Ang-1. The expression of mature ADAM10, ADAM17, and cleaved Notch 1 proteins in ARPE/EBV cells was downregulated after treatment with sorafenib through the regulatory activity of nardilysin (NRD-1). Gene silencing of NRD-1 in ARPE/EBV cells attenuated secretion of EMT-related cytokines and expression of ADAM10 and 17 and upregulated epithelial markers., Conclusions: Sorafenib controls the mesenchymal characteristics of EBV-infected ARPE cells. Nardilysin and ADAM family proteins might be new targets for the prevention or control of EMT in retinal diseases.