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We investigated whether the severity of anxiety among children undergoing endoscopy could affect sedation. We prospectively recruited patients under 18 years of age who were scheduled to undergo esophagogastroduodenoscopy (EGD). Baseline anxiety was evaluated using the Spence Children’s Anxiety Scale (SCAS). Our analysis considered the type and dose of sedative drugs, degree of sedation, and complications associated with sedation according to the scale score. The mean total SCAS T-scores and each subscale score were significantly higher in the 6–12-year age group. The mean T-score among patients who exhibited irritability during EGD was higher than that among other patients, with significant differences in the Total, Obsessive Disorder, Panic Agoraphobia and General Anxiety subscales. The midazolam doses of children exhibiting irritability were more likely to need higher ( p = 0.006). Other sedation-related complications were not associated with the T-scores in each subscale. Total sleep time was not associated with any T-score subscales. Anxiety levels were significantly higher among 6–12-year-olds and children exhibiting irritability. Anxiety was often associated with irritability, difficulties achieving adequate sedation during EGD, and additional sedative drug administration. We recommend higher doses or more potent drugs to facilitate endoscopy for children with high anxiety levels.

Heterologous protein production in Saccharomyces cerevisiae is a useful and effective strategy with many advantages, including the secretion of proteins that require posttranslational processing. However, heterologous proteins in S. cerevisiae are often secreted at comparatively low levels. To improve the production of the heterologous protein, human granulocyte colony-stimulating factor (hG-CSF) in S. cerevisiae, a secretion-enhancing peptide cassette including an hIL-1β-derived pro-peptide, was added and used as a secretion enhancer to alleviate specific bottlenecks in the yeast secretory pathway. The effects of three key parameters-N-glycosylation, net negative charge balance, and glycine-rich flexible linker-were investigated in batch cultures of S. cerevisiae. Using a three-stage design involving screening, selection, and optimization, the production and secretion of hG-CSF by S. cerevisiae were significantly increased. The amount of extracellular mature hG-CSF produced by the optimized pro-peptide after the final stage increased by 190% compared to that of the original pro-peptide. Although hG-CSF was used as the model protein in the current study, this strategy is applicable to the enhanced production of other heterologous proteins, using S. cerevisiae as the host.

Proteins are fundamentally the most important macromolecules for biochemical, mechanical, and structural functions in living organisms. Therefore, they provide us with diverse structural building blocks for constructing various types of biomaterials, including an important class of such materials, hydrogels. Since natural peptides and proteins are biocompatible and biodegradable, they have features advantageous for their use as the building blocks of hydrogels for biomedical applications. They display constitutional and mechanical similarities with the native extracellular matrix (ECM), and can be easily bio-functionalized via genetic and chemical engineering with features such as bio-recognition, specific stimulus-reactivity, and controlled degradation. This review aims to give an overview of hydrogels made up of recombinant proteins or synthetic peptides as the structural elements building the polymer network. A wide variety of hydrogels composed of protein or peptide building blocks with different origins and compositions - including β-hairpin peptides, α-helical coiled coil peptides, elastin-like peptides, silk fibroin, and resilin - have been designed to date. In this review, the structures and characteristics of these natural proteins and peptides, with each of their gelation mechanisms, and the physical, chemical, and mechanical properties as well as biocompatibility of the resulting hydrogels are described. In addition, this review discusses the potential of using protein- or peptide-based hydrogels in the field of biomedical sciences, especially tissue engineering.

As recent advancements in the chimeric antigen receptor-T cells have revolutionized the way blood cancers are handled, potential benefits from producing off-the-shelf, standardized immune cells entail the need for development of allogeneic immune cell therapy. However, host rejection driven by HLA disparity in adoptively transferred allogeneic T cells remains a key obstacle to the universal donor T cell therapy. To evade donor HLA-mediated immune rejection, we attempted to eliminate T cell’s HLA through the CRISPR/Cas9 gene editing system. First, we screened 60 gRNAs targeting B2M and multiple sets of gRNA each targeting α chains of HLA-II (DPA, DQA and DRA, respectively) using web-based design tools, and identified specific gRNA sequences highly efficient for target deletion without carrying off-target effects. Multiplex genome editing of primary human T cells achieved by the newly discovered gRNAs yielded HLA-I- or HLA-I/II-deficient T cells that were phenotypically unaltered and functionally intact. The overnight mixed lymphocyte reactions demonstrated the HLA-I-negative cells induced decreased production of IFN-γ and TNF-α in alloreactive T cells, and deficiency of HLA-I/II in T cells further dampened the inflammatory responses. Taken together, our approach will provide an efficacious pathway toward the universal donor cell generation by manipulating HLA expression in therapeutic T cells.

TIGIT is an immune checkpoint receptor that is expressed on subsets of activated T cells and natural killer (NK) cells. Several ligands for TIGIT, including poliovirus receptor (PVR), are expressed on cancer cells and mediate inhibitory signaling to suppress antitumor activities of the immune cells. Many studies support that the TIGIT signaling is a potential target for cancer immunotherapy. We developed an IgG4-type monoclonal antibody against human TIGIT, designated as MG1131, using a phage display library of single-chain variable fragments (scFvs). MG1131 interacts with TIGIT much more tightly than PVR does. The crystal structure of a scFv version of MG1131 bound to TIGIT was determined, showing that MG1131 could block the PVR-TIGIT interaction and thus the immunosuppressive signaling of TIGIT. Consistently, MG1131 is bound to TIGIT-expressing cells and interferes with PVR binding to these cells. Moreover, MG1131 increased NK cell-mediated tumor killing activities, inhibited immunosuppressive activity of regulatory T (Treg) cells from healthy donors, and restored interferon-γ secretion from peripheral blood mononuclear cells derived from multiple myeloma patients. MG1131 also increased T cell infiltration to the tumor site and inhibited tumor growth in mice. Collectively, these data indicate that MG1131 modulates the effector functions of T cells and NK cells positively and Treg cells negatively.

Since the coronavirus disease outbreak in 2019, several antibody therapeutics have been developed to treat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. Antibody therapeutics are effective in neutralizing the virus and reducing hospitalization in patients with mild and moderate infections. These therapeutics target the spike protein of SARS-CoV-2; however, emerging mutations in this protein reduce their efficiency. In this study, we developed a universal SARS-CoV-2 neutralizing antibody. We generated a humanized monoclonal antibody, MG1141A, against the receptor-binding domain of the spike protein through traditional mouse immunization. We confirmed that MG1141A could effectively neutralize live viruses, with an EC50 of 92 pM, and that it exhibited effective Fc-mediated functions. Additionally, it retained its neutralizing activity against the alpha (UK), beta (South Africa), and gamma (Brazil) variants of SARS-CoV-2. Taken together, our study contributes to the development of a novel antibody therapeutic approach, which can effectively combat emerging SARS-CoV-2 mutations.

Cancer targeting nanoparticles have been extensively studied, but stable and applicable agents have yet to be developed. Here, we report stable nanoparticles based on hepatitis B core antigen (HBcAg) for cancer therapy. HBcAg monomers assemble into spherical capsids of 180 or 240 subunits. HBcAg was engineered to present an affibody for binding to human epidermal growth factor receptor 1 (EGFR) and to present histidine and tyrosine tags for binding to gold ions. The HBcAg engineered to present affibody and tags (HAF) bound specifically to EGFR and exterminated the EGFR-overexpressing adenocarcinomas under alternating magnetic field (AMF) after binding with gold ions. Using cryogenic electron microscopy (cryo-EM), we obtained the molecular structures of recombinant HAF and found that the overall structure of HAF was the same as that of HBcAg, except with the affibody on the spike. Therefore, HAF is viable for cancer therapy with the advantage of maintaining a stable capsid form. If the affibody in HAF is replaced with a specific sequence to bind to another targetable disease protein, the nanoparticles can be used for drug development over a wide spectrum.

Background Despite heterogeneity of older people in suicidal behavior, research identifying characteristics by age groups is scarce. We examined baseline features of older community-dwelling suicidal ideators by dichotomized age groups and the 6-month trajectory of their suicidal ideation along with its related psychopathology. Predictors of suicidal ideation within each group were investigated. Methods Older community-dwelling suicidal ideators enrolled in the Korean Cohort for the Model Predicting a Suicide and Suicide-related Behavior study were subdivided into the “young-old (65–74 years)” and “old-old (≥75 years)” group. Baseline, 1-, and 6-month assessments were compared. Within each group, multiple regression analysis using rating scales (Patient Health Questionnaire-9, Beck Anxiety Inventory, Alcohol Use Disorders Identification Test, Stress Questionnaire for Korean National Health and Nutrition Examination Survey-Short Form, and Social Relationships Scale) was conducted to identify predictors of suicidal ideation measured with the intensity subscale of the Columbia-Suicide Severity Rating Scale. Two-way repeated-measures analysis of variance (RM-ANOVA) was used to compare changes in suicidal ideation, depression, anxiety between age groups over time, and one-way RM-ANOVA to examine changes within each age group. Results Among 29 “young-old” and 53 “old-old” ideators, the latter were less likely to be receiving psychiatric treatment (odds ratio [OR] = 4.065) and make suicide attempts (OR = 2.874), whereas the former revealed greater levels of anxiety and stress. Baseline depression and stress in the “young-old” group and the “old-old” group, respectively, predicted the intensity of suicidal ideation at both baseline and 1-month assessments. No significant age group x time interactions on suicidal ideation and depression were found. However, within each age group, both suicidal ideation and depression significantly decreased only during the first month with no further improvement. Conclusion We speculate cautiously that more attention may need to be paid to the “old-old” ideators in the evaluation of psychiatric issues and for referral to psychiatrists. To decrease suicidal ideation, tailored approaches involving proactive, timely management of depression in the “young-old” and interventions focusing on stress reduction in the “old-old,” would be helpful.

Background: The Korean Cohort for the Model Predicting a Suicide and Suicide-related Behavior (K-COMPASS) study is a prospective, naturalistic, observational cohort study, aiming to identify predictors of suicide attempt and suicide characteristics in the Korean suicidal population. The findings intend to contribute to a thorough understanding of suicidal phenomena and development of suicide prevention guidelines. The present cross-section study examines the study rationale, methodology, and baseline characteristics of the participants. Methods: Participants were enrolled via the hospital and community gateways, establishing the hospital-based cohort (HC) and community-based cohort (CC), respectively. Baseline assessment was conducted on sociodemographic, clinical, diagnostic, and psychopathological aspects. The Columbia-Suicide Severity Rating Scale was used to investigate suicidality. Results: A total of 800 suicidal people aged 15 years or older were enrolled from 8 university hospitals and 8 community mental health welfare centers (CMHWCs), among whom 480 (60%) were suicidal ideators and 320 (40%) were attempters. The ideators comprised 207 CC and 273 HC participants, whereas the attempters, 34 CC and 286 HC participants. Despite their lower severity in some measures, including suicidal ideation, compared with their HC counterparts, the CC participants within each group of ideators or attempters presented clinically significant psychopathology. Moreover, alcohol use problems and past suicide attempt were more likely to be found in CC participants. Only 11.1% to 21.6% of the participants in each of the four groups (defined by the cohorts and the ideators/attempters) were on any type of psychiatric treatment. Conclusions: Suicidal visitors to CMHWCs need to be as closely monitored as suicidal patients in university hospitals, especially considering their association with problem drinking and past suicide attempt. A cautious assumption is that the high suicide rate in Korea might be partly attributable to the low proportion of patients receiving psychiatric services. Keywords: Suicide, Suicide attempt, Cohort studies, Hospitals, Community mental health centers, Korea

Background Evidence continues to accumulate that the presence or absence of early trauma (ET) implies unique characteristics in the relationships between suicidal ideation and its risk factors. We examined the relationships among recent stress, depressive symptoms, anxiety symptoms, and suicidal ideation in Korean suicidal women with or without such a history. Methods Using data on suicidal adult females, 217 victims and 134 non-victims of ET, from the Korean Cohort for the Model Predicting a Suicide and Suicide-related Behavior, we performed structural equation modeling to investigate the contribution of recent stress, depressive symptoms, and anxiety symptoms on suicidal ideation within each group according to the presence or absence of a history of ET. Results Structural equation modeling with anxiety and depressive symptoms as potential mediators showed a good fit. Recent stress had a direct effect on both depressive symptoms and anxiety symptoms in both groups. Only anxiety symptoms for victims of ET (standardized regression weight, 0.281; P = 0.005) and depressive symptoms for non-victims of ET (standardized regression weight, 0.326; P = 0.003) were full mediators that increased suicidal ideation. Thus, stress contributed to suicidal ideation by increasing the level of anxiety and depressive symptoms for victims and non-victims, respectively. Conclusion Tailored strategies to reduce suicidal ideation should be implemented according to group type, victims or non-victims of ET. Beyond educating suicidal women in stress-management techniques, it would be effective to decrease anxiety symptoms for those with a history of ET and decrease depressive symptoms for those without such a history., Graphical Abstract

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Few studies have compared the three suicidality groups-suicidal ideators (SIs), single suicide attempters (SSAs), and multiple suicide attempters (MSAs)-in relation to the suicidal process. This cross-sectional study investigated trends and differences in suicide risk factors across suicidality groups. Using the baseline data of the Korean Cohort for the Model Predicting a Suicide and Suicide-related Behavior, we analyzed trends (Jonckheere-Terpstra or Mantel-Haenszel χ2 test) and differences (analysis of covariance or logistic regression) in sociodemographic and clinical factors, psychiatric diagnoses, as well as clinical rating scores on psychopathology (suicidal ideation, depressive symptoms, anxiety symptoms, and problem drinking), trait impulsiveness, and stress across suicidality groups. Across suicidality groups comprising 193 SIs, 207 SSAs, and 376 MSAs, we observed a decreasing trend in age and increasing trends in history of early trauma, familial histories of suicide attempts and suicide, most diagnoses and psychopathologies (suicidal ideation, anxiety symptoms, and problem drinking), trait impulsiveness, and stress-with MSAs more likely to have histories of early trauma and familial suicide, almost uniformly higher proportions of diagnoses, and higher psychopathology rating scores. Overall, increasing trends in suicide risk factors were found across all suicidality groups. Notably, MSAs presented greater proportions of most psychiatric diagnoses and higher degrees of most psychopathologies, motor impulsiveness, and stress, indicating they were at more severe clinical states and were closer to suicide. Mental health professionals should ascertain the number of suicide attempts to identify MSAs, implement more thorough evaluations, and employ additional measures for reducing motor impulsiveness.

This study developed a new method for forensic saliva identification using three oral bacteria, Streptococcus salivarius, Streptococcus sanguinis, and Neisseria subflava, combined with a real-time polymerase chain reaction (RT-PCR) system we called OB mRT-PCR. Analytical sensitivity results showed that the target bacteria were amplified at 102–107 copies/reaction, and analytical specificity was assessed using 24 other viruses, bacteria, and protozoa. To evaluate the OB mRT-PCR kit for forensic applications, saliva from 140 Korean individuals was tested, and at least two target bacteria were detected in all the samples. Additional studies on non-saliva samples demonstrated the specificity of the kit. Comparison of the kit with two conventional saliva test methods, the SALIgAE and RSID-Saliva assays, indicated that it was more sensitive and applicable to saliva samples in long-term storage (up to 14 weeks). Additionally, through amplification of mock forensic items and old DNA samples (isolated without lysis of the bacterial cells, regardless of their Gram-positivity), we found that the kit was applicable to not only saliva swabs, but also DNA samples. We suggest that this simple RT-PCR-based experimental method is feasible for rapid on-site analysis, and we expect this kit to be useful for saliva detection in old forensic DNA samples.

The endocytosis-mediating performances of two types of peptide ligands, cell receptor binding peptide (CRBP) and cell membrane penetrating peptide (CMPP), were analyzed and compared using a common carrier of peptide ligands-human ferritin heavy chain (hFTH) nanoparticle. Twenty-four copies of a CMPP(human immunodeficiency virus-derived TAT peptide) and/or a CRBP (peptide ligand with strong and specific affinity for either human integrin(αv β3 ) or epidermal growth factor receptor I (EGFR) that is overexpressed on various cancer cells) were genetically presented on the surface of each hFTH nanopariticle. The quantitative level of endocytosis and intracellular localization of fluorescence dye-labeled CRBP- and CMPP-presenting nanoparticles were estimated in the in vitro cultures of integrin- and EGFR-overexpressing cancer and human dermal fibroblast cells(control). From the cancer cell cultures treated with the CMPP- and CRBP-presenting nanoparticles, it was notable that CRBPs resulted in quantitatively higher level of endocytosis than CMPP (TAT) and successfully transported the nanoparticles to the cytosol of cancer cells depending on concentration and treatment period of time, whereas TAT-mediated endocytosis localized most of the nanoparticles within endosomal vesicles under the same conditions. These novel findings provide highly useful informations to many researchers both in academia and in industry who are interested in developing anticancer drug delivery systems/carriers.

In this paper, we describe the development of a novel method to detect oral bacteria by combining direct polymerase chain reaction (direct PCR) with an immunochromatographic strip (ICS), enabling the identification of saliva in forensic samples. Direct PCR was first used to directly amplify specific oral bacterial sequences (from Streptococcus sanguinis and Streptococcus salivarius) from swab samples, circumventing the need for tedious sample preparation steps such as cell lysis and DNA extraction and purification. The resultant amplicons were then colorimetrically detected on an ICS, a much more convenient, cost-effective, and user-friendly detection method than those currently available, thereby allowing the presence or absence of the target oral bacteria to be determined with the naked eye. Moreover, the entire analysis process was performed rapidly and with ease using this combination of direct PCR amplification from swab samples and ICS-based amplicon detection. This method successfully detected S. sanguinis and S. salivarius in most of the saliva swab samples tested, and returned negative results using blood, semen, urine, and vaginal fluid swab samples. Furthermore, S. sanguinis and S. salivarius were detected in a large number of mock forensic samples using this technique, which suggests that direct PCR and ICS-based detection of oral bacteria is sufficient to demonstrate the presence of saliva. Thus, we believe that the proposed method could be very useful for the identification of saliva in forensic applications.

The objective of this study is to develop an accurate, rapid, simple, and label-free assay technology that enables point-of-care diagnosis of AIDS. For this, 3-dimensional (3D) probes to sensitively detect anti-HIV antibodies were designed and synthesized by genetically presenting a HIV antigen (gp41) on the surface of engineered human ferritin nanoparticles. The 3D probes also present multi-copies of the hexa-histidine peptide (H6) on their surface to chemisorb gold ions (Au3+), which is essential for the generation and self-enhancement of assay signals. The developed new assay technology (named “one-step-immunoassay”) quickly produced clear optical signals through a simple and convenient one-step procedure. The diagnostic performance of the one-step-immunoassay was compared with that of the conventional lateral flow assay (LFA) using 30 AIDS patient and 20 healthy sera. The sensitivity of LFA was only 63% when a single antigen (gp41) was used but enhanced to 90% when three different antigens (gp41, p24, and gp120) were used together as the assay probes. In contrast, the one-step-immunoassay using only gp41 produced strong optical signals within 15 min without causing any false negative/positive signals, showing 100% sensitivity and 100% specificity and holding promising potential for clinical point-of-care diagnosis of AIDS.

Recently, a method of identifying body fluids using DNA methylation has been developed (Frumkin et al., 2011). An existing multiplex assay using 9 CpG markers could differentiate 5 body fluids: semen, blood, saliva, menstrual blood, and vaginal fluid. To validate this technique, we evaluated the previously described body fluid identification method by means of single base extension (SBE). DNA methylation was applied to 22 samples in 18 forensic cases; seven of these were semen, three were blood, eight were saliva, three were vaginal fluid, and one was menstrual blood. Total of 18 samples were tested, the DNA methylation profiles were coincident from preliminary tests (acid phosphatase (AP), leucomalachite green (LMG, Sigma Aldrich, St Louis, MO, USA) and SALIgAE®) except one sample which displayed an all-negative result. After applying the DNA methylation method to forensic samples, we determined that it could be very useful for differentiating vaginal secretions from menstrual blood, for which there is no conventional preliminary testing method.

We evaluated the clinical characteristics, cytokine/chemokine concentrations, viral shedding and antibody kinetics in 30 patients with Middle East respiratory syndrome (MERS), including 6 non-survivors admitted to 3 MERS-designated hospitals. Old age, low albumin, altered mentality and high pneumonia severity index score at admission were risk factors for mortality. In addition, severe signs of inflammation at initial presentation (at hospital days 1-4), such as high inducible protein-10 (p=0.0013), monocyte chemoattractant protein-1 (p=0.0007) and interleukin 6 (p=0.0007) concentrations, and poor viral control (high viral load at hospital days 5–10, p

The purpose of the present double-blind randomized placebo-controlled clinical study was to investigate the effects of Passionflower on polysomnographic sleep parameters in subjects with insomnia disorder. A total number 110 adult participants (mean age = 40.47 ± 11.68, Female = 53.6%) met the inclusion criteria of insomnia disorder according to the DSM-5 (Diagnostic and Statistical Manual of Mental Disorders). After randomization, patients received either the Passionflower extract or the placebo for 2 weeks. Patients underwent an overnight polysomnography and completed sleep diaries, Insomnia Severity Index, and Pittsburgh Sleep Quality Index. Within group comparisons were analyzed with paired t-tests or Wilcoxon's signed rank tests, and between-group comparisons were analyzed with independent t-tests or Mann-Whitney U Tests, as appropriate. Total sleep time (TST) was significantly increased in the Passionflower group compared with placebo (Passionflower vs placebo, 23.05 ± 54.26 vs -0.16 ± 53.12; P = 0.049). Sleep efficiency and wake after sleep onset (WASO) significantly improved after 2 weeks in the Passionflower group but there was no difference compared with the placebo group. The current study demonstrated the positive effects of Passionflower on objective sleep parameters including TST on polysomnography in adults with insomnia disorder. Further study is needed to investigate the clinical efficacy of Passionflower on insomnia.

Regulatory T cells (Treg) are enriched in the tumor microenvironment (TME) and suppress antitumor immunity; however, the molecular mechanism underlying the accumulation of Tregs in the TME is poorly understood. In various tumor models, tumor-infiltrating Tregs were highly enriched in the TME and had significantly higher expression of immune checkpoint molecules. To characterize tumor-infiltrating Tregs, we performed bulk RNA sequencing (RNA-seq) and found that proliferation-related genes, immune suppression–related genes, and cytokine/chemokine receptor genes were upregulated in tumor-infiltrating Tregs compared with tumor-infiltrating CD4+Foxp3− conventional T cells or splenic Tregs from the same tumor-bearing mice. Single-cell RNA-seq and T-cell receptor sequencing also revealed active proliferation of tumor infiltrating Tregs by clonal expansion. One of these genes, ST2, an IL33 receptor, was identified as a potential factor driving Treg accumulation in the TME. Indeed, IL33-directed ST2 signaling induced the preferential proliferation of tumor-infiltrating Tregs and enhanced tumor progression, whereas genetic deletion of ST2 in Tregs limited their TME accumulation and delayed tumor growth. These data demonstrated the IL33/ST2 axis in Tregs as one of the critical pathways for the preferential accumulation of Tregs in the TME and suggests that the IL33/ST2 axis may be a potential therapeutic target for cancer immunotherapy.

Beta amyloid (Aβ) deposition is a pathological milestone of Alzheimer's disease (AD). This is facilitated by an isoform of Apolipoprotein E4 (ApoE4), which is a dominant risk factor for AD. However, current in vitro Aβ aggregation assays were performed in extreme conditions not linked to physiological conditions, to understand the mechanism of Aβ induced neurotoxicity. Here, we present a simple method for the ApoE4-mediated Aβ aggregation at physiological conditions using single gold nanoparticle based on localized surface plasmon resonance (LSPR). It can be directly observed by dark-field microscope or even by the naked eye. Following LSPR principles, we used ApoE4 inducing Aβ42 self-assemblies on gold nanoparticles (AuNPs) surface via their surface charge interaction. Using physiologically mimic cerebrospinal fluid, we determined a detection limit of 1.5 pM for Aβ42 corresponding to the ~2.9 nm LSPR-peak shift under ApoE4. Interestingly, the result also shows that ApoE4 induces the aggregation of Aβ42 more specifically and rapidly than that of Aβ40. This is the first biomimetic platform for real-time detection of Aβ aggregation, mimicking biological conditions, which can be used to investigate AD directly.

Albumin has been used as a popular material for carrying imaging probes and/or drugs to provide efficient biomedical imaging and therapy. It is considered as an ideal material for in vivo applications, because albumin is naturally abundant in serum to show non-toxicity and non-immunogenicity. In addition, based on the convenience of chemical modifications, it is widely used for the delivery of diverse molecules including chemicals, proteins/peptides, and oligonucleotides. Albumin nanoparticles carrying these molecules have shown improved pharmacokinetic properties by providing longer circulation time and more disease-specific accumulation, and they are emerging as a promising carrier system for in vivo imaging and therapy. Constant efforts to improve the properties of albumin nanoparticles have led to a great progress in medical application, and recent examples of market approval and success are brightening the prospect of albumin-based nanocarrier formulations in the future. This article will summarize the developments in albumin nanocarriers for biomedical imaging and targeted drug delivery. This review will give an account of the different applications of albumin carriers with examples of some recent innovative works.

Aim The immune system may play an important role in the pathogenesis of cardiovascular disease. T cell-driven inflammation in human hypertension and atherosclerosis has recently been revealed. In the present study, we evaluated the association between serum levels of the C-X-C chemokine receptor type 3 chemokines and the carotid intima media thickness (IMT) in humans. Methods One hundred sixty-four consecutive patients undergoing baseline and 2-year follow-up carotid IMT (110 men, 62.4±10.0 years) were enrolled. The maximum carotid IMT (max-IMT) and the mean carotid IMT (mean-IMT) were measured at baseline and after 24 months. Clinical and laboratory variables, including serum levels of the monokine induced by gamma interferon (MIG) and interferon gamma-induced protein 10 (IP-10), were analyzed at the time of initial enrollment. Results The baseline max- and mean-IMT were 0.992±0.235 and 0.793±0.191 mm, respectively. The serum levels of MIG and IP-10 significantly correlated with the carotid IMT. However, there was no significant correlation between the serum levels of MIG or IP-10 and IMT changes. A multivariate regression analysis revealed the serum MIG to be independently associated with the carotid IMT (max-IMT: β=0.194, p=0.010; mean-IMT: β=0.184, p=0.016) when controlled for age, sex, diabetes mellitus history, smoking history, body mass index, blood pressure, total cholesterol, high-density lipoprotein cholesterol, high-sensitivity C-reactive protein, and aspirin and statin medication. Conclusions Circulating MIG levels are independently associated with the carotid IMT, after adjusting for confounding factors and medications. These findings indicate the potential clinical implication of MIG with respect to early atherosclerosis in humans.

Previously, an age-predictive method based on DNA-methylation patterns in semen was developed, using three CpG sites (cg06304190 in the TTC7B gene, cg12837463, and cg06979108 in the NOX4 gene). Before considering the routine use of a new method in forensics, validation studies such as concordance and sensitivity tests are essential for obtaining expanded and more reliable forensic information. Here, we evaluated a previously described age-predictive method for semen for routine forensic use. Concordance testing showed a high correlation between the predicted and chronological age, with a mean absolute deviation from the chronological age of 4.8 years. Sensitivity testing suggested that age prediction with reliable accuracy and consistency was possible with >5 ng of bisulfite-converted DNA. We also confirmed the applicability of the age-predictive method in forensic casework, using forensic samples. Thus, the proposed method could serve as a very valuable forensics tool for accurate age prediction with semen samples.

Current immunoassays are in general performed through time-consuming multi-step procedures that depend on the use of premade signal-producing reporters and often cause assay inaccuracy. Here we report an advanced immunoassay technology that resolves the delayed, complex, and inaccurate assay problems of conventional immunoassays. We have developed an accurate, rapid, simple, and label-free one-step-immunoassay based on the self-enhancement of sensitive immunoassay signals in an assay solution. The nano-scale protein particles (hepatitis B virus capsid and human ferritin heavy chain particles) were genetically engineered to present many well-oriented antibody (or antigen) probes and multi-copies of poly-histidine peptides on their surface, resulting in the construction of 3-dimensional (3D) bioprobes that chemisorb gold ions via coordination bonding and sensitively detect both antigen and antibody analytes. Systematic numerical and experimental analyses show that the signal self-enhancement happens through two coupled reactions under reducing conditions: (1) 3D bioprobe-based sensitive immuno-detection of analytes and (2) coordinated assembly of free and chemisorbed gold nanoparticles around the 3D bioprobe-analyte-associated complexes, which is followed by the quick generation of apparent optical signals. This advanced one-step-immunoassay was successfully applied to diagnostic assays requiring high accuracy and/or speed, i.e. diagnosis of acute myocardial infarction and hepatitis C through detecting a cardiac protein (troponin I) and anti-hepatitis C virus antibodies in patient sera, indicating that it is applicable to the accurate and rapid detection of both antigen and antibody markers of a wide range of diseases.

DNA methylation has important biological roles, such as gene expression regulation, as well as practical applications in forensics, such as in body fluid identification and age estimation. DNA methylation often occurs in the CpG site, and methylation within the CpG islands affects various cellular functions and is related to tissue-specific identification. Several programs have been developed to identify CpG islands; however, the size, location, and number of predicted CpG islands are not identical due to different search algorithms. In addition, they only provide structural information for predicted CpG islands without experimental information, such as primer design. We developed an analysis pipeline package, CpGPNP, to integrate CpG island prediction and primer design. CpGPNP predicts CpG islands more accurately and sensitively than other programs, and designs primers easily based on the predicted CpG island locations. The primer design function included standard, bisulfite, and methylation-specific PCR to identify the methylation of particular CpG sites. In this study, we performed CpG island prediction on all chromosomes and compared CpG island search performance of CpGPNP with other CpG island prediction programs. In addition, we compared the position of primers designed for a specific region within the predicted CpG island using other bisulfite PCR primer programs. The primers designed by CpGPNP were used to experimentally verify the amplification of the target region of markers for body fluid identification and age estimation. CpGPNP is freely available at http://forensicdna.kr/cpgpnp/.

Here we report a novel assembly structure of near-infrared plasmonic gold nanoparticles (AuNPs), possessing both photoacoustic (PA) and photothermal (PT) properties. The template for the plasmonic AuNP assembly is a bioconjugate between short double-strand DNA (sh-dsDNA) and human methyl binding domain protein 1 (MBD1). MBD1 binds to methylated cytosine-guanine dinucleotides (mCGs) within the sequence of sh-dsDNA. Hexahistidine peptides on the engineered MBD1 function as a nucleation site for AuNP synthesis, allowing the construction of hybrid conjugates, sh-dsDNA-MBD1-AuNPs (named DMAs). By varying the length of sh-dsDNA backbone and the spacer between two adjacent mCGs, we synthesized three different DMAs (DMA_5mCG, DMA_9mCG, and DMA_21mCG), among which DMA_21mCG exhibited a comparable photothermal and surprisingly a higher photoacoustic signals, compared to a plasmonic gold nanorod. Further, epidermal growth factor receptor I (EGFR)-binding peptides are genetically attached to the MBD1 of DMA_21mCG, enabling its efficient endocytosis into EGFR-overexpressing cancer cells. Notably, the denaturation of MBD1 disassembled the DMA and accordingly released the individual small AuNPs (

When used as an N-terminal fusion expression partner, the Escherichia coli stress-responsive protein, CysQ dramatically increased the cytoplasmic solubility of various aggregation-prone heterologous proteins: Pseudomonas putida cutinase (CUT), human granulocyte colony-stimulating factor (hG-CSF), human ferritin light chain (hFTN-L), arginine deiminase (ADI), human interleukin-2 (IL2), human activation induced cytidine deaminase (AID), and deletion mutant of human glutamate decarboxylase (GAD448-585). As compared with well-known fusion tags such as glutathione-S-transferase (GST) and maltose-binding protein (MBP), the performance of CysQ as solubility enhancer was evidently better than GST and was similar to or better than MBP for the seven heterologous proteins above. This is likely due to the intrinsic ability of CysQ to form its native conformation, probably promoting the binding of molecular chaperones during the folding of CysQ-fusion protein. When used as a substrate, p-nitrophenyl butyrate (PNB) was successfully hydrolyzed to p-nitrophenol by CysQ-CUT fusion mutant. Even after CysQ was removed, the solubility of hFTN-L and hG-CSF, the secondary structure of hG-CSF, and self-assembly activity of hFTN-L were successfully maintained. Conclusively, it seems that CysQ is a highly effective solubility enhancer and fusion expression partner for the production of a variety of bio-active recombinant proteins.

Background and Aims CD4 + CD25 + Foxp3 + T-regulatory (Treg) cells control immune responses and maintain immune homeostasis. However, under inflammatory conditions, Treg cells produce cytokines that promote inflammation. We investigated production of tumor necrosis factor (TNF) by Treg cells in patients with acute hepatitis A (AHA), and examined the characteristics of these cells and association with clinical factors. Methods We analyzed blood samples collected from 63 patients with AHA at the time of hospitalization (and some at later time points) and 19 healthy donors in South Korea. Liver tissues were collected from patients with fulminant AHA during liver transplantation. Peripheral blood mononuclear cells were isolated from whole blood and lymphocytes were isolated from liver tissues and analyzed by flow cytometry. Cytokine production from Treg cells (CD4 + CD25 + Foxp3 + ) was measured by immunofluorescence levels following stimulation with anti-CD3 and anti-CD28. Epigenetic stability of Treg cells was determined based on DNA methylation patterns. Phenotypes of Treg cells were analyzed by flow cytometry and an RORγt inhibitor, ML-209, was used to inhibit TNF production. Treg cell suppression assay was performed by co-culture of Treg-depleted peripheral blood mononuclear cells s and isolated Treg cells. Results A higher proportion of CD4 + CD25 + Foxp3 + Treg cells from patients with AHA compared with controls produced TNF upon stimulation with anti-CD3 and anti-CD28 (11.2% vs 2.8%). DNA methylation analysis confirmed the identity of the Treg cells. TNF-producing Treg cells had features of T-helper 17 cells, including up-regulation of RORγt, which was required for TNF production. The Treg cells had reduced suppressive functions compared with Treg cells from controls. The frequency of TNF-producing Treg cells in AHA patients' blood correlated with their serum level of alanine aminotransferase. Conclusions Treg cells from patients with AHA have altered functions compared with Treg cells from healthy individuals. Treg cells from patients with AHA produce higher levels of TNF, gain features of T-helper 17 cells, and have reduced suppressive activity. The presence of these cells is associated with severe liver injury in patients with AHA.

Acute hepatitis A caused by hepatitis A virus (HAV) infection is accompanied by severe liver injury in adult patients, and the liver injury is associated with the production of chemokines. Herein, we investigated the mechanism of how HAV infection induces the production of CXCR3 and CCR5 chemokines, such as CXCL10, CCL4 and CCL5. The production of CXCL10, CCL4 and CCL5 was markedly increased by HAV (HM-175/18f) infection in the culture of primary human hepatocytes and HepG2 cells. In particular, CXCL10 was produced in HAV-infected cells, not in neighboring uninfected cells. Moreover, these chemokines were significantly increased in the sera of acute hepatitis A patients. The production of IFN-λs was also robustly induced by HAV infection, and the blocking of secreted IFN-λs partially abrogated the production of CCL4 and CCL5 in HAV-infected cells. However, CXCL10 production was not decreased by the blocking of IFN-λs. Instead, CXCL10 production was reduced by silencing the expression of RIG-I-like receptor (RLR) signal molecules, such as mitochondrial antiviral signaling protein and interferon regulatory factor 3, in HAV-infected cells. In conclusion, HAV infection strongly induces the production of helper 1 T cell-associated chemokines, particularly CXCL10 via RLR signaling, even without secreted IFNs.

Human ferritin heavy-chain nanoparticle (hFTH) is genetically engineered to present tumor receptor-binding peptides (affibody and/or RGD-derived cyclic peptides, named 4CRGD here) on its surface. The affibody and 4CRGD specifically and strongly binds to human epidermal growth factor receptor I (EGFR) and human integrin αvβ3, respectively, which are overexpressed on various tumor cells. Through in vitro culture of EGFR-overexpressing adenocarcinoma (MDA-MB-468) and integrin-overexpressing glioblastoma cells (U87MG), it is clarified that specific interactions between receptors on tumor cells and receptor-binding peptides on engineered hFTH is critical in active tumor cell targeting. After labeling with the near-infrared fluorescence dye (Cy5.5) and intravenouse injection into MDA-MB-468 or U87MG tumor-bearing mice, the recombinant hFTHs presenting either peptide or both of affibody and 4CRGD are successfully delivered to and retained in the tumor for a prolonged period of time. In particular, the recombinant hFTH presenting both affibody and 4CRGD notably enhances in vivo detection of U87MG tumors that express heterogeneous receptors, integrin and EGFR, compared to the other recombinant hFTHs presenting either affibody or 4CRGD only. Like affibody and 4CRGD used in this study, other multiple tumor receptor-binding peptides can be also genetically introduced to the hFTH surface for actively targeting of in vivo tumors with heterogenous receptors.

Down syndrome is the most common genetic disorder and is characterized by three copies of chromosome 21. Regulator of calcineurin 1 (RCAN1) is located close to the Down syndrome critical region (distal part of chromosome 21), and its product functions as an endogenous inhibitor of calcineurin signaling. RCAN1 protein stability is regulated by several inflammatory signaling factors, though the underlying mechanisms remain incompletely understood. Here, we report that RCAN1 interacts with the inflammation-linked transcription factor, signal transducer and activator of transcription 2 (STAT2) in mammalian cells. STAT2 overexpression decreased levels of RCAN1 protein. Decreases in RCAN1 were blocked by a proteasome inhibitor, indicating that STAT2 regulates RCAN1 degradation via the ubiquitin-proteasome system. Co-immunoprecipitation/immunoblot analyses showed that STAT2 enhanced RCAN1 ubiquitination through the ubiquitin E3 ligase FBW7. This pathway appeared to be physiologically relevant, as treatment of cells with interferon-α reduced RCAN1 levels through the activation of STAT2 and FBW7. Together, these results suggest that STAT2 influences diverse cellular processes linked to RCAN1 by negatively affecting RCAN1 protein stability.

We synthesized a three-dimensional nanorod structure of zinc oxide (ZnO) using a simple sol-gel process and systematically investigated properties of the ZnO nanorods regarding protein adsorption and effect on fluorescence emission. As compared to conventional polystyrene plate that has been widely used for strong protein adsorption, the ZnO nanorods had a superior protein adsorption capacity and significantly amplified fluorescence emission, suggesting the ZnO nanorods are attractive for fluorescence-based biomolecular detection assays. When applied to diagnostic assay of rheumatoid arthritis (RA) using cyclic citrullinated peptide (CCP) probe with a RCGRS motif that reportedly has a strong affinity for ZnO, the ZnO nanorods gave apparently high positive signals for all the RA-positive standards and patient sera, whereas upon the detection using conventional polystyrene plate, all the detection signals were relatively negligible. Moreover, the streptavidin-mediated immobilization of well oriented CCP further enhanced sensitivity, even for a 5000-times diluted patient serum. A highly sensitive detection of a very small amount of RA autoantibodies is important because individuals at high risk of developing RA can be identified several years before the clinical onset. Consequently, the fluorescence-based sensitive assay of RA was successfully performed using the three-dimensional ZnO nanorods, owing to the fluorescence amplification and protein/peptide adsorption properties and dimensionality of ZnO nanorods that in turn increases probe accessibility to anti-CCP RA autoantibodies. Although RA was assayed here for proof-of-concept, the ZnO nanorods-based assay can be applied in general to sensitive detection of a wide variety of antibody or protein targets.

As a fusion partner to express aggregation-prone heterologous proteins, we investigated the efficacy of Escherichia coli phosphoglycerate kinase (ePGK) that consists of two functional domains (N- and C-domain) and reportedly has a high structural stability. When the full-length ePGK (F-ePGK) was used as a fusion partner, the solubility of the heterologous proteins increased, but some of them still had a large fraction of insoluble aggregates. Surprisingly, the fusion expression using the N-domain of ePGK (N-ePGK) made the insoluble fraction significantly reduce to less than 10% for all the heterologous fusion proteins tested. Also, we evaluated the efficacy of N-ePGK in making the target proteins be expressed with their own native function or structure. It was found that of human ferritin light chain, bacterial arginine deiminase, human granulocyte colony stimulating factor were synthesized evidently with the self-assembly function, L-arginine-degrading activity, and the correct secondary structure, respectively, through the fusion expression using N-ePGK. These results indicate that N-ePGK is a highly potent fusion partner that can be widely used for the synthesis of a variety of heterologous proteins in E. coli. Biotechnol. Bioeng. 2012; 109:325–335. © 2011 Wiley Periodicals, Inc.

Through the proteome analysis of Escherichia coli BL21(DE3), we previously identified the stress-responsive protein, arsenate reductase (ArsC), that showed a high cytoplasmic solubility and a folding capacity even in the presence of stress-inducing reagents. In this study, we used ArsC as an N-terminal fusion partner to synthesize nine aggregation-prone proteins as water-soluble forms. As a result, solubility of the aggregation-prone proteins increased dramatically by the fusion of ArsC, due presumably to its tendency to facilitate the folding of target proteins. Also, we evaluated and confirmed the efficacy of ArsC-fusion expression in making the fusion-expressed target proteins have their own native function or structure. That is, the self-assembly function of human ferritin light chain, l-arginine-degrading function of arginine deiminase, and the correct secondary structure of human granulocyte colony stimulating factor were clearly observed through transmission electron microscope analysis, colorimetric enzyme activity assay, and circular dichroism, respectively. It is strongly suggested that ArsC can be in general an efficient fusion expression partner for the production of soluble and active heterologous proteins in E. coli.

Protein nanoparticles (PNPs) that are nanostructured biomaterials with intrinsic biological function have been widely employed as three-dimensional nanobiomaterials for sensitive bioassays, MRI contrast, semiconductor devices, template for hybrid materials, etc., and stable and long-term maintenance of PNPs seems to be of crucial importance. We evaluated the stability of PNPs and the efficacy of lyophilization for the long-term stability of PNPs, especially using green fluorescent protein nanoparticles (gFPNPs) as a model PNP. Fluorescence intensities and TEM images of gFPNPs were analyzed to monitor their functional and structural stabilities. Unlike the green fluorescent protein monomers (eGFP) that were gradually inactivated in aqueous solution, gFPNP in the same aqueous solution retained the initial fluorescence activity and spherical nanoparticle structure even for 2 weeks at 4°C. To ensure stable and long-term maintenance of gFPNPs, gFPNPs in aqueous solution were converted to the dried solid forms through lyophilization. It is notable that fluorescence activity and nanoparticle structure of gFPNPs that were lyophilized with both Tween 80 and sucrose were very stably maintained even for 10weeks at various storage temperatures (-20°C, 4°C, 25°C, and 37°C). During the period of 10weeks, the fluorescence of gFPNP was always more than 80% level of initial fluorescence at a wide range of temperature. Although this stability study was focused on gFPNPs, the developed optimal lyophilization conditions for gFPNPs can be applied in general to stable and long-term maintenance of many other PNP-derived biomaterials.

Existing methods to enrich target regions of genomic DNA based on PCR, hybridization capture, or molecular inversion probes have various drawbacks, including long experiment times and low throughput and/or enrichment quality. We developed CRISPR-Cap, a simple and scalable CRISPR-based method to enrich target regions of dsDNA, requiring only two short experimental procedures that can be completed within two hours. We used CRISPR-Cap to enrich 10 target genes 355.7-fold on average from Escherichia coli genomic DNA with a maximum on-target ratio of 81% and high enrichment uniformity. We also used CRISPR-Cap to measure gene copy numbers and detect rare alleles with frequencies as low as 1%. Finally, we enriched coding sequence regions of 20 genes from the human genome. We envision that CRISPR-Cap can be used as an alternative to other widely used target-enrichment methods, which will broaden the scope of CRISPR applications to the field of target enrichment field.

We previously reported that under the stress condition caused by the addition of 2-hydroxyethyl disulfide, a thiol-specific oxidant, to growing cultures of Escherichia coli BL21(DE3), a population of stress-responsive proteins [peptidyl-prolyl cis–trans isomerase B (PpiB), bacterioferritin (Bfr), putative HTH-type transcriptional regulator yjdC (YjdC), dihydrofolate reductase (FolA), chemotaxis protein cheZ (CheZ), and glutathione synthetase (GshB)] were significantly upregulated when compared with the nonstress condition. When those stress-responsive proteins were used as fusion partners for the expression of human granulocyte colony-stimulating factor (hG-CSF), the solubility of hG-CSF was dramatically enhanced in E. coli cytoplasm, whereas almost all of the directly expressed hG-CSF were aggregated to inclusion bodies. In addition, the spectra of circular dichroism measured with the purified hG-CSF were identical to that of standard hG-CSF, implying that the synthesized hG-CSF has native conformation. These results indicate that the bacterial stress-responsive proteins could be potent fusion expression partners for aggregation-prone heterologous proteins in E. coli cytoplasm.

Early detection of the protein marker troponin I in patients with a higher risk of acute myocardial infarction can reduce the risk of death from heart attacks. Most troponin assays are currently based on the conventional enzyme linked immunosorbent assay and have detection limits in the nano- and picomolar range. Here, we show that by combining viral nanoparticles, which are engineered to have dual affinity for troponin antibodies and nickel, with three-dimensional nanostructures including nickel nanohairs, we can detect troponin levels in human serum samples that are six to seven orders of magnitude lower than those detectable using conventional enzyme linked immunosorbent assays. The viral nanoparticle helps to orient the antibodies for maximum capture of the troponin markers. High densities of antibodies on the surfaces of the nanoparticles and nanohairs lead to greater binding of the troponin markers, which significantly enhances detection sensitivities. The nickel nanohairs are re-useable and can reproducibly differentiate healthy serum from unhealthy ones. We expect other viral nanoparticles to form similar highly sensitive diagnostic assays for a variety of other protein markers.

Recently, we reported on the dual function of human ferritin heavy chain (hFTN-H) used for the fusion expression and solubility enhancement of various heterologous proteins: (1) high-affinity interaction with HSP70 chaperone DnaK and (2) formation of self-assembled supramolecules with limited and constant sizes. Especially the latter, the self-assembly function of hFTN-H is highly useful in avoiding the undesirable formation of insoluble macroaggregates of heterologous proteins in bacterial cytoplasm. In this study, using enhanced green fluorescent protein (eGFP) and several deletion mutants of Mycoplasma arginine deiminase (ADI(132-410)) as reporter proteins, we confirmed through TEM image analysis that the recombinant fusion proteins (hFTN-H::eGFP and hFTN-H::ADI(132-410)) formed intracellular spherical particles with nanoscale diameter ( approximately 10 nm), i.e., noncovalently cross-linked supramolecules. Surprisingly, the supramolecular eGFP and ADI showed much enhanced stability in bioactivity. That is, the activity level was much more stably maintained for the prolonged period of time even at high temperature, at high concentration of Gdn-HCl, and in wide range of pH. The stability enhancement by supramolecular self-assembly may make it possible to utilize the protein supramolecules as novel means for drug delivery, enzymatic material conversion (biotransformation), protein chip/sensor, etc. where the maintenance of protein/enzyme stability is strictly required.

The length of bioreceptors plays an important role in signal enhancement of surface-enhanced Raman scattering (SERS) due to amplification of electromagnetic fields generated by the excitation of localized surface plasmons. Herein, intact antibodies (IgG) and Fab fragments conjugated onto gold nanostar were used to fabricate two kinds of immunosensors for measurement of their SERS signals. Using CA125 as the antigen and Rhodamine-6G (R6G)-conjugated immunogolds, a SERS immunosensor was self-assembled by antigen-antibody interaction. The results showed that the SERS signal from the Fab immunosensor was 2.4 times higher than that of the IgG immunosensor. Furthermore, increased hot-spots by silver atom deposition onto the IgG and Fab immunosensor showed 2.1 and 1.4 times higher signals than before enhancement, respectively. For application, based on the Fab immunosensor, a SERS-compatible microfluidic system was designed for multiplex assays to overcome the drawbacks of conventional assays. This system can measure biological specimens directly from bio fluids instead of using a complex microfluidic device containing separation and detection elements. Four approved biomarkers of breast cancer, including cancer antigen (CA125), HER2, epididymis protein (HE4), and Eotaxin-1, were detected from patient-mimicked serum with limits of 15 fM, 17 fM, 21 fM, and 6.5 fM, respectively. The results indicated that the lengths and geometry of the bioreceptors determined the intensity of SERS signal from the interface and cavity of the sandwich immunosensor. Silver atom deposition at the cavity of the immunosensor increased the SERS signal. Finally, the SERS immunosensor built-in microfluidic system improved the performance of multiplex diagnostics.

Lateral flow assay (LFA) is an attractive method for rapid, simple, and cost-effective point of care diagnosis. For LFA-based multiplex diagnosis of three viral intractable diseases (acquired immune deficiency syndrome and hepatitis C and A), here we developed proteinticle-based 7 different 3D probes that display different viral antigens on their surface, which were synthesized in Escherichia coli by self-assembly of human ferritin heavy chain that was already engineered by genetically linking viral antigens to its C-terminus. Each of the three test lines on LFA strip contains the proteinticle probes to detect disease-specific anti-viral antibodies. Compared to peptide probes, the proteinticle probes were evidently more sensitive, and the proteinticle probe-based LFA successfully diagnosed all the 20 patient sera per each disease without a false negative signal, whereas the diagnostic sensitivities in the peptide probe-based LFAs were 65–90%. Duplex and triplex assays performed with randomly mixed patient sera gave only true positive signals for all the 20 serum mixtures without any false positive signals, indicating 100% sensitivity and 100% specificity. It seems that on the proteinticle surface the antigenic peptides have homogeneous orientation and conformation without inter-peptide clustering and hence lead to the enhanced diagnostic performance with solving the problems of traditional diagnostic probes. Although the multiplex diagnosis of three viral diseases above was demonstrated as proof-of-concept here, the proposed LFA system can be applied to multiplex point of care diagnosis of other intractable diseases.

The recombinant ferritin heavy chain (FTN-H) formed self-assembled spherical nanoparticles with the size comparable to native one. We tried to express the GAD65 COOH-terminal fragments, i.e., 448-585 (GAD65(448-585)), 487-585 (GAD65(487-585)), and 512-585 (GAD65(512-585)) amino acid fragments, using FTN-H as N-terminus fusion expression partner in Escherichia coli. All of recombinant fusion proteins (FTN-H::GAD65(448-585), FTN-H::GAD65(487-585), and FTN-H::GAD65(512-585)) also formed spherical nanoparticles due probably to the self-assembly function of the fused ferritin heavy chain. The antigenic epitopes within GAD65(448-585), GAD65(487-585), and GAD65(512-585) against insulin-dependent diabetes mellitus (IDDM) marker (autoantibodies against GAD65) were localized at the surface of the spherical protein nanoparticles so that anti-GAD65 Ab could recognize them. Protein nanoparticles like FTN-H seem to provide distinct advantages over other inorganic nanoparticles (e.g., Au, Ag, CdSe, etc.) in that through the bacterial synthesis, the active capture probes can be located at the nanoparticle surface with constant orientation/conformation via covalent cross-linking without complex chemistry. Also it is possible for the protein nanoparticles to have uniform particle size, which is rarely achieved in the chemical synthesis of inorganic nanoparticles. Thus, the recombinant ferritin particles can be used as a three-dimensional (spherical) and nanometer-scale probe structure that is a key component in ultra-sensitive protein chip for detecting protein-small molecule interactions and protein-protein interactions.

Three types of recombinant pre-S antigens (i.e., pre-S1S2) of hepatitis B virus (HBV) were synthesized in Saccharomyces cerevisiae and secreted into extracellular medium: wild type (pre-S1S2) and two mutant antigens, pre-S1 degrees S2 (Asn15Gln) and pre-S1 degrees S2 degrees (Asn15Gln and Asn123Gln). An N-terminus sequence (Ser5-Ala28) of human interleukin 1 beta (hIL-1 beta) was used as synthetic prosequence of recombinant HBV surface antigen (pre-S), secreted from S. cerevisiae. The expression cassette comprised the signal peptide of the killer toxin of Kluyveromyces lactis, the synthetic prosequence above, KEX2 dibasic endopeptidase cleavage site (-Lys-Arg-), and the surface antigen. The recombinant pre-S1S2 and pre-S1 degrees S2 were secreted in the hyper-mannosylated form, while the recombinant pre-S1 degrees S2 degrees was produced without N-glycosylation. It has been demonstrated that the two particular N-linked glycans at Asn15 and Asn123 interfered with the B-cell response to the HBV-derived pre-S1S2, resulting in low titers of pre-S1S2-neutralizing antibodies. This problem was overcome by eliminating both of the N-glycosylation signals. Despite enhanced immunogenicity, the recombinant pre-S1 degrees S2 degrees showed two major problems: (1) inefficient Kex2 cleavage process in the secretory pathway and (2) the severe proteolytic degradation by yeast proteases. The efficiency of Kex2 cleavage increased dramatically by removing N-glycosylation signal in the synthetic prosequence, but the proteolysis of pre-S1 degrees S2 degrees was somewhat inevitable. Further systematic approaches including modulation of degree of N-glycosylation or relocation of N-glycosylation sites in the recombinant pre-S1S2 may make it possible to achieve both enhanced immunogenicity and resistance towards proteolytic degradation of the secreted pre-S antigen.

As a novel feeding strategy for optimizing human epidermal growth factor (hEGF) production with a recombinant Hansenula polymorpha DL-1 using the methanol oxidase (MOX) promoter in H. polymorpha DL-1, independent exponential feeding of two substrates was used. A simple kinetic model considering the cell growth on two substrates was established and used to calculate the respective feeding rates of glycerol and methanol. In the fedbatch culture with methanol-only feeding, the optimal set point of specific growth rate on methanol was found to be 0.10 h-1. When the fed-batch cultures were conducted by the independent feeding of glycerol and methanol, the actual specific growth rate on glycerol and methanol was slightly lower than the set point of specific growth rate. By the uncoupled feeding of glycerol and methanol the volumetric productivity of hEGF increased from 6.4 to 8.0 mg/(L.h), compared with methanol-only feeding.

PGCS-NPs (40 nm) with excellent photo-thermal activity are developed, on the surface of which affibody peptides with specific affinity for EGFR and many small gold dots (1-3 nm) are densely presented. The IV-injected PGCS-NPs into EGFR-expressing tumor-bearing mice successfully perform targeted and photothermal therapy of cancer. It seems that the small gold dots released from disassembled PGCS-NPs are easily removed and never cause in vivo toxicity problems.

Two different protein nanoparticles that are totally different in shape and surface structure, i.e. Escherichia coli DNA-binding protein (eDPS) (spherical, 10 nm) and Thermoplasma acidophilum proteasome (tPTS) (cylindrical, 12 × 15 nm) were engineered for in vivo optical tumor detection: arginine-glycine-aspartic acid (RGD) peptide (CDCRGDCFC) was genetically inserted to the surface of each protein nanoparticle, and also near-infrared fluorescence dye was chemically linked to the surface lysine residues. The specific affinity of RGD for integrin (αvβ3) facilitated the uptake of RGD-presenting protein nanoparticles by integrin-expressing tumor cells, and also the protein nanoparticles neither adversely affected cell viability nor induced cell damage. After intravenously injected to tumor-bearing mice, all the protein nanoparticles successfully reached tumor with negligible renal clearance, and then the surface RGD peptides caused more prolonged retention of protein nanoparticles in tumor and accordingly higher fluorescence intensity of tumor image. In particular, the fluorescence of tumor image was more intensive with tPTS than eDPS, which is due presumably to longer in vivo half-life and circulation of tPTS that originates from thermophilic and acidophilic bacterium. Although eDPS and tPTS were used as proof-of-concept in this study, it seems that other protein nanoparticles with different size, shape, and surface structure can be applied to effective in vivo tumor detection.

Regulator of calcineurin 1 (RCAN1; also referred as DSCR1 or MCIP1) is located in close proximity to a Down syndrome critical region of human chromosome 21. Although RCAN1 is an endogenous inhibitor of calcineurin signaling that controls lymphocyte activation, apoptosis, heart development, skeletal muscle differentiation, and cardiac function, it is not yet clear whether RCAN1 might be involved in other cellular activities. In this study, we explored the extra-functional roles of RCAN1 by searching for novel RCAN1-binding partners. Using a yeast two-hybrid assay, we found that RCAN1 (RCAN1-1S) interacts with histone deacetylase 3 (HDAC3) in mammalian cells. We also demonstrate that HDAC3 deacetylates RCAN1. In addition, HDAC3 increases RCAN1 protein stability by inhibiting its poly-ubiquitination. Furthermore, HDAC3 promotes RCAN1 nuclear translocation. These data suggest that HDAC3, a new binding regulator of RCAN1, affects the protein stability and intracellular localization of RCAN1.

Though human L-chain ferritin has been known to be more resistant to physical denaturation than H-type ferritin, its stability characteristics and kinetic information have not been reported in detail. Overexpressed recombinant ferritin (FTN) in Escherichia coli formed inclusion bodies through noncovalent molecular interaction and easily dissolved with regaining the iron-uptake activity by a simple pH-shift process at high protein concentration (>600 mg l(-1)). FTN was relatively thermostable at low protein concentration (0.2 g l(-1)), but it became extremely thermolabile at high protein concentration (1.3 g l(-1)), i.e., more than 80% of FTN was coprecipitated within 5 min under the same heat-induced denaturation condition. Aggregation rate constant for initial 5 min at high protein concentration was 6.04 x 10(-3) s(-1) for FTN. Surprisingly, glucagon. ferritin mutant (GFTN), consisting of an N-terminus fusion partner, human glucagon (29-residue alpha-helical peptide), showed significantly enhanced thermal stability even at high protein concentration. That is, in spite of 40-min heat treatment, more than 50% of GFTN the still remained soluble with maintaining the same functional properties. The aggregation rate constants were 2.75 x 10(-4) and 2.80 x 10(-4) s(-1) at low and high concentration, respectively, for GFTN. These results suggest a critical participation of the N-terminal domain of ferritin in the temperature-induced aggregation pathway. Presumably, partially denatured amino terminus of FTN is involved in nonspecific molecular interaction resulting in the off-pathway aggregation. It is notable that the purified GFTN showed the same molar capacity of iron (Fe(+3)) storage as standard ferritin. From the analysis of fluorescence emission spectrum, the physical stability of GFTN was also very comparable to that of standard ferritin under the various denaturation conditions induced by GdnHCl.