Translational Detection of Nonproteinogenic Amino Acids Using an Engineered Complementary Cell-Free Protein Synthesis Assay.

We developed a simple and rapid method for analyzing nonproteinogenic amino acids that does not require conventional chromatographic equipment. In this technique, nonproteinogenic amino acids were first converted to a proteinogenic amino acid through in vitro metabolism in a cell extract. The proteinogenic amino acid generated from the nonproteinogenic precursors were then incorporated into a reporter protein using a cell-free protein synthesis system. The titers of the nonproteinogenic amino acids could be readily quantified by measuring the activity of reporter proteins. This method, which combines the enzymatic conversion of target amino acids with translational analysis, makes amino acid analysis more accessible while minimizing the cost and time requirements. We anticipate that the same strategy could be extended to the detection of diverse biochemical molecules with clinical and industrial implications.

Small molecule inhibitors of IκB kinase ß: A chip-based screening and molecular docking simulation.

A chip-based screening system for IκB kinase ß (IKKß) has been developed by physically immobilizing the substrate IκBα on a glass matrix using a calixarene linker. Phosphorylation of IκBα by IKKß and ATP was quantitated using a fluorescently labeled antibody. Using this efficient assay system a chemical library of 2000 bioactive compounds was screened against IKKß and four were identified as good inhibitors, namely, aurintricarboxylic acid, diosmin, ellagic acid, and hematein. None of them have been reported to be an inhibitor of IKKß although they were implicated in various NFκB-mediated biological processes. Our enzyme-based assay showed that IC50 of the four inhibitors is comparable with that of IKK-16, a previously known strong inhibitor. Molecular docking simulation shows that the hydrophobic moiety of an inhibitor interacts with the four hydrophobic residues (Leu21, Val29, Val152, and Ile165) of the active site. The MM-PBSA calculation suggests that these hydrophobic interactions appear to be the predominant contributor to the binding free energy. As IKKß is ubiquitously expressed in various cell types and executes many biological functions, the enzyme and cell specificity of the four inhibitors need to be rigorously tested before accepted as a drug candidate.

Exosomes derived from human umbilical cord blood mesenchymal stem cells stimulates rejuvenation of human skin.

Human umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) play an important role in cutaneous wound healing, and recent studies suggested that MSC-derived exosomes activate several signaling pathways, which are conducive in wound healing and cell growth. In this study, we investigated the roles of exosomes that are derived from USC-CM (USC-CM Exos) in cutaneous collagen synthesis and permeation. We found that USC-CM has various growth factors associated with skin rejuvenation. Our in vitro results showed that USC-CM Exos integrate in Human Dermal Fibroblasts (HDFs) and consequently promote cell migration and collagen synthesis of HDFs. Moreover, we evaluated skin permeation of USC-CM Exos by using human skin tissues. Results showed that Exo-Green labeled USC-CM Exos approached the outermost layer of the epidermis after 3 h and gradually approached the epidermis after 18 h. Moreover, increased expressions of Collagen I and Elastin were found after 3 days of treatment on human skin. The results showed that USC-CM Exos is absorbed into human skin, it promotes Collagen I and Elastin synthesis in the skin, which are essential to skin rejuvenation and shows the potential of USC-CM integration with the cosmetics or therapeutics.

Prospective, Multicenter Study on Tinnitus Changes after Cochlear Implantation.

OBJECTIVE: To investigate the time course of tinnitus changes in patients receiving cochlear implantation (CI) in a prospective, multicenter setting and to determine related factors. MATERIALS AND METHODS: A total of 79 adult patients who underwent CI were included in this study. We used the same questionnaires sequentially 5 times. The questionnaires included the Visual Analog Scale (VAS) for tinnitus severity, the Tinnitus Handicap Inventory (THI), Beck's Depression Index (BDI), and the Brief Encounter Psychosocial Instrument (BEPSI) for stress assessment. RESULTS: Tinnitus was present in 59 (74.7%) of the 79 study subjects. After CI, tinnitus was eliminated in 10 patients (25%) and improved in 16 patients (40%) of the 40 patients who completed the final questionnaires, and most of the tinnitus reduction occurred in the early period of CI use. In an analysis of psychological functioning with CI, BDI was reduced significantly after CI. Multiple linear regression analysis revealed that preoperative auditory steady-state response (ASSR), THI, and final BDI score were significantly associated with the changes in tinnitus after CI. CONCLUSIONS: Most of the tinnitus reduction occurred within 1 month after CI use, and the changes were significantly associated with THI, ASSR, and BDI scores 6 months after CI. CI is a valuable therapeutic modality in tinnitus of a deafened ear.

Effects of stimulating interleukin -2/anti- interleukin -2 antibody complexes on renal cell carcinoma.

BACKGROUND: Current therapies for advanced renal cell carcinoma (RCC) have low cure rates or significant side effects. It has been reported that complexes composed of interleukin (IL)-2 and stimulating anti-IL-2 antibody (IL-2C) suppress malignant melanoma growth. We investigated whether it could have similar effects on RCC. METHODS: A syngeneic RCC model was established by subcutaneously injecting RENCA cells into BALB/c mice, which were administered IL-2C or phosphate-buffered saline every other day for 4 weeks. RCC size was measured serially, and its weight was assessed 4 weeks after RENCA injection. Immune cell infiltration into RCC lesions and spleen was assessed by flow cytometry and immunohistochemistry. RESULTS: IL-2C treatment increased the numbers of CD8(+) memory T and natural killer (NK) cells in healthy BALB/c mice (P < 0.01). In the spleen of RCC mice, IL-2C treatment also increased the number of CD8(+) memory T, NK cells, and macrophages as compared to PBS-treated controls (P < 0.01). The number of interferon-γ- and IL-10-producing splenocytes increased and decreased, respectively after 4 weeks in the IL-2C-treated mice (P < 0.01). Tumor-infiltrating immune cells including CD4(+) T, CD8(+) T, NK cells as well as macrophages were increased in IL-2C-treated mice than controls (P < 0.05). Pulmonary edema, the most serious side effect of IL-2 therapy, was not exacerbated by IL-2C treatment. However, IL-2C had insignificant inhibitory effect on RCC growth (P = 0.1756). CONCLUSIONS: IL-2C enhanced immune response without significant side effects; however, this activity was not sufficient to inhibit RCC growth in a syngeneic, murine model.

Renin-Angiotensin-Aldosterone System Blockade in Critically Ill Patients Is Associated with Increased Risk for Acute Kidney Injury.

Acute kidney injury (AKI) is a major clinical problem and a predictor of outcomes in critically ill patients who frequently required treatments in the intensive care unit (ICU). Renin-angiotensin-aldosterone system (RAAS) blockers are commonly used for treating hypertension but demands caution because of accompanying illnesses including AKI. The aim of this study was to evaluate whether the use of RAAS blockers affected the incidence of AKI in ICU patients. From a total of 26,287 patients who were admitted to the ICU from January 2003 to December 2013 were included in the final analyses. The primary outcome was the incidence of AKI based on the prescription of RAAS blockers. The secondary outcomes were all-cause mortality. RAAS blocker users were more likely to develop AKI (P < 0.001) and remained an independent risk factor for AKI (odds ratio [OR], 1.56; 95% confidence interval [CI], 1.37-1.79; P < 0.001) after adjusting confounding factors. There was no significant difference in the cumulative 90-day survival rate between the RAAS blocker users and non-users (P = 0.381). However, the adjusted mortality risk associated with AKI was 1.38 (95% CI, 1.22 to 1.56; P < 0.001) and increased as the severity of AKI stage increased from 1 to 3: 1.17 (1.02 to 1.36), 1.77 (1.45 to 2.16), and 1.93 (1.55 to 2.41; P < 0.01 for the trend). RAAS blockers may have a harmful influence to increase the incidence of AKI and temporary withholding of these medications may deserve careful consideration in ICU patients.

Prevention of cisplatin-induced ototoxicity by the inhibition of gap junctional intercellular communication in auditory cells.

Cis-diamminedichloroplatinum (cisplatin) is an effective chemotherapeutic drug for cancer therapy. However, most patients treated with cisplatin are at a high risk of ototoxicity, which causes severe hearing loss. Inspired by the "Good Samaritan effect" or "bystander effect" from gap junction coupling, we investigated the role of gap junctions in cisplatin-induced ototoxicity as a potential therapeutic method. We showed that connexin 43 (Cx43) was highly expressed in House Ear Institute-Organ of Corti 1 (HEI-OC1) cells, mediating cell-cell communication. The viability of HEI-OC1 cells was greatly decreased by cisplatin treatment, and cisplatin-treated HEI-OC1 cells showed lower Cx43 expression compared to that of untreated HEI-OC1 cells. In particular, high accumulation of Cx43 was observed around the nucleus of cisplatin-treated cells, whereas scattered punctuate expression of Cx43 was observed in the cytoplasm and membrane in normal cells, suggesting that cisplatin may interrupt the normal gap junction communication by inhibiting the trafficking of Cx43 to cell membranes in HEI-OC1 cells. Interestingly, we found that the inhibition of gap junction activity reduced cisplatin-induced apoptosis of auditory hair cells. Cx43 siRNA- or 18α-GA-treated HEI-OC1 cells showed higher cell viability compared to control HEI-OC1 cells during cisplatin treatment; this was also supported by fluorescence recovery after photobleaching studies. Inhibition of gap junction activity reduced recovery of calcein acetoxymethyl ester fluorescence compared to control cells. Additionally, analysis of the mechanisms involved demonstrated that highly activate extracellular signal-regulated kinase and protein kinase B, combined with inhibition of gap junctions may promote cell viability during cisplatin treatment.

Soluble CD25 is increased in patients with sepsis-induced acute kidney injury.

AIM: Sepsis has been shown to induce the expansion of CD4+CD25+ regulatory T cells (Tregs), and this paradoxical immune suppression has been suggested to be closely associated with the development of sepsis-induced organ dysfunction. In the present study, we aimed to investigate the possible link between immune suppression and the development of septic acute kidney injury (AKI). METHODS: We prospectively enrolled patients with a diagnosis of sepsis, with or without AKI and as well as patients with AKI but without sepsis. Serum and urine samples at the time of the diagnosis were collected to measure neutrophil gelatinase-associated lipocalin (NGAL), cytokines, and soluble CD25 (sCD25). RESULTS: Of the 82 patients enrolled, 44, 18, and 20 patients were classified into septic-AKI, sepsis-non AKI and non-septic AKI groups. There were no differences in the baseline characteristics in all three groups and the severity of infection in the two sepsis groups. Serum levels of interleukin (IL)-10 were significantly elevated in patients with septic-AKI compared to the other two groups. Serum and urine NGAL levels and the level of serum sCD25, a marker of regulatory T cells, were significantly elevated in patients with septic AKI group, indicating the potential association of paradoxical immune suppression and the development of septic-AKI. CONCLUSIONS: These results suggest that immune suppression in sepsis may be closely linked to the development of AKI and that sCD25 or IL-10 may be useful as novel biomarkers for the development of septic AKI.

Successful pregnancy in a patient with autosomal dominant polycystic kidney disease on long-term hemodialysis.

Recent advances in dialysis and a multidisciplinary approach to pregnant patients with advanced chronic kidney disease provide a better outcome. A 38-yr-old female with autosomal dominant polycystic kidney disease (ADPKD) became pregnant. She was undergoing hemodialysis (HD) and her kidneys were massively enlarged, posing a risk of intrauterine fetal growth restriction. By means of intensive HD and optimal management of anemia, pregnancy was successfully maintained until vaginal delivery at 34.5 weeks of gestation. We discuss the special considerations involved in managing our patient with regard to the underlying ADPKD and its influence on pregnancy.

Comparative measurement of aortic root by transthoracic echocardiography in normal Korean population based on two different guidelines.

BACKGROUND: Aortic root size is an important parameter in vascular diseases and can be easily assessed by transthoracic echocardiography. However, measurements values may vary according to cardiac cycle and the definition used for edge. This study aimed to define normal values according to the measurement method specified by two different guidelines to determine the influence of the different methods on echocardiographic measurements. METHODS: Healthy Korean adults were enrolled. The aortic root diameters were measured twice at four levels (aortic annulus, sinuses of Valsalva, sinotubular junction, and ascending aorta) by the 2005 American Society of Echocardiography (ASE) guidelines (measured from leading edge to leading edge during diastole) and the 2010 ASE pediatric guidelines (measured from inner edge to inner edge during systole). RESULTS: One hundred twelve subjects aged 20-69 years were enrolled. The aortic diameters (cm) determine by the aforementioned two guidelines showed significant difference. Measurements were larger in 2005 ASE guideline at aortic annuls, sinuses of Valsalva, and sinotubular junction level, but smaller at ascending aortic level with 2-3mm of differences. Intraobserver variability was similarly good, but interobserver variability was slightly higher than intraobserver variability in both measurement methods. BSA and age was most important determinant for aortic root size. CONCLUSIONS: The measurement method of aortic root can affect the echocardiographic result. The measurement method should be noted when assessing clinical significance of aortic root measurement.

Clinical and histological characteristics of melanocytic nevus in external auditory canals and auricles.

Nevi, which consist of nevus cells arising from external auditory canals (EACs) and auricles, are rare and their characteristics are not thoroughly understood. The purpose of this study was to analyze the clinicopathological characteristics of melanocytic nevus (MN) in EACs and auricles. Medical records were reviewed in 35 cases with junctional, compound and intradermal nevi treated in Ajou University Hospital, Korea between 2001 and 2011. Patient demographic, location, shape, and diameter of nevi, and pathologic results were analyzed according to the location, EACs (23 cases) and auricles (12 cases). Female predominance was noted in both EAC (60.9 %, 14 cases) and auricular (75 %, 9 cases) nevi. The mean age of EAC nevi (37.1 years) was younger than that of auricular nevi (42.2 years). The chief complaint was a symptomless mass in both groups, mostly in dome-like gross appearances. The mean diameter of EAC and auricular nevi was 9.6 (3-16) mm and 12.2 (3-25) mm, respectively. Histological findings chiefly presented intradermal nevi in EACs (78.3 %) and auricles (83.3 %) which showed preference to older patients, in contrast to the compound type. All nevi including five cases with skin grafts were completely excised without any recurrence within the follow-up period (average 5.3 months). A possible dysplastic nevus was detected in only one case. All MNs in EACs or auricles reveal similar characteristics. Early and complete excision is recommended to avoid skin graft, functional problems, and the risk of malignant melanoma.

On-chip microfluidic dual detection of amino acid metabolism disorders using cell-free protein synthesis.

Various metabolic diseases are associated with the accumulation of specific amino acids due to abnormal metabolic pathways, and thus can be diagnosed by measuring the level of amino acids in body fluids. However, present methods for amino acid analysis are not readily accessible because they require a complex experimental setup, expensive equipment, and a long processing time. Here, we present a dual sensing microfluidic device that enables fast, portable, and quantitative analysis of target amino acids, harnessing the biological mechanism of protein synthesis. In this device, the working principle of a finger-actuated pumping unit is applied, and the microchannels are designed to perform cell-free synthesis of a reporter protein in response to the target amino acids in the assay samples. Multiple steps required for the translational assay are controlled by the simple operation of two pushbuttons on the device. It is demonstrated that the developed microfluidic device provides precise quantification of two amino acids (methionine and phenylalanine) within 30 min at room temperature. We expect that the application of the presented device can be readily extended to the point-of-care testing of other metabolic compounds.

Customized valorization of waste streams by Pseudomonas putida: State-of-the-art, challenges, and future trends.

Preventing catastrophic climate events warrants prompt action to delay global warming, which threatens health and food security. In this context, waste management using engineered microbes has emerged as a long-term eco-friendly solution for addressing the global climate crisis and transitioning to clean energy. Notably, Pseudomonas putida can valorize industry-derived synthetic wastes including plastics, oils, food, and agricultural waste into products of interest, and it has been extensively explored for establishing a fully circular bioeconomy through the conversion of waste into bio-based products, including platform chemicals (e.g., cis,cis-muconic and adipic acid) and biopolymers (e.g., medium-chain length polyhydroxyalkanoate). However, the efficiency of waste pretreatment technologies, capability of microbial cell factories, and practicability of synthetic biology tools remain low, posing a challenge to the industrial application of P. putida. The present review discusses the state-of-the-art, challenges, and future prospects for divergent biosynthesis of versatile products from waste-derived feedstocks using P. putida.

Quantitative Analysis of γ-Aminobutyric Acid by Combined Cell-Free Protein Synthesis and Transamination Reactions.

The synthetic power of cells can be harnessed for assaying important analytes, as well as for producing biomolecules. In particular, cell-free protein synthesis (CFPS) can be implemented as a signal amplification module for bioassays, while avoiding many problems associated with whole cell-based microbial biosensors. Here, we developed a method for analyzing γ-aminobutyric acid (GABA) by combining the enzymatic conversion of GABA and amino-acid-dependent CFPS. In this method, GABA molecules in the assay sample are used to generate alanine, which is incorporated into signal-generating proteins in the subsequent cell-free synthesis reaction. The activity of cell-free synthesized proteins was successfully used to estimate the GABA concentration in the assay sample. In principle, the developed method could be extended for the analyses of other important bioactive compounds.

Cell-free synthesis of industrial chemicals and biofuels from carbon feedstocks.

The power of biological systems can be harnessed with higher efficiency when biosynthetic reactions are decoupled from cellular physiology. This can be achieved by cell-free synthesis, which relies on the in vitro use of cellular machinery under optimized reaction conditions. As exemplified by the recent development of mRNA vaccines and therapeutics, the cell-free synthesis of biomolecules is fast, efficient and flexible. Cell-free synthesis of industrial chemicals and biofuels is drawing considerable attention as a promising alternative to microbial fermentation processes, which currently show low conversion yields and toxicity to host cells. Here, we provide a brief overview of the history of cell-free synthesis systems and the state-of-the-art cell-free technologies used to produce diverse chemicals and biofuels. We also discuss the future directions of cell-free synthesis that can fully harness the synthetic power of biological systems.

Valorization of lignocellulosic biomass for polyhydroxyalkanoate production: Status and perspectives.

With the increasing concerns regarding climate, energy, and plastic crises, bio-based production of biodegradable polymers has become a dire necessity. Significant progress has been made in biotechnology for the production of biodegradable polymers from renewable resources to achieve the goal of zero plastic waste and a net-zero carbon bioeconomy. In this review, an overview of polyhydroxyalkanoate (PHA) production from lignocellulosic biomass (LCB) was presented. Having established LCB-based biorefinery with proper pretreatment techniques, various PHAs could be produced from LCB-derived sugars, hydrolysates, and/or aromatic mixtures employing microorganisms. This provides a clue for addressing the current environmental crises because "biodegradable polymers" could be produced from one of the most abundant resources that are renewable and sustainable in a "carbon-neutral process". Furthermore, the potential future of LCB-to-non-natural PHA production was discussed with particular reference to non-natural PHA biosynthesis methods and LCB-derived aromatic mixture biofunnelling systems.

Microbial cell factories for the production of three-carbon backbone organic acids from agro-industrial wastes.

At present, mass production of basic and valuable commodities is dependent on linear petroleum-based industries, which ultimately makes the depletion of finite natural reserves and accumulation of non-biodegradable and hazardous wastes. Therefore, an ecofriendly and sustainable solution should be established for a circular economy where infinite resources, such as agro-industrial wastes, are fully utilized as substrates in the production of target value-added chemicals. Hereby, recent advances in metabolic engineering strategies and techniques used in the development of microbial cell factories for enhanced production of three-carbon platform chemicals such as lactic acid, propionic acid, and 3-hydroxypropionic acid are discussed. Further developments and future perspectives in the production of these organic acids from agro-industrial wastes from the dairy, sugar, and biodiesel industries are also highlighted to demonstrate the importance of waste-based biorefineries for organic acid production.

Consolidated microbial production of four-, five-, and six-carbon organic acids from crop residues: Current status and perspectives.

The production of platform organic acids has been heavily dependent on petroleum-based industries. However, petrochemical-based industries that cannot guarantee a virtuous cycle of carbons released during various processes are now facing obsolescence because of the depletion of finite fossil fuel reserves and associated environmental pollutions. Thus, the transition into a circular economy in terms of the carbon footprint has been evaluated with the development of efficient microbial cell factories using renewable feedstocks. Herein, the recent progress on bio-based production of organic acids with four-, five-, and six-carbon backbones, including butyric acid and 3-hydroxybutyric acid (C4), 5-aminolevulinic acid and citramalic acid (C5), and hexanoic acid (C6), is discussed. Then, the current research on the production of C4-C6 organic acids is illustrated to suggest future directions for developing crop-residue based consolidated bioprocessing of C4-C6 organic acids using host strains with tailor-made capabilities.

Bio-specific immobilization of enzymes on electrospun PHB nanofibers.

Enzyme immobilization provides substantial advantages in terms of improving the efficiency of enzymatic process as well as enhancing the reusability of enzymes. Phasins (PhaPs) are naturally occurring polyhydroxyalkanoate (PHA)-binding proteins, and thus can potentially be used as a fusion partner for oriented immobilization of enzymes onto PHA supports. However, presently available granular PHA supports have low surface-area-to-volume ratio and limited configurational flexibility of enzymatic reactions. In this study, we explored the use of electrospun polyhydroxybutyrate (PHB) nanofibers as an alternative support for high density immobilization of a PhaP-fused lipase. As envisioned, the electrospun PHB nanofibers could anchor 120-fold more enzyme than PHB granules of the same weight. Furthermore, the enzymes immobilized onto the PHB nanofibers exhibited markedly higher stability and activity compared to when immobilized on conventional immobilization supports. Our approach combines the advantageous features of nanofibrous material and specificity of biomolecular interaction for the efficient use of enzymes, which can be widely adopted in the development of various enzymatic processes.

Cell-Free Metabolic Engineering: Recent Developments and Future Prospects.

Due to the ongoing crises of fossil fuel depletion, climate change, and environmental pollution, microbial processes are increasingly considered as a potential alternative for cleaner and more efficient production of the diverse chemicals required for modern civilization. However, many issues, including low efficiency of raw material conversion and unintended release of genetically modified microorganisms into the environment, have limited the use of bioprocesses that rely on recombinant microorganisms. Cell-free metabolic engineering is emerging as a new approach that overcomes the limitations of existing cell-based systems. Instead of relying on metabolic processes carried out by living cells, cell-free metabolic engineering harnesses the metabolic activities of cell lysates in vitro. Such approaches offer several potential benefits, including operational simplicity, high conversion yield and productivity, and prevention of environmental release of microorganisms. In this article, we review the recent progress in this field and discuss the prospects of this technique as a next-generation bioconversion platform for the chemical industry.