High-level production and purification of bioactive recombinant human activin A in Chinese hamster ovary cells.

Activin A, a member of the TGF-ß superfamily, is a homodimer of the inhibin ßΑ subunit that plays a diversity of roles in biological processes. Because of its multiple functions, significant efforts have been made to produce activin A, however, unsatisfactory results were obtained due to its low level of expression. In this study, a stable CHO cell line exhibiting high expression of rhActivin A was isolated and production of rhActivin A was achieved using the cell line from 11-day fed-batch cultures in a 7.5 L bioreactor. The production rate was 0.22 g/L, substantially higher than those reported in previous studies. The culture supernatant of the bioreactor was used to purify rhActivin A (purity: >99%, recovery rate: 47%). The purified rhActivin A exhibited biological activity, with an EC50 of 3.893 ng/mL and a specific activity of 1.38 × 103 IU/mg. The control of process-related impurities in the purified rhActivin A was successful and met the USP recommendations for use in cell therapy. Thus, our production and purification methods were appropriate for large-scale GMP-grade rhActivin A production, which can be used for various purposes including cell therapy.

Ribosomal Protein S6: A Potential Therapeutic Target against Cancer?

Ribosomal protein S6 (RPS6) is a component of the 40S small ribosomal subunit and participates in the control of mRNA translation. Additionally, phospho (p)-RPS6 has been recognized as a surrogate marker for the activated PI3K/AKT/mTORC1 pathway, which occurs in many cancer types. However, downstream mechanisms regulated by RPS6 or p-RPS remains elusive, and the therapeutic implication of RPS6 is underappreciated despite an approximately half a century history of research on this protein. In addition, substantial evidence from RPS6 knockdown experiments suggests the potential role of RPS6 in maintaining cancer cell proliferation. This motivates us to investigate the current knowledge of RPS6 functions in cancer. In this review article, we reviewed the current information about the transcriptional regulation, upstream regulators, and extra-ribosomal roles of RPS6, with a focus on its involvement in cancer. We also discussed the therapeutic potential of RPS6 in cancer.

Efficient production process of bioactive recombinant human leukemia inhibitory factor in Chinese hamster ovary cells.

The rhLIF is widely used as an essential factor in stem cell cultures for cell therapies. However, all the recombinant LIFs commercially available are expensive, and no commercially available rhLIF meet the standards recommended by USP for use in cell therapies. The current study reports the efficient production of N-glycosylated and bioactive rhLIF in CHO cells. The production rate of established rhLIF-expressing rCHO cells was approximately 0.85 g/l in 12-day fed-batch cultures using a 7.5 l bioreactor. The rhLIF protein was purified via a four-step purification procedure with approximately 57% recovery rate and greater than 99% purity. The purified rhLIF was N-glycosylated and biologically active with an EC50 of 0.167 ng/ml and a specific activity of 0.86 × 103 IU/mg. The purification procedure controlled the levels of process-related impurities below critical levels recommended by USP for cytokines used in cell therapies. The current work is the first production process of N-glycosylated and bioactive rhLIF, which can be applied to large-scale manufacture of GMP-grade rhLIF for use as an ancillary material in cell therapy.

Generalized Nonlinear Least Squares Method for the Calibration of Complex Computer Code Using a Gaussian Process Surrogate.

The approximated nonlinear least squares (ALS) method has been used for the estimation of unknown parameters in the complex computer code which is very time-consuming to execute. The ALS calibrates or tunes the computer code by minimizing the squared difference between real observations and computer output using a surrogate such as a Gaussian process model. When the differences (residuals) are correlated or heteroscedastic, the ALS may result in a distorted code tuning with a large variance of estimation. Another potential drawback of the ALS is that it does not take into account the uncertainty in the approximation of the computer model by a surrogate. To address these problems, we propose a generalized ALS (GALS) by constructing the covariance matrix of residuals. The inverse of the covariance matrix is multiplied to the residuals, and it is minimized with respect to the tuning parameters. In addition, we consider an iterative version for the GALS, which is called as the max-minG algorithm. In this algorithm, the parameters are re-estimated and updated by the maximum likelihood estimation and the GALS, by using both computer and experimental data repeatedly until convergence. Moreover, the iteratively re-weighted ALS method (IRWALS) was considered for a comparison purpose. Five test functions in different conditions are examined for a comparative analysis of the four methods. Based on the test function study, we find that both the bias and variance of estimates obtained from the proposed methods (the GALS and the max-minG) are smaller than those from the ALS and the IRWALS methods. Especially, the max-minG works better than others including the GALS for the relatively complex test functions. Lastly, an application to a nuclear fusion simulator is illustrated and it is shown that the abnormal pattern of residuals in the ALS can be resolved by the proposed methods.

Expectation-Maximization Algorithm for the Calibration of Complex Simulator Using a Gaussian Process Emulator.

The approximated non-linear least squares (ALS) tunes or calibrates the computer model by minimizing the squared error between the computer output and real observations by using an emulator such as a Gaussian process (GP) model. A potential defect of the ALS method is that the emulator is constructed once and it is no longer re-built. An iterative method is proposed in this study to address this difficulty. In the proposed method, the tuning parameters of the simulation model are calculated by the conditional expectation (E-step), whereas the GP parameters are updated by the maximum likelihood estimation (M-step). These EM-steps are alternately repeated until convergence by using both computer and experimental data. For comparative purposes, another iterative method (the max-min algorithm) and a likelihood-based method are considered. Five toy models are tested for a comparative analysis of these methods. According to the toy model study, both the variance and bias of the estimates obtained from the proposed EM algorithm are smaller than those from the existing calibration methods. Finally, the application to a nuclear fusion simulator is demonstrated.

Pharmacokinetic and bioequivalence study of a fixed-dose combination of amlodipine besylate and rosuvastatin calcium compared to co-administration of separate tablets in healthy Korean subjects
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CONTEXT: A fixed-dose combination (FDC) tablet of amlodipine and rosuvastatin was recently developed for the treatment of concomitant hypertension and dyslipidemia and is anticipated to improve medication compliance. OBJECTIVE: This study was performed to compare the single-dose pharmacokinetic properties and safety of DP-R212 (FDC of amlodipine and rosuvastatin) to those of each agent co-administered in healthy Korean subjects. MATERIALS AND METHODS: A total of 36 healthy Korean subjects were enrolled in this randomized, open-label, single-dose, two-treatment, two-way crossover study. During each treatment period, subjects received the test drug (FDC tablet containing amlodipine and rosuvastatin) or reference drugs (individual tablets). Plasma samples were collected pre-dose and at 0.5, 1, 2, 3, 4, 5, 6, 8, 10, 12, 24, 48, and 72 hours post-dose. Safety was assessed by the evaluation of adverse events (AEs), laboratory assessments, 12-lead electrocardiograms (ECGs), physical examinations, and vital sign measurements. RESULTS: The 90% confidence intervals (CIs) of the geometric least-square mean ratios of AUClast and Cmax were 0.9796 - 1.0590 and 1.0135 - 1.0981 for amlodipine, and 0.9156 - 1.0490 and 0.8400 - 1.0306 for rosuvastatin, respectively. All AEs were of mild to moderate intensity, and no significant difference was observed in the incidence of AEs between the treatments. Moreover, the pharmacokinetic properties of the test and reference drugs were bioequivalent to each other, satisfying the regulatory criteria (0.8 - 1.25). DISCUSSION AND CONCLUSION: Both drugs were safe and well tolerated, and the pharmacokinetic profiles were comparable between the treatments.

Mowing: A cause of invasion, but also a potential solution for management of the invasive, alien plant species Erigeron annuus (L.) Pers.

Erigeron annuus is one of the major invasive, alien plants in Korea, and therefore research to manage (control) this invasive plant is essential. In this research, studies were conducted to determine the mechanisms by which E. annuus became the dominant plant at a landfill site and to develop management strategies for this alien plant. Because the seeds and seedling stage did not have superior adaptations to disturbed soil, demonstrate allelopathy, outcompete other species, or show rapid growth, the disturbance from mowing was likely the primary reason for the dominance of E. annuus. The areas without mowing showed a significant decrease in the coverage of E. annuus, whereas the mowed (managed) areas showed a significant increase. Additionally, mowing once increased the weight of reproductive organs by 50% and suppressed the growth of native species. Thus, the primary factor in the invasion of the alien species E. annuus was mowing, and, to control such an invasion, areas should be protected from mowing. Additionally, with selective mowing that targeted only E. annuus, mowing three times produced only approximately 10% of the reproductive organ biomass compared with that of the control. Because the flower stalk of E. annuus was relatively tall compared with that of native species in early summer, selective mowing might also provide a solution to control invasions of E. annuus. Therefore, with improved ecological understanding of the site and species, mowing of the right target during the optimal season and at an appropriate frequency is an environmental friendly solution to the management of E. annuus.

The ageing systemic milieu negatively regulates neurogenesis and cognitive function.

In the central nervous system, ageing results in a precipitous decline in adult neural stem/progenitor cells and neurogenesis, with concomitant impairments in cognitive functions. Interestingly, such impairments can be ameliorated through systemic perturbations such as exercise. Here, using heterochronic parabiosis we show that blood-borne factors present in the systemic milieu can inhibit or promote adult neurogenesis in an age-dependent fashion in mice. Accordingly, exposing a young mouse to an old systemic environment or to plasma from old mice decreased synaptic plasticity, and impaired contextual fear conditioning and spatial learning and memory. We identify chemokines--including CCL11 (also known as eotaxin)--the plasma levels of which correlate with reduced neurogenesis in heterochronic parabionts and aged mice, and the levels of which are increased in the plasma and cerebrospinal fluid of healthy ageing humans. Lastly, increasing peripheral CCL11 chemokine levels in vivo in young mice decreased adult neurogenesis and impaired learning and memory. Together our data indicate that the decline in neurogenesis and cognitive impairments observed during ageing can be in part attributed to changes in blood-borne factors.

Radiogenomic correlation in lung adenocarcinoma with epidermal growth factor receptor mutations: Imaging features and histological subtypes.

OBJECTIVES: To correlate imaging features of resected lung adenocarcinoma with epidermal growth factor receptor (EGFR) mutation and the IASLC/ATS/ERS classification histological subtypes. METHODS: In 250 consecutive patients with resected lung adenocarcinoma, EGFR mutation status was correlated with demographics, imaging features including ground-glass opacity (GGO) proportion and the IASLC/ATS/ERS classification histological subtypes. RESULTS: EGFR mutations were significantly more frequent in women (54.5 % vs. 38.1 %, p = 0.011) and in never-smokers (54.7 % vs. 35.3 %, p = 0.003). GGO proportion was significantly higher in tumours with EGFR mutation than in those without (30.3 ± 33.8 % vs. 19.0 ± 29.3 %, p = 0.005). EGFR mutation was significantly more frequent in tumours with GGO ≥ 50 % and tumours with any GGO (p = 0.026 and 0.008, respectively). Adenocarcinomas with exon 19 or 21 mutation showed significantly higher GGO proportion than that in EGFR wild-type tumours (p = 0.009 and 0.029, respectively). Absence of GGO was an independent predictor of negative EGFR mutation (odds ratio, 1.81; 95 % confidence interval, 1.16-3.04; p = 0.018). CONCLUSIONS: GGO proportion in adenocarcinomas with EGFR mutation was significantly higher than that in EGFR wild-type tumours, and the absence of GGO on CT was an independent predictor of negative EGFR mutation. KEY POINTS: • Ground-glass opacity (GGO) proportion is significantly higher in EGFR-mutated adenocarcinomas • Exon 19 or 21 mutated adenocarcinomas shows significantly higher GGO proportion • GGO absence is an independent predictor of negative EGFR mutation in lung adenocarcinomas.

A Randomized Comparison of Remifentanil Target-Controlled Infusion Versus Dexmedetomidine Single-Dose Administration: A Better Method for Smooth Recovery From General Sevoflurane Anesthesia.

Remifentanil target-controlled infusion and dexmedetomidine single-dose administration are known to reduce airway response and hemodynamic stimulation during anesthetic recovery. We compared the effects of 2 drugs on the prevention of cough during emergence. We enrolled 70 female patients aged 20-60 years with American Society of Anesthesiologists (ASA) I-II who underwent general anesthesia for elective thyroidectomy. The patients were randomly assigned to remifentanil (group R) or dexmedetomidine (group D). Anesthesia was maintained with sevoflurane and effect-site target-controlled infusion of remifentanil. In group D, remifentanil was discontinued, and dexmedetomidine 0.5 µg/kg was given 10 minutes before the end of surgery. In group R, remifentanil target-controlled infusion at an effective-site concentration of 2.0 ng/mL was maintained during emergence until extubation. The cough grade, hemodynamic values, respiration, and other recovery profiles were evaluated during the periextubation period. The proportion of patients with no cough or just a single cough during extubation was significantly higher in group R than in group D (96.8% vs. 55.9%). The change of mean arterial pressure and heart rate were not significantly different during extubation in both groups. Respiratory rate and the incidence of residual sedation after extubation were lower in group R. There were no desaturation events and no differences in time to extubation or duration of postanesthesia care unit stay in both groups. Remifentanil target-controlled infusion reduces emergence cough from general anesthesia more effectively than single-dose dexmedetomidine. However, a single-dose of dexmedetomidine has the effect with respect to respiratory and hemodynamic stability during emergence.

The minimum number of cord blood units needed for Koreans is 51,000.

BACKGROUND: The inventory size for cord blood (CB) depends on the ethnic diversity of human leukocyte antigen (HLA) and the size estimation is important for public health in each ethnicity. STUDY DESIGN AND METHODS: We estimate the CB inventory size in Koreans with stored CB units (CBUs) and patients who underwent allogeneic hematopoietic stem cell transplantation. Two-digit HLA specificities were determined using intermediate DNA typing. From 17,508 stored Korean CBUs, 1460 haplotypes with a frequency greater than 0.001% were used for reconstitution of the HLA. A total of 1002 transplanted patients' HLA was used for matching probability calculation. RESULTS: The best probability for 6/6 matching is 47% in 500,000 hypothetical size. Ninety-five percent probability is achieved with 51,000 CBUs in 5/6, and 2150 in 4/6 matching condition. Because 4/6 matched CB is rarely selected in the Korean situation, 51,000 units is the lowest limit of CBUs required and the number will be adjusted depending on the cell number required for patients and the resolution of HLA typing. CONCLUSION: Approximately 51,000 units could provide the minimum requirement for hematopoietic transplantation in Korea.

The 27 to 29-year outcomes of the PCA total hip arthroplasty in patients younger than 50 years old.

We previously reported our six and 19.4-year results of arthroplasty with the PCA total hip prosthesis. We now report on the performance of this prosthesis at 27-29years. Eighty-eight consecutive primary THAs using a PCA total hip system were performed in 70 patients (mean age, 45.6±11.1years). The mean follow-up was 28.4years (27-29). The mean Harris hip score was 89 points at final follow-up. Thigh pain was reported in 22 hips (25%). The mean annual polyethylene wear was 0.182±0.03mm. There were 75 acetabular (85%) and 40 femoral (45%) osteolysis. The rate of survival after 28.4years as the end point of revision was 66% for the acetabular component and 90% for the femoral component.

Rapid deep learning-assisted predictive diagnostics for point-of-care testing.

Prominent techniques such as real-time polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), and rapid kits are currently being explored to both enhance sensitivity and reduce assay time for diagnostic tests. Existing commercial molecular methods typically take several hours, while immunoassays can range from several hours to tens of minutes. Rapid diagnostics are crucial in Point-of-Care Testing (POCT). We propose an approach that integrates a time-series deep learning architecture and AI-based verification, for the enhanced result analysis of lateral flow assays. This approach is applicable to both infectious diseases and non-infectious biomarkers. In blind tests using clinical samples, our method achieved diagnostic times as short as 2 minutes, exceeding the accuracy of human analysis at 15 minutes. Furthermore, our technique significantly reduces assay time to just 1-2 minutes in the POCT setting. This advancement has the potential to greatly enhance POCT diagnostics, enabling both healthcare professionals and non-experts to make rapid, accurate decisions.

Overproduction of recombinant human mannose-binding lectin (MBL) in Chinese hamster ovary cells.

Mannose-binding lectin (MBL) is an important serum protein that functions in the innate immune system and has been considered to have therapeutic potential in MBL replacement therapies for patients with deficient or low levels of MBL. In this study, we established a Chinese hamster ovary (CHO) cell line that overexpresses the recombinant human MBL (rhMBL) protein. In an 11-day batch culture process using a 30-L bioreactor (20-L working volume) and serum-free medium, these cells could produce over 226 mg/L of rhMBL protein. The recombinant protein was then purified to homogeneity from the culture supernatant using a three-step chromatographic procedure that resulted in a recovery rate of approximately 55%. This purified rhMBL protein adopted oligomeric bouquet-like structures that were similar to those of native MBL present in human blood, and these oligomeric structures were reported to be critical in MBL functions. We further demonstrated in carbohydrate binding and complementation activation assays that this rhMBL protein was functionally active with very similar dissociation constants and half maximal effective concentrations to those of native MBL.

Unstable expression of recombinant antibody during long-term culture of CHO cells is accompanied by histone H3 hypoacetylation.

The gradual loss of recombinant protein expression in CHO cell lines during prolonged subculture is a common issue, referred to as instability, which seriously affects the industrial production processes of therapeutic proteins. Loss of recombinant gene copies, due to the genetic instability of CHO cells, and epigenetic silencing of transgene sequences, are the main reported causes of production instability. To increase our understanding on the molecular mechanisms inherent to CHO cells involved in production instability, we explored the molecular features of stable and unstable antibody producing cell lines obtained without gene amplification, to exclude the genetic instability induced by the gene amplification process. The instability of recombinant antibody production during long-term culture was caused by a 48-53% decrease in recombinant mRNA levels without significant loss of recombinant gene copies, but accompanied by a ~45% decrease in histone H3 acetylation (H3ac). Thus, our results suggest a critical role of H3ac in the stability of recombinant protein production.

Predicting the spatio-temporal distribution of the invasive alien plant Andropogon virginicus, in the South Korean peninsula considering long-distance dispersal capacities.

The spread of invasive alien species is a major threat to biodiversity. Estimating the long-distance dispersal capacity of invasive alien plants is vital for understanding their population dynamics and community composition. We predicted the spatial-temporal distribution of the alien plant Andropogon virginicus, in the Korean peninsula under climate change scenario using Random Forest (RF) and Cellular Automaton (CA) methods. Land use, barriers to dispersal, long-distance dispersal frequency, and maximum long-distance dispersal range were considered in our analysis. Our results showed that, among the five selected environmental variables, annual mean temperature and Human Foot-Printing (HFP) were positively associated with the occurrence probability of A. virginicus. This suggests that A. virginicus is likely to spread to the disturbed northern part of the Korean Peninsula due to climate change and habitat preference. When comparing modeling results for dispersal to field survey data, the modeling raster sets drawn from the long-distance dispersal frequency of 0.05 and maximum long-distance dispersal distance of 30 km y-1 had the most similar spatial expansion among the six long-distance dispersal parameter sets. The dispersal directions were associated with the landscape. Specifically, seeds dispersed by wind (anemochorous seeds) could propagate into open landscapes more easily than in forests. Regarding A. virginicus management, this grass can quickly invade bare ground with their wind-dispersed seeds, therefore habitat destruction, such as excessive logging and weeding, should be restrained.

Affordable on-site COVID-19 test using non-powered preconcentrator.

A simple, affordable point of care test (POCT) is necessary for on-site detection of coronavirus disease 2019 (COVID-19). The lateral flow assay (LFA) has great potential for use in POCT mainly because of factors such as low time consumption, low cost, and ease of use. However, it lacks sensitivity and limits of detection (LOD), which are essential for early diagnostics. In this study, we proposed a non-powered preconcentrator (NPP) based on nanoelectrokinetics for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Antigen (Ag) lateral flow assay. The non-powered preconcentrator is composed of glass fiber-based composite paper and ion permselective material, and it can be simply operated by force balancing gravitational, capillary, and depletion-induced forces. The proposed approach helps enrich the SARS-CoV-2 viral nucleocapsid (N) proteins based on a 10-min operation, and it improved the LOD by up to 10-fold. The corresponding virus enrichment, which was evaluated using the reverse-transcriptase polymerase chain reaction (RT-PCR), revealed an improvement in ΔCt values > 3. We successfully demonstrated the enhancement of the NPP-assisted LFA, we extended to applying it to clinical samples. Further, we demonstrated an affordable, easy-to-implement form of LFA by simply designing NPP directly on the LFA buffer tube.

Sample-to-answer platform for the clinical evaluation of COVID-19 using a deep learning-assisted smartphone-based assay.

Since many lateral flow assays (LFA) are tested daily, the improvement in accuracy can greatly impact individual patient care and public health. However, current self-testing for COVID-19 detection suffers from low accuracy, mainly due to the LFA sensitivity and reading ambiguities. Here, we present deep learning-assisted smartphone-based LFA (SMARTAI-LFA) diagnostics to provide accurate decisions with higher sensitivity. Combining clinical data learning and two-step algorithms enables a cradle-free on-site assay with higher accuracy than the untrained individuals and human experts via blind tests of clinical data (n = 1500). We acquired 98% accuracy across 135 smartphone application-based clinical tests with different users/smartphones. Furthermore, with more low-titer tests, we observed that the accuracy of SMARTAI-LFA was maintained at over 99% while there was a significant decrease in human accuracy, indicating the reliable performance of SMARTAI-LFA. We envision a smartphone-based SMARTAI-LFA that allows continuously enhanced performance by adding clinical tests and satisfies the new criterion for digitalized real-time diagnostics.

PCR-like performance of rapid test with permselective tunable nanotrap.

Highly sensitive rapid testing for COVID-19 is essential for minimizing virus transmission, especially before the onset of symptoms and in asymptomatic cases. Here, we report bioengineered enrichment tools for lateral flow assays (LFAs) with enhanced sensitivity and specificity (BEETLES2), achieving enrichment of SARS-CoV-2 viruses, nucleocapsid (N) proteins and immunoglobulin G (IgG) with 3-minute operation. The limit of detection is improved up to 20-fold. We apply this method to clinical samples, including 83% with either intermediate (35%) or low viral loads (48%), collected from 62 individuals (n = 42 for positive and n = 20 for healthy controls). We observe diagnostic sensitivity, specificity, and accuracy of 88.1%, 100%, and 91.9%, respectively, compared with commercial LFAs alone achieving 14.29%, 100%, and 41.94%, respectively. BEETLES2, with permselectivity and tunability, can enrich the SARS-CoV-2 virus, N proteins, and IgG in the nasopharyngeal/oropharyngeal swab, saliva, and blood serum, enabling reliable and sensitive point-of-care testing, facilitating fast early diagnosis.

Penalized likelihood approach for the four-parameter kappa distribution.

The four-parameter kappa distribution (K4D) is a generalized form of some commonly used distributions such as generalized logistic, generalized Pareto, generalized Gumbel, and generalized extreme value (GEV) distributions. Owing to its flexibility, the K4D is widely applied in modeling in several fields such as hydrology and climatic change. For the estimation of the four parameters, the maximum likelihood approach and the method of L-moments are usually employed. The L-moment estimator (LME) method works well for some parameter spaces, with up to a moderate sample size, but it is sometimes not feasible in terms of computing the appropriate estimates. Meanwhile, using the maximum likelihood estimator (MLE) with small sample sizes shows substantially poor performance in terms of a large variance of the estimator. We therefore propose a maximum penalized likelihood estimation (MPLE) of K4D by adjusting the existing penalty functions that restrict the parameter space. Eighteen combinations of penalties for two shape parameters are considered and compared. The MPLE retains modeling flexibility and large sample optimality while also improving on small sample properties. The properties of the proposed estimator are verified through a Monte Carlo simulation, and an application case is demonstrated taking Thailand's annual maximum temperature data.