Effective Invasiveness Recognition of Imbalanced Data by Semi-Automated Segmentations of Lung Nodules.

Over the past few decades, recognition of early lung cancers was researched for effective treatments. In early lung cancers, the invasiveness is an important factor for expected survival rates. Hence, how to effectively identify the invasiveness by computed tomography (CT) images became a hot topic in the field of biomedical science. Although a number of previous works were shown to be effective on this topic, there remain some problems unsettled still. First, it needs a large amount of marked data for a better prediction, but the manual cost is high. Second, the accuracy is always limited in imbalance data. To alleviate these problems, in this paper, we propose an effective CT invasiveness recognizer by semi-automated segmentation. In terms of semi-automated segmentation, it is easy for doctors to mark the nodules. Just based on one clicked pixel, a nodule object in a CT image can be marked by fusing two proposed segmentation methods, including thresholding-based morphology and deep learning-based mask region-based convolutional neural network (Mask-RCNN). For thresholding-based morphology, an initial segmentation is derived by adaptive pixel connections. Then, a mathematical morphology is performed to achieve a better segmentation. For deep learning-based mask-RCNN, the anchor is fixed by the clicked pixel to reduce the computational complexity. To incorporate advantages of both, the segmentation is switched between these two sub-methods. After segmenting the nodules, a boosting ensemble classification model with feature selection is executed to identify the invasiveness by equalized down-sampling. The extensive experimental results on a real dataset reveal that the proposed segmentation method performs better than the traditional segmentation ones, which can reach an average dice improvement of 392.3%. Additionally, the proposed ensemble classification model infers better performances than the compared method, which can reach an area under curve (AUC) improvement of 5.3% and a specificity improvement of 14.3%. Moreover, in comparison with the models with imbalance data, the improvements of AUC and specificity can reach 10.4% and 33.3%, respectively.

Association of CD34+ Cell Dose with Progression-free Survival after Allogeneic Peripheral Blood Hematopoietic Cell Transplantation in Children with Hematologic Malignancies.

BACKGROUND: A higher CD34+ cell dose is associated with improved engraftment but may also be associated with an increased risk of complications after allogeneic hematopoietic stem cell transplantation, including graft-versus-host disease (GVHD). METHODS: We retrospectively analyze the impact of CD34+ cell dose on OS, PFS, neutrophil engraftment, platelet engraftment, treatment-related mortality, and GVHD grading. RESULTS: For analyses, CD34+ cell dose was stratified into low (< 8.5 × 106/kg) and high (> 8.5 × 106/kg). A subgroup analysis of higher CD34+ cell dose leads to prolonged OS and PFS, but statistical significance was achieved only for PFS (OR 0.36; 95%CI 0.14-0.95; P = 0.04). CONCLUSIONS: This study reinforced that CD34+ cell dose at the time of allo-HSCT retained a positive impact on PFS.

Melatonin protects against methotrexate hepatotoxicity in young rats: Impact of PI3K/Akt/mTOR signaling.

With the improvement in children's acute lymphoblastic leukemia (ALL) care, the survival rate in children ALL has improved much. Methotrexate (MTX) plays an essential role in the success of children's ALL treatment. Since hepatotoxicity is commonly reported in individuals treated with intravenous or oral MTX, our study further examined the hepatic effect following intrathecal MTX treatment, which is an essential treatment for leukemia patients. Specifically, we examined the pathogenesis of MTX hepatotoxicity in young rats and explored the impact of melatonin treatment in protection against MTX hepatotoxicity. Successfully, we found that melatonin was able to protect against MTX hepatotoxicity.

Downregulation of AANAT by c-Fos in tubular epithelial cells with membranous nephropathy.

Melatonin is a hormone majorly secreted by the pineal gland and contributes to a various type of physiological functions in mammals. The melatonin production is tightly limited to the AANAT level, yet the most known molecular mechanisms underlying AANAT gene transcription is limited in the pinealocyte. Here, we find that c-Fos and cAMP-response element-binding protein (CREB) decreases and increases the AANAT transcriptional activity in renal tubular epithelial cell, respectively. Notably, c-Fos knockdown significantly upregulates melatonin levels in renal tubular cells. Functional results indicate that AANAT expression is decreased by c-Fos and resulted in enhancement of cell damage in albumin-injury cell model. We further find an inverse correlation between c-Fos and AANAT levels in renal tubular cells from experimental membranous nephropathy (MN) samples and clinical MN specimens. Our finding provides the molecular basis of c-Fos in transcriptionally downregulating expression of AANAT and melatonin, and elucidate the protective role of AANAT in preventing renal tubular cells death in albumin-injury cell model and MN progression.

Pediatric thalassemic patients have higher incidence of asthma: A nationwide population-based retrospective cohort study.

INTRODUCTION: Patients with hemoglobinopathies have been reported to have higher rates of pulmonary complications. Few studies have investigated the association between thalassemia and asthma in children. METHODS: We used the data of one million individuals randomly selected from the Registry for Beneficiaries of the National Health Insurance Research Database. One thalassemic child was matched with four control children without thalassemia according to sex, birth year, birth season, prematurity, and previous enteroviral infection. RESULTS: A total of 800 hundred thalassemic children and 3200 controls were included. Children with thalassemia had higher rates of developing asthma (41.81 vs 25.70 per 1000 person-years, P < 0.001) than the non-thalassemia controls with an adjusted hazard ratio of 1.37 (95% confidence interval [CI] = 1.19-1.58). Boys in the thalassemia cohort had a significantly higher adjusted incidence hazard ratio (IRR) of asthma than those in the non-thalassemia cohort (adjusted IRR = 1.45, 95% CI = 1.02-1.73). The risk of atopic and nonatopic asthma was higher in the thalassemia cohort than in the non-thalassemia cohort (IRR = 1.3, 1.61, respectively). CONCLUSIONS: Children with thalassemia were more likely to develop asthma. More attention should be paid to the early diagnosis of asthma and prevention of asthma attacks.

Neonatal mortality among outborn versus inborn babies.

BACKGROUND: Most previous studies reported there were higher survival rates if low birth weight babies were born in tertiary perinatal centers (inborn) than elsewhere (outborn). The objective of this study is to examine whether the number and ratio of outborn babies decrease and the neonatal mortality differs between inborn and outborn babies. METHODS: We used the pooled data of the Taiwan Clinical Effectiveness Index for the years 2011-2016 obtained from the Joint Commission of Taiwan to study the outborn/inborn number and neonatal mortality rate. RESULTS: We found that the number of outborn babies did not decrease year by year. The ratio of outborn to total babies was lower in the groups of birth body weight 750-999 g and ≧ 2500 g than the other groups. The neonatal mortality rate in outborns was significantly higher than the inborns in the groups of birth body weight 1000-1499 g, 2000-2499 g and ≧ 2500 g (6.9 ± 2.4 vs. 3.8 ± 0.9, P = 0.009, 2.6 ± 0.6 vs. 0.6 ± 0.3, P = 0.002 and 1.52 ± 0.67 vs. 0.08 ± 0.02, P = 0.002, respectively) in medical centers. CONCLUSION: Improved maternal transport which promotes in utero transfer of patients may further improve neonatal outcome.

The MTNR1A mRNA is stabilized by the cytoplasmic hnRNPL in renal tubular cells.

The downregulation of melatonin receptor 1A (MTNR1A) is associated with a range of pathological conditions, including membranous nephropathy. Knowledge of the mechanism underlying MTNR1A expression has been limited to the transcriptional regulation level. Here, RNA interference screening in human kidney cells revealed that heterogeneous nuclear ribonucleoprotein L (hnRNPL) upregulated MTNR1A RNA post-transcriptionally. hnRNPL knockdown or overexpression led to increased or decreased levels of cyclic adenosine monophosphate-responsive element-binding protein phosphorylation, respectively. Molecular studies showed that cytoplasmic hnRNPL exerts a stabilizing effect on the MTNR1A transcript through CA-repeat elements in its coding region. Further studies revealed that the interaction between hnRNPL and MTNR1A serves to protect MNTR1A RNA degradation by the exosome component 10 protein. MTNR1A, but not hnRNPL, displays a diurnal rhythm in mouse kidneys. Enhanced levels of MTNR1A recorded at midnight correlated with robust binding activity between cytoplasmic hnRNPL and the MTNR1A transcript. Both hnRNPL and MTNR1A were decreased in the cytoplasm of tubular epithelial cells from experimental membranous nephropathy kidneys, supporting their clinical relevance. Collectively, our data identified cytoplasmic hnRNPL as a novel player in the upregulation of MTNR1A expression in renal tubular epithelial cells, and as a potential therapeutic target.

The "Societalization" of pandemic unpreparedness: lessons from Taiwan's COVID response.

Adopting a Civil Sphere Theory framework, we argue that Taiwan's efforts at containing COVID-19 resulted from its "societalization" of pandemic unpreparedness, which was triggered by the 2003 SARS outbreak and resumed during the COVID-19 pandemic. Societalization refers to the process through which institutional failures are transformed into societal crises, with the civil sphere mobilized to discuss institutional dysfunctions, push for reforms, and attempt to democratize or otherwise transform institutional cultures. The societalization of pandemic unpreparedness in Taiwan led to reforms of the public health administration and the medical profession, thereby establishing state mechanisms for encouraging early responses and coordinating centralized command during outbreaks, and healthcare infrastructures for coordinating patient transfer and ensuring supplies of personal protective equipment. Reflections upon past uncivil acts among citizens motivated the civil sphere to foster a discourse of interdependence, redefining the boundaries between individual choices and civic virtues. Meanwhile, unaddressed challenges remained, including threats related to Taiwan's political polarization. Our paper challenges the thesis of "authoritarian advantage," highlighting how democratic societies can foster social preparedness to respond to crises. By illustrating how societalization can reach temporary closures but become reactivated subsequently, our study extends the theory of societalization by explicating its historical dimension.

Maternal Iron Deficiency Programs Offspring Cognition and Its Relationship with Gastrointestinal Microbiota and Metabolites.

Iron is an essential micronutrient for the brain development of the fetus. Altered intestinal microbiota might affect behavior and cognition through the so-called microbiota-gut-brain axis. We used a Sprague-Dawley rat model of a maternal low-iron diet to explore the changes in cognition, dorsal hippocampal brain-derived neurotrophic factor (BDNF) and related pathways, gut microbiota, and related metabolites in adult male offspring. We established maternal iron-deficient rats by feeding them a low-iron diet (2.9 mg/kg), while the control rats were fed a standard diet (52.3 mg/kg). We used a Morris water maze test to assess spatial learning and long-term memory. Western blot (WB) assays and a quantitative reverse-transcription polymerase chain reaction (qRT-PCR) were used to detect the BDNF concentration and related signaling pathways. We collected fecal samples for microbiota profiling and measured the concentrations of plasma short-chain fatty acids. The adult male offspring of maternal rats fed low-iron diets before pregnancy, during pregnancy and throughout the lactation period had (1) spatial deficits, (2) a decreased BDNF mRNA expression and protein concentrations, accompanied by a decreased TrkB protein abundance, (3) a decreased plasma acetate concentration, and (4) an enrichment of the Bacteroidaceae genus Bacteroides and Lachnospiraceae genus Marvinbryantia. Maternal iron deficiency leads to an offspring spatial deficit and is associated with alternations in gastrointestinal microbiota and metabolites.

Triple-Junction Optoelectronic Sensor with Nanophotonic Layer Integration for Single-Molecule Level Decoding.

Interest in developing a rapid and robust DNA sequencing platform has surged over the past decade. Various next-/third-generation sequencing mechanisms have been employed to replace the traditional Sanger sequencing method. In sequencing by synthesis, a signal is monitored by a scanning charge-coupled device (CCD) to identify thousands to millions of incorporated dNTPs with distinctive fluorophores on a chip. Because one reaction site usually occupies dozens of pixels on a CCD detector, a bottleneck related to the bandwidth of CCD imaging limits the throughputs of the sequencing performance and causes trade-offs among speed, accuracy, read length, and the numbers of reaction sites in parallel. Thus, current research aims to align one reaction site to a few pixels by directly stacking nanophotonic layers onto a CMOS detector to minimize the size of the sequencing platforms and accelerate the processing procedures. This article reports a custom integrated optoelectronic device based on a triple-junction photodiode (TPD) CMOS sensor in conjunction with NPL integration for real-time illumination and detection of fluorescent molecules.

Role of melatonin receptor 1A and pituitary homeobox-1 coexpression in protecting tubular epithelial cells in membranous nephropathy.

Membranous nephropathy (MN), a type of glomerular nephritis, is one of the most common causes of nephrotic syndrome in adults. Although it is known that melatonin plays a protective role in MN, the role of melatonin receptors in the pathophysiology of MN is unclear. Using an experimental MN model and clinical MN specimens, we studied melatonin receptor expression and found that melatonin receptor 1A (MTNR1A) expression was significantly downregulated in renal tubular epithelial cells. Molecular studies showed that the transcription factor pituitary homeobox-1 (PITX1) promoted MTNR1A expression via direct binding to its promoter. Treatment of a human tubular cell line with albumin to induce injury resulted in the stable reduction in MTNR1A and PITX1 expression. PITX1 levels were significantly downregulated in tubular epithelial cells from mice MN kidneys and MN renal specimens. Knockdown of MTNR1A, PITX1, or cyclic adenosine monophosphate-responsive element-binding protein (CREB) decreased E-cadherin (CDH1) expression, but upregulated Per2 and α-smooth muscle actin (αSMA) expression. Blockade of the MTNR1A receptor with luzindole in MN mice further impaired renal function; this was accompanied by CDH1 downregulation and Per2 and αSMA upregulation. Together, our results suggest that in injured tissue, decreased PITX1 expression at the MTNR1A promoter regions leads to decreased levels of MTNR1A in renal tubular epithelial cells, which increases the future risk of MN.

A DNA dot hybridization model for molecular diagnosis of parasitic keratitis.

PURPOSE: Developing a DNA dot hybridization model for diagnosing parasitic keratitis. METHODS: Newly designed oligonucleotide probes for detecting Acanthamoeba and microsporidia were tested with target reference strains of Acanthamoeba (n = 20) and microsporidia (n = 3), and non-target microorganisms, including bacteria (n = 20) and fungi (n = 20). These probes, which had passed the preliminary tests, were then assembled as a parasite dot hybridization (PDH) model for assessing 33 clinical samples from patients with clinically suspected Acanthamoeba and microsporidia keratitis, including eight positives for Acanthamoeba, 13 positives for microsporidia, and 12 negatives for both pathogens. RESULTS: Two probes for detecting Acanthamoeba and two for detecting microsporidia passed the tests using target and non-target strains and then were assembled in the PDH model. For clinical samples, one Acanthamoeba-positive sample (proved with pathology) was falsely negative according to the PDH assay. The sensitivity and specificity of the PDH assay for diagnosing Acanthamoeba keratitis were 87.5% and 100%, respectively, while the sensitivity and specificity for diagnosing microsporidia keratitis were 100%. The infectious agent of all clinical samples of microsporidia keratitis was identified as Vittaforma corneae with DNA sequencing, while those of Acanthamoeba keratitis were caused by four species of Acanthamoeba, with Acanthamoeba castellanii found in four samples (50%, 4/8). CONCLUSIONS: The PDH model has the potential to be a molecular assay for diagnosing Acanthamoeba and microsporidia keratitis. However, a prospective clinical study might be needed before the model is adopted in routine clinical practice.

Gradient Strain Chip for Stimulating Cellular Behaviors in Cell-laden Hydrogel.

Artificial guidance for cellular alignment is a hot topic in the field of tissue engineering. Most of the previous research has investigated single strain-induced cellular alignment on a cell-laden hydrogel by using complex experimental processes and mass controlling systems, which are usually associated with contamination issues. Thus, in this article, we propose a simple approach to building a gradient static strain using a fluidic chip with a plastic PDMS cover and a UV transparent glass substrate for the stimulation of cellular behavior in a 3D hydrogel. Overloading photo-patternable cell prepolymer in the fluidic chamber can generate a convex curved PDMS membrane on the cover. After UV crosslinking, through a concentric circular micropattern under the curved PDMS membrane, and buffer washing, a microenvironment for investigating cell behaviors under a variety of gradient strains is self-established in a single fluidic chip, without external instruments. NIH3T3 cells were demonstrated after observing the change in the cellular alignment trend under geometry guidance, in cooperation with strain stimulation, which varied from 15 - 65% on hydrogels. After a 3-day incubation, the hydrogel geometry dominated the cell alignment under low compressive strain, where cells aligned along the hydrogel elongation direction under high compressive strain. Between these, the cells showed random alignment due to the dissipation of the radical guidance of hydrogel elongation and the geometry guidance of the patterned hydrogel.

Inhibition of tumor necrosis factor signaling attenuates renal immune cell infiltration in experimental membranous nephropathy.

Idiopathic membranous nephropathy (MN) is an autoimmune-mediated glomerulonephritis and the most common cause of idiopathic nephrotic syndrome in adult humans. A tumor necrosis factor α (TNF-α)-mediated inflammatory response via TNF receptor 1 (TNFR1) and TNFR2 has been proposed as a pathogenic factor. In this study, we assessed the therapeutic response to blocking TNF signaling in experimental MN. Murine MN was induced experimentally by cationic bovine serum albumin (cBSA); phosphate-buffered saline was used in control mice. In MN mice, TNF was inhibited by etanercept blocking of TNFR1/TNFR2 or the preligand assembly domain fusion protein (PLAD.Fc), a small fusion protein that can preferentially block TNFR1 signaling. Disease severity and possible mechanisms were assessed by analyzing the metabolic and histopathology profiles, lymphocyte subsets, immunoglobulin production, oxidative stress, and apoptosis. cBSA-induced MN mice exhibited typical nephrotic syndrome and renal histopathology. MN mice given etanercept or PLAD.Fc did not exhibit significant reduction of proteinuria, amelioration of glomerular lesions, or attenuation of immune complex deposition. Immune cell subsets, serum immunoglobulin levels, production of reactive oxygen species, and cell apoptosis in the kidney were not altered by TNF inhibition. By contrast, MN mice receiving etanercept or PLAD.Fc exhibited significantly decreased infiltration of immune cells into the kidney. These results show that the therapeutic effects of blocking TNFR1 and/or TNFR2 signaling in experimental MN are not clinically effective. However, TNF signaling inhibition significantly attenuated renal immune cell infiltration in experimental MN.

Constructing 3D heterogeneous hydrogels from electrically manipulated prepolymer droplets and crosslinked microgels.

Formation of multifunctional, heterogeneous, and encoded hydrogel building blocks, or microgels, by crosslinking and assembly of microgels are two essential steps in establishing hierarchical, complicated, and three-dimensional (3D) hydrogel architectures that recapitulate natural and biological structures or originate new materials by design. However, for the variety of the hydrogel materials crosslinked differently and for the varied scales of microgels and architectures, the formation and assembly processes are usually performed separately, which increases the manufacturing complexity of designed hydrogel materials. We show the construction of hydrogel architectures through programmable formation and assembly on an electromicrofluidic platform, adopting two reciprocal electric manipulations (electrowetting and dielectrophoresis) to manipulate varied objects (i) in multiple phases, including prepolymer liquid droplets and crosslinked microgels, (ii) on a wide range of scales from micrometer functional particles or cells to millimeter-assembled hydrogel architectures, and (iii) with diverse properties, such as conductive and dielectric droplets that are photocrosslinkable, chemically crosslinkable, or thermally crosslinkable. Prepolymer droplets, particles, and dissolved molecules are electrically addressable to adjust the properties of the microgel building blocks in liquid phase that subsequently undergo crosslinking and assembly in a flexible sequence to accomplish heterogeneous and seamless hydrogel architectures. We expect the electromicrofluidic platform to become a general technique to obtain 3D complex architectures.

Rapid detection of dermatophytes and Candida albicans in onychomycosis specimens by an oligonucleotide array.

BACKGROUND: Onychomycosis is a fungal infection of nails, leading to the gradual destruction of the nail plate. Treatment of onychomycosis may need long-time oral antifungal therapy that can have potential side effects, thus accurate diagnosis of the disease before treatment is important. Culture for diagnosis of onychomycosis is time-consuming and has high false-negative rates. To expedite the diagnosis, an oligonucleotide array, based on hybridization between immobilized oligonucleotide probes and PCR products, for direct detection of dermatophytes and Candida albicans in clinical specimens was evaluated. METHODS: Species-specific oligonucleotide probes designed from the internal transcribed spacer (ITS) regions of the rRNA gene were immobilized on a nylon membrane. The assay procedures consisted of PCR amplification of the ITS using universal primers, followed by hybridization of the digoxigenin-labeled amplicons to probes on the array. Thirty two nail samples (29 patients) were analyzed by the array, and the results were compared with those obtained by culture. Array-positive but culture-negative samples were confirmed by cloning and re-sequencing of the amplified ITS and by reviewing patient's clinical data. The total recovery of culture and confirmed array-positive but culture-negative results was considered 100% and was used for performance evaluation of both methods. RESULTS: Concordant results were obtained in 21 samples (10 positives and 11 negatives) by both methods. Eleven samples were array-positive but culture-negative; among them, 9 samples were considered true positives after discrepant analysis. Comparing with culture, the array had significantly higher sensitivity [100% (95% CI 82.2% -100%) vs 52.6% (28.9% -75.5%), p <0.001] and negative predictive value [100% (71.3% -100%) vs 59.1% (36.4% -79.3%), p <0.05), while no significant differences were observed in specificity (84.6% vs 100%, p =0.48) and positive predictive value (90.5% vs 100%, p =1.0). The whole procedures of the array were about 24 h, whilst results from culture take 1 to 3 weeks. CONCLUSIONS: The array offers an accurate and rapid alternative to culture. Rapid diagnosis can expedite appropriate antifungal treatment of onychomycosis. However, the single site nature of this study conducted at a referral hospital invites caution.

To pre-challenge lactic acid bacteria with simulated gastrointestinal conditions is a suitable approach to studying potential probiotic properties.

The potential probiotic properties of lactic acid bacteria (LAB) after treatment with gastrointestinal (GI) conditions were investigated. Some LAB strains that survived simulated GI treatment retained their adhesiveness and antagonism against the pathogen. Therefore pre-challenging LAB with simulated GI conditions is a suitable way for potential probiotic studies.

Urinary Xist is a potential biomarker for membranous nephropathy.

Membranous nephropathy (MN), a type of glomerular nephritis, is the most common cause of nephrotic syndrome in human adults. Changes in gene expression as a result of epigenetic dysregulation through long noncoding RNAs (lncRNAs) are increasingly being recognized as important factors in disease. Using an experimental MN mouse model, we identify the first dysregulated lncRNAs, Xist and NEAT1, whose levels are significantly upregulated in both tubular epithelial and glomerular cells. MN is also often characterized by glomerular podocyte injury. Treatment of a mouse podocyte cell line with lipopolysaccharides to induce injury resulted in the stable elevation of Xist, but not NEAT1 levels. In mice, the observed changes in Xist levels are specific: Xist can be effectively detected in urine, with a strong correlation to disease severity, but not serum in MN samples. We find that regulation of Xist may be controlled by post-translational modifications. H3K27me3 levels are significantly downregulated in mouse MN kidney, where chromatin immunoprecipitation experiments also showed decreased H3K27me3 at Xist promoter regions. Finally, we show that our findings in mice can be extended to human clinical samples. Urinary Xist is significantly elevated in urine samples from patients with different types of glomerular nephritis, including MN, compared to normal counterparts. Together, our results suggest that a reduction of H3K27me3 at Xist promoter regions leads to elevated levels of urinary Xist, which may be used as a biomarker to detect MN.

Mutations in the non-structural protein region contribute to intra-genotypic evolution of enterovirus 71.

BACKGROUND: Clinical manifestations of enterovirus 71 (EV71) range from herpangina, hand-foot-and-mouth disease (HFMD), to severe neurological complications. Unlike the situation of switching genotypes seen in EV71 outbreaks during 1998-2008 in Taiwan, genotype B5 was responsible for two large outbreaks in 2008 and 2012, respectively. In China, by contrast, EV71 often persists as a single genotype in the population and causes frequent outbreaks. To investigate genetic changes in viral evolution, complete EV71 genome sequences were used to analyze the intra-genotypic evolution pattern in Taiwan, China, and the Netherlands. RESULTS: Genotype B5 was predominant in Taiwan's 2008 outbreak and was re-emergent in 2012. EV71 strains from both outbreaks were phylogenetically segregated into two lineages containing fourteen non-synonymous substitutions predominantly in the non-structural protein coding region. In China, genotype C4 was first seen in 1998 and caused the latest large outbreak in 2008. Unlike shifting genotypes in Taiwan, genotype C4 persisted with progressive drift through time. A majority of non-synonymous mutations occurred in residues located in the non-structural coding region, showing annual increases. Interestingly, genotype B1/B2 in the Netherlands showed another stepwise evolution with dramatic EV71 activity increase in 1986. Phylogeny of the VP1 coding region in 1971-1986 exhibited similar lineage turnover with genotype C4 in China; however, phylogeny of the 3D-encoding region indicated separate lineage appearing after 1983, suggesting that the 3D-encoding region of genotype B2 was derived from an unidentified ancestor that contributed to intra-genotypic evolution in the Netherlands. CONCLUSIONS: Unlike VP1 coding sequences long used for phylogenetic study of enteroviruses due to expected host immune escape, our study emphasizes a dominant role of non-synonymous mutations in non-structural protein regions that contribute to (re-)emergent genotypes in continuous stepwise evolution. Dozens of amino acid substitutions, especially in non-structural proteins, were identified via genetic changes driven through intra-genotypic evolution worldwide. These identified substitutions appeared to increase viral fitness in the population, affording valuable insights not only for viral evolution but also for prevention, control, and vaccine against EV71 infection.

Gradient static-strain stimulation in a microfluidic chip for 3D cellular alignment.

Cell alignment is a critical factor to govern cellular behavior and function for various tissue engineering applications ranging from cardiac to neural regeneration. In addition to physical geometry, strain is a crucial parameter to manipulate cellular alignment for functional tissue formation. In this paper, we introduce a simple approach to generate a range of gradient static strains without external mechanical control for the stimulation of cellular behavior within 3D biomimetic hydrogel microenvironments. A glass-supported microfluidic chip with a convex flexible polydimethylsiloxane (PDMS) membrane on the top was employed for loading the cells suspended in a prepolymer solution. Following UV crosslinking through a photomask with a concentric circular pattern, the cell-laden hydrogels were formed in a height gradient from the center (maximum) to the boundary (minimum). When the convex PDMS membrane retracted back to a flat surface, it applied compressive gradient forces on the cell-laden hydrogels. The concentric circular hydrogel patterns confined the direction of hydrogel elongation, and the compressive strain on the hydrogel therefore resulted in elongation stretch in the radial direction to guide cell alignment. NIH3T3 cells were cultured in the chip for 3 days with compressive strains that varied from ~65% (center) to ~15% (boundary) on hydrogels. We found that the hydrogel geometry dominated the cell alignment near the outside boundary, where cells aligned along the circular direction, and the compressive strain dominated the cell alignment near the center, where cells aligned radially. This study developed a new and simple approach to facilitate cellular alignment based on hydrogel geometry and strain stimulation for tissue engineering applications. This platform offers unique advantages and is significantly different from the existing approaches owing to the fact that gradient generation was accomplished in a miniature device without using an external mechanical source.