Correct Closure of the Left Atrial Appendage Reduces Stagnant Blood Flow and the Risk of Thrombus Formation: A Proof-of-Concept Experimental Study Using 4D Flow Magnetic Resonance Imaging.

OBJECTIVE: The study was conducted to investigate the effect of correct occlusion of the left atrial appendage (LAA) on intracardiac blood flow and thrombus formation in patients with atrial fibrillation (AF) using four-dimensional (4D) flow magnetic resonance imaging (MRI) and three-dimensional (3D)-printed phantoms. MATERIALS AND METHODS: Three life-sized 3D-printed left atrium (LA) phantoms, including a pre-occlusion (i.e., before the occlusion procedure) model and correctly and incorrectly occluded post-procedural models, were constructed based on cardiac computed tomography images from an 86-year-old male with long-standing persistent AF. A custom-made closed-loop flow circuit was set up, and pulsatile simulated pulmonary venous flow was delivered by a pump. 4D flow MRI was performed using a 3T scanner, and the images were analyzed using MATLAB-based software (R2020b; Mathworks). Flow metrics associated with blood stasis and thrombogenicity, such as the volume of stasis defined by the velocity threshold (|V̅| < 3 cm/s), surface-and-time-averaged wall shear stress (WSS), and endothelial cell activation potential (ECAP), were analyzed and compared among the three LA phantom models. RESULTS: Different spatial distributions, orientations, and magnitudes of LA flow were directly visualized within the three LA phantoms using 4D flow MRI. The time-averaged volume and its ratio to the corresponding entire volume of LA flow stasis were consistently reduced in the correctly occluded model (70.82 mL and 39.0%, respectively), followed by the incorrectly occluded (73.17 mL and 39.0%, respectively) and pre-occlusion (79.11 mL and 39.7%, respectively) models. The surface-and-time-averaged WSS and ECAP were also lowest in the correctly occluded model (0.048 Pa and 4.004 Pa-1 , respectively), followed by the incorrectly occluded (0.059 Pa and 4.792 Pa-1 , respectively) and pre-occlusion (0.072 Pa and 5.861 Pa-1 , respectively) models. CONCLUSION: These findings suggest that a correctly occluded LAA leads to the greatest reduction in LA flow stasis and thrombogenicity, presenting a tentative procedural goal to maximize clinical benefits in patients with AF.

Regeneration of the Eyespot and Flagellum in Euglena gracilis during Cell Division.

Cell division of unicellular microalgae is a fascinating process of proliferation, at which whole organelles are regenerated and distributed to two new lives. We performed dynamic live cell imaging of Euglena gracilis using optical microscopy to elucidate the mechanisms involved in the regulation of the eyespot and flagellum during cell division and distribution of the organelles into the two daughter cells. Single cells of the wild type (WT) and colorless SM-ZK cells were confined in a microfluidic device, and the appearance of the eyespot (stigma) and emergent flagellum was tracked in sequential video-recorded images obtained by automatic cell tracking and focusing. We examined 12 SM-ZK and 10 WT cells and deduced that the eyespot diminished in size and disappeared at an early stage of cell division and remained undetected for 26-97 min (62 min on average, 22 min in deviation). Subsequently, two small eyespots appeared and were distributed into the two daughter cells. Additionally, the emergent flagellum gradually shortened to zero-length, and two flagella emerged from the anterior ends of the daughter cells. Our observation revealed that the eyespot and flagellum of E. gracilis are degraded once in the cell division, and the carotenoids in the eyespot are also decomposed. Subsequently, the two eyespots/flagella are regenerated for distribution into daughter cells. As a logical conclusion, the two daughter cells generated from a single cell division possess the equivalent organelles and each E. gracilis cell has eternal or non-finite life span. The two newly regenerated eyespot and flagellum grow at different rates and mature at different timings in the two daughter cells, resulting in diverse cell characteristics in E. gracilis.

Temporal Evolution of the Gravitaxis of Euglena gracilis from a Single Cell.

Gravitaxis is one of the most important issues in the growth of microalgae in the water column; it determines how easily cells receive sunlight with a comfortable intensity that is below the damaging threshold. We quantitatively investigated and analyzed the gravitaxis and cell multiplication of Euglena gracilis using vertically placed microchambers containing a single cell. A temporal change in gravitaxis and cell multiplication was observed after transferring the cells to fresh culture medium for 9 days. We performed 29 individual experiments with 2.5 mm × 2.5 mm × 0.1 mm square microchambers and found that the cells showed positive, negative, and moderate gravitaxis in 8, 7, and 14 cases, respectively, after transferring to fresh culture medium. A common trend was observed for the temporal change in gravitaxis for the eight initially positive gravitaxis cases. The cells with initially positive gravitaxis showed a higher rate of cell multiplication than those with initially negative gravitaxis. We also discussed the gravitaxis mechanism of E. gracilis from the observed trend of gravitaxis change and swimming traces. In addition, bioconvection in a larger and thicker chamber was investigated at a millimeter scale and visualized.

Flow control in a laminate capillary-driven microfluidic device.

Capillary-driven microfluidic devices are of significant interest for on-site analysis because they do not require external pumps and can be made from inexpensive materials. Among capillary-driven devices, those made from paper and polyester film are among the most common and have been used in a wide array of applications. However, since capillary forces are the only driving force, flow is difficult to control, and passive flow control methods such as changing the geometry must be used to accomplish various analytical applications. This study presents several new flow control methods that can be utilized in a laminate capillary-driven microfluidic device to increase available functionality. First, we introduce push and burst valve systems that can stop and start flow. These valves can stop flow for >30 min and be opened by either pressing the channel or inflowing other fluids to the valve region. Next, we propose flow control methods for Y-shaped channels that enable more functions. In one example, we demonstrate the ability to accurately control concentration to create laminar, gradient, and fully mixed flows. In a second example, flow velocity in the main channel is controlled by adjusting the length of the inlet channel. In addition, the flow velocity is constant as the inlet length increases. Finally, the flow velocity in the Y-shaped device as a function of channel height and fluid properties such as viscosity and surface tension was examined. As in previous studies on capillary-driven channels, the flow rate was affected by each parameter. The fluidic control tools presented here will enable new designs and functions for low cost point of need assays across a variety of fields.

U.S. Healthcare Insurance Market Concentration from 2001 to 2016: Increased Growth in Direct Written Premiums and Overall Decreased Market Consolidation.

With the establishment of state-based health insurance marketplaces, how U.S. health insurers are responding to market pressures and influencing premiums have represented important questions. We made novel use of the Standard and Poor's (S&P) Financial, a Wall Street financial dataset platform, to analyze trends in market capitalization and total direct written premiums (DWPs) from 2001 to 2016 of the top 5, 10, and 25 health insurance companies. Our results indicate that the market concentration of publicly traded companies has remained relatively stable over the past decade. The top 5, 10, and 25 health insurance companies were 43.5%, 57.5%, and 78.6% of the total market share in 2001 and 39.4%, 52.9%, and 72.8% in 2016, respectively. DWPs have grown nearly four-fold from $177 billion to $631 billion at a compounded annual rate of 8.8%, consistent with overall healthcare sector growth. Aggregating state-specific data, the overall U.S. health insurance market has become slightly less consolidated over recent years, as measured using the population-weighted Herfindahl-Hirschman index, a measure for market concentration, falling from 3,817 to 2,174 during this time period. As health insurance costs place a growing burden on American families, additional efforts are needed to study the impact on choice, quality, access, cost, and value to patients and providers from evolving health insurance markets.

Patient-specific Hemodynamics of Severe Carotid Artery Stenosis Before and After Endarterectomy Examined by 4D Flow MRI.

Carotid endarterectomy (CEA) influences the carotid endoluminal anatomy, which results in hemodynamic changes before and after surgery. We investigated the hemodynamics of severe carotid artery stenosis before and after conventional endarterectomy with/without patch repair. An in vitro experiment utilizing carotid phantoms, which underwent a procedure that emulated CEA with/without the patch repair, was performed with a high-spatiotemporal resolution using 4D flow MRI. We evaluated an abnormal region of carotids, which consists of the normalized time-averaged wall shear stress (NTA|WSS|) and the oscillatory shear index (OSI), to account for continuous high-shear regions (high NTA|WSS| and low OSI) and chaotic low-shear regions, i.e., stenosis-prone regions (low NTA|WSS| and high OSI). The use of normalized hemodynamic parameters (e.g., NTA|WSS|) allowed comparison of diverse cases with different conditions of hemodynamics and vessel geometry. We observed that the stenosis-prone regions of the carotids with patches were noticeably larger than the corresponding regions in no-patch carotids. A large recirculating flow zone found in the stenosis-prone region of the internal carotid artery (ICA) of the postoperative carotids with patches partially blocks the flow path into ICA, and consequently the flow rate was not recovered after surgery unlike an expectation.

Development of a Paper-Based Viscometer for Blood Plasma Using Colorimetric Analysis.

The viscosity of biofluids can be used to acquire meaningful medical information on the conditions of a patient but has seldom been utilized in clinical practices owing to cumbersome measurement procedures and the need for large sample volumes. We present a colorimetric method to measure the viscosity of blood plasma using a paper-based viscometer developed in this study specifically for clinical diagnosis. The proposed analytical device consists of multilayered papers with fluid-loading, -mixing, and -measuring regions, and it can be fabricated readily in a simple manner using three-layered paper channels and tape. Moreover, the colorimetric analysis enables viscosity estimations by analyzing a single optical image. To validate the device performance, we measured the viscosities of fluids such as glycerin aqueous solutions, bovine-serum-albumin solution, dimethyl sulfoxide, and blood plasma. We found that the measured viscosities were in good agreement with the reference values. Finally, we developed a simple smartphone application for the viscosity measurements that helped enhance the convenience and utility of the paper-based viscometer while maintaining the measurement accuracy.

Novel and facile criterion to assess the accuracy of WSS estimation by 4D flow MRI.

Four-dimensional flow magnetic resonance imaging (4D flow MRI) is a versatile tool to obtain hemodynamic information and anatomic information simultaneously. The wall shear stress (WSS), a force exerted on a vessel wall in parallel, is one of the hemodynamic parameters available with 4D flow MRI and is thought to play an important role in clinical applications such as assessing the development of atherosclerosis. Nevertheless, the accuracy of WSS obtained with 4D flow MRI is rarely evaluated or reported in literature, especially in the in vivo studies. We propose a novel and facile criterion called Reynolds resolution to assess the accuracy of WSS estimation in 4D flow MRI studies. Reynolds resolution consists of a spatial resolution, encoding velocity, kinematic viscosity of a working fluid, and signal-to-noise ratio, which are readily accessible information in 4D flow MRI measurements. We explored the relationship between Reynolds resolution and the WSS error. To include diverse and extensive cases, we measured three circular tubing flows with a diameter of 40, 8, and 2 mm. The 40 mm tubing flow was measured by 3 Tesla (T) human MR scanner with a knee coil and spatial resolution of 0.5 mm. The 8 and 2 mm tubing flows were both measured by 4.7 T MR scanner, but the scans were performed with a conventional birdcage coil (8 mm tubing) and a custom-made solenoid coil (2 mm tubing), respectively. The spatial resolution was varied from 0.2, 0.4 or 0.8 mm for the 8 mm tubing flow, but was fixed at 0.090 mm for 2 mm tubing flow. In addition, the near-wall velocity gradient, required to be determined prior to the WSS, was calculated using two methods; these included assuming a linear velocity profile or quadratic velocity profile near wall. The accuracy of WSS obtained using each method and tubing flow was evaluated against the theoretical WSS value. As a result, we found that Reynolds resolution is in logarithmic relation to the WSS error.

Consolidation and maturation of the orthopaedic medical device market between 1999 and 2015.

Orthopaedic surgeons often require highly specialized medical devices, implants, and equipment, which are usually offered by several vendors/companies. This study assesses long-term market trends for orthopaedic medical device companies and examines various implications for healthcare cost. Using S&P Capital IQ, a Wall Street database, financial data were gathered on orthopaedic device companies, ranked by worldwide sales, from 1999 to 2015. Annual sales were aggregated to calculate market share and compounded annual growth rates (CAGRs). Overall, the global orthopaedic device market grew at 12.0% CAGR from 1999 to 2008, before slowing to 2.8% from 2009 to 2015. Between 1999 and 2015, the top 5 companies increased total market share from 52.8 to 62.2%. The orthopaedic device market is not only consolidating under a few dominant players, but also growing at a decreasing rate, both of which signal a maturing industry. These trends are likely to shape patient care and healthcare costs in orthopaedic surgery in years to come.

Water-repellent Hybrid Nanowire and Micro-scale Denticle Structures on Flexible Substrates of Effective Air Retention.

The air retention capability of a superhydrophobic surface plays the crucial role of drag reduction in an aqueous environment. Here, fabrication of water-repellent hybrid structural surfaces by synthesizing superhydrophobic nanowires with a high aspect ratio on micro-scale denticle structures to improve their air holding capacity in water is reported. The hybrid structure is realized by carrying out polymer molding of denticle structures on flexible substrates, hydrothermal growth of nanowires, and subsequent ultra-thin film coating. This technique is readily applicable to large areas, and the fabricated substrates are attachable onto curved surfaces. Our engineered, super water-repellent hybrid structures are found to effectively maintain air bubbles on their surfaces in a highly shear flow condition with a wall shear stress of up to 33.4 Pa, due to the combined effects of the micro-scale denticle structure, which reduces flow resistance, and the superhydrophobic, high-aspect-ratio nanowire structure, which enhances the capillary force to maintain the air bubbles. Our results show the importance of developing superhydrophobic structures of improved air retention capability.

Stem cell industry update: 2012 to 2016 reveals accelerated investment, but market capitalization and earnings lag.

Treatments based on stem cells have long been heralded for their potential to drive the future of regenerative medicine and have inspired increasing medical and business interest. The stem cell therapy market has been expanding since 2012, but earnings and profitability still lag the broader health care sector (compounded annual growth rate in annual financing of 31.5% versus 13.4%, respectively). On the basis of historical financial data, approximately $23 billion has been invested in stem cell companies since 1994, with more than 80% of this raised from 2011 through 2016. This reflects a marked acceleration in capital investment, as companies began late-stage clinical trials, initiate partnerships or are acquired by large pharmaceutical companies. All of these data reflect a field that is emerging from infancy, which will demand more time and capital to mature. This update is relevant to researchers, clinicians and investors who wish to quantify the potential in this field.

Estimation of the thermocapillary force and its applications to precise droplet control on a microfluidic chip.

Droplet control through the use of light-induced thermocapillary effects has recently garnered attention due to its non-intrusive and multifunctional nature. An important issue in droplet control is the estimation of the thermocapillary force. The purpose of the present study is to estimate the thermocapillary force and propose empirical equations between the force and simply measurable key parameters such as droplet diameter and power of heat source. In addition, we aim to shift the droplet trajectory and develop an on-demand droplet routing system based on the estimation of the thermocapillary force. We illuminated a continuous phase with a 532 nm laser beam to minimize possible damage or property changes to target molecules contained within droplets. A mixture of light-absorbing material and oleic acid was used for the continuous phase fluid, while deionized water (DI water) was used for the dispersed phase fluid. We proposed empirical equations to estimate the thermocapillary force, which was then applied to precise droplet shifting and routing. We found that the shifting distance was linearly proportional to the thermocapillary force, and that an on-demand droplet routing system resulted in a success rate greater than 95%.

The Mitochondrial Genome of the Guanaco Louse, Microthoracius praelongiceps: Insights into the Ancestral Mitochondrial Karyotype of Sucking Lice (Anoplura, Insecta).

Fragmented mitochondrial (mt) genomes have been reported in 11 species of sucking lice (suborder Anoplura) that infest humans, chimpanzees, pigs, horses, and rodents. There is substantial variation among these lice in mt karyotype: the number of minichromosomes of a species ranges from 9 to 20; the number of genes in a minichromosome ranges from 1 to 8; gene arrangement in a minichromosome differs between species, even in the same genus. We sequenced the mt genome of the guanaco louse, Microthoracius praelongiceps, to help establish the ancestral mt karyotype for sucking lice and understand how fragmented mt genomes evolved. The guanaco louse has 12 mt minichromosomes; each minichromosome has 2-5 genes and a non-coding region. The guanaco louse shares many features with rodent lice in mt karyotype, more than with other sucking lice. The guanaco louse, however, is more closely related phylogenetically to human lice, chimpanzee lice, pig lice, and horse lice than to rodent lice. By parsimony analysis of shared features in mt karyotype, we infer that the most recent common ancestor of sucking lice, which lived ∼75 Ma, had 11 minichromosomes; each minichromosome had 1-6 genes and a non-coding region. As sucking lice diverged, split of mt minichromosomes occurred many times in the lineages leading to the lice of humans, chimpanzees, and rodents whereas merger of minichromosomes occurred in the lineage leading to the lice of pigs and horses. Together, splits and mergers of minichromosomes created a very complex and dynamic mt genome organization in the sucking lice.

Temporal change of photophobic step-up responses of Euglena gracilis investigated through motion analysis.

The adaptation to a strong light is one of the essential characteristics of green algae, yet lacking relatively the information about the photophobic responses of Eukaryotic microalgae. We investigated the photophobic step-up responses of Euglena gracilis over a time course of several hours with alternated repetition of blue-light pulse illumination and spatially patterned blue-light illumination. Four distinctive photophobic motions in response to strong blue light were identified in a trace image analysis, namely on-site rotation, running and tumbling, continuous circular swimming, and unaffected straightforward swimming. The cells cultured in autotrophic conditions under weak light showed mainly the on-site rotation response at the beginning of blue-light illumination, but they acquired more blue-light tolerant responses of running and tumbling, circular swimming, or straightforward swimming. The efficiency of escaping from a blue-light illuminated area improved markedly with the development of these photophobic motions. Time constant of 3.0 h was deduced for the evolution of photophobic responses of E. gracilis. The nutrient-rich metabolic status of the cells resulting from photosynthesis during the experiments, i.e., the accumulation of photosynthesized nutrient products in balance between formation and consumption, was the main factor responsible for the development of photophobic responses. The reduction-oxidation status in and around E. gracilis cells did not affect their photophobic responses significantly, unlike the case of photophobic responses and phototaxis of Chlamydomonas reinhardtii cells. This study shows that the evolution of photophobic motion type of E. gracilis is dominated mainly by the nutrient metabolic status of the cells. The fact suggests that the nutrient-rich cells have a higher threshold for switching the flagellar motion from straightforward swimming to rotation under a strong light.

Autonomous oscillation/separation of cell density artificially induced by optical interlink feedback as designed interaction between two isolated microalgae chips.

We demonstrate a designed interaction between two isolated cell populations of Euglena gracilis and Chlamydomonas reinhardtii, separately confined in two 25-square micro-aquariums of lab-on-chip size. The interaction was realized by interlinking two identical optical feedback systems, which measured the cell distribution. To analyze the cell populations, we measured the cell distribution in the 25 squares and irradiated the cells with a blue light pattern as an external stimulus. The cell distribution dataset was exchanged between the two systems. Governed by a designed interaction algorithm, the feedback systems produced a dynamic blue light illumination pattern that evoked the photophobic responses of both species. We also induced autonomous cell density oscillation and cell distribution separation and clustering, and analyzed how the types and diversities of the photophobic responses affected the oscillation period and separation and clustering. We conclude that artificial interlink feedback is a promising method for investigating diverse cell-cell interactions in ecological communities, and for developing soft-computing applications with living cells.

Fragmented mitochondrial genomes in two suborders of parasitic lice of eutherian mammals (Anoplura and Rhynchophthirina, Insecta).

Parasitic lice (order Phthiraptera) infest birds and mammals. The typical animal mitochondrial (mt) genome organization, which consists of a single chromosome with 37 genes, was found in chewing lice in the suborders Amblycera and Ischnocera. The sucking lice (suborder Anoplura) known, however, have fragmented mt genomes with 9-20 minichromosomes. We sequenced the mt genome of the elephant louse, Haematomyzus elephantis - the first species of chewing lice investigated from the suborder Rhynchophthirina. We identified 33 mt genes in the elephant louse, which were on 10 minichromosomes. Each minichromosome is 3.5-4.2 kb in size and has 2-6 genes. Phylogenetic analyses of mt genome sequences confirm that the elephant louse is more closely related to sucking lice than to the chewing lice in the Amblycera and Ischnocera. Our results indicate that mt genome fragmentation is shared by the suborders Anoplura and Rhynchophthirina. Nine of the 10 mt minichromosomes of the elephant louse differ from those of the sucking lice (Anoplura) known in gene content and gene arrangement, indicating that distinct mt karyotypes have evolved in Anoplura and Rhynchophthirina since they diverged ~92 million years ago.

Facile and precise flow control for a paper-based microfluidic device through varying paper permeability.

Paper-based microfluidic devices have recently attracted attention for their ability to utilize the inherent capillary force, or absorptivity, of paper to generate flows instead of requiring an external force. This allows for a simple sensor system that can be readily manufactured at low costs; however, sophisticated flow control is still necessary to implement and analyze diverse functions. In this paper, we propose a facile flow rate control method by varying the permeability of chromatography paper using a wax printing method frequently used to make channel walls in paper-based microfluidic devices. We found that the flow rates in paper channels can be precisely controlled by varying either the brightness of wax patterns, which is inversely proportional to the amount of printed wax droplets, or the length of wax patterns printed on the paper channel. Based on these results, we developed a paper-based micromixer that minutely controls the mixing ratio of two dye flows as prescribed in printed wax patterns. We found experimental mixing ratio values that were in excellent agreement with predicted values.

Flow characterization of electroconvective micromixer with a nanoporous polymer membrane in-situ fabricated using a laser polymerization technique.

Electroconvection is known to cause strong convective mixing in a microchannel near a nanoporous membrane or a nanochannel in contact with an electrolyte solution due to the external electric field. This study addresses micromixer behavior subject to electroconvection occurring near a nanoporous membrane in-situ fabricated by a laser polymerization technique on a microfluidic chip. We found that the micromixer behavior can be categorized into three regimes. Briefly, the weak electroconvection regime is characterized by weak mixing performance at a low applied voltage and KCl concentration, whereas the strong electroconvection regime has a high mixing performance when the applied voltage and KCl concentration are moderately high. Finally, the incomplete electroconvection regime has an incomplete electric double-layer overlap in the nanopores of the membrane when the electrolyte concentration is very high. The mixing index reached 0.92 in the strong electroconvection regime. The detailed fabrication methods for the micromixer and characterization results are discussed in this paper.

Sequential phosphorylation analysis using dye-tethered peptides and microfluidic isoelectric focusing electrophoresis.

We report a simple method for analyzing sequential phosphorylation by protein kinases using fluorescent peptide substrates and microfluidic isoelectric focusing (µIEF) electrophoresis. When a dye-labeled peptide substrate was sequentially phosphorylated by two consecutive protein kinases (mitogen-activated protein kinase (MAPK) and glycogen synthase kinase 3 (GSK3)), its differently phosphorylated forms were easily separated and visualized by fluorescent focusing zones in the µIEF channel based on a change in the isoelectric point (pI) by phosphorylation. As a result, ratiometric and quantitative analysis of the fluorescent focusing regions shifted by phosphorylation enabled the analysis of phosphorylation efficiency and the relevant inhibition of protein kinases (MAPK and GSK3) with high simplicity and selectivity. Furthermore, the GSK3 activity in the cell lysates was elucidated by µIEF electrophoresis in combination with immunoprecipitation. Our results suggest that this method has great potential for analyzing the sequential phosphorylation of multiple protein kinases that are implicated in cellular signaling pathways.

Size-Controlled Fabrication of Polyaniline Microfibers Based on 3D Hydrodynamic Focusing Approach.

Owing to the relatively high conductivity and unique redox behavior, polyaniline (PANI) has been one of the most technologically promising conducting polymers. Although various methodologies have been developed, fabrication of PANI microfibers has been a challenging task owing to the poor solubility in most organic solvents. By taking advantage of a microfluidic technology and organic soluble acid labile t-Boc-protected PANI (t-Boc-PANI) as the conducting polymer precursor, fabrication of PANI microfibers in a size-controlled manner is possible. Introduction of a THF solution containing t-Boc-PANI, and dodecylbenzenesulfonic acid (DBSA) as a core flow, and water as a sheath flow into a microfluidic channel with a 3D hydrodynamic focusing effect results in crystallization of the polymer fiber. By changing the flow rate, linear PANI microfibers that range from 16.2 to 39.4 µm in diameter are readily obtained.