First Report of Root rot Caused by Mucor circinelloides on Fructus forsythiae in Hubei，China.

Fructus forsythiae is a typical traditional Chinese medicinal herb with antibacterial and anti-inflammatory effects. Surveys for root rot of F. forsythiae were conducted from 2021 to 2022 in China's major planting areas (Daweiyuan Village, Sanguandong Forest Area, Yunxi County, Shiyan City, Hubei Province, 32°52'52"N, 110°19'29"E). The disease has occurred in several plantations. A total of 200 F. forsythiae were investigated, among which 112 were diseased, the incidence rate was more than 50%, and all the plants in the plantation were over 3 years old. The roots of diseased plants were fully covered by white mycelia. When the disease was severe, leaves curled and fell, roots withered, and some eventually died. A total of 22 isolates were isolated from the 18 infected tissues of F. forsythiae and purified by single spore cultures on PDA medium. The 22 isolates similared in morphology to isolate Lianmao (The name is one of the five sequenced samples in the lab) were selected to be representative of the group. The results showed that these samples belonged to the same pathogen. The isolates were characterized by yellowish colonies composed of tall and short sporangiophores 6 to 11 µm in width, terminal and globose sporangia, ellipsoidal sporangiospores with dimensions of 5 to 8 µm in length and 4 to 5 µm in width, and obovoid columellae. It was identified as Mucor circinelloides, based on morphological characteristics (Schipper 1976). The ITS and LSU sequences of the fungus were amplified, and sequenced with the primers ITS1/ITS4 and LROR/LR5 (White et al. 1990; Rehner et al. 1994). Sequences from isolate Lianmao were deposited in GenBank (accession nos. OQ359158 for ITS, OQ359157 for LSU). Analysis using the BLAST algorithm of the two amplified sequences showed 99.69 to 100% similarity with the sequences (KY933391 and MH868051) of M. circinelloides. The isolated M. circinelloides was prepared into 150ml spore suspension (The method was to filter the PDB after 10 days of culture using gauze to get spore suspension. Then the concentration of spore suspension was diluted to 1.0×106 spores/ml using sterile water). The spore suspension was subsequently inoculated into healthy potted F. forsythiae plants. Uninoculated potted F. forsythiae plants served as controls. All the potted F. forsythiae plants were incubated at 25°C under 12h light and 12h dark conditions. The symptoms of the infected plants were similar to those observed in the field; The control plants were symptomless. The pathogen reisolated from symptomatic roots and morphologically identified as M. circinelloides. M. circinelloides has been reported as a pathogen of Morinda citrifolia, Aconitum carmichaelii and so on (Cui et al. 2021; Nishijima et al. 2011), but it has never been reported on F. forsythiae before. This is the first report of root rot caused by M.circinelloides on F. forsythiae. This pathogen may present a threat to the production of F. forsythiae in China.

Multiple-Streams Focusing-Based Cell Separation in High Viscoelasticity Flow.

Viscoelastic flow has been widely used in microfluidic particle separation processes, in which particles get focused on the channel center in diluted viscoelastic flow. In this paper, the transition from single-stream focusing to multiple-streams focusing (MSF) in high viscoelastic flow is observed, which is applied for cell separation processes. Particle focusing stream bifurcation is caused by the balance between elastic force and viscoelastic secondary flow drag force. The influence of cell physical properties, such as cell dimension, shape, and deformability, on the formation of multiple-streams focusing is studied in detail. Particle separation is realized utilizing different separation criteria. The size-based separation of red (RBC) and white (WBC) blood cells is demonstrated in which cells get focused in different streams based on their dimension difference. Cells with different deformabilities get stretched in the viscoelastic flow, leading to the change of focusing streams, and this property is harnessed to separate red blood cells infected with the malaria parasite, Plasmodium falciparum. The achieved results promote our understanding of particle movement in the high viscoelastic flow and enable new particle manipulation and separation processes for sample treatment in biofluids.

Arbuscular mycorrhizal fungi and phosphorus supply accelerate main medicinal component production of Polygonum cuspidatum.

The medicinal plant Polygonum cuspidatum Sieb. Et Zucc is rich in stilbenes (e.g., polygonin and resveratrol) and anthraquinones (e.g., emodin) for the therapy of human diseases, while how to increase the growth and medicinal composition concentrations of P. cuspidatum has become an urgent issue. The aim of the present study was to evaluate the effects of inoculation with an arbuscular mycorrhizal (AM) fungus, Funneliformis mosseae, on plant growth, phosphorus (P) acquisition, medicinal component concentrations, and expressions of resveratrol synthesis-associated enzyme genes of P. cuspidatum at two P levels (0 M and 0.2 M). P supply (0.2 M) stimulated root AM fungal colonization rate. F. mosseae inoculation significantly improved growth performance (height, diameter, and biomass) and root morphology (diameter, length, and projected area), irrespectively of substrate P levels. P supply and F. mosseae distinctly increased soil acid and neutral phosphatase activities, as well as root P concentrations. P supply increased root physcion and resveratrol concentrations in inoculated and uninoculated plants, along with up-regulated expressions of PcCHS1, PcCRS1, PcRS11, and PcSTS. AM plants represented significantly higher root aloe-emodin, chrysophanol, emodin, physcion, polydatin, and resveratrol concentrations than non-AM plants irrespective of P levels, coupled with up-regulated expressions of PcCHS1, PcCHS2, PcRS11, PcRS, and PcSTS. It is concluded that 0.2 M P supply and F. mosseae inoculation promoted chrysophanol, physcion, polydatin, and resveratrol concentrations of P. cuspidatum, with the increase in resveratrol associated with up-regulated expressions of related genes.

Root Endophytic Fungi Regulate Changes in Sugar and Medicinal Compositions of Polygonum cuspidatum.

Polygonum cuspidatum Sieb. et Zucc is an important industrial crop because it contains a large amount of medicinal secondary metabolites (such as polydatin, resveratrol, chrysophanol, and emodin). However, it is unclear whether root endophytic fungi increase the content of secondary metabolites in the plant. This study aimed to analyze the effects of Funneliformis mosseae (Fm) and Piriformospora indica (Pi) alone or in combination on plant growth, root morphology, thirteen sugars concentrations, and six secondary metabolites (physcion, chrysophanol, emodin, aloe-emodin, polydatin, and resveratrol) concentrations of P. cuspidatum. After 11 weeks of the fungal inoculation, the roots could be colonized by Fm and Pi single or in combination, along with the higher root colonization frequency of Fm > Pi > Fm + Pi in the descending order. In addition, Fm and Pi improved plant growth performance (plant height, stem diameter, leaf number, and shoot and root biomass) and root morphology (average diameter, maximum diameter, total length, area, and volume) to varying degrees, depending on fungal inoculations, in which Pi displayed a relatively better effect on plant growth. Single Fm and Pi inoculation significantly increased three disaccharides (sucrose, maltose, and trehalose) accumulation, while dual inoculum (Fm + Pi) only elevated sucrose concentrations. Most monosaccharides concentrations, such as D-arabinose, D-galactose, D-sorbitol, D-fructose, glucose, and L-rhamnose were not altered or inhibited by the endophytic fungi, except the increase in L-fucose and inositol. All fungal treatments significantly increased root chrysophanol and resveratrol concentrations, while decreased aloe-emodin concentrations. In addition, single Pi and dual Fm + Pi increased emodin concentrations, and single Fm and dual Fm + Pi elevated physcion and polydatin concentrations. It was concluded that Fm and Pi promoted the growth of P. cuspidatum, and the combination of Fm and Pi was more conducive to the production of some secondary metabolites than single inoculation.

Separation of U87 glioblastoma cell-derived small and medium extracellular vesicles using elasto-inertial flow focusing (a spiral channel).

Nanoscale and microscale cell-derived extracellular vesicle types and subtypes are of significant interest to researchers in biology and medicine. Extracellular vesicles (EVs) have diagnostic and therapeutic potential in terms of biomarker and nanomedicine applications. To enable such applications, EVs must be isolated from biological fluids or separated from other EV types. Developing methods to fractionate EVs is of great importance to EV researchers. Our goal was to begin to develop a device that would separate medium EVs (mEVs, traditionally termed microvesicles or shedding vesicles) and small EVs (sEVs, traditionally termed exosomes) by elasto-inertial effect. We sought to develop a miniaturized technology that works similar to and provides the benefits of differential ultracentrifugation but is more suitable for EV-based microfluidic applications. The aim of this study was to determine whether we could use elasto-inertial focusing to re-isolate and recover U87 mEVs and sEVs from a mixture of mEVs and sEVs isolated initially by one round of differential ultracentrifugation. The studied spiral channel device can continuously process 5 ml of sample fluid per hour. Using the channel, sEVs and mEVs were recovered and re-isolated from a mixture of U87 glioma cell-derived mEVs and sEVs pre-isolated by one round of differential ultracentrifugation. Following two passes through the spiral channel, approximately 55% of sEVs were recovered with 6% contamination by mEVs (the recovered sEVs contained 6% of the total mEVs). In contrast, recovery of U87 mEVs and sEVs re-isolated using a typical second centrifugation wash step was only 8% and 53%, respectively. The spiral channel also performed similar to differential ultracentrifugation in reisolating sEVs while significantly improving mEV reisolation from a mixture of U87 sEVs and mEVs. Ultimately this technology can also be coupled to other microfluidic EV isolation methods in series and/or parallel to improve isolation and minimize loss of EV subtypes.

Viscoelastic Particle Focusing and Separation in a Spiral Channel.

As one type of non-Newtonian fluid, viscoelastic fluids exhibit unique properties that contribute to particle lateral migration in confined microfluidic channels, leading to opportunities for particle manipulation and separation. In this paper, particle focusing in viscoelastic flow is studied in a wide range of polyethylene glycol (PEO) concentrations in aqueous solutions. Polystyrene beads with diameters from 3 to 20 µm are tested, and the variation of particle focusing position is explained by the coeffects of inertial flow, viscoelastic flow, and Dean flow. We showed that particle focusing position can be predicted by analyzing the force balance in the microchannel, and that particle separation resolution can be improved in viscoelastic flows.

Multiple Linear Regression Modeling of Nanosphere Self-Assembly via Spin Coating.

Nanosphere lithography employs single- or multilayer self-assembled nanospheres as a template for bottom-up nanoscale patterning. The ability to produce self-assembled nanospheres with minimal packing defects over large areas is critical to advancing applications of nanosphere lithography. Spin coating is a simple-to-execute, high-throughput method of nanosphere self-assembly. The wide range of possible process parameters for nanosphere spin coating, however-and the sensitivity of nanosphere self-assembly to these parameters-can lead to highly variable outcomes in nanosphere configuration by this method. Finding the optimum process parameters for nanosphere spin coating remains challenging. This work adopts a design-of-experiments approach to investigate the effects of seven factors-nanosphere wt%, methanol/water ratio, solution volume, wetting time, spin time, maximum revolutions per minute, and ramp rate-on two response variables-percentage hexagonal close packing and macroscale coverage of nanospheres. Single-response and multiple-response linear regression models identify main and two-way interaction effects of statistical significance to the outcomes of both response variables and enable prediction of optimized settings. The results indicate a tradeoff between the high ramp rates required for large macroscale coverage and the need to minimize high shear forces and evaporation rates to ensure that nanospheres properly self-assemble into hexagonally packed arrays.

Thermally tunable hydrogel crosslinking mediated by temperature sensitive liposome.

Hydrogel crosslinking by external stimuli is a versatile strategy to control and modulate hydrogel properties. Besides photonic energy, thermal energy is one of the most accessible external stimuli and widely applicable for many biomedical applications. However, conventional thermal crosslinking systems require a relatively high temperature (over 100 °C) to initiate covalent bond formation. To our knowledge, there has not been a thermally tunable hydrogel crosslinking system suitable for biological applications. This work demonstrates a unique approach to utilize temperature sensitive liposomes to control and modulate hydrogel crosslinking over mild temperature range (below 50 °C). Temperature sensitive liposomes were used to control the release of chemical crosslinkers by moderate temperature changes. The thermally controlled crosslinker release resulted in tunable mechanical and transport properties of the hydrogel. No significant inflammable response observed in the histology results ensured the biocompatibility of the liposome-mediated crosslinkable hydrogel. This work opens new opportunities to implement thermal energy system for control and modulate hydrogel properties.

High efficiency rare sperm separation from biopsy samples in an inertial focusing device.

Immotile and rare sperm isolation from a complex cell background is an essential process for infertility treatment. The traditional sperm collection process from a biopsy sample requires long, tedious searches, yet still results in low sperm retrieval. In this work, a high recovery, high throughput sperm separation process is proposed for the clinical biopsy sperm retrieval process. It is found that sperm have different focusing positions compared with non-sperm cells in the inertial flow, which is explained by a sperm alignment phenomenon. Separation in the spiral channel device results in a 95.6% sperm recovery in which 87.4% of non-sperm cells get removed. Rare sperm isolation from a clinical biopsy sample is performed with the current approach. The chance of finding sperm is shown to increase 8.2 fold in the treated samples. The achieved results highly support this method being used for the development of a rapid biopsy sperm sorting process. In addition, the mechanism was proposed and can be applied for the high-efficiency separation of non-spherical particles in general.

SARS-CoV-2 pandemic: a review of molecular diagnostic tools including sample collection and commercial response with associated advantages and limitations.

The unprecedented global pandemic known as SARS-CoV-2 has exercised to its limits nearly all aspects of modern viral diagnostics. In doing so, it has illuminated both the advantages and limitations of current technologies. Tremendous effort has been put forth to expand our capacity to diagnose this deadly virus. In this work, we put forth key observations in the functionality of current methods for SARS-CoV-2 diagnostic testing. These methods include nucleic acid amplification-, CRISPR-, sequencing-, antigen-, and antibody-based detection methods. Additionally, we include analysis of equally critical aspects of COVID-19 diagnostics, including sample collection and preparation, testing models, and commercial response. We emphasize the integrated nature of assays, wherein issues in sample collection and preparation could impact the overall performance in a clinical setting.

Size and shape based chromosome separation in the inertial focusing device.

In this paper, we use a spiral channel inertial focusing device for isolation and purification of chromosomes, which are highly asymmetric. The method developed is proposed as a sample preparation process for transchromosomic research. The proposed microfluidics-based chromosome separation approach enables rapid, label-free isolation of bioactive chromosomes and is compatible with chromosome buffer. As part of this work, particle force analysis during the separation process is performed utilizing mathematic models to estimate the expected behavior of chromosomes in the channel and the model validated with experiments employing fluorescent beads. The chromosome sample is further divided into subtypes utilizing fluorescent activated cell sorting , including small condensed chromosomes, single chromosomes, and groups of two chromosomes (four sister chromatids). The separation of chromosome subtypes is realized based on their shape differences in the spiral channel device under high flow rate conditions. When chromosomes become aligned in the shear flow, the balance between the inertial focusing force and the Dean flow drag force is determined by the chromosome projection area and aspect ratio, or shape difference, leading to different focusing locations in the channel. The achieved results indicate a new separation regime in inertial microfluidics that can be used for the separation of non-spherical particles based on particle aspect ratios, which could potentially be applied in fields such as bacteria subtype separation and chromosome karyotyping.

An automated instrument for intrauterine insemination sperm preparation.

Sperm preparation is critical to achieving a successful intrauterine insemination and requires the processing of a semen sample to remove white blood cells, wash away seminal plasma, and reduce sample volume. We present an automated instrument capable of performing a sperm preparation starting with a diluted semen sample. We compare our device against a density gradient centrifugation by processing 0.5 mL portions of patient samples through each treatment. In 5 min of operating time, the instrument recovers an average of 86% of all sperm and 82% of progressively motile sperm from the original sample while removing white blood cells, replacing the seminal plasma, and reducing the volume of the sample to the clinically required level. In 25 min of operating time, density gradient centrifugation recovers an average of 33% of all sperm and 41% of progressively motile sperm. The automated instrument could improve access to IUI as a treatment option by allowing satellite doctor's offices to offer intrauterine insemination as an option for patients without the clinical support required by existing methods.

Optimization of a microfluidic spiral channel used to separate sperm from blood cells.

Assisted reproductive technology includes medical procedures that confront the problem of infertility. In some cases of male infertility, blood cells are present in the sperm containing samples and must be removed. Spiral-channel devices have been developed to perform this task, but there is a strong need to increase their throughput. In this work, the theory behind the separation is employed to optimize the device for increased throughput. An existing device that is known to separate sperm and blood cells with a rectangular cross section of 600 × 100 µm2 was used as the baseline. Using its physics, theoretical models were generated to explore theoretical performances of larger-size channels. The models suggested that a channel of size 800 × 133 µm2 would likely work. This geometry enabled the throughput to be increased by 50%, from 2 ml/min in the case of the baseline-size to 3 ml/min in the designed device. Experiments using the larger device resulted in a recovery of more than 90% of sperm cells while removing 89% of red blood cells (RBCs). In comparison, the reference device results in a 90% recovery of sperm cells while removing 74% of white blood cells (WBCs). The length of the channel was also reduced to reduce the pressure required to operate the chip. Literature has shown the removal of WBCs to be higher than that of RBCs due to their larger size, spherical shape, and comparatively low deformability, suggesting that the revised chip would be faster and better for the separation of sperm and all blood cells.

Social Cost of Blindness Due to AMD and Diabetic Retinopathy in the United States in 2020.

BACKGROUND AND OBJECTIVE: To estimate the social cost of blindness due to wet age-related macular degeneration (wAMD), diabetic macular edema (DME), and proliferative diabetic retinopathy (PDR) in the United States in 2020. PATIENTS AND METHODS: Excess costs that occur because of blindness were estimated as the difference in costs in blind versus non-blind individuals. Per-patient costs were aggregated using the number of cases of blindness due to wAMD, DME, and PDR projected in 2020. RESULTS: Associated annual excess direct costs, indirect costs, and quality-adjusted life year loss per blind individual were $4,944, $54,614, and 0.214, respectively. Combining estimates with 246,423 projected cases of blindness due to wAMD, DME, and PDR translated to total societal costs of $20 billion in 2020, estimated to triple by 2050. CONCLUSION: Excess social costs associated with blindness in individuals with wAMD, DME, and PDR are substantial, with more than half of the burden attributed to indirect costs. [Ophthalmic Surg Lasers Imaging Retina. 2020;51:S6-S14.].

Microfluidic System for Rapid Isolation of Sperm From Microdissection TESE Specimens.

OBJECTIVES: To demonstrate a novel prototype microfluidic system for rapid isolation of sperm from real and simulated microdissection testicular sperm extraction samples. METHODS: The novel microfluidic system was tested using minced testicular biopsies from patients with nonobstructive azoospermia. The samples were split into 2 portions, conventional processing vs microfluidic. The embryologists were blinded to the processing protocol and searched the specimens for sperm after processing. We recorded the number of sperm found and the time to sperm identification and compared the sperm retrieval rates. RESULTS: When compared to conventional methods, samples processed through the microfluidic system were cleaner (decreased somatic cells/debris), with the average number of sperm identified per minute improving from 1.52 sperm per minute for the control and 13.5 sperm per minute with the device yielding an 8.88 fold improvement in the sperm found per minute for the device as compared to the control. Preliminary viability and morphology tests show a minimal impact on sperm processed through the microfluidic system. CONCLUSION: The presented microfluidic system can facilitate rapid and efficient isolation of sperm from microdissection testicular sperm extraction samples. A prospective clinical trial to verify these results is needed to confirm this preliminary data.

Enhanced chromosome extraction from cells using a pinched flow microfluidic device.

Extraction and purification of intact chromosomes are critical sample preparation steps for transchromosomic research and other applications. The commonly used sample preparation methods lead to too few chromosomes with chromosome deactivation and degradation. In this paper, a "mild" chromosome extraction process that combines a chemical and mechanical lysis approach is introduced for the preparation of intact chromosomes that can readily be used for downstream processing. Metaphase cells are treated by chemical lysis buffer and pushed through a microfluidic pinched flow device. Cells are ruptured, and chromosomes are released by a combination of shear stress and chemical reagents. Chromosomes are released intact from the cell membrane into the solution. Simulations and experiments are performed to optimize the microfluidic device geometry and operation parameters. Cell rupture and chromosome release are found to be improved by the shear stress in the pinched flow device. Simulation results indicate that the maximum shear stress appears in the channel constriction region, and the narrow channel maintains constant shear stress. It is concluded that the constriction design, narrow channel width, and operation flow rate have a significate influence on chromosome release. Utilizing an optimized device, near-complete cell lysis is achieved and 4 times as many chromosomes are released (8% in control experiments to 25% in optimized pinched flow devices). Sample treatment time can also be reduced utilizing this combined chemical-mechanical chromosome release method.

Viscoelastic second normal stress difference dominated multiple-stream particle focusing in microfluidic channels.

Particle focusing in viscoelastic fluid flow is a promising approach for inducing particle separations in microfluidic devices. The results from theoretical studies indicated that multiple stream particle focusing can be realized with a large magnitude of the elastic second normal stress difference (N2). For dilute polymer solutions, theoretical and experimental studies show that the magnitude of N2 is never large, no matter how large the polymer molecular weight nor how high the shear rate. However, for concentrated entangled polymer solutions, the magnitude of N2 becomes large at high shear rates. Therefore, in order to test the hypothesis that N2 can be used to induce multiple particle stream focusing behavior, we perform the systematic study of the effects of increasing carrier fluid polymer concentrations in a microchannel containing fluorescent particles. In a dilute polymer solution, multiple particle stream focusing is not observed, even at high shear rates and large dimensionless Weissenberg number values (Wi ≈ 30) at which the elastic first normal stress difference (N1) and the viscosity shear-thinning should be very large, while in a concentrated entangled polymer solution, we observe that particle streams focused upon the channel centerline bifurcate to form two symmetric off-channel particle streams at higher shear rates. This particle focusing behavior is different from previous multiple-stream focusing phenomena, and that we attribute to the influence of the second normal stress difference N2. This N2 induced multiple stream focusing phenomenon provides a different approach for manipulating the particle trajectory and separation in a microchannel.

Microfluidic-based sperm sorting & analysis for treatment of male infertility.

Microfluidics technology has emerged as an enabling technology for different fields of medicine and life sciences. One such field is male infertility where microfluidic technologies are enabling optimization of sperm sample preparation and analysis. In this chapter we review how microfluidic technology has been used for sperm quantification, sperm quality analysis, and sperm manipulation and isolation with subsequent use of the purified sperm population for treatment of male infertility. As we discuss demonstrations of microfluidic sperm sorting/manipulation/analysis, we highlight systems that have demonstrated feasibility towards clinical adoption or have reached commercialization in the male infertility market. We then review microfluidic-based systems that facilitate non-invasive identification and sorting of viable sperm for in vitro fertilization. Finally, we explore commercialization challenges associated with microfluidic sperm sorting systems and provide suggestions and future directions to best overcome them.

Multiple coupled landscapes and non-adiabatic dynamics with applications to self-activating genes.

Many physical, chemical and biochemical systems (e.g. electronic dynamics and gene regulatory networks) are governed by continuous stochastic processes (e.g. electron dynamics on a particular electronic energy surface and protein (gene product) synthesis) coupled with discrete processes (e.g. hopping among different electronic energy surfaces and on and off switching of genes). One can also think of the underlying dynamics as the continuous motion on a particular landscape and discrete hoppings among different landscapes. The main difference of such systems from the intra-landscape dynamics alone is the emergence of the timescale involved in transitions among different landscapes in addition to the timescale involved in a particular landscape. The adiabatic limit when inter-landscape hoppings are fast compared to continuous intra-landscape dynamics has been studied both analytically and numerically, but the analytical treatment of the non-adiabatic regime where the inter-landscape hoppings are slow or comparable to continuous intra-landscape dynamics remains challenging. In this study, we show that there exists mathematical mapping of the dynamics on 2(N) discretely coupled N continuous dimensional landscapes onto one single landscape in 2N dimensional extended continuous space. On this 2N dimensional landscape, eddy current emerges as a sign of non-equilibrium non-adiabatic dynamics and plays an important role in system evolution. Many interesting physical effects such as the enhancement of fluctuations, irreversibility, dissipation and optimal kinetics emerge due to non-adiabaticity manifested by the eddy current illustrated for an N = 1 self-activator. We further generalize our theory to the N-gene network with multiple binding sites and multiple synthesis rates for discretely coupled non-equilibrium stochastic physical and biological systems.