Dimensional Fidelity and Orientation Effects of PolyJet Technology in 3D Printing of Negative Features for Microfluidic Applications.

Negative features in microdevices find a wide range of applications. The process of 3D printing has revolutionized their fabrication due to the combination of good resolution and integration capability. Herein, we report on a systematic study of the effects of materials and print directions on the 3D printing of microfluidic channels as negative features under PolyJet technology. Specifically, the Statasys Objet500 printer was used for this study. We printed two sets of chips (n=10 each), each of which contains channel pairs of a high-contrast reference material and a sacrificial material, respectively. Both materials were embedded in a clear photopolymer resin. The channel pairs ranged in planned width from 64 to 992 µm. To explore the effect on print orientation, channels were printed either parallel or perpendicular with respect to the jetting head's movement. The width of each channel of a pair was compared for each planned width and each combination of materials. The effect of print orientation on channel morphology was also investigated. We found that reproducibility and accuracy were highest at a planned channel width of approximately ≥600 µm and that channel morphology was most suitable when the jetting head of the printer moved parallel to the channel's longitudinal axis. The results should be of interest to any users who wish to create negative features using PolyJet 3D technology.

Mono-UFMylation promotes misfolding-associated secretion of α-synuclein.

Stressed cells secret misfolded proteins lacking signaling sequence via an unconventional protein secretion (UcPS) pathway, but how misfolded proteins are targeted selectively in UcPS is unclear. Here, we report that misfolded UcPS clients are subject to modification by a ubiquitin-like protein named ubiquitin-fold modifier 1 (UFM1). Using α-synuclein (α-Syn) as a UcPS model, we show that mutating the UFMylation sites in α-Syn or genetic inhibition of the UFMylation system mitigates α-Syn secretion, whereas overexpression of UFBP1, a component of the endoplasmic reticulum-associated UFMylation ligase complex, augments α-Syn secretion in mammalian cells and in model organisms. UFM1 itself is cosecreted with α-Syn, and the serum UFM1 level correlates with that of α-Syn. Because UFM1 can be directly recognized by ubiquitin specific peptidase 19 (USP19), a previously established UcPS stimulator known to associate with several chaperoning activities, UFMylation might facilitate substrate engagement by USP19, allowing stringent and regulated selection of misfolded proteins for secretion and proteotoxic stress alleviation.

Comparative In Vivo Imaging of Retinal Structures in Tree Shrews, Humans, and Mice.

Rodent models, such as mice and rats, are commonly used to examine retinal ganglion cell damage in eye diseases. However, as nocturnal animals, rodent retinal structures differ from primates, imposing significant limitations in studying retinal pathology. Tree shrews (Tupaia belangeri) are small, diurnal paraprimates that exhibit superior visual acuity and color vision compared with mice. Like humans, tree shrews have a dense retinal nerve fiber layer (RNFL) and a thick ganglion cell layer (GCL), making them a valuable model for investigating optic neuropathies. In this study, we applied high-resolution visible-light optical coherence tomography to characterize the tree shrew retinal structure in vivo and compare it with that of humans and mice. We quantitatively characterize the tree shrew's retinal layer structure in vivo, specifically examining the sublayer structures within the inner plexiform layer (IPL) for the first time. Next, we conducted a comparative analysis of retinal layer structures among tree shrews, mice, and humans. We then validated our in vivo findings in the tree shrew inner retina using ex vivo confocal microscopy. The in vivo and ex vivo analyses of the shrew retina build the foundation for future work to accurately track and quantify the retinal structural changes in the IPL, GCL, and RNFL during the development and progression of human optic diseases.

Visual Acuity in Aniridia and WAGR Syndrome.

Purpose: Our purpose was to evaluate visual acuity in aniridia subjects and the more severely affected phenotype in WAGR syndrome subjects, and to assess potential impact on visual function. Materials and Methods: This was a retrospective comparative study of 25 aniridia subjects with nonsense mutations of PAX6 (50 eyes) and 25 WAGR syndrome subjects with large deletion mutations involving PAX6 (50 eyes). Aniridia subjects were age- and gender-matched with WAGR syndrome subjects in the Coordination of Rare Diseases at Sanford (CoRDS) database. Best-corrected ETDRS visual acuity measurements were converted to LogMAR visual acuity values, which were used to perform statistical analyses. Results: The age and gender distribution of the subjects was not statistically significantly different. The mean LogMAR values in aniridia and WAGR syndrome subjects were 0.95±0.53 and 1.51±0.99, respectively (P<0.001). In the better-seeing eye, mean LogMAR values were 0.78±0.15 in aniridia subjects and 1.40±0.88 in WAGR syndrome subjects (P=0.001). The mean LogMAR values for the better-seeing eye corresponded to Snellen visual acuity of 20/125 in aniridia subjects and 20/500 in WAGR syndrome subjects. This average visual acuity was worse than the threshold for profound visual impairment (WHO criteria) and legal blindness (AAO criteria) in WAGR syndrome but not in aniridia subjects. In analysis of both eyes, the visual efficiency was 34% in aniridia subjects and 2% in WAGR syndrome subjects. Conclusion: Visual acuity was significantly worse in WAGR subjects with multi-gene deletion mutations compared with aniridia subjects with nonsense mutations, which corresponded to differences in standard visual function thresholds. Our results suggest that visual acuity may indicate severity of ocular involvement and variability of phenotype in aniridia and WAGR syndrome.

Longitudinal Analysis of Retinal Ganglion Cell Damage at Individual Axon Bundle Level in Mice Using Visible-Light Optical Coherence Tomography Fibergraphy.

Purpose: We developed a new analytic tool based on visible-light optical coherence tomography fibergraphy (vis-OCTF) to longitudinally track individual axon bundle transformation as a new in vivo biomarker for retinal ganglion cell (RGC) damage. Methods: After acute optic nerve crush injury (ONC) in mice, we analyzed four parameters: lateral bundle width, axial bundle height, cross-sectional area, and the shape of individual bundles. We next correlated the morphological changes in RGC axon bundles with RGC soma loss. Results: We showed that axon bundles became wider and taller at three days post ONC (pONC), which correlated with about 15% RGC soma loss. At six days pONC, axon bundles showed a significant reduction in lateral width and cross-sectional area, followed by a reduction in bundle height at nine days pONC. Bundle shrinking at nine days pONC correlated with about 68% RGC soma loss. Both experimental and simulated results suggested that the cross-sectional area of individual RGC axon bundles is more sensitive than bundle width and height to indicate RGC soma loss. Conclusions: This study is the first to track and quantify individual RGC axon bundles in vivo after ONC injury. Translational Relevance: Recognizing RGC loss at its earliest stage is crucial for disease diagnosis and treatment. However, current clinical methods to detect the functional and structural changes in the inner retina are not sensitive enough to directly assess RGC health. In this study, we developed vis-OCTF-based parameters to track RGC damage, making possible to establishing a quantifiable biomarker for glaucoma.

Laser-based sample preparation for high-resolution X-ray-computed tomography of glasses and glass ceramics.

X-ray-computed tomography with sub-micron resolution (nano-CT) is one of the most useful techniques to examine the 3D microstructure of materials down to voxel sizes 10 nm. However, since size and shape of samples have considerable influence on acquisition time and data quality, adapted and universally applicable workflows are needed. Three novel workflows for sample preparation using ultra-short pulsed lasers are presented which allow for reproducible fabrication, safe extraction and mounting of samples. Their application potential is illustrated via nano-CT measurements of glass ceramics as well as a laser-modified glass. Since the according sample geometries take also the requirements of other analytical techniques such as transmission electron microscopy into account, samples prepared according to the new workflows can be furthermore seen as a starting point for correlative microstructural analyses involving multiple techniques.

Efficient preparation of microtip arrays for atom probe tomography using fs-laser processing.

Microtip arrays, also called microtip coupons, are routinely used in atom probe tomography (APT) as specimen carriers. They are commercially available consumables, usually made of Si with high electrical conductivity, produced via dedicated shaping techniques. Their purpose is to act as a specimen mount after focused ion beam (FIB) based lift-out procedures. Within this work, an alternative approach to prefabricated microtip coupons is presented, by directly creating a microtip array on the sample to be investigated utilizing fs-laser processing. An exemplary array of microtip posts was fs-laser processed from a TiN coating on Si substrate and subjected to final preparation via annular FIB milling. Subsequently, APT specimen of the TiN coating as well as of the Si substrate were successfully measured in laser assisted mode, using a commercial local electrode APT system. To further emphasize the versatility of the proposed approach, additional voltage measurements of highly conductive B doped Si arrays as well as exemplarily fs-laser processed microtip arrays of various other materials are provided as supplementary material to this article. The presented methodology bypasses the lift-out and avoids the necessity of a Pt weld between specimens and coupon posts which is frequently considered to represent a weak spot. It reduces consumables consumption and provides a high number of specimens in short time, while it is applicable for a wide range of materials and has thus the potential to revolutionize APT specimen preparation.

Characterization of ophthalmology virtual visits during the COVID-19 pandemic.

OBJECTIVES: To characterize the use of virtual visits, as well as compare the characteristics to in-person visits during the pandemic period. METHODS: This retrospective study included patients who had virtual and in-person ophthalmology visits from March 19, 2020, to July 31, 2020, in a large multispecialty ophthalmic center. Exclusion criteria included patients aged less than 18 years old; canceled, incomplete, mislabelled, and duplicated visits. 2943 virtual and 56,174 in-person visits were identified. A random sample of 3000 in-person visits was created. Each visit was analyzed as an individual data point. RESULTS: 2,266 virtual visits (2,049 patients, 64.3% female, mean [SD] age 64.3 [16.6] years old) and 2590 in-person visits (2509 patients, 59.5% female, 65.9 [15.8] years old) were included. Most virtual visits were classified as comprehensive ophthalmology (34.6%), optometry-related (19.5%), and oculoplastics (13.0%). For in-person visits, the most common specialties were optometry (29.8%), comprehensive ophthalmology (23.9%), and retina and uveitis (17.3%). The most common diagnoses in the virtual group were from the eyelids, lacrimal system, and orbits group (26.9%), while in the in-person groups were choroid and retina conditions (19.3%). CONCLUSIONS: Numerous ocular conditions were evaluated and managed through virtual visits, and external complaints and oculoplastic consults appear to be well-suited to the virtual format. Further studies focusing on visual outcomes and patient experience will be beneficial.

Reference Intervals in Coagulation Analysis.

Blood coagulation analysis is characterized by the application of a variety of materials, reagents, and analyzers for the determination of the same parameter, or analyte, by different laboratories worldwide. Accordingly, the application of common reference intervals, that, by definition, would represent a "range of values (of a certain analyte) that is deemed normal for a physiological measurement in healthy persons," is difficult to implement without harmonization of procedures. In fact, assay-specific reference intervals are usually established to allow for the discrimination of normal and abnormal values during evaluation of patient results. While such assay-specific reference intervals are often determined by assay manufacturers and subsequently adopted by customer laboratories, verification of transferred values is still mandatory to confirm applicability on site. The same is true for reference intervals that have been adopted from other laboratories, published information, or determined by indirect data mining approaches. In case transferable reference intervals are not available for a specific assay, a direct recruiting approach may or needs to be applied. In comparison to transferred reference interval verification, however, the direct recruiting approach requires a significantly higher number of well-defined samples to be collected and analyzed. In the present review, we aim to give an overview on the above-mentioned aspects and procedures, also with respect to relevant standards, regulations, guidelines, but also challenges for both, assay manufacturers and coagulation laboratories.

O-GlcNAc transferase plays a non-catalytic role in C. elegans male fertility.

Animal behavior is influenced by the competing drives to maintain energy and to reproduce. The balance between these evolutionary pressures and how nutrient signaling pathways intersect with mating remains unclear. The nutrient sensor O-GlcNAc transferase, which post-translationally modifies intracellular proteins with a single monosaccharide, is responsive to cellular nutrient status and regulates diverse biological processes. Though essential in most metazoans, O-GlcNAc transferase (ogt-1) is dispensable in Caenorhabditis elegans, allowing genetic analysis of its physiological roles. Compared to control, ogt-1 males had a four-fold reduction in mean offspring, with nearly two thirds producing zero progeny. Interestingly, we found that ogt-1 males transferred sperm less often, and virgin males had reduced sperm count. ogt-1 males were also less likely to engage in mate-searching and mate-response behaviors. Surprisingly, we found normal fertility for males with hypodermal expression of ogt-1 and for ogt-1 strains with catalytic-dead mutations. This suggests OGT-1 serves a non-catalytic function in the hypodermis impacting male fertility and mating behavior. This study builds upon research on the nutrient sensor O-GlcNAc transferase and demonstrates a role it plays in the interplay between the evolutionary drives for reproduction and survival.

Influencing Factors and Differences in Born Aggregometry in Specialized Hemostaseological Centers: Results of a Multicenter Laboratory Comparison.

Introduction Light transmission aggregometry (LTA) is regarded as the gold standard in platelet function diagnostics. However, there is a relevant degree of interlaboratory variability in practical applications. Objective The aim of the present study was to develop a practicable laboratory comparison on LTA and to analyze differences and influencing factors in regard to standardization in five specialized hemostaseological centers. Methods The study was performed on 30 patients in total. Each center performed LTA on blood samples from six healthy volunteers (three men and three women) using the inductors collagen (Col), adenosine diphosphate (ADP), arachidonic acid (ARA), and ristocetin. The LTA was performed three times using different methods as follows: (1) International Society on Thrombosis and Haemostasis recommendations with identical reagents, (2) in-house protocols and the identical reagents; and (3) in-house protocols and in-house reagents. Results A total of 396 measurements of 30 probands were performed. Even after standardization of the protocol and using identical reagents, there were significant differences between the centers regarding the final and maximum aggregation ( p = 0.002 and <0.001) and further significant differences in the maximum and final aggregation according to the wavelength of the device used to measure the LTA (PAP-8: 430 nm, APACT 4004: 740 nm [ p < 0.001 each]). Using identical reagents but individual inductor concentrations and laboratory protocols also resulted in different maximum and final aggregation. The largest differences were seen with Col and ristocetin; there were significant influences from the reagents' manufacturers in the results of aggregometry for the inductor Col ( p < 0.01) but not for ADP, ARA, and ristocetin. Conclusion In this study, we proved that there are significant influences from the used aggregometers, inductors concentrations, and manufacturers. These results illustrate the challenges and importance of standardization of LTA.

On-Chip Preconcentration Microchip Capillary Electrophoresis Based CE-PRM-LIVE for High-Throughput Selectivity Profiling of Deubiquitinase Inhibitors.

The family of deubiquitinases (DUBs) comprises ∼100 enzymes that cleave ubiquitin from substrate proteins and thereby regulate key aspects of human physiology. DUBs have recently emerged as disease-relevant and chemically tractable, although currently there are no approved DUB-targeting drugs and most preclinical small molecules are low-potency and/or multitargeted. We paired a novel capillary electrophoresis microchip containing an integrated, "on-chip" C18 bed (SPE-ZipChip) with a TMT version of our recently described PRM-LIVE acquisition scheme on a timsTOF Pro mass spectrometer to facilitate rapid activity-based protein profiling of DUB inhibitors. We demonstrate the ability of the SPE-ZipChip to improve proteome coverage of complex samples as well as the quantitation integrity of CE-PRM-LIVE for TMT labeled samples. These technologies provide a platform to accurately quantify competitive binding of covalent and reversible inhibitors in a multiplexed assay that spans 49 endogenous DUBs in less than 15 min.

Material-specific high-resolution table-top extreme ultraviolet microscopy.

Microscopy with extreme ultraviolet (EUV) radiation holds promise for high-resolution imaging with excellent material contrast, due to the short wavelength and numerous element-specific absorption edges available in this spectral range. At the same time, EUV radiation has significantly larger penetration depths than electrons. It thus enables a nano-scale view into complex three-dimensional structures that are important for material science, semiconductor metrology, and next-generation nano-devices. Here, we present high-resolution and material-specific microscopy at 13.5 nm wavelength. We combine a highly stable, high photon-flux, table-top EUV source with an interferometrically stabilized ptychography setup. By utilizing structured EUV illumination, we overcome the limitations of conventional EUV focusing optics and demonstrate high-resolution microscopy at a half-pitch lateral resolution of 16 nm. Moreover, we propose mixed-state orthogonal probe relaxation ptychography, enabling robust phase-contrast imaging over wide fields of view and long acquisition times. In this way, the complex transmission of an integrated circuit is precisely reconstructed, allowing for the classification of the material composition of mesoscopic semiconductor systems.

Long-Term Follow-Up of Mechanical Circulatory Support in Peripartum Cardiomyopathy (PPCM) Refractory to Medical Management: A Multicenter Study.

BACKGROUND: Peripartum cardiomyopathy (PPCM) is a rare, life-threatening form of heart disease, frequently associated with gene alterations and, in some cases, presenting with advanced heart failure. Little is known about ventricular assist device (VAD) implantation in severe PPCM cases. We describe long-term follow-up of PPCM patients who were resistant to medical therapy and received mechanical circulatory support or heart transplant. METHODS AND RESULTS: A total of 13 patients were included with mean follow-up of eight years. Mean age of PPCM onset was 33.7 ± 7.7 years. All patients were initially treated with angiotensin-converting enzyme inhibitors and beta-blockers, and four received bromocriptine. Overall, five patients received VADs (three biventricular, two isolated left ventricular) at median 27 days (range: 3 to 150) following childbirth. Two patients developed drive line infection. Due to the short support time, none of those patients had a stroke or VAD thrombosis. In total, five patients underwent heart transplantation, of which four previously had implanted VADs. Median time to transplantation from PPCM onset was 140 days (range: 43 to 776), and time to transplantation from VAD implantation were 7, 40, 132, and 735 days, respectively. All patients survived until most recent follow up, with the exception of one patient who died following unrelated abdominal surgery two years after PPCM recovery. CONCLUSIONS: In patients with severe, life-threatening PPCM refractory to medical management, mechanical circulatory support with or without heart transplantation is a safe therapeutic option.

Analysis of drug residue in needle-exchange syringes in Washington, D.C.

For the first time in Washington, D.C., an analysis of drug residue from used needle-exchange syringes has been performed. This analysis is part of a larger initiative to understand the District of Columbia's illicit drug supply and its intravenous (IV) user's consumption trends as our nation faces the opioid epidemic. The goal of this study is to develop a more comprehensive monitoring program that provides real-time analysis necessary for public health organizations, in addition to providing initial observations of drugs detected. A total of 1187 syringes were analyzed over a period of nine months. Of these, 732 syringes (61.7%) were confirmed to contain a controlled dangerous substance (CDS). Fentanyl was detected in 490 syringes, the most observed CDS in all syringes analyzed. Heroin was the second most detected CDS, observed in 192 syringes. The third most detected CDS was cocaine, which was observed in 132 syringes, followed by the fourth most detected CDS, methamphetamine, observed in 82 syringes. Novel findings of this study include the first reported detections of methamphetamine, synthetic cathinones, and synthetic cannabinoids in used syringes in D.C. Ninety-seven syringes that contained no CDS contained a non-controlled substance of interest, such as diphenhydramine, xylazine, and etizolam. One limitation of this study is that this method cannot determine whether mixtures present in syringes stem from mixtures present prior to injection, back-to-back usage, or sharing of needles. This preliminary study illustrates the strength of surveillance to monitor drug trends and can be used to detect emerging novel dangerous substances in the future.

PRM-LIVE with Trapped Ion Mobility Spectrometry and Its Application in Selectivity Profiling of Kinase Inhibitors.

Parallel reaction monitoring (PRM) has emerged as a popular approach for targeted protein quantification. With high ion utilization efficiency and first-in-class acquisition speed, the timsTOF Pro provides a powerful platform for PRM analysis. However, sporadic chromatographic drift in peptide retention time represents a fundamental limitation for the reproducible multiplexing of targets across PRM acquisitions. Here, we present PRM-LIVE, an extensible, Python-based acquisition engine for the timsTOF Pro, which dynamically adjusts detection windows for reproducible target scheduling. In this initial implementation, we used iRT peptides as retention time standards and demonstrated reproducible detection and quantification of 1857 tryptic peptides from the cell lysate in a 60 min PRM-LIVE acquisition. As an application in functional proteomics, we use PRM-LIVE in an activity-based protein profiling platform to assess binding selectivity of small-molecule inhibitors against 220 endogenous human kinases.

Sample preparation for analytical scanning electron microscopy using initial notch sectioning.

A novel method for broad ion beam based sample sectioning using the concept of initial notches is presented. An adapted sample geometry is utilized in order to create terraces with a well-define d step in erosion depth from the surface. The method consists of milling a notch into the surface, followed by glancing-angle ion beam erosion, which leads to preferential erosion at the notch due to increased local surface elevation. The process of terrace formation can be utilized in sample preparation for analytical scanning electron microscopy in order to get efficient access to the depth-dependent microstructure of a material. It is demonstrated that the method can be applied to both conducting and non-conducting specimens. Furthermore, experimental parameters influencing the preparation success are determined. Finally, as a proof-of-concept, an electron backscatter diffraction study on a surface crystallized diopside glass ceramic is performed, where the method is used to analyze orientation dependent crystal growth phenomena occurring during growth of surface crystals into the bulk.

Detection of simple and complex de novo mutations with multiple reference sequences.

The characterization of de novo mutations in regions of high sequence and structural diversity from whole-genome sequencing data remains highly challenging. Complex structural variants tend to arise in regions of high repetitiveness and low complexity, challenging both de novo assembly, in which short reads do not capture the long-range context required for resolution, and mapping approaches, in which improper alignment of reads to a reference genome that is highly diverged from that of the sample can lead to false or partial calls. Long-read technologies can potentially solve such problems but are currently unfeasible to use at scale. Here we present Corticall, a graph-based method that combines the advantages of multiple technologies and prior data sources to detect arbitrary classes of genetic variant. We construct multisample, colored de Bruijn graphs from short-read data for all samples, align long-read-derived haplotypes and multiple reference data sources to restore graph connectivity information, and call variants using graph path-finding algorithms and a model for simultaneous alignment and recombination. We validate and evaluate the approach using extensive simulations and use it to characterize the rate and spectrum of de novo mutation events in 119 progeny from four Plasmodium falciparum experimental crosses, using long-read data on the parents to inform reconstructions of the progeny and to detect several known and novel nonallelic homologous recombination events.

New Roles for the Heterochronic Transcription Factor LIN-29 in Cuticle Maintenance and Lipid Metabolism at the Larval-to-Adult Transition in Caenorhabditis elegans.

Temporal regulation of gene expression is a crucial aspect of metazoan development. In the roundworm Caenorhabditis elegans, the heterochronic pathway controls multiple developmental events in a time-specific manner. The most downstream effector of this pathway, the zinc-finger transcription factor LIN-29, acts in the last larval stage (L4) to regulate elements of the larval-to-adult switch. Here, we explore new LIN-29 targets and their implications for this developmental transition. We used RNA-sequencing to identify genes differentially expressed between animals misexpressing LIN-29 at an early time point and control animals. Among 230 LIN-29-activated genes, we found that genes encoding cuticle collagens were overrepresented. Interestingly, expression of lin-29 and some of these collagens was increased in adults with cuticle damage, suggesting a previously unknown function for LIN-29 in adult cuticle maintenance. On the other hand, genes involved in fat metabolism were enriched among 350 LIN-29-downregulated targets. We used mass spectrometry to assay lipid content in animals overexpressing LIN-29 and observed reduced fatty acid levels. Many LIN-29-repressed genes are normally expressed in the intestine, suggesting cell-nonautonomous regulation. We identified several LIN-29 upregulated genes encoding signaling molecules that may act as mediators in the regulation of intestinally expressed genes encoding fat metabolic enzymes and vitellogenins. Overall, our results support the model of LIN-29 as a major regulator of adult cuticle synthesis and integrity, and as the trigger for metabolic changes that take place at the important transition from rapid growth during larval life to slower growth and offspring production during adulthood.

Plasmodium vivax chloroquine resistance links to pvcrt transcription in a genetic cross.

Mainstay treatment for Plasmodium vivax malaria has long relied on chloroquine (CQ) against blood-stage parasites plus primaquine against dormant liver-stage forms (hypnozoites), however drug resistance confronts this regimen and threatens malaria control programs. Understanding the basis of P. vivax chloroquine resistance (CQR) will inform drug discovery and malaria control. Here we investigate the genetics of P. vivax CQR by a cross of parasites differing in drug response. Gametocytogenesis, mosquito infection, and progeny production are performed with mixed parasite populations in nonhuman primates, as methods for P. vivax cloning and in vitro cultivation remain unavailable. Linkage mapping of progeny surviving >15 mg/kg CQ identifies a 76 kb region in chromosome 1 including pvcrt, an ortholog of the Plasmodium falciparum CQR transporter gene. Transcriptional analysis supports upregulated pvcrt expression as a mechanism of CQR.