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SARS-CoV-2 vaccine-acquired immunity provides robust cross-variant recognition, while infection-acquired immunity can be heterogenous, with disease severity often modulating post-recovery responses. We assessed antibody waning dynamics between infection- and vaccination-acquired immunity across variants of concern (VOC). mRNA vaccination induced potent, cross-VOC Spike recognition and functional responses, but waned more rapidly for Omicron Spike. Hospitalized individuals developed more durable functional responses with lower peaks compared to mRNA vaccination, while outpatients exhibited slower decay than inactivated vaccine recipients. Humoral decay for the receptor binding domain tracked with neutralizing antibody titers, while S2-directed responses tracked with antibody-dependent myeloid cellular phagocytosis. Boosting the recovered patients with mRNA or inactivated vaccines expanded humoral breadth, durability, and restored functional responses, eliminating the severity- and platform-associated decay differences. Therefore, post-recovery hybrid immunization compensates for this distinction and broadens humoral breadth, highlighting the value of boosting immunity in previously infected individuals., Competing Interests: Competing Interests: The authors declare the following competing interests; Galit Alter is a founder/equity holder in Seroymx Systems and Leyden Labs. G.A. has served as a scientific advisor for Sanofi Vaccines. G.A. has collaborative agreements with GSK, Merck, Abbvie, Sanofi, Medicago, BioNtech, Moderna, BMS, Novavax, SK Biosciences, Gilead, and Sanaria. R.A.M. has served as a scientific advisor for Valneva SE. The remaining authors declare no competing interests.

Mounting evidence indicates that proteotoxic stress is a primary activator of the CARD8 inflammasome, but the complete array of signals that control this inflammasome have not yet been established. Notably, we recently discovered that several hydrophobic radical-trapping antioxidants (RTAs), including JSH-23, potentiate CARD8 inflammasome activation through an unknown mechanism. Here, we report that these RTAs directly alkylate several cysteine residues in the N-terminal disordered region of CARD8. These hydrophobic modifications destabilize the repressive CARD8 N-terminal fragment and accelerate its proteasome-mediated degradation, thereby releasing the inflammatory CARD8 C-terminal fragment from autoinhibition. Consistently, we also found that unrelated (non-RTA) hydrophobic electrophiles as well as genetic mutation of the CARD8 cysteine residues to isoleucines similarly potentiate inflammasome activation. Overall, our results not only provide further evidence that protein folding stress is a key CARD8 inflammasome-activating signal, but also indicate that the N-terminal cysteines can play key roles in tuning the response to this stress., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: Candida is one of the common pathogens in nosocomial infections. Culture is the gold standard for diagnosing candidemia. Candida albicans is identified via the germ tube test, which uses serum as the culture medium, which is costly and time-consuming. This study was conducted to evaluate and compare a relatively simple, fast, and reliable method for the detection of Candida albicans ., Methods: We conducted this randomized case study at Taipei City Hospital (TCH) from January 2023 to August 2023, with a total of 30 specimen culture reports collected and confirmed to be cases of Candida albicans infection. A germ tube test was performed in a 37°C water bath using serum, plasma, and safe plasma products (Fresh Frozen Plasma, FFP). Further, the same procedures were repeated with the addition of 22% bovine serum albumin (BSA) to the identification/culture., Results: By adding BSA, more than 50% of the budding phenomenon was observed in 40 minutes, which shortened the diagnosis time compared with the traditional method (2-3 hours). Using BSA can shorten the identification time for early clinical medication and improve the quality of medical care., Conclusion: Using safer plasma products for germ tube test of candidiasis not only reduced the risk of infection for medical technicians but could also replace the serum used in traditional methods to increase convenience and save time. This study proposed BSA as a germ tube induction medium enhancer, which reduced the culture time, thereby enabling quicker diagnosis of C. albicans infections., Competing Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2024.)

Mounting evidence indicates that antibodies can contribute towards control of tuberculosis (TB). However, the underlying mechanisms of humoral immune protection and whether antibodies can be exploited in therapeutic strategies to combat TB are relatively understudied. Here we engineered the receptor-binding Fc (fragment crystallizable) region of an antibody recognizing the Mycobacterium tuberculosis (Mtb) capsule, to define antibody Fc-mediated mechanism(s) of Mtb restriction. We generated 52 Fc variants that either promote or inhibit specific antibody effector functions, rationally building antibodies with enhanced capacity to promote Mtb restriction in a human whole-blood model of infection. While there is likely no singular Fc profile that universally drives control of Mtb, here we found that several Fc-engineered antibodies drove Mtb restriction in a neutrophil-dependent manner. Single-cell RNA sequencing analysis showed that a restrictive Fc-engineered antibody promoted neutrophil survival and expression of cell-intrinsic antimicrobial programs. These data show the potential of Fc-engineered antibodies as therapeutics able to harness the protective functions of neutrophils to promote control of TB., (© 2024. The Author(s).)

NLRP1 is an innate immune receptor that detects pathogen-associated signals, assembles into a multiprotein structure called an inflammasome, and triggers a proinflammatory form of cell death called pyroptosis. We previously discovered that the oxidized, but not the reduced, form of thioredoxin-1 directly binds to NLRP1 and represses inflammasome formation. However, the molecular basis for NLRP1's selective association with only the oxidized form of TRX1 has not yet been established. Here, we leveraged AlphaFold-Multimer, site-directed mutagenesis, thiol-trapping experiments, and mass spectrometry to reveal that a specific cysteine residue (C427 in humans) on NLRP1 forms a transient disulfide bond with oxidized TRX1. Overall, this work demonstrates how NLRP1 monitors the cellular redox state, further illuminating an unexpected connection between the intracellular redox potential and the innate immune system., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Nuclear factor kappa B (NF-κB) is a key regulator in immune signaling and is known to exhibit a digital activation pattern. Yet the molecular basis underlying the heterogeneity in NF-κB activation at single-cell level is not entirely understood. Here, we show that NF-κB activation in single cells is largely regulated by intrinsic differences at the receptor level. Using the genome editing and time-lapse imaging, we directly characterize endogenous TNFR1 dynamics and NF-κB activation from the same single cells. Total internal reflection fluorescence (TIRF) microscopy shows that endogenous TNFR1 forms pre-ligand clusters in the resting cells. Upon tumor necrosis factor (TNF) stimulation, the diffusion coefficient of membrane TNFR1 was significantly decreased and a substantial level of TNFR1 undergoes oligomerization to form trimers and hexamers. Moreover, multi-color cell imaging reveals that both digital and graded information processing regulate NF-κB activation across different TNFR1 expression levels. Our results indicate that single-cell NF-κB activation potential strongly correlates with its TNFR1 characteristics., Competing Interests: The authors declare no competing interests., (© 2024 The Author(s).)

Multicolor labeling for monitoring the intracellular localization of the same target type in the native environment using chemical fluorescent dyes is a challenging task. This approach requires both bioorthogonal and biocompatible ligations with an excellent fluorescence signal-to-noise ratio. Here, we present a metabolic glycan labeling technique that uses homemade fluorogenic dyes to investigate glycosylation patterns in live cells. These dyes allowed us to demonstrate rapid and efficient simultaneous multilabeling of glycoconjugates with minimum fluorescence noise. Our results demonstrate that this approach is capable of not only probing sialylation and GlcNAcylation in cells but also specifically labeling the cell-surface and intracellular sialylated glycoconjugates in live cells. In particular, we performed site-specific dual-channel fluorescence imaging of extra and intracellular sialylated glycans in HeLa and PC9 cancer cells as well as identified fluorescently labeled sialylated glycoproteins and glycans by a direct enrichment approach combined with an MS-based proteomic analysis in the same experiment. In conclusion, this study provides multilabeling tools in cellular systems for simultaneous site-specific glycan imaging and glycoproteomic analysis to study potential cancer- and disease-associated glycoconjugates., (© 2023 Wiley-VCH GmbH.)

The study evaluated the prevalence of gonorrhoea and chlamydia infections and find out other non-infectious diseases in sexually active young males with urethritis-like symptoms and their treatment outcomes. We retrospectively reviewed the young adult males (aged 20-50 years) who visited our clinic with urethritis symptoms from March 2019 to April 2022. All patients underwent urinalysis, urine culture, and urinary polymerase chain reaction (PCR) testing for gonorrhoea and chlamydia. Student's t-test and Pearson's chi-square test were used to compare the differences between the triple-negative group (i.e., negative results in urinalysis, urine culture, and urinary PCR) and the any-positive group. Logistic regression analyses were used to evaluate the predictive factors for positive PCR results for gonorrhoea or chlamydia in patients with negative urinalysis and urine culture. Of the 365 participants with urethritis-like symptoms, 139 patients were diagnosed of gonococcal or chlamydia urethritis. Among the 202 patients with negative urinalysis and urine culture, 60 patients were diagnosed with gonorrhoea or chlamydia using PCR. Urethral discharge was an independent predictor. 142 patients with triple negative results were attributed to other non-infectious diseases. Empirical antibiotic treatment is recommended for patients with urethritis symptoms showing positive or negative urinalysis results but with urethral discharge., (© 2023. Springer Nature Limited.)

Secondary mutation, T790M, conferring tyrosine kinase inhibitors (TKIs) resistance beyond oncogenic epidermal growth factor receptor (EGFR) mutations presents a challenging unmet need. Although TKI-resistant mechanisms are intensively investigated, the underlying responses of cancer cells adapting drug perturbation are largely unknown. To illuminate the molecular basis linking acquired mutation to TKI resistance, affinity purification coupled mass spectrometry was adopted to dissect EGFR interactome in TKI-sensitive and TKI-resistant non-small cell lung cancer cells. The analysis revealed TKI-resistant EGFR-mutant interactome allocated in diverse subcellular distribution and enriched in endocytic trafficking, in which gefitinib intervention activated autophagy-mediated EGFR degradation and thus autophagy inhibition elevated gefitinib susceptibility. Alternatively, gefitinib prompted TKI-sensitive EGFR translocating toward cell periphery through Rab7 ubiquitination which may favor efficacy to TKIs suppression. This study revealed that T790M mutation rewired EGFR interactome that guided EGFR to autophagy-mediated degradation to escape treatment, suggesting that combination therapy with TKI and autophagy inhibitor may overcome acquired resistance in non-small cell lung cancer., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: The decision between local and free tissue coverage for distal lower leg defects has long been dictated by the location and size of defects. Recent reports of distal defects treated successfully with pedicled perforator flaps demonstrate equivalent clinical outcomes; however, the complication rate can be high. The goal of this study was to evaluate the cost equivalence of free versus pedicled perforator flap to assist decision-making and guide clinical care., Methods: The institutional database was searched for patients with acute injury over the distal lower extremity requiring free or pedicled perforator flap. Demographic, clinical, and total resource cost was gathered. Patients were matched to Gustilo-Anderson or Arbeitsgemeinschaft fur Osteosynthesefragen classification as well as size of defect and outcomes, and cost compared., Results: We have included 108 free flaps and 22 pedicled perforator flaps in the study. There was no difference in complication rate between groups. Free flaps had significantly more reoperations, required longer operative time, and had longer intensive care unit (ICU) care with higher cost of surgery and overall cost than pedicled flaps. When controlling for size of defect, surgical cost remained significantly different between groups ( p  = 0.013), but overall cost did not. Multivariable regression analysis indicated flap type to be the primary driver of cost of surgery, while body mass index elevated the total cost., Conclusion: Pedicled perforator flap coverage for small to medium-sized defects (< 70 cm 2 ) is a viable and cost-effective option for distal lower leg soft tissue reconstruction after acute traumatic injury with similar clinical outcomes and shorter operative duration and ICU stay., Competing Interests: None declared., (Thieme. All rights reserved.)

Efficiently delivering liposomal content to cells in a relatively uniform dose and patterned fashion, especially bypassing the degradative endocytosis pathway, is an important technology in cell culture and potentially to tissue engineering that still remains challenging. We developed a "nano-on-nano" platform technology that consists of the following three material features: (1) high density silicon nanopillars to create a pseudo-3-dimensional nanoenvironment for cell culturing, (2) thermoresponsive polymer grafted onto silicon nanopillars to form a responsive nanosubstrate, and (3) immobilized liposomes using a biotin-streptavidin-biotin conjugation. The working principle is that the liposomes are detached for cellular uptake upon thermal stimulation and high local liposome concentration between the cells and substrates drives the cellular uptake with nonendocytic pathways. Cryo-EM images confirms that liposomes are attached to form liposome-warped nanopillars. Upon thermal stimulation, an 8 times higher increase in the liposomal fluorescence intensity is observed compared to the conventional solution-phase liposome delivery, indicating that high local concentration drives liposome uptake with greater efficiency. Moreover, preliminary mechanistic studies reveal that these liposomes are taken up by nonendocytic pathways. The ability of our nano-on-nano delivery system that achieves efficient dose-uniform cellular delivery can open a unique era in cell and tissue engineering by controlling cell behaviors with the delivery of bioactive ingredient-loaded liposomes.

Inflammasomes are innate immune signaling platforms that trigger pyroptotic cell death. NLRP1 and CARD8 are related human inflammasomes that detect similar danger signals, but NLRP1 has a higher activation threshold and triggers a more inflammatory form of pyroptosis. Both sense the accumulation of intracellular peptides with Xaa-Pro N-termini, but Xaa-Pro peptides on their own without a second danger signal only activate the CARD8 inflammasome. We recently reported that a dual inhibitor of the Xaa-Pro-cleaving M24B aminopeptidases PEPD and XPNPEP1 called CQ31 selectively activates the CARD8 inflammasome by inducing the build-up of Xaa-Pro peptides. Here, we performed structure-activity relationship studies on CQ31 to develop the optimized dual PEPD/XPNPEP1 inhibitor CQ80 that more effectively induces CARD8 inflammasome activation. We anticipate that CQ80 will become a valuable tool to study the basic biology and therapeutic potential of selective CARD8 inflammasome activation.

The danger signals that activate the related nucleotide-binding domain leucine-rich repeat pyrin domain-containing 1 (NLRP1) and caspase activation and recruitment domain-containing 8 (CARD8) inflammasomes have not been fully established. We recently reported that the oxidized form of TRX1 binds to NLRP1 and represses inflammasome activation. These findings suggested that intracellular reductive stress, which would reduce oxidized TRX1 and thereby abrogate the NLRP1-TRX1 interaction, is an NLRP1 inflammasome-activating danger signal. However, no agents that induce reductive stress were known to test this premise. Here, we identify and characterize several radical-trapping antioxidants, including JSH-23, that induce reductive stress. We show that these compounds accelerate the proteasome-mediated degradation of the repressive N-terminal fragments of both NLRP1 and CARD8, releasing the inflammasome-forming C-terminal fragments from autoinhibition. Overall, this work validates chemical probes that induce reductive stress and establishes reductive stress as a danger signal sensed by both the NLRP1 and CARD8 inflammasomes., Competing Interests: Declaration of interests MSKCC has filed for patent protection on behalf of D.A.B. and Q.W. for inventions relating to the induction of reductive stress., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Aeromonas necrotizing fasciitis (NF) causes high rates of amputation and mortality, even after aggressive surgical debridement and antibacterial therapy. This study investigated the effects of rational use of antibiotics and education by infectious disease (ID) physicians on Aeromonas NF treatment outcomes., Methods: Retrospective review for conducted for four years (period I, without an ID physician, December 2001 to December 2005) and 15 years (period II, with an ID physician, January 2006 to March 2021). In period II, the hospital-wide computerized antimicrobial approval system (HCAAS) was also implemented. A pretest-posttest time series analysis compared the two periods. Differences in clinical outcomes, demographics, comorbidities, signs and symptoms, laboratory findings, Aeromonas antibiotic susceptibility, and antibiotic regimens were compared between the two periods., Results: There were 19 patients in period I and 53 patients in period II. Patients had a lower rate of amputation or mortality in period II (35.8%) compared with period I (63.2%). Forty-four patients (61.1%) had polymicrobial infections. In the emergency room, the rate of misdiagnosis decreased from 47.4% in period I to 28.3% in period II, while effective empiric antibiotic usage increased from 21.1% in period I to 66.0% in period II. After the ID physician's adjustment, 69.4% received monotherapy in period II compared to 33.3% in period I., Conclusions: Because Aeromonas NF had a high mortality rate and was often polymicrobial, choosing an antibiotic regimen was difficult. Using the HCAAS by an experienced ID physician can improve rational antibiotic usage and clinical outcomes in Aeromonas NF.

Bacterial and viral pathogens can modulate the glycosylation of key host proteins to facilitate pathogenesis by using various glycosidases, particularly sialidases. Epidermal growth factor receptor (EGFR) signaling is activated by ligand-induced receptor dimerization and oligomerization. Ligand binding induces conformational changes in EGFR, leading to clusters and aggregation. However, information on the relevance of EGFR clustering in the pattern of glycosylation during bacterial and viral invasion remains unclear. In this study, (1) we established CRISPR/Cas9-mediated GFP knock-in (EGFP-KI) HeLa cells expressing fluorescently tagged EGFR at close to endogenous levels to study EGF-induced EGFR clustering and molecular dynamics; (2) We studied the effect of sialylation on EGF-induced EGFR clustering and localization in live cells using a high content analysis platform and raster image correlation spectroscopy (RICS) coupled with a number and brightness (N&B) analysis; (3) Our data reveal that the removal of cell surface sialic acids by sialidase treatment significantly decreases EGF receptor clustering with reduced fluorescence intensity, number, and area of EGFR-GFP clusters per cell upon EGF stimulation. Sialylation appears to mediate EGF-induced EGFR clustering as demonstrated by the change of EGFR-GFP clusters in the diffusion coefficient and molecular brightness, providing new insights into the role of sialylation in EGF-induced EGFR activation; and (4) We envision that the combination of CRISPR/Cas9-mediated fluorescent tagging of endogenous proteins and fluorescence imaging techniques can be the method of choice for studying the molecular dynamics and interactions of proteins in live cells.

CARD8 is a pattern-recognition receptor that forms a caspase-1-activating inflammasome. CARD8 undergoes constitutive autoproteolysis, generating an N-terminal (NT) fragment with a disordered region and a ZU5 domain and a C-terminal (CT) fragment with UPA and CARD domains. Dipeptidyl peptidase 8 and dipeptidyl peptidase 9 inhibitors, including Val-boroPro, accelerate the degradation of the NT fragment via a poorly characterized proteasome-mediated pathway, thereby releasing the inflammatory CT fragment from autoinhibition. Here, we show that the core 20S proteasome, which degrades disordered and misfolded proteins independent of ubiquitin modification, controls activation of the CARD8 inflammasome. In unstressed cells, we discovered that the 20S proteasome degrades just the NT disordered region, leaving behind the folded ZU5, UPA, and CARD domains to act as an inhibitor of inflammasome assembly. However, in Val-boroPro-stressed cells, we show the 20S proteasome degrades the entire NT fragment, perhaps due to ZU5 domain unfolding, freeing the CT fragment from autoinhibition. Taken together, these results show that the susceptibility of the CARD8 NT domain to 20S proteasome-mediated degradation controls inflammasome activation., Competing Interests: Conflicts of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Accurate glioma subtype classification is critical for the treatment management of patients with brain tumors. Developing an automatically computer-aided algorithm for glioma subtype classification is challenging due to many factors. One of the difficulties is the label constraint. Specifically, each case is simply labeled the glioma subtype without precise annotations of lesion regions information. In this paper, we propose a novel hybrid fully convolutional neural network (CNN)-based method for glioma subtype classification using both whole slide imaging (WSI) and multiparametric magnetic resonance imagings (mpMRIs). It is comprised of two methods: a WSI-based method and a mpMRIs-based method. For the WSI-based method, we categorize the glioma subtype using a 2D CNN on WSIs. To overcome the label constraint issue, we extract the truly representative patches for the glioma subtype classification in a weakly supervised fashion. For the mpMRIs-based method, we develop a 3D CNN-based method by analyzing the mpMRIs. The mpMRIs-based method consists of brain tumor segmentation and classification. Finally, to enhance the robustness of the predictions, we fuse the WSI-based and mpMRIs-based results guided by a confidence index. The experimental results on the validation dataset in the competition of CPM-RadPath 2020 show the comprehensive judgments from both two modalities can achieve better performance than the ones by solely using WSI or mpMRIs. Furthermore, our result using the proposed method ranks the third place in the CPM-RadPath 2020 in the testing phase. The proposed method demonstrates a competitive performance, which is creditable to the success of weakly supervised approach and the strategy of label agreement from multi-modality data., (© 2022. The Author(s).)

Solvatofluorochromism, a solvation effect on the fluorescence color of an organic dye, is a property generally limited to fluid solutions. We demonstrate herein the concept of solid-state solvatofluorochromism by using an organogelator ( 1 -SG), which consists of a solvatofluorochromic green fluorescence protein (GFP) chromophore ( 1 ) and a sugar gelator (SG). While 1 -SG could be located in the liquid phase or in the fibrous solid matrix of the SG gel, our results show that the one in the solid matrix but near the liquid interface has superior fluorescence stability and quantum efficiency as well as solvatofluorochromicity than the one in the liquid phase. In addition, the phenomenon of fluorescence turn-on occurs when the gel is formed in protic solvents. These features have been applied to perform multicolor fluorescence patterning, chemical vapor sensing, data encryption and decryption, and real-time fluorescence cell imaging.

Respiratory syncytial virus (RSV) infection is a major cause of respiratory illness in infants and the elderly. Although several vaccines have been developed, none have succeeded in part due to our incomplete understanding of the correlates of immune protection. While both T cells and antibodies play a role, emerging data suggest that antibody-mediated mechanisms alone may be sufficient to provide protection. Therefore, to map the humoral correlates of immunity against RSV, antibody responses across six different vaccines were profiled in a highly controlled nonhuman primate-challenge model. Viral loads were monitored in both the upper and lower respiratory tracts, and machine learning was used to determine the vaccine platform-agnostic antibody features associated with protection. Upper respiratory control was associated with virus-specific IgA levels, neutralization, and complement activity, whereas lower respiratory control was associated with Fc-mediated effector mechanisms. These findings provide critical compartment-specific insights toward the rational development of future vaccines., Competing Interests: Declaration of interests Galit Alter is a founder of SeromYx Systems., (Copyright © 2021. Published by Elsevier Inc.)

Thermoelectric materials are considered promising candidates for thermal energy conversion. This study presents the fabrication of Zn- and Ce-alloyed In 2 O 3 with a porous structure. The electrical conductivity was improved by the alloying effect and an ultra-low thermal conductivity was observed owing to the porous structure, which concomitantly provide a distinct enhancement of ZT . However, SiO 2 nanoparticle additives react with the matrix to form a third-phase impurity, which weakens the electrical conductivity and increases the thermal conductivity. A thermoelectric module was constructed for the purpose of thermal heat energy conversion. Our experimental results proved that both an enhancement in electrical conductivity and a suppression in thermal conductivity could be achieved through nano-engineering. This approach presents a feasible route to synthesize porous thermoelectric oxides, and provides insight into the effect of additives; moreover, this approach is a cost-effective method for the fabrication of thermoelectric oxides without traditional hot-pressing and spark-plasma-sintering processes., (© 2022 IOP Publishing Ltd.)

Microfluidic lab-on-a-chip devices are changing the way that in vitro diagnostics and drug development are conducted, based on the increased precision, miniaturization and efficiency of these systems relative to prior methods. However, the full potential of microfluidics as a platform for therapeutic medical devices such as extracorporeal organ support has not been realized, in part due to limitations in the ability to scale current designs and fabrication techniques toward clinically relevant rates of blood flow. Here we report on a method for designing and fabricating microfluidic devices supporting blood flow rates per layer greater than 10 mL min -1 for respiratory support applications, leveraging advances in precision machining to generate fully three-dimensional physiologically-based branching microchannel networks. The ability of precision machining to create molds with rounded features and smoothly varying channel widths and depths distinguishes the geometry of the microchannel networks described here from all previous reports of microfluidic respiratory assist devices, regarding the ability to mimic vascular blood flow patterns. These devices have been assembled and tested in the laboratory using whole bovine or porcine blood, and in a porcine model to demonstrate efficient gas transfer, blood flow and pressure stability over periods of several hours. This new approach to fabricating and scaling microfluidic devices has the potential to address wide applications in critical care for end-stage organ failure and acute illnesses stemming from respiratory viral infections, traumatic injuries and sepsis.

Advances in microfluidics technologies have spurred the development of a new generation of microfluidic respiratory assist devices, constructed using microfabrication techniques capable of producing microchannel dimensions similar to those found in human capillaries and gas transfer films in the same thickness range as the alveolar membrane. These devices have been tested in laboratory settings and in some cases in extracorporeal animal experiments, yet none have been advanced to human clinical studies. A major challenge in the development of microfluidic oxygenators is the difficulty in scaling the technology toward high blood flows necessary to support adult humans; such scaling efforts are often limited by the complexity of the fabrication process and the manner in which blood is distributed in a three-dimensional network of microchannels. Conceptually, a central advantage of microfluidic oxygenators over existing hollow-fiber membrane-based configurations is the potential for shallower channels and thinner gas transfer membranes, features that reduce oxygen diffusion distances, to result in a higher gas transfer efficiency defined as the ratio of the volume of oxygen transferred to the blood per unit time to the active surface area of the gas transfer membrane. If this ratio is not significantly higher than values reported for hollow fiber membrane oxygenators (HFMO), then the expected advantage of the microfluidic approach would not be realized in practice, potentially due to challenges encountered in blood distribution strategies when scaling microfluidic designs to higher flow rates. Here, we report on scaling of a microfluidic oxygenator design from 4 to 92 mL/min blood flow, within an order of magnitude of the flow rate required for neonatal applications. This scaled device is shown to have a gas transfer efficiency higher than any other reported system in the literature, including other microfluidic prototypes and commercial HFMO cartridges. While the high oxygen transfer efficiency is a promising advance toward clinical scaling of a microfluidic architecture, it is accompanied by an excessive blood pressure drop in the circuit, arising from a combination of shallow gas transfer channels and equally shallow distribution manifolds. Therefore, next-generation microfluidic oxygenators will require novel design and fabrication strategies to minimize pressure drops while maintaining very high oxygen transfer efficiencies., (© 2021 International Center for Artificial Organs and Transplantation and Wiley Periodicals LLC.)

The aim of this study was to determine the relationship between relative peripheral refraction and retinal shape by 2-D magnetic resonance imaging in high myopes. Thirty-five young adults aged 20 to 30 years participated in this study with 16 high myopes (spherical equivalent < -6.00 D) and 19 emmetropes (+0.50 to -0.50 D). An open field autorefractor was used to measure refractions from the center out to 60° in the horizontal meridian and out to around 20° in the vertical meridian, with a step of 3 degrees. Axial length was measured by using A-scan ultrasonography. In addition, images of axial, sagittal, and tangential sections were obtained using 2-D magnetic resonance imaging. The highly myopic group had a significantly relative peripheral hyperopic refraction and showed a prolate ocular shape compared to the emmetropic group. The highly myopic group had relative peripheral hyperopic refraction and showed a prolate ocular form. Significant differences in the ratios of height/axial (1.01 ± 0.02 vs. 0.94 ± 0.03) and width/axial (0.99 ± 0.17 vs. 0.93 ± 0.04) were found from the MRI images between the emmetropic and the highly myopic eyes ( p < 0.001). There was a negative correlation between the retina's curvature and relative peripheral refraction for both temporal (Pearson r = -0.459; p < 0.01) and nasal (Pearson r = -0.277; p = 0.011) retina. For the highly myopic eyes, the amount of peripheral hyperopic defocus is correlated to its ocular shape deformation. This could be the first study investigating the relationship between peripheral refraction and ocular dimension in high myopes, and it is hoped to provide useful knowledge of how the development of myopia changes human eye shape.

Glycine-alanine dipeptide repeats (GA DPRs) translated from the mutated C9orf72 gene have recently been correlated with amyotrophic lateral sclerosis (ALS). While GA DPRs aggregates have been suggested as amyloid, the biophysical features and cytotoxicity of GA DPRs oligomers has not been explored due to its unstable nature. In this study, we develop a photoinducible platform based on methoxynitrobenzene chemistry to enrich GA DPRs that allows monitoring the oligomerization process of GA DPRs in cells. By applying advanced microscopies, we examined the GA DPRs oligomerization process nanoscopically in a time-dependent manner. We provided direct evidences to demonstrate GA DPRs oligomers rather than nanofibrils disrupt nuclear membrane. Moreover, we found GA DPRs hamper nucleocytoplasmic transport in cells and cause cytosolic retention of TAR DNA-binding protein 43 in cortical neurons. Our results highlight the toxicity of GA DPRs oligomers, which is a key step toward elucidating the pathological roles of C9orf72 DPRs., (© 2021. The Author(s).)

CARD8 detects intracellular danger signals and forms a caspase-1 activating inflammasome. Like the related inflammasome sensor NLRP1, CARD8 autoprocesses into noncovalently associated N-terminal (NT) and C-terminal (CT) fragments and binds the cellular dipeptidyl peptidases DPP8 and 9 (DPP8/9). Certain danger-associated signals, including the DPP8/9 inhibitor Val-boroPro (VbP) and HIV protease, induce proteasome-mediated NT degradation and thereby liberate the inflammasome-forming CT. Here, we report cryoelectron microscopy (cryo-EM) structures of CARD8 bound to DPP9, revealing a repressive ternary complex consisting of DPP9, full-length CARD8, and CARD8-CT. Unlike NLRP1-CT, CARD8-CT does not interact with the DPP8/9 active site and is not directly displaced by VbP. However, larger DPP8/9 active-site probes can directly weaken this complex in vitro, and VbP itself nevertheless appears to disrupt this complex, perhaps indirectly, in cells. Thus, DPP8/9 inhibitors can activate the CARD8 inflammasome by promoting CARD8 NT degradation and by weakening ternary complex stability., Competing Interests: Declaration of interests H.W. is a co-founder of Ventus Therapeutics., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Enzyme-nanoparticle interactions can give rise to a range of new phenomena, most notably significant enzymatic rate enhancement. Accordingly, the careful study and optimization of such systems is likely to give rise to advanced biosensing applications. Herein, we report a systematic study of the interactions between nuclease enzymes and oligonucleotide-coated gold nanoparticles (spherical nucleic acids, SNAs), with the aim of revealing phenomena worthy of evolution into functional nanosystems. Specifically, we study two nucleases, an exonuclease (ExoIII) and an endonuclease (Nt.BspQI), via fluorescence-based kinetic experiments, varying parameters including enzyme and substrate concentrations, and nanoparticle size and surface coverage in non-recycling and a recycling formats. We demonstrate the tuning of nuclease activity by SNA characteristics and show that the modular units of SNAs can be leveraged to either accelerate or suppress nuclease kinetics. Additionally, we observe that the enzymes are capable of cleaving restriction sites buried deep in the oligonucleotide surface layer and that enzymatic rate enhancement occurs in the target recycling format but not in the non-recycling format. Furthermore, we demonstrate a new SNA phenomenon, we term 'target stacking', whereby nucleic acid hybridization efficiency increases as enzyme cleavage proceeds during the beginning of a reaction. This investigation provides important data to guide the design of novel SNAs in biosensing and in vitro diagnostic applications.

The design principle of establishing an intracellular protein gradient for asymmetric cell division is a long-standing fundamental question. While the major molecular players and their interactions have been elucidated via genetic approaches, the diversity and redundancy of natural systems complicate the extraction of critical underlying features. Here, we take a synthetic cell biology approach to construct intracellular asymmetry and asymmetric division in Escherichia coli, in which division is normally symmetric. We demonstrate that the oligomeric PopZ from Caulobacter crescentus can serve as a robust polarized scaffold to functionalize RNA polymerase. Furthermore, by using another oligomeric pole-targeting DivIVA from Bacillus subtilis, the newly synthesized protein can be constrained to further establish intracellular asymmetry, leading to asymmetric division and differentiation. Our findings suggest that the coupled oligomerization and restriction in diffusion may be a strategy for generating a spatial gradient for asymmetric cell division.

Background: The medial sural artery perforator flap is a versatile option in reconstructive microsurgery. However, most reports of this flap have been limited by sample size. This study reviews the experience of a single large-volume center using the medial sural artery perforator flap and its variants for a variety of reconstructive purposes., Methods: A retrospective review of all patients who received the medial sural artery perforator flap reconstruction between March of 2006 and July of 2014 was performed., Results: Two-hundred medial sural artery perforator flaps were used at three anatomical regions: head and neck (n = 129), upper extremity (n = 47), and lower extremity (n = 24). Flaps were transferred as free (n = 189) and pedicled (n = 11). Overall flap survival was 96 percent. Flap variants used included chimeric flap (n = 11) and dual skin paddle (n = 2). Plantaris tendon and saphenous/sural nerve graft were harvested in conjunction in 20 and seven patients, respectively. In 5 percent of the cases harvested in the conventional manner, a reliable perforator could not be found., Conclusions: The medial sural artery perforator flap is a reliable, alternative workhorse flap for nearly any small- to medium-size defect. It can be harvested as free or pedicled and the option to include multiple tissue components further expands its versatility., Clinical Question/level of Evidence: Therapeutic, IV.

Several cytosolic pattern-recognition receptors (PRRs) form multiprotein complexes called canonical inflammasomes in response to intracellular danger signals. Canonical inflammasomes recruit and activate caspase-1 (CASP1), which in turn cleaves and activates inflammatory cytokines and gasdermin D (GSDMD), inducing pyroptotic cell death. Inhibitors of the dipeptidyl peptidases DPP8 and DPP9 (DPP8/9) activate both the human NLRP1 and CARD8 inflammasomes. NLRP1 and CARD8 have different N-terminal regions but have similar C-terminal regions that undergo autoproteolysis to generate two non-covalently associated fragments. Here, we show that DPP8/9 inhibition activates a proteasomal degradation pathway that targets disordered and misfolded proteins for destruction. CARD8's N terminus contains a disordered region of ∼160 amino acids that is recognized and destroyed by this degradation pathway, thereby freeing its C-terminal fragment to activate CASP1 and induce pyroptosis. Thus, CARD8 serves as an alarm to signal the activation of a degradation pathway for disordered and misfolded proteins., Competing Interests: Declaration of Interests The authors declare no competing interests., (Published by Elsevier Inc.)

Breastfeeding during the first week postpartum is recognised as essential, affecting the mother's mental health. The human milk from early breastfeeding also protects against infections via the secretory IgA antibodies and plays an important role in the newborn's development. At a Baby-Friendly Hospital, early breastfeeding was promoted to increase the benefits for the newborn, but few focussed on the mother's health. The association between breastfeeding and postpartum depression (PPD) has not been fully addressed. This study aimed to investigate the relationship between breastfeeding and postpartum depression (PPD) and it included 333 Taiwanese pregnant women. Women who had a higher score of Edinburgh Postnatal Depression Scale (EPDS) were significant older (age ≥ 35, p  < .037), had a caesarean section delivery ( p  < .001), and a higher score of Brief Symptom Rating Scale-5 ( p  < .001). Higher scores on EPDS were significantly associated with lower rates of breastfeeding, which reflected cessation of breastfeeding as an important risk factor for PPD (Odds ratio: 2.159, CI: 1.220-3.821, p  = .008). Other risk factors included caesarean section delivery (Odds ratio: 2.814, CI: 1.617-4.899, p  < .001) and age ≥35 years (Odds ratio: 1.821, CI: 1.057-3.137, p  = .031).IMPACT STATEMENT What is already known on this subject: Breastfeeding during the first week postpartum is recognised as essential, affecting the mother's mental health. The human milk from early breastfeeding also protects against infections via the secretory IgA antibodies and plays an important role in the newborn's development. The detail association between breastfeeding and postpartum depression has not yet been fully addressed. What the results of this study add: Higher scores on EPDS were significantly associated with lower rates of breastfeeding, which reflected cessation of breastfeeding as an important risk factor for PPD (Odds ratio: 2.159, CI: 1.220-3.821, p  = .008). Other risk factors included caesarean section delivery (Odds ratio: 2.814, CI: 1.617-4.899, p  < .001) and age ≥35 years (Odds ratio: 1.821, CI: 1.057-3.137, p  = .031). What the implications are of these findings for clinical practice and/or further research? The early breastfeeding was significantly associated with postpartum depression. Other risk factors included caesarean section delivery and age ≥35 years.

We report the design and operation of a surface-emitting surface acoustic wave (SAW) acousto-optical modulator which behaves as a cm-scale linear hologram in response to an applied electronic waveform. The modulator is formed by an optical waveguide, transducer, and out-coupling surface grating on a 1 mm-thick lithium niobate substrate. We demonstrate the ability to load and illuminate a 9-region linear hologram into the modulator's 8 mm-long interaction region using applied waveforms of 280-320 MHz. To the best of the authors' knowledge, this is the first demonstration of a monolithically-integrated, surface-emitting SAW modulator fabricated using lithographic techniques. Applications include practical implementations of a holographic display.

Background: Patient-reported outcome measures are an important metric in evaluating treatment efficacy of reconstructive surgery. Toe-to-hand transfer can restore vital prehensile function; however, this surgery is complex, extensive rehabilitation is required, and there are concerns about donor-site morbidity. This study longitudinally explores the benefits of this procedure, from the patient's perspective, using patient-reported outcome measures., Methods: Twenty-three patients who underwent free toe-to-hand transfers from 2012 to 2015 were evaluated preoperatively and postoperatively using the following validated questionnaires: the Michigan Hand Outcomes Questionnaire, the 36-Item Short-Form Health Survey, and the Lower Limb Outcomes Questionnaire. Subgroup analysis was performed between dominant and nondominant reconstructed hands., Results: Mechanism of injury was crush in 83 percent; the remainder sustained cutting, avulsion, and burn injuries. Thirty-four toes were transferred: nine great toes, 20 second toes, and five third toes. Michigan Hand Outcomes Questionnaire results showed significant improvement in overall activities of daily living, work, aesthetics, and patient satisfaction (p < 0.05). The results of the 36-Item Short-Form Health Survey showed significant improvements in physical and emotional roles (p < 0.05). The Lower Limb Outcomes Questionnaire showed no deterioration of foot function (p = 0.55). Subgroup analysis showed significant improvement in patient-reported outcome measures for patients undergoing dominant hand reconstruction but no difference between thumb reconstruction and finger-only reconstruction., Conclusions: Patient-reported outcome measures demonstrate the significant utility of toe-to-hand transfer procedures in both functional and psychosocial domains in that there are relatively greater benefits in reconstructing the dominant hand, and that donor-site morbidity is well tolerated., Clinical Question/level of Evidence: Therapeutic, IV.

Background: The medial sural artery perforator (MSAP) flap has become increasingly popular because it is thin and pliable for small to moderate defect soft tissue reconstruction. Furthermore, chimeric MSAP flap, which includes a skin paddle and a separated piece of medial gastrocnemius muscle, allowed more freedom for flap insetting, especially in 3-dimensional defect reconstruction. Here we describe our experience regarding this clinical application., Patients and Methods: From 2007 to 2016, 14 male patients (average age, 46.9 ± 14.4 years) who received either a free or pedicled chimeric MSAP flap were included. Of these 14 patients, 7 received this flap for reconstruction in the head and neck, 2 in the upper extremities, and 5 in the lower extremities. Demographic data were collected and analyzed, and a literature review was performed., Results: Ten patients received free chimeric MSAP flap, and 4 received the pedicled type. Thirteen of the 14 flaps (92.6%) survived, and 1 failed 2 days later owing to venous insufficiency. Venous congestion-related partial loss occurred in another case., Conclusions: The chimeric MSAP flap is a good alternative for deep space obliteration or reconstruction of adjacent but separate defects in both free and pedicled flap design. Donor site morbidity is limited. However, the perforator needs to be mobilized carefully to prevent postoperative venous compromise.

Noninvasive tools that target tumor cells could improve the management of glioma. Cancer generally has a high demand for Fe(III), an essential nutrient for a variety of biochemical processes. We tested whether 68Ga-citrate, an Fe(III) biomimetic that binds to apo-transferrin in blood, detects glioma in preclinical models and patients using hybrid PET/MRI. Mouse PET/CT studies showed that 68Ga-citrate accumulates in subcutaneous U87MG xenografts in a transferrin receptor-dependent fashion within 4 hours after injection. Seventeen patients with WHO grade III or IV glioma received 3.7-10.2 mCi 68Ga-citrate and were imaged with PET/MR 123-307 minutes after injection to establish that the radiotracer can localize to human tumors. Multiple contrast-enhancing lesions were PET avid, and tumor to adjacent normal white matter ratios were consistently greater than 10:1. Several contrast-enhancing lesions were not PET avid. One minimally enhancing lesion and another tumor with significantly reduced enhancement following bevacizumab therapy were PET avid. Advanced MR imaging analysis of one patient with contrast-enhancing glioblastoma showed that metabolic hallmarks of viable tumor spatially overlaid with 68Ga-citrate accumulation. These early data underscore that high-grade glioma may be detectable with a radiotracer that targets Fe(III) transport.

The swell of experimental imaging technologies to noninvasively measure immune checkpoint protein expression presents the opportunity for rigorous comparative studies toward identifying a gold standard. 89 Zr-atezolizumab is currently in man, and early data show tumor targeting but also abundant uptake in several normal tissues. Therefore, we conducted a reverse translational study both to understand if tumor to normal tissue ratios for 89 Zr-atezolizumab could be improved and to make direct comparisons to 89 Zr-C4, a radiotracer that we showed can detect a large dynamic range of tumor-associated PD-L1 expression. PET/CT and biodistribution studies in tumor bearing immunocompetent and nu/nu mice revealed that high specific activity 89 Zr-atezolizumab (∼2 μCi/μg) binds to PD-L1 on tumors but also results in very high uptake in many normal mouse tissues, as expected. Unexpectedly, 89 Zr-atezolizumab uptake was generally higher in normal mouse tissues compared to 89 Zr-C4 and lower in H1975, a tumor model with modest PD-L1 expression. Also unexpectedly, reducing the specific activity at least 15-fold suppressed 89 Zr-atezo uptake in normal mouse tissues but increased tumor uptake to levels observed with high specific activity 89 Zr-C4. In summary, these data reveal that low specific activity 89 Zr-atezo may be necessary for accurately measuring PD-L1 in the tumor microenvironment, assuming a threshold can be identified that preferentially suppresses binding in normal tissues without reducing binding to tumors with abundant expression. Alternatively, high specific activity approaches like 89 Zr-C4 PET may be simpler to implement clinically to measure the broad dynamic range of PD-L1 expression known to manifest among tumors.

Microelectromechanical systems (MEMS) have enabled the fabrication of silicon nanopore membranes (SNM) with uniform non-overlapping "slit shaped" pores. The application of SNM has been suggested for high selectivity of biomolecules in a variety of medical filtration applications. The aim of this study was to rigorously quantify the differences in sieving between slit pore SNM and more commonly modeled cylindrical pore membranes, including effects of the extended Derjaguin, Landau, Verwey, and Overbeek (XDLVO) interactions. Applying equations derived for SNM in previous work, we compare the partition coefficient of slit and cylindrical pore membranes while accounting for both steric and XDLVO interactions. Simple, steric approximations demonstrate that slit pore membranes exhibit significantly lower partition coefficients than cylindrical pore models. Incorporating XDLVO interactions results in an even more marked difference between slit pore and cylindrical pore membranes. These partition coefficients were used to evaluate changes in beta-2-microglobulin (B2M) selectivity. The data demonstrate that XDLVO interactions increase the selectivity advantage that slit pores possess over cylindrical pores, particularly for larger values of the acid-base decay constant. Finally, the bovine serum albumin (BSA) to B2M selectivity ratio was investigated. The selectivity ratio appears larger in slit pores than cylindrical pores for all cases, indicating that slit pores are particularly well suited for hemofiltration applications. The results of this study have significant implications for the application of SNM in membrane processes where highly selective separation of biomolecules is desirable., (Copyright © 2017. Published by Elsevier Inc.)

A central conserved arginine, first identified as a clinical mutation leading to sulfite oxidase deficiency, is essential for catalytic competency of sulfite oxidizing molybdoenzymes, but the molecular basis for its effects on turnover and substrate affinity have not been fully elucidated. We have used a bacterial sulfite dehydrogenase, SorT, which lacks an internal heme group, but transfers electrons to an external, electron accepting cytochrome, SorU, to investigate the molecular functions of this arginine residue (Arg78). Assay of the SorT Mo centre catalytic competency in the absence of SorU showed that substitutions in the central arginine (R78Q, R78K and R78M mutations) only moderately altered SorT catalytic properties, except for R78M which caused significant reduction in SorT activity. The substitutions also altered the Mo-centre redox potentials (Mo VI/V potential lowered by ca. 60-80mV). However, all Arg78 mutations significantly impaired the ability of SorT to transfer electrons to SorU, where activities were reduced 17 to 46-fold compared to SorT WT , precluding determination of kinetic parameters. This was accompanied by the observation of conformational changes in both the introduced Gln and Lys residues in the crystal structure of the enzymes. Taking into account data collected by others on related SOE mutations we propose that the formation and maintenance of an electron transfer complex between the Mo centre and electron accepting heme groups is the main function of the central arginine, and that the reduced turnover and increases in K Msulfite are caused by the inefficient operation of the oxidative half reaction of the catalytic cycle in enzymes carrying these mutations., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Microelectromechanical systems (MEMS), a technology that resulted from significant innovation in semiconductor fabrication, have recently been applied to the development of silicon nanopore membranes (SNM). In contrast to membranes fabricated from polymeric materials, SNM exhibit slit-shaped pores, monodisperse pore size, constant surface porosity, zero pore overlap, and sub-micron thickness. This development in membrane fabrication is applied herein for the validation of the XDLVO (extended Derjaguin, Landau, Verwey, and Overbeek) theory of membrane transport within the context of hemofiltration. In this work, the XDLVO model has been derived for the unique slit pore structure of SNM. Beta-2-microglobulin (B2M), a clinically relevant "middle molecular weight" solute in kidney disease, is highlighted in this study as the solute of interest. In order to determine interaction parameters within the XDLVO model for B2M and SNM, goniometric measurements were conducted, yielding a Hamaker constant of 4.61× 10 -21 J and an acid-base Gibbs free energy at contact of 41 mJ/m 2 . The XDLVO model was combined with existing models for membrane sieving, with predictions of the refined model in good agreement with experimental data. Furthermore, the results show a significant difference between the XDLVO model and the simpler steric predictions typically applied in membrane transport. The refined model can be used as a tool to tailor membrane chemistry and maximize sieving or rejection of different biomolecules.

Oral delivery of therapeutics is the preferred route for systemic drug administration due to ease of access and improved patient compliance. However, many therapeutics suffer from low oral bioavailability due to low pH and enzymatic conditions, poor cellular permeability, and low residence time. Microfabrication techniques have been used to create planar, asymmetric microdevices for oral drug delivery to address these limitations. The geometry of these microdevices facilitates prolonged drug exposure with unidirectional release of drug toward gastrointestinal epithelium. While these devices have significantly enhanced drug permeability in vitro and in vivo , loading drug into the micron-scale reservoirs of the devices in a low-waste, high-capacity manner remains challenging. Here, we use picoliter-volume inkjet printing to load topotecan and insulin into planar microdevices efficiently. Following a simple surface functionalization step, drug solution can be spotted into the microdevice reservoir. We show that relatively high capacities of both topotecan and insulin can be loaded into microdevices in a rapid, automated process with little to no drug waste.

A new fluorescence-forming probe, coumOCT, designed by fusing cyclooctyne with a coumarin fluorophore was successfully used for the imaging of azido-glycoconjugates in living HeLa cells. This probe is cell-permeable and generates fluorescence after triazole formation, thus minimizing the background signal and enabling the real-time intracellular imaging of glycoconjugate trafficking.

In pre-clinical safety studies, drug-induced vascular injury (DIVI) is defined as an adverse response to a drug characterized by degenerative and hyperplastic changes of endothelial cells and vascular smooth muscle cells. Inflammation may also be seen, along with extravasation of red blood cells into the smooth muscle layer (i.e., hemorrhage). Drugs that cause DIVI are often discontinued from development after considerable cost has occurred. An in vitro vascular model has been developed using endothelial and smooth muscle cells in co-culture across a porous membrane mimicking the internal elastic lamina. Arterial flow rates of perfusion media within the endothelial chamber of the model induce physiologic endothelial cell alignment. Pilot testing with a drug known to cause DIVI induced extravasation of red blood cells into the smooth muscle layer in all devices with no extravasation seen in control devices. This engineered vascular model offers the potential to evaluate candidate drugs for DIVI early in the discovery process. The physiologic flow within the co-culture model also makes it candidate for a wide variety of vascular biology investigations.

Unlabelled: Vaccinia virus, the prototype of the Orthopoxvirus genus in the family Poxviridae, infects a wide range of cell lines and animals. Vaccinia mature virus particles of the WR strain reportedly enter HeLa cells through fluid-phase endocytosis. However, the intracellular trafficking process of the vaccinia mature virus between cellular uptake and membrane fusion remains unknown. We used live imaging of single virus particles with a combination of various cellular vesicle markers, to track fluorescent vaccinia mature virus particle movement in cells. Furthermore, we performed functional interference assays to perturb distinct vesicle trafficking processes in order to delineate the specific route undertaken by vaccinia mature virus prior to membrane fusion and virus core uncoating in cells. Our results showed that vaccinia virus traffics to early endosomes, where recycling endosome markers Rab11 and Rab22 are recruited to participate in subsequent virus trafficking prior to virus core uncoating in the cytoplasm. Furthermore, we identified WASH-VPEF/FAM21-retromer complexes that mediate endosome fission and sorting of virus-containing vesicles prior to virus core uncoating in the cytoplasm., Importance: Vaccinia mature virions of the WR strain enter HeLa cells through fluid phase endocytosis. We previously demonstrated that virus-containing vesicles are internalized into phosphatidylinositol 3-phosphate positive macropinosomes, which are then fused with Rab5-positive early endosomes. However, the subsequent process of sorting the virion-containing vesicles prior to membrane fusion remains unclear. We dissected the intracellular trafficking pathway of vaccinia mature virions in cells up to virus core uncoating in cytoplasm. We show that vaccinia mature virions first travel to early endosomes. Subsequent trafficking events require the important endosome-tethered protein VPEF/FAM21, which recruits WASH and retromer protein complexes to the endosome. There, the complex executes endosomal membrane fission and cargo sorting to the Rab11-positive and Rab22-positive recycling pathway, resulting in membrane fusion and virus core uncoating in the cytoplasm., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The vaccinia viral protein A27 in mature viruses specifically interacts with heparan sulfate for cell surface attachment. In addition, A27 associates with the viral membrane protein A17 to anchor to the viral membrane; however, the specific interaction between A27 and A17 remains largely unclear. To uncover the active binding sites and the underlying binding mechanism, we expressed and purified the N-terminal (18-50 residues) and C-terminal (162-203 residues) fragments of A17, which are denoted A17-N and A17-C. Through surface plasmon resonance, the binding affinity of A27/A17-N (KA = 3.40 × 10(8) m(-1)) was determined to be approximately 3 orders of magnitude stronger than that of A27/A17-C (KA = 3.40 × 10(5) m(-1)), indicating that A27 prefers to interact with A17-N rather than A17-C. Despite the disordered nature of A17-N, the A27-A17 interaction is mediated by a specific and cooperative binding mechanism that includes two active binding sites, namely (32)SFMPK(36) (denoted as F1 binding) and (20)LDKDLFTEEQ(29) (F2). Further analysis showed that F1 has stronger binding affinity and is more resistant to acidic conditions than is F2. Furthermore, A27 mutant proteins that retained partial activity to interact with the F1 and F2 sites of the A17 protein were packaged into mature virus particles at a reduced level, demonstrating that the F1/F2 interaction plays a critical role in vivo. Using these results in combination with site-directed mutagenesis data, we established a computer model to explain the specific A27-A17 binding mechanism.

Species detection is an important topic in the text mining field. According to the importance of the research topics (e.g., species assignment to genes and document focus species detection), some studies are dedicated to an individual topic. However, no researcher to date has discussed species detection as a general problem. Therefore, we developed a multi-scope species detection model to identify the focus species for different scopes (i.e., gene mention, sentence, paragraph, and global scope of the entire article). Species assignment is one of the bottlenecks of gene name disambiguation. In our evaluation, recognizing the focus species of a gene mention in four different scopes improved the gene name disambiguation. We used the species cue words extracted from articles to estimate the relevance between an article and a species. The relevance score was calculated by our proposed entities frequency-augmented invert species frequency (EF-AISF) formula, which represents the importance of an entity to a species. We also defined a relation guide factor (RGF) to normalize the relevance score. Our method not only achieved better performance than previous methods but also can handle the articles that do not specifically mention a species. In the DECA corpus, we outperformed previous studies and obtained an accuracy of 88.22 percent.

Objective: To identify factors associated with death before the start of anti-tuberculosis treatment, and early and late during treatment, among adult Taiwanese with culture-positive pulmonary tuberculosis (PTB)., Method: All adult culture-positive PTB patients in Taipei, Taiwan, were included in a retrospective cohort study in 2005-2010., Results: Of 4438 patients (mean age 64.6 years, 70.6% male), 76.8% were successfully treated, 5.4% died before start of treatment, 9.0% died within 8 weeks of treatment initiation and 8.8% died >8 weeks after treatment initiation. After controlling for potential confounders, age ≥ 65 years and male sex were associated with higher risks of death at all time periods investigated. High school education or higher reduced the risk of death before the start of and during treatment, while unemployment increased the risk of mortality during treatment. Cavity on chest X-ray and positivity for acid-fast bacilli were associated with lower risk of mortality before the start of treatment., Conclusion: To lower mortality among adult culture-positive PTB patients, it is imperative for clinicians to maintain high awareness of TB and provide more intensive care early, especially for men, the elderly and people with lower socio-economic status (e.g., the unemployed and less educated).

Microfluidic fabrication technologies are emerging as viable platforms for extracorporeal lung assist devices and oxygenators for cardiac surgical support and critical care medicine, based in part on their ability to more closely mimic the architecture of the human vasculature than existing technologies. In comparison with current hollow fiber oxygenator technologies, microfluidic systems have more physiologically-representative blood flow paths, smaller cross section blood conduits and thinner gas transfer membranes. These features can enable smaller device sizes and a reduced blood volume in the oxygenator, enhanced gas transfer efficiencies, and may also reduce the tendency for clotting in the system. Several critical issues need to be addressed in order to advance this technology from its current state and implement it in an organ-scale device for clinical use. Here we report on the design, fabrication and characterization of multilayer microfluidic oxygenators, investigating scaling effects associated with fluid mechanical resistance, oxygen transfer efficiencies, and other parameters in multilayer devices. Important parameters such as the fluidic resistance of interconnects are shown to become more predominant as devices are scaled towards many layers, while other effects such as membrane distensibility become less significant. The present study also probes the relationship between blood channel depth and membrane thickness on oxygen transfer, as well as the rate of oxygen transfer on the number of layers in the device. These results contribute to our understanding of the complexity involved in designing three-dimensional microfluidic oxygenators for clinical applications.

One of the principal challenges in artificial lung technology has been the ability to provide levels of oxygen and carbon dioxide exchange that rival those of the natural human lung, while mitigating the deleterious interaction between blood and the surface of the synthetic gas exchange membrane. This interaction is exacerbated by the large oxygenator surface area required to achieve sufficient levels of gas transfer. In an effort to address this challenge, microfluidics-based artificial lung technologies comprising stacked microchannel networks have been explored by several groups. Here we report the design, fabrication and initial testing of a parallel plate multilayered silicone-based microfluidic construct containing ultrathin gas exchange membranes, aimed at maximizing gas transfer efficiency while minimizing membrane-blood contact area. The device comprises a branched microvascular network that provides controlled wall shear stress and uniform blood flow, and is designed to minimize blood damage, thrombosis and inflammatory responses seen in current oxygenators. Initial testing indicates that flow distribution through the multilayer structure is uniform and that the thin membrane can withstand pressures equivalent to those expected during operation. Oxygen transfer using phosphate buffered saline as the carrier fluid has also been assessed, demonstrating a sharp increase in oxygen transfer as membrane thickness is reduced, consistent with the expected values of oxygen permeance for thin silicone membranes.

Polymer scaffolds that direct elongation and orientation of cultured cells can enable tissue engineered muscle to act as a mechanically functional unit. We combined micromolding and microablation technologies to create muscle tissue engineering scaffolds from the biodegradable elastomer poly(glycerol sebacate). These scaffolds exhibited well defined surface patterns and pores and robust elastomeric tensile mechanical properties. Cultured C2C12 muscle cells penetrated the pores to form spatially controlled engineered tissues. Scanning electron and confocal microscopy revealed muscle cell orientation in a preferential direction, parallel to micromolded gratings and long axes of microablated anisotropic pores, with significant individual and interactive effects of gratings and pore design.