Correction: Direct laser writing-enabled 3D printing strategies for microfluidic applications.

Correction for 'Direct laser writing-enabled 3D printing strategies for microfluidic applications' by Olivia M. Young et al., Lab Chip, 2024, DOI: https://doi.org/10.1039/D3LC00743J.

Direct laser writing-enabled 3D printing strategies for microfluidic applications.

Over the past decade, additive manufacturing-or "three-dimensional (3D) printing"-has attracted increasing attention in the Lab on a Chip community as a pathway to achieve sophisticated system architectures that are difficult or infeasible to fabricate via conventional means. One particularly promising 3D manufacturing technology is "direct laser writing (DLW)", which leverages two-photon (or multi-photon) polymerization (2PP) phenomena to enable high geometric versatility, print speeds, and precision at length scales down to the 100 nm range. Although researchers have demonstrated the potential of using DLW for microfluidic applications ranging from organ on a chip and drug delivery to micro/nanoparticle processing and soft microrobotics, such scenarios present unique challenges for DLW. Specifically, microfluidic systems typically require macro-to-micro fluidic interfaces (e.g., inlet and outlet ports) to facilitate fluidic loading, control, and retrieval operations; however, DLW-based 3D printing relies on a micron-to-submicron-sized 2PP volume element (i.e., "voxel") that is poorly suited for manufacturing these larger-scale fluidic interfaces. In this Tutorial Review, we highlight and discuss the four most prominent strategies that researchers have developed to circumvent this trade-off and realize macro-to-micro interfaces for DLW-enabled microfluidic components and systems. In addition, we consider the possibility that-with the advent of next-generation commercial DLW printers equipped with new dynamic voxel tuning, print field, and laser power capabilities-the overall utility of DLW strategies for Lab on a Chip fields may soon expand dramatically.

A 3D-MICROPRINTED COAXIAL NOZZLE FOR FABRICATING LONG, FLEXIBLE MICROFLUIDIC TUBING.

A variety of emerging applications, particularly those in medical and soft robotics fields, are predicated on the ability to fabricate long, flexible meso/microfluidic tubing with high customization. To address this need, here we present a hybrid additive manufacturing (or "three-dimensional (3D) printing") strategy that involves three key steps: (i) using the "Vat Photopolymerization (VPP) technique, "Liquid-Crystal Display (LCD)" 3D printing to print a bulk microfluidic device with three inlets and three concentric outlets; (ii) using "Two-Photon Direct Laser Writing (DLW)" to 3D microprint a coaxial nozzle directly atop the concentric outlets of the bulk microdevice, and then (iii) extruding paraffin oil and a liquid-phase photocurable resin through the coaxial nozzle and into a polydimethylsiloxane (PDMS) channel for UV exposure, ultimately producing the desired tubing. In addition to fabricating the resulting tubing-composed of polymerized photomaterial-at arbitrary lengths (e.g., > 10 cm), the distinct input pressures can be adjusted to tune the inner diameter (ID) and outer diameter (OD) of the fabricated tubing. For example, experimental results revealed that increasing the driving pressure of the liquid-phase photomaterial from 50 kPa to 100 kPa led to fluidic tubing with IDs and ODs of 291±99 µm and 546±76 µm up to 741±31 µm and 888±39 µm, respectively. Furthermore, preliminary results for DLW-printing a microfluidic "M" structure directly atop the tubing suggest that the tubing could be used for "ex situ DLW (esDLW)" fabrication, which would further enhance the utility of the tubing.

Fine tuning of CpG spatial distribution with DNA origami for improved cancer vaccination.

Multivalent presentation of ligands often enhances receptor activation and downstream signalling. DNA origami offers a precise nanoscale spacing of ligands, a potentially useful feature for therapeutic nanoparticles. Here we use a square-block DNA origami platform to explore the importance of the spacing of CpG oligonucleotides. CpG engages Toll-like receptors and therefore acts to activate dendritic cells. Through in vitro cell culture studies and in vivo tumour treatment models, we demonstrate that square blocks induce Th1 immune polarization when CpG is spaced at 3.5 nm. We observe that this DNA origami vaccine enhances DC activation, antigen cross-presentation, CD8 T-cell activation, Th1-polarized CD4 activation and natural-killer-cell activation. The vaccine also effectively synergizes with anti-PD-L1 for improved cancer immunotherapy in melanoma and lymphoma models and induces long-term T-cell memory. Our results suggest that DNA origami may serve as a platform for controlling adjuvant spacing and co-delivering antigens in vaccines.

FABRICATION OF MULTILUMEN MICROFLUIDIC TUBING FOR EX SITU DIRECT LASER WRITING.

Among the numerous additive manufacturing or "three-dimensional (3D) printing" techniques, two-photon Direct Laser Writing (DLW) is distinctively suited for applications that demand high geometric versatility with micron-to-submicron-scale feature resolutions. Recently, "ex situ DLW (esDLW)" has emerged as a powerful approach for printing 3D microfluidic structures directly atop meso/macroscale fluidic tubing that can be manipulated by hand; however, difficulties in creating custom esDLW-compatible multilumen tubing at such scales has hindered progress. To address this impediment, here we introduce a novel methodology for fabricating submillimeter multilumen tubing for esDLW 3D printing. Preliminary fabrication results demonstrate the utility of the presented strategy for resolving 743 µm-in-diameter tubing with three lumens-each with an inner diameter (ID) of 80 µm. Experimental results not only revealed independent flow of discrete fluorescently labelled fluids through each of the three lumens, but also effective esDLW-printing of a demonstrative 3D "MEMS" microstructure atop the tubing. These results suggest that the presented approach could offer a promising pathway to enable geometrically sophisticated microfluidic systems to be 3D printed with input and/or output ports fully sealed to multiple, distinct lumens of fluidic tubing for emerging applications in fields ranging from drug delivery and medical diagnostics to soft surgical robotics.

DNA origami vaccine (DoriVac) nanoparticles improve both humoral and cellular immune responses to infectious diseases.

Current SARS-CoV-2 vaccines have demonstrated robust induction of neutralizing antibodies and CD4+ T cell activation, however CD8+ responses are variable, and the duration of immunity and protection against variants are limited. Here we repurposed our DNA origami vaccine platform, DoriVac, for targeting infectious viruses, namely SARS-CoV-2, HIV, and Ebola. The DNA origami nanoparticle, conjugated with infectious-disease-specific HR2 peptides, which act as highly conserved antigens, and CpG adjuvant at precise nanoscale spacing, induced neutralizing antibodies, Th1 CD4+ T cells, and CD8+ T cells in naïve mice, with significant improvement over a bolus control. Pre-clinical studies using lymph-node-on-a-chip systems validated that DoriVac, when conjugated with antigenic peptides or proteins, induced promising cellular immune responses in human cells. These results suggest that DoriVac holds potential as a versatile, modular vaccine platform, capable of inducing both humoral and cellular immunities. The programmability of this platform underscores its potential utility in addressing future pandemics.

Catch your breath: The protective role of the angiotensin AT2 receptor for the treatment of idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease whereby excessive deposition of extracellular matrix proteins (ECM) ultimately leads to respiratory failure. While there have been advances in pharmacotherapies for pulmonary fibrosis, IPF remains an incurable and irreversible disease. There remains an unmet clinical need for treatments that reverse fibrosis, or at the very least have a more tolerable side effect profile than currently available treatments. Transforming growth factor ß1(TGFß1) is considered the main driver of fibrosis in IPF. However, as our understanding of the role of the pulmonary renin-angiotensin system (PRAS) in the pathogenesis of IPF increases, it is becoming clear that targeting angiotensin receptors represents a potential novel treatment strategy for IPF - in particular, via activation of the anti-fibrotic angiotensin type 2 receptor (AT2R). This review describes the current understanding of the pathophysiology of IPF and the mediators implicated in its pathogenesis; focusing on TGFß1, angiotensin II and related peptides in the PRAS and their contribution to fibrotic processes in the lung. Preclinical and clinical assessment of currently available AT2R agonists and the development of novel, highly selective ligands for this receptor will also be described, with a focus on compound 21, currently in clinical trials for IPF. Collectively, this review provides evidence of the potential of AT2R as a novel therapeutic target for IPF.

Modulation of Mitochondrial Metabolic Parameters and Antioxidant Enzymes in Healthy and Glaucomatous Trabecular Meshwork Cells with Hybrid Small Molecule SA-2.

Oxidative stress (OS)-induced mitochondrial damage is a risk factor for primary open-angle glaucoma (POAG). Mitochondria-targeted novel antioxidant therapies could unearth promising drug candidates for the management of POAG. Previously, our dual-acting hybrid molecule SA-2 with nitric oxide-donating and antioxidant activity reduced intraocular pressure and improved aqueous humor outflow in rodent eyes. Here, we examined the mechanistic role of SA-2 in trabecular meshwork (TM) cells in vitro and measured the activity of intracellular antioxidant enzymes during OS. Primary human TM cells isolated from normal (hNTM) or glaucomatous (hGTM) post-mortem donors and transformed glaucomatous TM cells (GTM-3) were used for in vitro assays. We examined the effect of SA-2 on oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in vitro using Seahorse Analyzer with or without the oxidant, tert-butyl hydroperoxide (TBHP) treatment. Concentrations of total antioxidant enzymes, catalase (CAT), malondialdehyde (MDA), and glutathione peroxidase (GPx) were measured. We observed significant protection of both hNTM and hGTM cells from TBHP-induced cell death by SA-2. Antioxidant enzymes were elevated in SA-2-treated cells compared to TBHP-treated cells. In addition, SA-2 demonstrated an increase in mitochondrial metabolic parameters. Altogether, SA-2 protected both normal and glaucomatous TM cells from OS via increasing mitochondrial energy parameters and the activity of antioxidant enzymes.

Toward a novel soft robotic system for minimally invasive interventions.

PURPOSE: During minimally invasive surgery, surgeons maneuver tools through complex anatomies, which is difficult without the ability to control the position of the tools inside the body. A potential solution for a substantial portion of these procedures is the efficient design and control of a pneumatically actuated soft robot system. METHODS: We designed and evaluated a system to control a steerable catheter tip. A macroscale 3D printed catheter tip was designed to have two separately pressurized channels to induce bending in two directions. A motorized hand controller was developed to allow users to control the bending angle while manually inserting the steerable tip. Preliminary characterization of two catheter tip prototypes was performed and used to map desired angle inputs into pressure commands. RESULTS: The integrated robotic system allowed both a novice and a skilled surgeon to position the steerable catheter tip at the location of cylindrical targets with sub-millimeter accuracy. The novice was able to reach each target within ten seconds and the skilled surgeon within five seconds on average. CONCLUSION: This soft robotic system enables its user to simultaneously insert and bend the pneumatically actuated catheter tip with high accuracy and in a short amount of time. These results show promise concerning the development of a soft robotic system that can improve outcomes in minimally invasive interventions.

Folate Receptor as a Biomarker and Therapeutic Target in Solid Tumors.

Folate is a B vitamin necessary for basic biological functions, including rapid cell turnover occurring in cancer cell proliferation. Though the role of folate as a causative versus protective agent in carcinogenesis is debated, several studies have indicated that the folate receptor (FR), notably subtype folate receptor alpha (FRα), could be a viable biomarker for diagnosis, progression, and prognosis. Several cancers, including gastrointestinal, gynecological, breast, lung, and squamous cell head and neck cancers overexpress FR and are currently under investigation to correlate receptor status to disease state. Traditional chemotherapies have included antifolate medications, such as methotrexate and pemetrexed, which generate anticancer activity during the synthesis phase of the cell cycle. Increasingly, the repertoire of pharmacotherapies is expanding to include FR as a target, with a heterogenous pool of directed therapies. Here we discuss the FR, expression and effect in cancer biology, and relevant pharmacologic inhibitors.

A universal glycoenzyme biosynthesis pipeline that enables efficient cell-free remodeling of glycans.

The ability to reconstitute natural glycosylation pathways or prototype entirely new ones from scratch is hampered by the limited availability of functional glycoenzymes, many of which are membrane proteins that fail to express in heterologous hosts. Here, we describe a strategy for topologically converting membrane-bound glycosyltransferases (GTs) into water soluble biocatalysts, which are expressed at high levels in the cytoplasm of living cells with retention of biological activity. We demonstrate the universality of the approach through facile production of 98 difficult-to-express GTs, predominantly of human origin, across several commonly used expression platforms. Using a subset of these water-soluble enzymes, we perform structural remodeling of both free and protein-linked glycans including those found on the monoclonal antibody therapeutic trastuzumab. Overall, our strategy for rationally redesigning GTs provides an effective and versatile biosynthetic route to large quantities of diverse, enzymatically active GTs, which should find use in structure-function studies as well as in biochemical and biomedical applications involving complex glycomolecules.

Deterministic Lateral Displacement Using Hexagonally Arranged, Bottom-Up-Inspired Micropost Arrays.

Size-based separation of particles in microfluidic devices can be achieved using arrays of micro- or nanoscale posts using a technique known as deterministic lateral displacement (DLD). To date, DLD arrays have been limited to parallelogram or rotated-square arrangements of posts, with various post shapes having been explored in these two principal arrangements. This work examines a new DLD geometry based on patterning obtainable through self-assembly of single-layer nanospheres, which we call hexagonally arranged triangle (HAT) geometry. Finite element simulations are used to characterize the DLD separation properties of the HAT geometry. The relationship between the array angle, the gap spacing, and the critical diameter for separation is derived for the HAT geometry and expressed in a similar mathematical form as conventional parallelogram and rotated-square DLD arrays. At array angles <7°, HAT structures demonstrate smaller particle sorting capability (smaller critical diameter-to-gap spacing ratio) compared to published experimental results for parallelogram-type DLD arrays with circular posts. Experimental validation of DLD separation confirms the separation ability of the HAT array geometry. It is envisioned that this work will provide the first step toward future implementation of nanoscale DLD arrays fabricated by low-cost, bottom-up self-assembly approaches.

A 3D adult zebrafish brain atlas (AZBA) for the digital age.

Zebrafish have made significant contributions to our understanding of the vertebrate brain and the neural basis of behavior, earning a place as one of the most widely used model organisms in neuroscience. Their appeal arises from the marriage of low cost, early life transparency, and ease of genetic manipulation with a behavioral repertoire that becomes more sophisticated as animals transition from larvae to adults. To further enhance the use of adult zebrafish, we created the first fully segmented three-dimensional digital adult zebrafish brain atlas (AZBA). AZBA was built by combining tissue clearing, light-sheet fluorescence microscopy, and three-dimensional image registration of nuclear and antibody stains. These images were used to guide segmentation of the atlas into over 200 neuroanatomical regions comprising the entirety of the adult zebrafish brain. As an open source, online (azba.wayne.edu), updatable digital resource, AZBA will significantly enhance the use of adult zebrafish in furthering our understanding of vertebrate brain function in both health and disease.

Engineering orthogonal human O-linked glycoprotein biosynthesis in bacteria.

A major objective of synthetic glycobiology is to re-engineer existing cellular glycosylation pathways from the top down or construct non-natural ones from the bottom up for new and useful purposes. Here, we have developed a set of orthogonal pathways for eukaryotic O-linked protein glycosylation in Escherichia coli that installed the cancer-associated mucin-type glycans Tn, T, sialyl-Tn and sialyl-T onto serine residues in acceptor motifs derived from different human O-glycoproteins. These same glycoengineered bacteria were used to supply crude cell extracts enriched with glycosylation machinery that permitted cell-free construction of O-glycoproteins in a one-pot reaction. In addition, O-glycosylation-competent bacteria were able to generate an antigenically authentic Tn-MUC1 glycoform that exhibited reactivity with antibody 5E5, which specifically recognizes cancer-associated glycoforms of MUC1. We anticipate that the orthogonal glycoprotein biosynthesis pathways developed here will provide facile access to structurally diverse O-glycoforms for a range of important scientific and therapeutic applications.

Development of a Dietary Management Care Map for Metabolic Syndrome.

Metabolic syndrome (MetS) refers to a particular cluster of metabolic abnormalities (hypertension, dyslipidemia, type 2 diabetes, and visceral fat deposition) that can lead to a 1.5- to 2-fold increased relative risk of cardiovascular disease. Various combinations of healthier eating patterns and increased physical activity have been shown to improve metabolic abnormalities and reduce MetS prevalence. Dietitians who counsel MetS patients are challenged to integrate guidance from various medical management guidelines and research studies with effective behavioural change strategies and specific advice on what food and eating pattern changes will be most effective, feasible, and acceptable to clients. As part of a demonstration project that is currently underway, we developed a care map (decision aid) that represents the key decision processes involved in diet counselling for MetS. The care map is based on evidence from both clinical and health behaviour change studies and expert consensus and has undergone limited dietitian review. It is being used to help project dietitians clearly articulate their specific food intake change goals. Additional studies to directly compare counselling strategies could inform future development of the map. In the meantime, dietitians may find this care map helpful in clarifying counselling goals and strategies in this client group.