Search Results (14)

Three-dimensional (3D) bioprinting has emerged as a versatile biofabrication strategy that enables the precise, spatiotemporally controlled co-deposition of living cells, biomaterials, and bioactive agents, including growth factors, cytokines, and extracellular matrix (ECM) components, into geometrically defined 3D constructs. By translating digital design models derived from computed tomography (CT), magnetic resonance imaging (MRI), or computational modeling directly into physical tissue architectures, 3D bioprinting facilitates the assembly of hierarchically organized constructs that closely recapitulate the structural, mechanical, and functional characteristics of native tissues. The principal 3D bioprinting strategies are broadly classified into scaffold-based and scaffold-free approaches. Engineered bioinks, whether formulated as cell-laden natural, synthetic, or composite polymer hydrogels, tissue-derived decellularized ECM (dECM) components, or pure cellular spheroids and organoids, constitute the cornerstone of these biofabrication platforms. Scaffold-based 3D bioprinting comprises extrusion-based, droplet-based (inkjet and drop-on-demand), light-based vat photopolymerization (stereolithography and digital light processing), and laser-assisted bioprinting based on laser-induced forward transfer (LIFT). Each of these modalities imposes distinct constraints on bioink rheology, crosslinking mechanisms, spatial resolution, throughput, and post-printing cell viability; consequently, a specific 3D bioprinting strategy is selected according to the specific requirements of the target tissue application. Scaffold-free 3D bioprinting and bioassembly techniques, including the Kenzan method, aspiration-assisted bioprinting, magnetic bioprinting, and other field-directed tissue assembly approaches, enable the fabrication of spheroid- and organoid-based constructs without the necessity for exogenous biomaterial scaffolds. Because native tissues exhibit diversity in cellular composition, ECM architecture, mechanical properties, and physiological function, no individual bioprinting platform or bioink formulation serves as a universal 3D bioprinting solution. The engineering of biomimetic tissue constructs, therefore, requires the selection of application-tailored fabrication approaches. Under this biofabrication paradigm, 3D bioprinting has been applied across a wide range of tissue engineering targets, including skin, bone, cartilage, osteochondral interfaces, cardiac and vascular tissue, neural structures, ocular, dental, and adipose tissue. This review discusses recent advances in scaffold-based and scaffold-free 3D bioprinting applications for tissue engineering and regenerative medicine across diverse tissue systems. Full article

Background: Multiple myeloma (MM) is characterised by clonal expansion of plasma cells (PCs) in the bone marrow (BM). Disease assessment and monitoring typically rely on invasive, single-site procedures, such as BM biopsies (BMBs), which may inadequately capture intra- and extra-medullary spatial heterogeneity. Circulating plasma cells (CPCs), enriched from peripheral blood (PB), may represent a minimally invasive alternative or adjunct for molecular profiling. Objectives: This study aimed to evaluate the feasibility of using CPCs, enriched from PB, for mRNA analysis in plasma cell dyscrasia, including MM. A secondary objective was to assess whether mRNA expression levels of the endoplasmic reticulum (ER) stress sensors X-box-binding protein 1 (uXBP1) and activating transcription factor 6 (ATF6), and the chaperone-mediated autophagy marker Lysosomal-Associated Membrane Protein 2 (LAMP2A) by droplet digital PCR (ddPCR), were associated with resistance to the second-generation proteasome inhibitor (PI) carfilzomib (Cfz). Methods: Multiple myeloma (MM) cell lines (H929 and U266) and their carfilzomib-adapted derivatives were used to establish and validate droplet digital PCR (ddPCR) assays targeting ER stress (uXBP1, ATF6) and autophagy-related (LAMP2A) transcripts. Solid tumour cell lines, including serum-starved HeLa cells, served as biological controls to support assay specificity and sensitivity. Total RNA was extracted and reverse-transcribed to complementary DNA prior to analysis. Transcript levels were normalised to those of β-actin or GAPDH, as appropriate. ddPCR was performed using the BioRad QX200 system, with results reported as the normalised transcript copy number per microlitre of reaction. Matched bone marrow aspirate (BMA) and peripheral blood (PB) samples were collected at a single clinical time point from adults undergoing investigation for plasma cell dyscrasia between January 2021 and December 2023. Samples were obtained as part of standard clinical care and/or during treatment with Bortezomib (Btz) or Cfz. Mononuclear cells were isolated by density gradient centrifugation, and CD138⁺ plasma cells were enriched by fluorescence-activated cell sorting. Enrichment purity was assessed qualitatively by immunofluorescence microscopy using CD138 and CD117 markers. Samples yielding fewer than 1000 CD138⁺ plasma cells were excluded, resulting in 10 evaluable matched patient pairs. Results: Carfilzomib-adapted MM cell lines demonstrated reduced levels of uXBP1, ATF6, and LAMP2A mRNA compared to treatment-naïve cells. In matched BM and PB samples, uXBP1 mRNA levels were consistently lower in circulating PCs than in BM-derived PCs, whereas ATF6 mRNA levels were concordant between compartments. LAMP2A mRNA levels exhibited marked inter-patient heterogeneity. Conclusions: This study demonstrates the feasibility of using CPCs as a minimally invasive source for mRNA-based biomarker assessment and highlights ddPCR as a sensitive platform for quantifying ER stress and chaperone-mediated autophagy related transcripts in CPCs. Cfz adaptation was associated with reduced levels of uXBP1 and LAMP2A mRNA in MM cell lines. Future prospective studies evaluating the clinical utility of ER stress and chaperone-mediated autophagy associated transcripts in CPCs as predictors of resistance to PI are warranted. Full article

Enterovirus-D68 (EV-D68) is a reemerging virus that has been associated with numerous outbreaks in children in the past 10 years. Most assays examining viral infection kinetics have relied on the use of quantitative RT-PCR (qRT-PCR) assays as an assay of choice. Though valuable, there are inherent limitations that introduce variability, thereby reducing its value when comparing results across the field. Unlike the qRT-PCR assay that uses a standard curve to determine the copy number of viral RNA, the droplet digital PCR assay (ddPCR) directly quantifies the absolute number of copies within a given sample, which in turn makes the assay highly sensitive and accurate. Here, we have developed an EV-D68-specific ddPCR assay that effectively quantifies EV-D68 RNA copies in both cells and supernatants within a dynamic range of 6.7 × 10⁻³ copies/μL to 1.2 × 10⁴ copies/μL of the sample. The assay was highly specific for a broad range of EV-D68 isolates (Fermon, US/MO/14-18947, US/MO/14-18949, US/KY/14-18953, USA/2018-23088, USA/2020-23336 and EV-D68-infected human nasal turbinate samples from the 2022 outbreak) without cross-reactivity to other viruses such as Enterovirus-A71 (EV-A71), Human Parechovirus (HPeV)-1 and -2, Coxsackievirus (CV)-B1, Human Coronavirus (HCoV)-NL63, SARS-CoV-2, Influenza-A and B, Rhinovirus, and Respiratory Syncytial Virus (RSV)-A2, which are known to cause infection in children. The assay was able to readily quantify EV-D68 in infected cells and supernatants along with nasal turbinate samples collected from children during the 2022 outbreak. Our results suggest that the assay can be readily translated to accurately quantify viral loads in tissues and body fluids such as plasma and lung or nasal aspirates. Full article

The rapid quantification of nitrate nitrogen concentration plays a pivotal role in monitoring soil nutrient content. Nevertheless, the low detection efficiency limits the application of traditional methods in rapid testing. For this investigation, we utilized a digital microfluidic platform and 3D-printed microfluidics to accomplish automated detection of soil nitrate nitrogen with high sensitivity across numerous samples. The system combines digital microfluidics (DMF), 3D-printed microfluidics, a peristaltic pump, and a spectrometer. The soil solution, obtained after extraction, was dispensed onto the digital microfluidic platform using a micropipette. The digital microfluidic platform regulated the movement of droplets until they reached the injection area, where they were then aspirated into the 3D-printed microfluidic device for absorbance detection. Implementing this approach allows for the convenient sequential testing of multi-samples, thereby enhancing the efficiency of nitrate nitrogen detection. The results demonstrate that the device exhibits rapid detection (200 s for three samples), low reagent consumption (40 µL per sample), and low detection limit (95 µg/L). In addition, the relative error between the detected concentration and the concentration measured by ultraviolet spectrophotometry is kept within 20%, and the relative standard deviation (RSD) of the measured soil samples is between 0.9% and 4.7%. In the foreseeable future, this device will play a significant role in improving the efficiency of soil nutrient detection and guiding fertilization practices. Full article

As the demand for powerful, light energy sources continues to grow, traditional electrochemical batteries are no longer sufficient and combustion-based power generation devices have become an attractive alternative due to their high energy density, compact size, fast recharging time and long service life. While most research on miniature-scale combustors has focused on gaseous fuels, the use of commonly available liquid fuels has the potential to be highly portable and economical. However, the complexity of droplet atomization, evaporation, mixing and burning in a limited volume and short residence time has presented significant challenges for researchers. This review focuses on various methodologies proposed by researchers (like flow burring injector, fuel film injection, injecting into porous media, electrospray and some self-aspirating designs) to overcome these challenges, the combustion behaviour and different instabilities associated with liquid fuels at small scales. The current review intends to present a clear direction to channel the efforts made by researchers to overcome the difficulties associated with liquid fuel combustion at small scales for power generation applications. Additionally, this review aims to give an overview of power systems at the micro and meso scales that operate using liquid fuels. The methodologies introduced like electrospray requires external power, which again makes the system complex. Towards the development of standalone type power generators, the self-aspirating design which makes use of hydrostatic pressure, fuel film injection or taking advantage of exhaust gas enthalpy to preheat and evaporate the liquid fuel are the promising methodologies. Full article

Feline Immunodeficiency Virus (FIV) causes progressive immune dysfunction in cats similar to human immunodeficiency virus (HIV) in humans. Although combination antiretroviral therapy (cART) is effective against HIV, there is no definitive therapy to improve clinical outcomes in cats with FIV. This study therefore evaluated pharmacokinetics and clinical outcomes of cART (2.5 mg/kg Dolutegravir; 20 mg/kg Tenofovir; 40 mg/kg Emtricitabine) in FIV-infected domestic cats. Specific pathogen free cats were experimentally infected with FIV and administered either cART or placebo treatments (n = 6 each) for 18 weeks, while n = 6 naïve uninfected cats served as controls. Blood, saliva, and fine needle aspirates from mandibular lymph nodes were collected to quantify viral and proviral loads via digital droplet PCR and to assess lymphocyte immunophenotypes by flow cytometry. cART improved blood dyscrasias in FIV-infected cats, which normalized by week 16, while placebo cats remained neutropenic, although no significant difference in viremia was observed in the blood or saliva. cART-treated cats exhibited a Th2 immunophenotype with increasing proportions of CD4⁺CCR4⁺ cells compared to placebo cats, and cART restored Th17 cells compared to placebo-treated cats. Of the cART drugs, dolutegravir was the most stable and long-lasting. These findings provide a critical insight into novel cART formulations in FIV-infected cats and highlight their role as a potential animal model to evaluate the impact of cART on lentiviral infection and immune dysregulation. Full article

Lab-in-syringe is a new approach for the integration of various analytical extraction steps inside a syringe. Fully automated dispersive liquid–liquid microextraction is carried out in-syringe using a very simple instrumental setup. Dispersion is achieved by aspiration of the organic phase and then the watery phase into the syringe as rapidly as possible. After aggregation of the solvent droplets, the organic phase is pushed towards the detector allowing a highly sensitive spectrophotometric or fluorimetric detection. This technique is very useful not only for the preconcentration of analyte, but also for the elimination of their interferences. In this work, its application is described using solvents that are lighter and denser than water. The magnetically assisted variant and its coupling to different instruments has been also described with the aim of increasing the resolution of complex samples, especially useful for the determination of emerging contaminants. Full article

Chronic kidney disease (CKD), which is defined as a condition causing the gradual loss of kidney function, shows renal lipid droplet (LD) accumulation that is associated with oxidative damage. There is a possibility that an LD abnormality in quality plays a role in CKD development. This study aimed to explore the chemical composition of LDs that are induced in human kidney cells during exposure to free fatty acids as an LD source and oxidized lipoproteins as oxidative stress. The LDs were aspirated directly from cells using nanotips, followed by in-tip microextraction, and the LD lipidomic profiling was conducted using nanoelectrospray mass spectrometry. As a result, the free fatty acids increased the LD lipid content and, at the same time, changed their composition significantly. The oxidized lipoproteins caused distorted proportions of intact lipids, such as triacylglycerols (TG), phosphatidylcholines (PC), phosphatidylethanolamines (PE), and cholesteryl esters (CE). Notably, the oxidized lipids, including the hydroperoxides of TG, PC, and PE, exhibited significant elevations in dose-dependent manners. Furthermore, the dysregulation of intact lipids was paralleled with the accumulation of lipid hydroperoxides. The present study has revealed that the oxidation of lipids and the dysregulation of the lipid metabolism coexisted in LDs in the kidney cells, which has provided a potential new target for diagnosis and new insights into CKD. Full article

Droplet-based microfluidic screening techniques can benefit from interfacing established microtiter plate-based screening and sample management workflows. Interfacing tools are required both for loading preconfigured microtiter-plate (MTP)-based sample collections into droplets and for dispensing the used droplets samples back into MTPs for subsequent storage or further processing. Here, we present a collection of Digital Microfluidic Pipetting Tips (DMPTs) with integrated facilities for droplet generation and manipulation together with a robotic system for its operation. This combination serves as a bidirectional sampling interface for sample transfer from wells into droplets (w2d) and vice versa droplets into wells (d2w). The DMPT were designed to fit into 96-deep-well MTPs and prepared from glass by means of microsystems technology. The aspirated samples are converted into the channel-confined droplets’ sequences separated by an immiscible carrier medium. To comply with the demands of dose-response assays, up to three additional assay compound solutions can be added to the sample droplets. To enable different procedural assay protocols, four different DMPT variants were made. In this way, droplet series with gradually changing composition can be generated for, e.g., 2D screening purposes. The developed DMPT and their common fluidic connector are described here. To handle the opposite transfer d2w, a robotic transfer system was set up and is described briefly. Full article

Background: Sarcopenic dysphagia, a swallowing disorder caused by sarcopenia, is prevalent in older patients and can cause malnutrition and aspiration pneumonia. This study aimed to develop a simple screening test using image recognition with a low risk of droplet transmission for sarcopenic dysphagia. Methods: Older patients admitted to a post-acute care hospital were enrolled in this cross-sectional study. As a main variable for the development of a screening test, we photographed the anterior neck to analyze the image features of sarcopenic dysphagia. The studied image features included the pixel values and the number of feature points. We constructed screening models using the image features, age, sex, and body mass index. The prediction performance of each model was investigated. Results: A total of 308 patients participated, including 175 (56.82%) patients without dysphagia and 133 (43.18%) with sarcopenic dysphagia. The area under the receiver operating characteristic curve (ROC-AUC), sensitivity, specificity, positive predictive value, negative predictive value, and area under the precision-recall curve (PR-AUC) values of the best model were 0.877, 87.50%, 76.67%, 66.67%, 92.00%, and 0.838, respectively. The model with image features alone showed an ROC-AUC of 0.814 and PR-AUC of 0.726. Conclusions: The screening test for sarcopenic dysphagia using image recognition of neck appearance had high prediction performance. Full article

More than a year ago, the coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was declared a pandemic by the World Health Organization, with the world approaching its fourth wave. During this period, vaccines were developed in a much shorter period than thought possible, with the initiation of the pertinent vaccination. However, oral cavities have come under renewed scrutiny worldwide because saliva, a mixture of salivary secretions, pharyngeal secretions, and gingival crevicular fluid, have not only been shown to contain infective viral loads, mediating the route of SARS-CoV-2 transmission via droplet, aerosol, or contagion, but also used as a sample for viral RNA testing with a usefulness comparable to the nasopharyngeal swab. The oral cavity is an important portal for ingress of SARS-CoV-2, being an entryway to the bronchi, alveoli, and rest of the lower respiratory tract, causing inflammation by viral infection. Moreover, angiotensin-converting enzyme 2, a host receptor for SARS-CoV-2, coupled with proteases responsible for viral entry have been found to be expressed on the tongue and other oral mucosae, suggesting that the oral cavity is the site of virus replication and propagation. Furthermore, there is a possibility that the aspiration of oral bacteria (such as periodontal pathogens) along with saliva into the lower respiratory tract may be a complicating factor for COVID-19 because chronic obstructive pulmonary disease and diabetes are known COVID-19 comorbidities with a greater risk of disease aggravation and higher death rate. These comorbidities have a strong connection to chronic periodontitis and periodontal pathogens, and an oral health management is an effective measure to prevent these comorbidities. In addition, oral bacteria, particularly periodontal pathogens, could be proinflammatory stimulants to respiratory epithelia upon its exposure to aspirated bacteria. Therefore, it may be expected that oral health management not only prevents comorbidities involved in aggravating COVID-19 but also has an effect against COVID-19 progression. This review discusses the significance of oral health management in SARS-CoV-2 infection in the era of “the new normal with COVID-19” and COVID-19 prevention with reference to the hypothetical mechanisms that the authors and the other researchers have proposed. Full article

Liquid crystal (LC) phases typically show anisotropic alignment-dependent properties, such as viscosity and dielectric permittivity, so it stands to reason that LCs also have anisotropic interfacial tensions. Measuring the interfacial tension $\gamma$ of an LC with conventional methods, such as pendant drops, can be challenging, however, especially when we need to know $\gamma$ for different LC aligning conditions, as is the case when we seek $\Delta \gamma$, the interfacial tension anisotropy. Here, we present measurements of $\Delta \gamma$ of the common synthetic nematic LC compound 5CB against water using a microfluidic droplet aspiration technique. To ensure tangential and normal alignment, respectively, we add poly(vinyl alcohol) (PVA) and sodium dodecylsulfate (SDS), respectively, as a stabilizer and measure $\gamma$ for different concentrations of stabilizer. By fitting the Szyszkowski equation to the data, we can extrapolate to zero-stabilizer concentration, obtaining the $\gamma$ of 5CB to pure water for each alignment. For normal alignment, we find ${\gamma }_{\perp }=31.9\pm 0.8$ mN·m⁻¹, on the order of 1 mN·m⁻¹ greater than ${\gamma }_{\left|\right|}=30.8\pm 5$ mN·m${}^{-1}$ for tangential alignment. This resonates with the empirical knowledge that 5CB aligns tangentially to an interface with pure water. The main uncertainty arises from the use of polymeric PVA as tangential-promoting stabilizer. Future improvements in accuracy may be expected if PVA can be replaced by a low molar mass stabilizer that ensures tangential alignment. Full article

Dental-care workers operate very close to the patient’s mouth and are at high risk of contamination by SARS-CoV-2. Droplets may be contaminated by patient’s saliva and exhaled breath particles. All asymptomatic patients should be considered as Coronavirus positive. All dental procedures must be revised after positive identification of SARS-Cov-2. Novel recommendations as the use of novel suction cannula designed for fast spray/saliva aspiration, use of Tyvek suits and innovative sprayhoods designed for dental-care worker protections are proposed to prevent virus transmission. New tailored operative and clinical procedures are being currently developed by university dental clinics and hospitals in attempt to reduce risk for dental workers and patients. Full article

Techniques, such as micropipette aspiration and optical tweezers, are widely used to measure cell mechanical properties, but are generally labor-intensive and time-consuming, typically involving a difficult process of manipulation. In the past two decades, a large number of microfluidic devices have been developed due to the advantages they offer over other techniques, including transparency for direct optical access, lower cost, reduced space and labor, precise control, and easy manipulation of a small volume of blood samples. This review presents recent advances in the development of microfluidic devices to evaluate the mechanical response of individual red blood cells (RBCs) and microbubbles flowing in constriction microchannels. Visualizations and measurements of the deformation of RBCs flowing through hyperbolic, smooth, and sudden-contraction microchannels were evaluated and compared. In particular, we show the potential of using hyperbolic-shaped microchannels to precisely control and assess small changes in RBC deformability in both physiological and pathological situations. Moreover, deformations of air microbubbles and droplets flowing through a microfluidic constriction were also compared with RBCs deformability. Full article