A rapid antibiotic susceptibility test (AST) is desperately needed in clinical settings for fast and appropriate antibiotic administration. Traditional ASTs, which rely on cell culture, are not suitable for urgent cases of bacterial infection and antibiotic resistance owing to their relatively long test times. We describe a novel AST called single-cell morphological analysis (SCMA) that can determine antimicrobial susceptibility by automatically analyzing and categorizing morphological changes in single bacterial cells under various antimicrobial conditions. The SCMA was tested with four Clinical and Laboratory Standards Institute standard bacterial strains and 189 clinical samples, including extended-spectrum β-lactamase-positive Escherichia coli and Klebsiella pneumoniae, imipenem-resistant Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus, and vancomycin-resistant Enterococci from hospitals. The results were compared with the gold standard broth microdilution test. The SCMA results were obtained in less than 4 hours, with 91.5% categorical agreement and 6.51% minor, 2.56% major, and 1.49% very major discrepancies. Thus, SCMA provides rapid and accurate antimicrobial susceptibility data that satisfy the recommended performance of the U.S. Food and Drug Administration.
Objective Studying the biology of the human placenta represents a major experimental challenge. Although conventional cell culture techniques have been used to study different types of placenta-derived cells, current in vitro models have limitations in recapitulating organ-specific structure and key physiological functions of the placenta. Here we demonstrate that it is possible to leverage microfluidic and microfabrication technologies to develop a microengineered biomimetic model that replicates the architecture and function of the placenta. Materials and methods A “Placenta-on-a-Chip” microdevice was created by using a set of soft elastomer-based microfabrication techniques known as soft lithography. This microsystem consisted of two polydimethylsiloxane (PDMS) microfluidic channels separated by a thin extracellular matrix (ECM) membrane. To reproduce the placental barrier in this model, human trophoblasts (JEG-3) and human umbilical vein endothelial cells (HUVECs) were seeded onto the opposite sides of the ECM membrane and cultured under dynamic flow conditions to form confluent epithelial and endothelial layers in close apposition. We tested the physiological function of the microengineered placental barrier by measuring glucose transport across the trophoblast-endothelial interface over time. The permeability of the barrier study was analyzed and compared to that obtained from acellular devices and additional control groups that contained epithelial or endothelial layers alone. Results Our microfluidic cell culture system provided a tightly controlled fluidic environment conducive to the proliferation and maintenance of JEG-3 trophoblasts and HUVECs on the ECM scaffold. Prolonged culture in this model produced confluent cellular monolayers on the intervening membrane that together formed the placental barrier. This in vivo-like microarchitecture was also critical for creating a physiologically relevant effective barrier to glucose transport. Quantitative investigation of barrier function was conducted by calculating permeability coefficients and metabolic rates in varying conditions of barrier structure. The rates of glucose transport and metabolism were consistent with previously reported in vivo observations. Conclusion The “Placenta-on-a-Chip” microdevice described herein provides new opportunities to simulate and analyze critical physiological responses of the placental barrier. This system may be used to address the major limitations of existing placenta model systems and serve to enable research platforms for reproductive biology and medicine.
Human epidermal cells grew and differentiated in vitro, provided that the pH of the culture medium was at 5.6-5.8, the seeding density was optimal (-2.5 X 105 cells per cm2), and the incubation temperature was maintained at 35-37°C. Under these conditions, epidermal cells from many different skin locations grew to confluency within 15-20 days and formed multi-layered sheets whose differentiated structure resembled that of the full depth of skin epidermis. Cell proliferation and differentiation did not require a feeder layer, a collagen substrate, a high concentration of fetal bovine serum, or added hormones. The sheets of differentiated epidermal cells could be dissociated from the plastic surfaces of the tissue culture flasks. The use of such cultured cells for wound dressing is proposed.The apparent dependence of human epidermal cell growth and differentiation in vitro on the presence of a mouse 3T3 cell feeder layer (1) or collagenized substrates (2, 3) has led to the supposition that epidermal cell growth and differentiation in vivo may depend on dermal cell products. Epidermal cell growth has also been observed in the absence of such supports, but then the medium had to be supplemented by hormone preparations such as pituitary extracts and a high concentration (20%) of fetal bovine serum (4). Thus, it has been difficult to study the role of cell-to-cell interactions, and the influences of substances such as hormones, chalones, etc. on the process of epidermal cell differentiation in vitro because of the obligatory presence of nonepidermal components in the system. We now present evidence tht epidermal growth in vitro demands neither dermal elements nor special nutrients, provided conditions of pH, seeding density, and incubation temperature have been optimized. MATERIALS AND METHODSTissue Culture. More than 200 human skin specimens from different skin locations (scalp, face, neck, arms, breast, foreskin, legs, and trunk) as well as skin shavings from burn victims and cadavers (4-6 hr after death) have been successfully cultivated. Both full and split-thickness skin have been used. Skin samples were freed from fatty tissue and washed in minimal essential medium (GIBCO) with Earle's salts, containing, per ml, 1000 units of penicillin, 1 mg of streptomycin, and 2.5 ug of Fungizone for 30 min, followed by two washes, of 10 min each, in the same medium. Two additional washes 10 min each were in minimal essential medium with 1/10th the concentration of antibiotics. Discs of tissue were cut from the epidermal side of each specimen with sharp curved scissors, including as little dermis as possible. After washing in 0.02% EDTA (Sigma), the pieces were transferred to 0.25% trypsin (1:250, Difco) at 4°C for 12-15 hr. Subsequent to this incubation, the cut pieces were transferred to a fresh dish and the epidermis of each piece was detached from its dermis with fine forceps. Isolated epidermal samples were pooled in a trypsin/EDTA solution (5) [8 g of NaCl, 0.4 g of KCI, 1 g of dextrose, 0.58 g of NaHCO3, 0.5 g ...
Alcoholic liver disease (ALD) causes significant morbidity and mortality, and pharmacological treatment options are limited. In this study, we evaluated the PCSK9 inhibitor alirocumab, a monoclonal antibody that robustly reduces low-density lipoprotein cholesterol (LDL-C), for the treatment of ALD using a rat model of chronic alcohol exposure. Alirocumab (50 mg/kg) or vehicle was administered weekly for 6 weeks to rats receiving a 12% alcohol liquid diet or an isocaloric control diet. At the end of the alcohol exposure protocol, serum and liver samples were obtained for molecular characterization and histopathological analysis. PCSK9 inhibition with alirocumab attenuated alcohol-induced hepatic triglyceride accumulation through regulation of lipid metabolism (mRNA expression of modulators of fatty acid synthesis (FAS) and catabolism (PPARα and CPT1)), hepatocellular injury (ALT), hepatic inflammation (mRNA expression of pro-inflammatory cytokines/chemokines (TNFa, IL-1β, IL-22, IL-33, IL-17α, IL-2, MIP-2, and MCP-1), and neutrophil infiltration (myeloperoxidase staining)). Alirocumab treatment also attenuated alcohol-induced PCSK9 mRNA elevation and upregulated LDL-receptor (LDL-R) via modulation of the transcription factors (SREBP-1, SREBP-2, and E2F1) in liver. We demonstrated that chronic anti-PCSK9 treatment using the monoclonal antibody alirocumab attenuated alcohol-induced steatohepatitis in the rat model. Given the large unmet clinical need for effective and novel treatments for ALD, anti-PCSK9 treatment with the monoclonal antibody that spares liver metabolism is a viable new therapeutic possibility. Future studies are needed to elucidate the exact role of PCSK9 in ALD and alcohol use disorder (AUD) and to evaluate efficacy and safety of anti-PCSK9 treatment in clinical populations with ALD/AUD.
Background. The innovative pure laparoscopic living donor right hepatectomy (LLDRH) procedure for liver transplantation has never been fully compared to open living donor right hepatectomy (OLDRH). We aimed to compare the donor safety and graft results of pure LLDRH to those of OLDRH. Methods. From May 2013 to July 2017, 288 consecutive donors underwent either OLDRH (n = 197) or pure LLDRH (n = 91). After propensity score matching, 72 donors were included in each group. The primary outcome was postoperative complications during a 90-day follow-up period. Comprehensive complication index, duration of hospital stay, need for additional pain control, readmission, and donor outcomes were also compared. Results. The incidence of major complication during the 90-day follow-up was higher in the LLDRH group than the OLDRH group (6.6% vs 15.4%, P = 0.017) but was not statistically significant in propensity-matched analysis (11.1% vs 13.9%, odds ratio [OR], 1.29; 95% confidence interval [CI], 0.47-3.51; P = 0.62). A right hepatic duct <1 cm was independently associated with complication in the pure LLDRH group (odds ratio, 4.01; 95% confidence interval, 1.08-14.99; P = 0.04). Conclusions. In the initial 91 pure LLDRH cases, incidence of major complication was higher than in the OLDRH group, but the difference was not significant in propensity-matched analysis. A right hepatic duct verified as <1 cm may be related to increased frequency of complications in pure LLDRH donors. Further analysis is needed.
The influences of particle size on the physicochemical, release, and cellular uptake properties of chitosan nanoparticles (CSNPs) were investigated. Ionotropic CSNPs of different sizes (200-1000 nm) loaded with two model core materials (resveratrol or coumarin-6) were prepared using tripolyphosphate and carrageenan as cross-linkers. With an increase of particle size, zeta potential (34.6 ± 0.5 to 51.1 ± 0.9) and entrapment efficiency (14.9 ± 1.4 to 40.9 ± 1.9) of the CSNPs were significantly (p < 0.05) increased and release rates were decreased. However, Caco-2 cellular uptake of CSNPs were significantly increased from 3.70 ± 0.03 to 5.24 ± 0.20 with an increase of particle size from 200 to 600 nm, whereas those significantly decreased from 5.24 ± 0.20 to 4.55 ± 0.2 for particles larger than 600 nm in transwell assay. Moreover, much the same uptake patterns were also observed in confocal microscopy and flow cytometry. Investigation of cellular uptake of CSNPs revealed positive correlations between ZP and EE and indicated the effects of complex factors of nanoparticles other than size. These results provide a better understanding of CSNPs absorption and raises the possibility of controlling alternative nanoparticle properties to enhance bioavailability.
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