The present studies were performed to determine subtype-specific roles of mitogen-activated protein kinase in chondrogenesis. Erk-1/2 activities, downstream of protein kinase C, decreased as chondrogenesis proceeded, whereas p38 activities, independent of protein kinase C, continuously increased during chondrogenesis. Inhibition of Erk-1/2 with PD98059 enhanced chondrogenesis up to 1.7-fold, whereas inhibition of p38 with SB203580 reduced it to about 30% of the control level. Inhibition of Erk-1/2 or p38 did not affect precartilage condensation. However, cartilage nodule formation was significantly blocked by the inhibition of p38, whereas Erk-1/2 inhibition did not affect it. Modulation of chondrogenesis by the inhibition of Erk-1/2 and p38 was accompanied by altered expression of adhesion molecules in an opposite way. Expression of N-cadherin was reduced as chondrogenesis proceeded. Inhibition of p38 caused sustained expression of N-cadherin, whereas Erk-1/2 inhibition accelerated the reduction of N-cadherin expression. Expression of integrin ␣5␤1 and fibronectin were found to transiently increase during chondrogenesis. Inhibition of p38 caused continuous increase of expression of these molecules, whereas Erk-1/2 inhibition accelerated the decrease of expression of these molecules at a later period of chondrogenesis. Because temporal expression of these adhesion molecules regulates chondrogenesis, the above results indicate that Erk-1/2 and p38 conversely regulate chondrogenesis at post-precartilage condensation stages by modulating expression of adhesion molecules.Formation of cartilage is initiated by differentiation of mesenchymes into chondrocytes. The differentiation process requires proliferation of chondrogenic competent cells that subsequently undergo precartilage condensation (1-3). Precartilage condensation is characterized by cells that are more densely packed in specific regions. This precartilage condensation is an important prerequisite for the initiation of chondrogenesis in mesenchymes in vivo. It becomes evident that several adhesion molecules including cadherins (4-7), integrins (8, 9), and extracellular matrix (ECM) 1 components (9-12) regulate precartilage condensation. Limb mesenchymes undergo spontaneous differentiation to chondrocytes in vitro when cultured at a high seeding density such as micromass culture in the presence of serum. Precartilage condensation similar to that formed in vivo during chondrogenesis occurs in micromass cultures of limb mesenchymes (13). The cellular aggregates that form in early micromass cultures subsequently differentiate into cartilage nodules, which express cartilage-specific molecules such as type II collagen (6, 14-16). However, the signal transduction pathways involved in precartilage condensation and initiated by condensation are not well characterized.We have previously shown that protein kinase C (PKC) positively regulates chondrogenesis of mesenchymes (17,18). PKC is a multigene family composed of 11 known isoforms (19,20). Multiple PKC isoforms su...

Conspectus Antimicrobial resistance (AMR) is one of the greatest threats faced by humankind. The development of resistance in clinical and hospital settings has been well documented ever since the initial discovery of penicillin and the subsequent introduction of sulfonamides as clinical antibiotics. In contrast, the environmental (i.e., community-acquired) dimensions of resistance dissemination have been only more recently delineated. The global spread of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) between air, water, soil, and food is now well documented, while the factors that affect ARB and ARG dissemination (e.g., water and air quality, antibiotic fluxes, urbanization, sanitation practices) in these and other environmental matrices are just now beginning to be more fully appreciated. In this Account, we discuss how the global perpetuation of resistance is dictated by highly interconnected socioeconomic risk factors and illustrate that development status should be more fully considered when developing global strategies to address AMR. We first differentiate low to middle income countries (LMICs) and high-income countries (HICs), then we summarize the modes of action of commercially available antibiotics, and then discuss the four primary mechanisms by which bacteria develop resistance to those antibiotics. Resistance is disseminated via both vertical gene transfer (VGT; parent to offspring) as well as by horizontal gene transfer (HGT; cell to cell transference of genetic material). A key challenge hindering attempts to control resistance dissemination is the presence of native, environmental bacteria that can harbor ARGs. Such environmental “resistomes” have potential to transfer resistance to pathogens via HGT. Of particular concern is the development of resistance to antibiotics of last-resort such as the cephalosporins, carbapenems, and polymyxins. We then illustrate how antibiotic use differs in LMICs relative to HICs in terms of the volumes of antibiotics used and their fate within local environments. Antibiotic use in HICs has remained flat over the past 15 years, while in LMICs use over the same period has increased substantially as a result of economic improvements and changes in diet. These use and fate differences impact local citizens and thus the local dissemination of AMR. Various physical, social, and economic circumstances within LMICs potentially favor AMR dissemination. We focus on three physical factors: changing population density, sanitation infrastructure, and solid-waste disposal. We show that high population densities in cities within LMICs that suffer from poor sanitation and solid-waste disposal can potentially impact the dissemination of resistance. In the final section, we discuss potential monitoring approaches to quantify the spread of resistance both within LMICs as well as in HICs. We posit that culture-based approaches, molecular approaches, and cutting-edge nanotechnology-based methods for monitoring ARB and ARGs should be considered both within ...

Pathogen resistance to clinical antimicrobial agents is an urgent problem. The fungus Aspergillus fumigatus causes 300,000 life-threatening infections in susceptible humans annually. Azoles, which are widely used in both clinical and agricultural settings, are currently the most effective treatment, but resistance to clinical azoles is emerging worldwide. Here we report the isolation and analysis of azole-sensitive and azole-resistant A. fumigatus from agricultural environments in the southeastern United States (USA) and show that the USA pan-azole-resistant isolates form a clade with pan-azole-resistant isolates from the United Kingdom, the Netherlands, and India. We show that several pan-azole-resistant isolates from agricultural settings in the USA and India also carry alleles with mutations conferring resistance to agricultural fungicides from the benzimidazole (MBC) and quinone outside inhibitor (QoI) classes. We further show that pan-azole-resistant A. fumigatus isolates from patients in clinical settings in the USA, India and the Netherlands also carry alleles conferring resistance to MBC and QoI agricultural fungicides. The presence of markers for resistance to agricultural-use fungicides in clinical A. fumigatus isolates is strong evidence for an agricultural origin of pan-azole resistance in patients. The presence of multiple fungicide-resistance alleles in agricultural and clinical isolates further suggests that the unique genetics of the pan-azole-resistant clade enables the evolution and/or persistence of antimicrobial resistance mutations leading to the establishment of multi-fungicide-resistant isolates.

The fungus Aspergillus fumigatus causes pulmonary invasive aspergillosis resulting in nearly 100,000 deaths each year. Patients are often treated with antifungal drugs such as amphotericin B (AmB) loaded into liposomes (AmB-LLs), but all antifungal drugs, including AmB-LLs, have serious limitations due to human toxicity and insufficient fungal cell killing. Even with the best current therapies, 1-year survival among patients with invasive aspergillosis is only 25 to 60%. Hence, there is a critical need for improved antifungal therapeutics. Dectin-1 is a mammalian protein that binds to beta-glucan polysaccharides found in nearly all fungal cell walls. We coated AmB-LLs with Dectin-1 to make DEC-AmB-LLs. DEC-AmB-LLs bound strongly to fungal cells, while AmB-LLs had little affinity. DEC-AmB-LLs killed or inhibited A. fumigatus 10 times more efficiently than untargeted liposomes, decreasing the effective dose of AmB. Dectin-1-coated drug-loaded liposomes targeting fungal pathogens have the potential to greatly enhance antifungal therapeutics.

Ultrasensitive surface-enhanced Raman spectroscopy (SERS) still faces difficulties in quantitative analysis because of its susceptibility to local optical field variations at plasmonic hotspots in metallo-dielectric nanostructures. Current SERS calibration approaches using Raman tags have inherent limitations due to spatial occupation competition with analyte molecules, spectral interference with analyte Raman peaks, and photodegradation. Herein, we report that plasmon-enhanced electronic Raman scattering (ERS) signals from metal can serve as an internal standard for spatial and temporal calibration of molecular Raman scattering (MRS) signals from analyte molecules at the same hotspots, enabling rigorous quantitative SERS analysis. We observe a linear dependence between ERS and MRS signal intensities upon spatial and temporal variations of excitation optical fields, manifesting the |E|4 enhancements for both ERS and MRS processes at the same hotspots in agreement with our theoretical prediction. Furthermore, we find that the ERS calibration’s performance limit can result from orientation variations of analyte molecules at hotspots.