Design, Synthesis, and Evaluation of N- and C-Terminal Protein Bioconjugates as G Protein-Coupled Receptor Agonists.

A G protein-coupled receptor (GPCR) agonist protein, thaumatin, was site-specifically conjugated at the N- or C-terminus with a fluorophore for visualization of GPCR:agonist interactions. The N-terminus was specifically conjugated using a synthetic 2-pyridinecarboxyaldehyde reagent. The interaction profiles observed for N- and C-terminal conjugates were varied; N-terminal conjugates interacted very weakly with the GPCR of interest, whereas C-terminal conjugates bound to the receptor. These chemical biology tools allow interactions of therapeutic proteins:GPCR to be monitored and visualized. The methodology used for site-specific bioconjugation represents an advance in application of 2-pyridinecarboxyaldehydes for N-terminal specific bioconjugations.

An analysis of the changes in soluble hydrogenase and global gene expression in Cupriavidus necator (Ralstonia eutropha) H16 grown in heterotrophic diauxic batch culture.

BACKGROUND: Soluble hydrogenases (SH) are enzymes that catalyse the oxidation of molecular hydrogen. The SH enzyme from Cupriavidus necator H16 is relatively oxygen tolerant and makes an attractive target for potential application in biochemical hydrogen fuel cells. Expression of the enzyme can be mediated by derepression of the hox promoter system under heterotrophic conditions. However, the overall impact of hox derepression, from a transcriptomic perspective, has never been previously reported. RESULTS: Derepression of hydrogenase gene expression upon fructose depletion was confirmed in replicate experiments. Using qRT-PCR, hoxF was 4.6-fold up-regulated, hypF2 was up-regulated in the cells grown 2.2-fold and the regulatory gene hoxA was up-regulated by a mean factor of 4.5. A full transcriptomic evaluation revealed a substantial shift in the global pattern of gene expression. In addition to up-regulation of genes associated with hydrogenase expression, significant changes were observed in genes associated with energy transduction, amino acid metabolism, transcription and translation (and regulation thereof), genes associated with cell stress, lipid and cell wall biogenesis and other functions, including cell motility. CONCLUSIONS: We report the first full transcriptome analysis of C. necator H16 grown heterotrophically on fructose and glycerol in diauxic batch culture, which permits expression of soluble hydrogenase under heterotrophic conditions. The data presented deepens our understanding of the changes in global gene expression patterns that occur during the switch to growth on glycerol and suggests that energy deficit is a key driver for induction of hydrogenase expression in this organism.

Polymersomes prepared from thermoresponsive fluorescent protein-polymer bioconjugates: capture of and report on drug and protein payloads.

Polymersomes provide a good platform for targeted drug delivery and the creation of complex (bio)catalytically active systems for research in synthetic biology. To realize these applications requires both spatial control over the encapsulation components in these polymersomes and a means to report where the components are in the polymersomes. To address these twin challenges, we synthesized the protein-polymer bioconjugate PNIPAM-b-amilFP497 composed of thermoresponsive poly(N-isopropylacrylamide) (PNIPAM) and a green-fluorescent protein variant (amilFP497). Above 37 °C, this bioconjugate forms polymersomes that can (co-)encapsulate the fluorescent drug doxorubicin and the fluorescent light-harvesting protein phycoerythrin 545 (PE545). Using fluorescence lifetime imaging microscopy and Förster resonance energy transfer (FLIM-FRET), we can distinguish the co-encapsulated PE545 protein inside the polymersome membrane while doxorubicin is found both in the polymersome core and membrane.

One-pot synthesis of high molecular weight synthetic heteroprotein dimers driven by charge complementarity electrostatic interactions.

Despite the importance of protein dimers and dimerization in biology, the formation of protein dimers through synthetic covalent chemistry has not found widespread use. In the case of maleimide-cysteine-based dimerization of proteins, we show here that when the proteins have the same charge, dimerization appears to be inherently difficult with yields around 1% or less, regardless of the nature of the spacer used or whether homo- or heteroprotein dimers are targeted. In contrast, if the proteins have opposing (complementary) charges, the formation of heteroprotein dimers proceeds much more readily, and in the case of one high molecular weight (>80 kDa) synthetic dimer between cytochrome c and bovine serum albumin, a 30% yield of the purified, isolated dimer was achieved. This represents at least a 30-fold increase in yield for protein dimers formed from proteins with complementary charges, compared to when the proteins have the same charge, under otherwise similar conditions. These results illustrate the role of ionic supramolecular interactions in controlling the reactivity of proteins toward bis-functionalized spacers. The strategy here for effective synthetic dimerization of proteins could be very useful for developing novel approaches to study the important role of protein-protein interactions in chemical biology.

Evaluation of alternate hosts for recombinant expression of a reductive dehalogenase.

Organohalides are recalcitrant, toxic environmental pollutants. Reductive dehalogenase enzymes (RDases) found in organohalide respiring bacteria (OHRB) utilise organohalides as electron acceptors for cellular energy and growth, producing lesser-halogenated compounds. Consequently, microbial reductive dehalogenation via organohalide respiration represents a promising solution for clean-up of organohalide pollutants. Dehalobacter sp. UNSWDHB is an OHRB capable of respiring highly toxic chloroform (CF) and converting it to dichloromethane (DCM). TmrA has been identified as an RDase responsible for this conversion and different strategies for generation of functional recombinant TmrA is the focus of this article. In this study, TmrA was recovered from inclusion bodies expressed in E. coli and refolded in the presence of FeCl3, Na2S and cobalamin to yield functional enzyme. TmrA has been previously expressed in a soluble and functional form in the corrinoid-producing Bacillus megaterium. Using a fractional experimental design for cultivation and induction combined with purification under anaerobic conditions resulted in substantially higher activity of recombinant and native TmrA than previously reported. TmrA was then expressed in a soluble and active form in Shimwellia blattae. Co-expression with two different putative chaperone proteins from the original host did not increase the level of soluble expression in S. blattae, however activity assays showed that removing the TAT signal from TmrA increases the dechlorination activity compared to when the TAT signal is present. Finally, TmrA was successfully expressed in a soluble and active form in the H2-oxidizing C. necator H16, a novel host for the expression of RDases.

Induced adaptation of Bacillus sp. to antimicrobial nanosilver.

The natural ability of Bacillus sp. to adapt to nanosilver cytotoxicity upon prolonged exposure is reported for the first time. The combined adaptive effects of nanosilver resistance and enhanced growth are induced under various intensities of nanosilver-stimulated cellular oxidative stress, ranging from only minimal cellular redox imbalance to the lethal levels of cellular ROS stimulation. An important implication of the present work is that such adaptive effects lead to the ultimate domination of nanosilver-resistant Bacillus sp. in the microbiota, to which nanosilver cytotoxicity is continuously applied.

Macrophage inhibitory cytokine-1 (MIC-1/GDF15) and mortality in end-stage renal disease.

BACKGROUND: Elevated macrophage inhibitory cytokine-1 (MIC-1/GDF15) levels in serum mediate anorexia and weight loss in some cancer patients and similarly elevated levels occur in chronic kidney disease (CKD). Serum MIC-1/GDF15 is also elevated in chronic inflammatory diseases and predicts atherosclerotic events independently of traditional risk factors. The relationship between chronic inflammation, decreasing body mass index (BMI) and increased mortality in CKD is not well understood and is being actively investigated. MIC-1/GDF15 may link these features of CKD. METHODS: Cohorts of incident dialysis patients from Sweden (n = 98) and prevalent hemodialysis patients from the USA (n = 381) had serum MIC-1/GDF15, C-reactive protein (CRP) levels and BMI measured at study entry. Additional surrogate markers of nutritional adequacy, body composition and inflammation were assessed in Swedish patients. Patients were followed for all-cause mortality. RESULTS: In the Swedish cohort, serum MIC-1/GDF15 was associated with decreasing BMI, measures of nutrition and markers of oxidative stress and inflammation. Additionally, high serum MIC-1/GDF15 levels identified patients with evidence of protein-energy wasting who died in the first 3 years of dialysis. The ability of serum MIC-1/GDF15 to predict mortality in the first 3 years of dialysis was confirmed in the USA cohort. In both cohorts, serum MIC-1/GDF15 level was an independent marker of mortality when adjusted for age, CRP, BMI, history of diabetes mellitus and/or cardiovascular disease and glomerular filtration rate or length of time on dialysis at study entry. CONCLUSIONS: MIC-1/GDF15 is a novel independent serum marker of mortality in CKD capable of significantly improving the mortality prediction of other established markers. MIC-1/GDF15 may mediate protein-energy wasting in CKD and represent a novel therapeutic target for this fatal complication.

A Tunable Tumor Microenvironment through Recombinant Bacterial Collagen-Hyaluronic Acid Hydrogels.

Laboratory models of the tumor microenvironment require control of mechanical and biochemical properties to ensure accurate mimicry of patient disease. In contrast to pure natural or synthetic materials, hybrid approaches that pair recombinant protein fragments with synthetic scaffolding show many advantages. Here we demonstrate production of a recombinant bacterial collagen-like protein (CLP) for thiol-ene pairing to norbornene functionalized hyaluronic acid (NorHA). The resultant hydrogel material shows an adjustable modulus with evidence for strain-stiffening behavior that resembles natural tumor matrices. Cysteine terminated peptide binding motifs are incorporated to adjust the cell-adhesion points. The modular hybrid gel shows good biocompatibility and was demonstrated to control cell adhesion, proliferation, and the invasive properties of MCF7 and MD-MBA-231 breast adenocarcinoma cells. The ease in which multiple structural and bioactive components can be integrated provides a robust framework to form models of the tumor microenvironment for fundamental studies and drug development.

The TGF-ß superfamily cytokine, MIC-1/GDF15: a pleotrophic cytokine with roles in inflammation, cancer and metabolism.

Macrophage inhibitory cytokine-1 (MIC-1/GDF15) is associated with cardiovascular disease, inflammation, body weight regulation and cancer. Its serum levels facilitate the diagnosis and prognosis of cancer and vascular disease. Furthermore, its serum levels are a powerful predictor of all-cause mortality, suggesting a fundamental role in biological processes associated with ageing. In cancer, the data available suggest that MIC-1/GDF15 is antitumorigenic, but this may not always be the case as disease progresses. Cancer promoting effects of MIC-1/GDF15 may be due, in part, to effects on antitumour immunity. This is suggested by the anti-inflammatory and immunosuppressive properties of MIC-1/GDF15 in animal models of atherosclerosis and rheumatoid arthritis. Furthermore, in late-stage cancer, large amounts of MIC-1/GDF15 in the circulation suppress appetite and mediate cancer anorexia/cachexia, which can be reversed by monoclonal antibodies in animals. Available data suggest MIC-1/GDF15 may be an important molecule mediating the interplay between cancer, obesity and chronic inflammation.

Interpretation of the scarf osteotomy by 10 surgeons.

BACKGROUND: The study compared three-dimensional (3D) changes in geometry of the first metatarsal following scarf osteotomy performed on standardised Sawbone® models by consultant foot and ankle surgeons. The study considered the inter-surgeon variances in interpretation and performance of the scarf osteotomy with respect to intra-surgeon variances. METHODS: The analysis used an accurate digitising system to measure and record points on the Sawbone® models in 3D space. Computer software performed vector analysis to calculate 3D rotations and translations of the first metatarsal head as well as the inter-metatarsal angle. Bone cut lengths and displacements were measured using a digital Vernier caliper. One surgeon performed the osteotomy 10 times to form an intra-surgeon control dataset, while 10 different surgeons each did one scarf osteotomy to form an inter-surgeon test dataset. RESULTS: Both surgical groups produced reductions in the 3D inter-metatarsal angle with non-significant differences between the groups (p>0.05). In contrast, the test group demonstrated highly significant (p=0.000) greater variance compared with the control dataset for all of the variables associated with surgical technique. In addition, there were highly significant (p=0.02 and 0.002) greater variances in the interpretation of the degree to which the metatarsal head should be translated medially (X) and inferiorly (Z). There was also a significant (p=0.001) increase in variances in the rotations about the dorsi/plantar-flexion (X) axis. The only significant differences (all p=0.000) attributable solely to differences in mean values were in proximal-distal (Y) translation, pronation (Y) rotation and medial (Z) rotation. The test group applied greater medial and plantar-flexion rotation of the metatarsal head than the control surgeon and significantly less (p=0.000) shortening of the first metatarsal than the control surgeon. CONCLUSIONS: The results of this geometric study demonstrate the versatility of the scarf osteotomy. As a result of the multi-planar nature of the osteotomy, there is a potential risk of producing unintended rotational mal-unions in all three planes. These rotational mal-unions may account for some of the poorer outcomes documented within the literature.

Assembly of polyethylenimine-based magnetic iron oxide vectors: insights into gene delivery.

The use of a nonviral magnetic vector, comprised of magnetic iron oxide nanoparticles (MNP), polyethylenimine (PEI), and plasmid DNA, for transfection of BHK21 cells under a magnetic field is presented. Four different vector configurations were studied by systematically varying the mixing order of MNP, PEI, and DNA. The assembly of the vector has significant effects on its vector size, surface charge, cellular uptake, and level of gene expression. Mixing MNP with PEI first improved MNP stability, giving a narrow aggregate size distribution and positive surface charge at physiological pH, which in turn facilitated DNA binding onto MNP. The presence of serum in culture media improves vector dispersion and alters the surface charge of all vectors to negative charge, indicating serum protein adsorption. Cellular uptake was greater for larger vectors than the smaller vectors due to enhanced gravitational and magnetic aided sedimentation onto the cells. High MNP uptake by the cells, however, does not inevitably lead to increase gene expression efficiency. It can be shown that besides vector uptake, gene expression is affected by extracellular factors such as premature DNA release from MNP and DNA degradation by serum as well as intracellular factors such as vector lysosomal degradation, inability of DNA to detach from MNP, and cytotoxic effects of MNP at high uptake. Some of these extra- and intracellular properties are shown to be mediated by the presence of PEI.

Polyethylenimine based magnetic iron-oxide vector: the effect of vector component assembly on cellular entry mechanism, intracellular localization, and cellular viability.

The order of assembly of a magnetic nanoparticle (MNP) vector comprised of the same components (MNP, PEI, and plasmid DNA) on entry mechanism, intracellular localization, and viability of BHK21 cells was investigated. Cellular uptake measurements under four different uptake inhibiting conditions, such as low temperature, depleted cellular ATP, nystatin treatment, and hypertonic environment, show that the cellular entry mechanism of the MNP vector was mediated via clathrin endocytosis. Despite different vector component assembly, all MNP vectors were taken up by the cells through the same mechanism. Labeling and intracellular tracking of the MNP vectors using epi-fluorescence and confocal laser scanning microscopy showed localization of MNP vector within the lysosomes when DNA was assembled on the outer layer of vector. Conversely, when PEI was on the surface of the vector, such that it enclosed both magnetic nanoparticles and the DNA, vector localization in the cell nucleus was observed. The microscopy results demonstrated that the configuration of the MNP vectors dictate the vector's final intracellular target location, and thus the efficiency of transfection. The cellular viability assessment using three different assays further showed that the cellular viability of MNP vector was dose-dependent and varied with the assembly of vector component. All viability assays found negligible toxicity when DNA was on the outer layer of MNP vector except at the highest vector loading. In contrast, attachment of PEI on MNP vector surface induced a significant decrease in cellular viability, due to the ability of PEI on the MNP vector to rupture the lysosomal vesicles.

Nanosilver Targets the Bacterial Cell Envelope: The Link with Generation of Reactive Oxygen Radicals.

The work describes the interactions of nanosilver (NAg) with bacterial cell envelope components at a molecular level and how this associates with the reactive oxygen species (ROS)-mediated toxicity of the nanoparticle. Major structural changes were detected in cell envelope biomolecules as a result of damages in functional moieties, such as the saccharides, amides, and phosphodiesters. NAg exposure disintegrates the glycan backbone in the major cell wall component peptidoglycan, causes complete breakdown of lipoteichoic acid, and disrupts the phosphate-amine and fatty acid groups in phosphatidylethanolamine, a membrane phospholipid. Consistent with the oxidative attacks, we propose that the observed cell envelope damages are inflicted, at least in part, by the reactive oxygen radicals being generated by the nanoparticle during its leaching process, abiotically, without cells. The cell envelope targeting, especially those on the inner membrane phospholipid, is likely to then trigger the rapid generation of lethal levels of cellular superoxide (O2•-) and hydroxyl (OH•) radicals herein seen with a model bacterium. The present study provides a better understanding of the antibacterial mechanisms of NAg, whereby ROS generation could be both the cause and consequence of the toxicity, associated with the initial cell envelope targeting by the nanoparticle.

Growth differentiation factor-15 slows the growth of murine prostate cancer by stimulating tumor immunity.

Growth Differentiation Factor-15 (GDF15) is a divergent TGF-beta superfamily cytokine that is overexpressed by most cancers and is induced by anticancer therapy. Transgenic and induced animal models suggest that it protects from cancer development but the mechanisms are uncertain. We investigated the role of immunity in GDF15 induced reduction in prostate cancer (PCa) growth. The C57BL/6 transgenic TRAMP prostate cancer prone mice were bred with mice that were immunodeficient and/or systemically overexpressed GDF15. We developed a novel orthotopic TRAMP PCa model in which primary TRAMP tumor cells were implanted into prostates of mice to reduce the study time. These mice were administered recombinant mouse GDF15, antibody to CD8, PD1 or their respective controls. We found that GDF15 induced protection from tumor growth was reversed by lack of adaptive immunity. Flow cytometric evaluation of lymphocytes within these orthotopic tumors showed that GDF15 overexpression was associated with increased CD8 T cell numbers and an increased number and proportion of recently activated CD8+CD11c+ T cells and a reduced proportion of "exhausted" CD8+PD1+ T cells. Further, depletion of CD8 T cells in tumor bearing mice abolished the GDF15 induced protection from tumor growth. Infusion of GDF15 into mice bearing orthotopic TRAMP tumor, substantially reduced tumor growth that was further reduced by concurrent PD1 antibody administration. GDF15 overexpression or recombinant protein protects from TRAMP tumor growth by modulating CD8 T cell mediated antitumor immunity and augments the positive effects of anti-PD1 blockers.

Antimicrobial activity of T4 bacteriophage conjugated indium tin oxide surfaces.

We report the antimicrobial activity of bare and surface functionalized indium tin oxide (ITO) conjugated with T4 bacteriophage towards E. coli. A ∼ 103-fold reduction (99.9%) in the bacterial concentration was achieved within 2 h exposure of E. coli to the bare as well as the amine, carboxylic and methyl functionalized ITO/T4 surfaces. Despite the known differences in bacteriophage loading of these ITO/T4 systems, the almost identical extent of antimicrobial activity of all of the ITO/T4 systems resulted from the release of a comparable amount of infective T4 from the systems. As anticipated, a single dose of immobilized bacteriophage was sufficient to eliminate further surge of bacterial population. Upon the 2 h eradication of the '1st batch' of E. coli population, all of the ITO/T4 systems, each system with 102-fold more suspended bacteriophage (due to propagation of the phage at the expense of the '1st batch' E. coli death), reduced the '2nd batch' of E. coli concentration by ∼104-fold in just 30 min, suggesting the potential of immobilized bacteriophage systems as solution to the issues of antimicrobial agent depletion. All of the ITO/T4 systems maintained their antimicrobial activity in the presence of model food components. The antimicrobial activity was however, affected by pH; at pH 5 whereby the bacteria's growth was physiologically inhibited, generally no reduction in E. coli concentration was detected. The present work provides an understanding of the mode of antimicrobial activity exhibited by an immobilized bacteriophage based substrate and demonstrates efficacy in the presence of food components.

Nanosilver and the microbiological activity of the particulate solids versus the leached soluble silver.

Nanosilver (Ag NPs) is currently one of the most commercialized antimicrobial nanoparticles with as yet, still unresolved cytotoxicity origins. To date, research efforts have mostly described the antimicrobial contribution from the leaching of soluble silver, while the undissolved solid Ag particulates are often considered as being microbiologically inert, serving only as source of the cytotoxic Ag ions. Here, we show the rapid stimulation of lethal cellular oxidative stress in bacteria by the presence of the undissolved Ag particulates. The cytotoxicity characteristics are distinct from those arising from the leached soluble Ag, the latter being locked in organic complexes. The work also highlights the unique oxidative stress-independent bacterial toxicity of silver salt. Taken together, the findings advocate that future enquiries on the antimicrobial potency and also importantly, the environmental and clinical impact of Ag NPs use, should pay attention to the potential bacterial toxicological responses to the undissolved Ag particulates, rather than just to the leaching of soluble silver. The findings also put into question the common use of silver salt as model material for evaluating bacterial toxicity of Ag NPs.

Heterologous Production and Purification of a Functional Chloroform Reductive Dehalogenase.

Reductive dehalogenases (RDases) are key enzymes involved in the respiratory process of anaerobic organohalide respiring bacteria (ORB). Heterologous expression of respiratory RDases is desirable for structural and functional studies; however, there are few reports of successful expression of these enzymes. Dehalobacter sp. strain UNSWDHB is an ORB, whose preferred electron acceptor is chloroform. This study describes efforts to express recombinant reductive dehalogenase (TmrA), derived from UNSW DHB, using the heterologous hosts Escherichia coli and Bacillus megaterium. Here, we report the recombinant expression of soluble and functional TmrA, using B. megaterium as an expression host under a xylose-inducible promoter. Successful incorporation of iron-sulfur clusters and a corrinoid cofactor was demonstrated using UV-vis spectroscopic analyses. In vitro dehalogenation of chloroform using purified recombinant TmrA was demonstrated. This is the first known report of heterologous expression and purification of a respiratory reductive dehalogenase from an obligate organohalide respiring bacterium.

An improved process for the production of highly purified recombinant thaumatin tagged-variants.

The sweetest tasting molecule known is the protein thaumatin, first isolated from the katemfe fruit, Thaumatococcus daniellii. Thaumatin is used in the food and beverage industry as a low-calorie sugar substitute. Thaumatin interacts with taste receptors in the oral cavity eliciting a persistent sweet taste and a bitter, liquorice flavor. Recombinant thaumatin was expressed in Pichia pastoris and through a co-expression strategy with a molecular chaperone, yields of one engineered thaumatin variant increased by greater than two-fold. A detailed purification strategy for thaumatin is reported resulting in a homogenous sample recovered at a yield of 42%. The recombinant thaumatins were extensively characterised using size exclusion chromatography for homogeneity, reversed-phase HPLC for purity (99%), peptide digest LC-MS/MS for sequence determination, and circular dichroism and tryptophan fluorescence spectroscopies for conformational characterisation. These new thaumatin variants are amenable for bioconjugation, providing chemical biology tools for thaumatin:taste receptor interaction studies.

T4 bacteriophage conjugated magnetic particles for E. coli capturing: Influence of bacteriophage loading, temperature and tryptone.

This work demonstrates the use of bacteriophage conjugated magnetic particles (Fe3O4) for the rapid capturing and isolation of Escherichia coli. The investigation of T4 bacteriophage adsorption to silane functionalised Fe3O4 with amine (NH2), carboxylic (COOH) and methyl (CH3) surface functional groups reveals the domination of net electrostatic and hydrophobic interactions in governing bacteriophage adsorption. The bare Fe3O4 and Fe3O4-NH2 with high T4 loading captured 3-fold more E. coli (∼70% capturing efficiency) compared to the low loading T4 on Fe3O4-COOH, suggesting the significance of T4 loading in E. coli capturing efficiency. Importantly, it is further revealed that E. coli capture is highly dependent on the incubation temperature and the presence of tryptone in the media. Effective E. coli capturing only occurs at 37°C in tryptone-containing media with the absence of either conditions resulted in poor bacteria capture. The incubation temperature dictates the capturing ability of Fe3O4/T4, whereby T4 and E. coli need to establish an irreversible binding that occurred at 37°C. The presence of tryptophan-rich tryptone in the suspending media was also critical, as shown by a 3-fold increase in E. coli capture efficiency of Fe3O4/T4 in tryptone-containing media compared to that in tryptone-free media. This highlights for the first time that successful bacteria capturing requires not only an optimum tailoring of the particle's surface physicochemical properties for favourable bacteriophage loading, but also an in-depth understanding of how factors, such as temperature and solution chemistry influence the subsequent bacteriophage-bacteria interactions.

Widespread and Indiscriminate Nanosilver Use: Genuine Potential for Microbial Resistance.

In this era of increasing antibiotic resistance, the use of alternative antimicrobials such as silver has become more widespread. Superior antimicrobial activity has been provided through fabrication of silver nanoparticles or nanosilver (NAg), which imparts cytotoxic actions distinct from those of bulk silver. In the wake of the recent discoveries of bacterial resistance to NAg and its rising incorporation in medical and consumer goods such as wound dressings and dietary supplements, we argue that there is an urgent need to monitor the prevalence and spread of NAg microbial resistance. In this Perspective, we describe how the use of NAg in commercially available products facilitates prolonged microorganism exposure to bioavailable silver, which underpins the development of resistance. Furthermore, we advocate for a judicial approach toward NAg use in order to preserve its efficacy and to avoid environmental disruption.