Embracing enzyme promiscuity with activity-based compressed biosensing.

The development of protease-activatable drugs and diagnostics requires identifying substrates specific to individual proteases. However, this process becomes increasingly difficult as the number of target proteases increases because most substrates are promiscuously cleaved by multiple proteases. We introduce a method-substrate libraries for compressed sensing of enzymes (SLICE)-for selecting libraries of promiscuous substrates that classify protease mixtures (1) without deconvolution of compressed signals and (2) without highly specific substrates. SLICE ranks substrate libraries using a compression score (C), which quantifies substrate orthogonality and protease coverage. This metric is predictive of classification accuracy across 140 in silico (Pearson r = 0.71) and 55 in vitro libraries (r = 0.55). Using SLICE, we select a two-substrate library to classify 28 samples containing 11 enzymes in plasma (area under the receiver operating characteristic curve [AUROC] = 0.93). We envision that SLICE will enable the selection of libraries that capture information from hundreds of enzymes using fewer substrates for applications like activity-based sensors for imaging and diagnostics.

Erratum: Variants in PHF8 cause a spectrum of X-linked neurodevelopmental disorders and facial dysmorphology.

[This corrects the article DOI: 10.1016/j.xhgg.2022.100102.].

Variants in PHF8 cause a spectrum of X-linked neurodevelopmental disorders and facial dysmorphology.

Loss-of-function variants in PHD Finger Protein 8 (PHF8) cause Siderius X-linked intellectual disability (ID) syndrome, hereafter called PHF8-XLID. PHF8 is a histone demethylase that is important for epigenetic regulation of gene expression. PHF8-XLID is an under-characterized disorder with only five previous reports describing different PHF8 predicted loss-of-function variants in eight individuals. Features of PHF8-XLID include ID and craniofacial dysmorphology. In this report we present 16 additional individuals with PHF8-XLID from 11 different families of diverse ancestry. We also present five individuals from four different families who have ID and a variant of unknown significance in PHF8 with no other explanatory variant in another gene. All affected individuals exhibited developmental delay and all but two had borderline to severe ID. Of the two who did not have ID, one had dyscalculia and the other had mild learning difficulties. Craniofacial findings such as hypertelorism, microcephaly, elongated face, ptosis, and mild facial asymmetry were found in some affected individuals. Orofacial clefting was seen in three individuals from our cohort, suggesting that this feature is less common than previously reported. Autism spectrum disorder and attention deficit hyperactivity disorder, which were not previously emphasized in PHF8-XLID, were frequently observed in affected individuals. This series expands the clinical phenotype of this rare ID syndrome caused by loss of PHF8 function.

Dimensionless parameter predicts bacterial prodrug success.

Understanding mechanisms of antibiotic failure is foundational to combating the growing threat of multidrug-resistant bacteria. Prodrugs-which are converted into a pharmacologically active compound after administration-represent a growing class of therapeutics for treating bacterial infections but are understudied in the context of antibiotic failure. We hypothesize that strategies that rely on pathogen-specific pathways for prodrug conversion are susceptible to competing rates of prodrug activation and bacterial replication, which could lead to treatment escape and failure. Here, we construct a mathematical model of prodrug kinetics to predict rate-dependent conditions under which bacteria escape prodrug treatment. From this model, we derive a dimensionless parameter we call the Bacterial Advantage Heuristic (BAH) that predicts the transition between prodrug escape and successful treatment across a range of time scales (1-104 h), bacterial carrying capacities (5 × 104 -105 CFU/µl), and Michaelis constants (KM = 0.747-7.47 mM). To verify these predictions in vitro, we use two models of bacteria-prodrug competition: (i) an antimicrobial peptide hairpin that is enzymatically activated by bacterial surface proteases and (ii) a thiomaltose-conjugated trimethoprim that is internalized by bacterial maltodextrin transporters and hydrolyzed by free thiols. We observe that prodrug failure occurs at BAH values above the same critical threshold predicted by the model. Furthermore, we demonstrate two examples of how failing prodrugs can be rescued by decreasing the BAH below the critical threshold via (i) substrate design and (ii) nutrient control. We envision such dimensionless parameters serving as supportive pharmacokinetic quantities that guide the design and administration of prodrug therapeutics.

DNA Gold Nanoparticle Motors Demonstrate Processive Motion with Bursts of Speed Up to 50 nm Per Second.

Synthetic motors that consume chemical energy to produce mechanical work offer potential applications in many fields that span from computing to drug delivery and diagnostics. Among the various synthetic motors studied thus far, DNA-based machines offer the greatest programmability and have shown the ability to translocate micrometer-distances in an autonomous manner. DNA motors move by employing a burnt-bridge Brownian ratchet mechanism, where the DNA "legs" hybridize and then destroy complementary nucleic acids immobilized on a surface. We have previously shown that highly multivalent DNA motors that roll offer improved performance compared to bipedal walkers. Here, we use DNA-gold nanoparticle conjugates to investigate and enhance DNA nanomotor performance. Specifically, we tune structural parameters such as DNA leg density, leg span, and nanoparticle anisotropy as well as buffer conditions to enhance motor performance. Both modeling and experiments demonstrate that increasing DNA leg density boosts the speed and processivity of motors, whereas DNA leg span increases processivity and directionality. By taking advantage of label-free imaging of nanomotors, we also uncover Lévy-type motion where motors exhibit bursts of translocation that are punctuated with transient stalling. Dimerized particles also demonstrate more ballistic trajectories confirming a rolling mechanism. Our work shows the fundamental properties that control DNA motor performance and demonstrates optimized motors that can travel multiple micrometers within minutes with speeds of up to 50 nm/s. The performance of these nanoscale motors approaches that of motor proteins that travel at speeds of 100-1000 nm/s, and hence this work can be important in developing protocellular systems as well next generation sensors and diagnostics.

Protease circuits for processing biological information.

Engineered biocircuits designed with biological components have the capacity to expand and augment living functions. Here we demonstrate that proteases can be integrated into digital or analog biocircuits to process biological information. We first construct peptide-caged liposomes that treat protease activity as two-valued (i.e., signal is 0 or 1) operations to construct the biological equivalent of Boolean logic gates, comparators and analog-to-digital converters. We use these modules to assemble a cell-free biocircuit that can combine with bacteria-containing blood, quantify bacteria burden, and then calculate and unlock a selective drug dose. By contrast, we treat protease activity as multi-valued (i.e., signal is between 0 and 1) by controlling the degree to which a pool of enzymes is shared between two target substrates. We perform operations on these analog values by manipulating substrate concentrations and combine these operations to solve the mathematical problem Learning Parity with Noise (LPN). These results show that protease activity can be used to process biological information by binary Boolean logic, or as multi-valued analog signals under conditions where substrate resources are shared.

Deconvolving multiplexed protease signatures with substrate reduction and activity clustering.

Proteases are multifunctional, promiscuous enzymes that degrade proteins as well as peptides and drive important processes in health and disease. Current technology has enabled the construction of libraries of peptide substrates that detect protease activity, which provides valuable biological information. An ideal library would be orthogonal, such that each protease only hydrolyzes one unique substrate, however this is impractical due to off-target promiscuity (i.e., one protease targets multiple different substrates). Therefore, when a library of probes is exposed to a cocktail of proteases, each protease activates multiple probes, producing a convoluted signature. Computational methods for parsing these signatures to estimate individual protease activities primarily use an extensive collection of all possible protease-substrate combinations, which require impractical amounts of training data when expanding to search for more candidate substrates. Here we provide a computational method for estimating protease activities efficiently by reducing the number of substrates and clustering proteases with similar cleavage activities into families. We envision that this method will be used to extract meaningful diagnostic information from biological samples.

Non-invasive early detection of acute transplant rejection via nanosensors of granzyme B activity.

The early detection of the onset of transplant rejection is critical for the long-term survival of patients. The diagnostic gold standard for detecting transplant rejection involves a core biopsy, which is invasive, has limited predictive power and carries a morbidity risk. Here, we show that nanoparticles conjugated with a peptide substrate specific for the serine protease granzyme B, which is produced by recipient T cells during the onset of acute cellular rejection, can serve as a non-invasive biomarker of early rejection. When administered systemically in mouse models of skin graft rejection, these nanosensors preferentially accumulate in allograft tissue, where they are cleaved by granzyme B, releasing a fluorescent reporter that filters into the recipient's urine. Urinalysis then discriminates the onset of rejection with high sensitivity and specificity before features of rejection are apparent in grafted tissues. Moreover, in mice treated with subtherapeutic levels of immunosuppressive drugs, the reporter signals in urine can be detected before graft failure. This method may enable routine monitoring of allograft status without the need for biopsies.

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics.

Proteases are multi-functional enzymes that specialize in the hydrolysis of peptide-bonds and control broad biological processes including homeostasis and allostasis. Moreover, dysregulated protease activity drives pathogenesis and is a functional biomarker of diseases such as cancer; therefore, the ability to detect protease activity in vivo may provide clinically relevant information for biomedical diagnostics. The goal of this protocol is to create nanosensors that probe for protease activity in vivo by producing a quantifiable signal in urine. These protease nanosensors consist of two components: a nanoparticle and substrate. The nanoparticle functions to increase circulation half-life and substrate delivery to target disease sites. The substrate is a short peptide sequence (6-8 AA), which is designed to be specific to a target protease or group of proteases. The substrate is conjugated to the surface of the nanoparticle and is terminated by a reporter, such as a fluorescent marker, for detection. As dysregulated proteases cleave the peptide substrate, the reporter is filtered into urine for quantification as a biomarker of protease activity. Herein we describe construction of a nanosensor for matrix metalloproteinase 9 (MMP9), which is associated with tumor progression and metastasis, for detection of colorectal cancer in a mouse model.

Aqueous Zinc Compounds as Residual Antimicrobial Agents for Textiles.

Textiles, especially those worn by patients and medical professionals, serve as vectors for proliferating pathogens. Upstream manufacturing techniques and end-user practices, such as transition-metal embedment in textile fibers or alcohol-based disinfectants, can mitigate pathogen growth, but both techniques have their shortcomings. Fiber embedment requires complete replacement of all fabrics in a facility, and the effects of embedded nanoparticles on human health remain unknown. Alcohol-based, end-user disinfectants are short-lived because they quickly volatilize. In this work, common zinc salts are explored as an end-user residual antimicrobial agent. Zinc salts show cost-effective and long-lasting antimicrobial efficacy when solution-deposited on common textiles, such as nylon, polyester, and cotton. Unlike common alcohol-based disinfectants, these zinc salt-treated textiles mitigate microbial growth for more than 30 days and withstand commercial drying. Polyester fabrics treated with ZnO and ZnCl2 were further explored because of their commercial ubiquity and likelihood for rapid commercialization. ZnCl2-treated textiles were found to retain their antimicrobial coating through abrasive testing, whereas ZnO-treated textiles did not. Scanning electron microscopy, Fourier transform infrared spectroscopy, and differential scanning calorimetry analyses suggest that ZnCl2 likely hydrolyzes and reacts with portions of the polyester fiber, chemically attaching to the fiber, whereas colloidal ZnO simply sediments and binds with weaker physical interactions.

Fc microparticles can modulate the physical extent and magnitude of complement activity.

The complement system is an integral component of the humoral immune system, and describes a cascade of interacting proteins responsible for the opsonization and lysis of foreign pathogens, in addition to the recruitment of immune cells. However, complement activation is also implicated in the progression and complication of immune dysfunctions such as sepsis. Microparticle (MP) biomaterials capable of tuning the local magnitude of serum complement activation could improve complement-mediated cytotoxicity to serum-resistant bacteria or calm an overactive immune response during sepsis. We demonstrate that model Fc-functionalized microparticles can be designed to either enhance or diminish the local cytotoxic effect of complement activation in human serum. The particles were formed with either the antibody Fc domains oriented outward from the particle surface or randomly adsorbed in a non-oriented fashion. In the oriented Fc form, complement products were directly sequestered to the particle surface, including C5a, a potent anaphylatoxin that, when elevated, is associated with poor sepsis prognosis. The oriented particle also lowered the cytotoxicity of serum and thus decreased the antibiotic effect when compared to serum alone. Conversely, the non-oriented microparticles were found to sequester similar levels of C5a, but much lower levels of iC3b and TCC on the microparticle surface, thereby increasing the amount of the soluble terminal complement complex. In addition, the non-oriented microparticles extend the distance over which TCC forms and enhance serum cytotoxicity to bacteria. Together, these two types of complement-modulating particles provide the first biomaterial that can functionally modify the range of complement activation at sites distant from the particle surface. Thus, biomaterials that exploit Fc presentation provide new possibilities to functionally modulate complement activation to achieve a desired clinical result.