Biomolecular condensates composed of proteins and RNA are one approach by which cells regulate post-transcriptional gene expression. Their formation typically involves the phase separation of intrinsically disordered proteins with a target mRNA, sequestering the mRNA into a liquid condensate. This sequestration regulates gene expression by modulating translation or facilitating RNA processing. Here we engineer synthetic condensates using a fusion of an RNA-binding protein, the human Pumilio2 homology domain (Pum2), and a synthetic intrinsically disordered protein, an elastin-like polypeptide (ELP), that can bind and sequester a target mRNA transcript. In protocells, sequestration of a target mRNA largely limits its translation. Conversely, in Escherichia coli, sequestration of the same target mRNA increases its translation. We characterize the Pum2-ELP condensate system using microscopy, biophysical and biochemical assays, and RNA sequencing. This approach enables the modulation of cell function via the formation of synthetic biomolecular condensates that regulate the expression of a target protein.

The formation of biomolecular condensates mediated by a coupling of associative and segregative phase transitions plays a critical role in controlling diverse cellular functions in nature. This has inspired the use of phase transitions to design synthetic systems. While design rules of phase transitions have been established for many synthetic intrinsically disordered proteins, most efforts have focused on investigating their phase behaviors in a test tube. Here, we present a rational engineering approach to program the formation and physical properties of synthetic condensates to achieve intended cellular functions. We demonstrate this approach through targeted plasmid sequestration and transcription regulation in bacteria and modulation of a protein circuit in mammalian cells. Our approach lays the foundation for engineering designer condensates for synthetic biology applications.

Elon University has committed to composting food waste, however currently this process requires biweekly visits from CompostNow: a company that takes compost from the community and turns it into fertilizer. Reducing the weight of the waste will result in fewer trips and therefore a reduction in carbon emissions for the composting process; according to U.N. Food and Agriculture Organization, every pound of food thrown away equates to 3.8 pounds of greenhouse gas emissions. The current process being used by Elon University requires compost to be stored in individual cans which hold 200 pounds each. Our analysis of the current composting process reveals potential for savings in cost and emissions. Since compost is comprised of 40-60% water, due to its high-water content, liquid removal from the compost would result in a more efficient process. Therefore, our team proposes a way to reduce the amount of water in compost which will in turn result in a reduction of costs for Elon University. Through observational visits, we established a process flow model of Lakeside dining hall. This included both pre-consumer and post-consumer waste. This model was used to identify potential sizes and locations for compaction as well as to specify our user needs. We additionally performed research into state-of-the-art compaction units as well as user preferences. Regulations and standards for liquid leachate disposal were also reviewed for potential effects on operations. Process flow information was used to specify and design a solution to reduce the weight of compostable food waste. We anticipate that the successful incorporation of this compaction technology into Elon University dining operations will result in decreased cost and carbon emissions associated with composting.

Sustainability and environmental ethics are major focuses of future developments in many fields of infrastructure and industry. One of these fields is the industry of garment dyeing. With only a handful of garment dyeing facilities in the country, TS Designs, located in Burlington, North Carolina, has developed a niche clientele and craft of the use of natural materials for use in commercial dyeing. Organic materials have been used in textile dyeing since the very beginning of documented history, but limited research has been done in the translation of these practices to industrial contexts. Natural dye can be derived from organic waste products and is a great way to incorporate eco-friendly methods in the industrial production of clothing. Unfortunately, due to the dyes being made from organic materials, the resulting color of the product may change over time as the material degrades, which is not preferable for the sale of a consistent product. It is important to extract dye from materials as they are available before they degrade in order to reduce waste. The goal of our research is to be able to test the dye stability of organic materials and determine proper practices for preserving each dye extract.

The demonstration of complete protection of macaques in a repeated low dose virus challenge by a tenofovir disoproxil fumarate (TDF) intravaginal ring (IVR) and the success of the dapivirine IVR in clinical trials highlighted the potential of IVRs as pre- exposure prophylaxis against HIV. Efficacy of TDF ring was not investigated in sexually active women. Our understanding of the mechanisms of protection is limited. To address this knowledge gap, we performed simultaneous pharmacokinetic and pharmacodynamic analysis of a TDF-IVR at the site of SIV challenge in pigtail macaques at the anatomical and cellular level. Specifically, we challenged TDF-IVR administered pigtail macaques with a single high dose of a non-replicative SIV-based vector containing a dual reporter system that helped us to identify the earliest targets of SIV infection within the mucosa. Two and three days after challenge, the macaques were euthanized and tenofovir (TFV) concentrations were measured in the female reproductive tract (FRT) by HPLC-MS/MS to correlate drug concentrations and SIV-vector transduction efficiency. TFV formed a gradient through the mucosal tissue, with the highest concentrations near the ring, in the upper vagina and endocervix. Despite this, several transduction events were identified with the most common sites being in the ovaries. Moreover, proviral DNA was detected in the cervix and vagina. Thus, our studies demonstrate an uneven distribution of TFV in the FRT of macaques after release from a TDF-IVR that leads to incomplete FRT protection from high viral dose challenge.

ABSTRACTWe describe the in vitro and in vivo evaluation of a subcutaneous reservoir implant delivering tenofovir alafenamide hemifumarate (TAF) for the prevention of HIV infection. These long-acting reservoir implants were able to deliver antiretroviral drug for over 90 days in vitro and in vivo. We evaluated the implants for implantation site histopathology and pharmacokinetics in plasma and tissues for up to 12 weeks in New Zealand White rabbits and rhesus macaque models. A dose-ranging study in rabbits demonstrated dose-dependent pharmacokinetics and local inflammation up to severe necrosis around the active implants. The matched placebos showed normal wound healing and fibrous tissue encapsulation of the implant. We designed a second implant with a lower release rate and flux of TAF and achieved a median cellular level of tenofovir diphosphate of 42 fmol per 106 rhesus macaque peripheral blood mononuclear cells at a dose of 10 µg/kg/day. This dose and flux of TAF also resulted in adverse local inflammation and necrosis near the implant in rhesus macaques. Inflammation in the primates was markedly lower in the placebo group than the active implant. The histological inflammatory response to the TAF implant at 4 and 12 weeks in primates was graded as a severe reaction. Thus, while we were able to achieve sustained target dose we observed unacceptable inflammatory response locally at the implant tissue interface.

Purpose Sexual transmission of HIV has been clinically proven to be preventable with a once-daily oral tablet; however, missed doses dramatically increase the risk of HIV infection. Long-acting subcutaneous implants do not allow the user to miss a dose. A desirable long-acting drug-eluting implant can deliver a constant amount of drug, adjust the delivered dose, and be readily manufactured. We present a long-acting, subcutaneous implant design composed of tenofovir alafenamide hemifumarate (TAF) pellets loaded in a sealed polyether urethane tube for the prevention of HIV transmission. Methods Implants were prepared with pressed drug pellets and extruded polyurethane tubing. In vitro release rate of implants using different pellet formulations, rate-controlling membranes, and geometries were measured. Results Tenofovir alafenamide release appeared to be governed by a pseudo-steady state and followed a mass transport model of release from a cylindrical drug reservoir. Implant seal integrity was tested and confirmed using mechanical testing. The inclusion of sodium chloride in the pellet increased the release rate and reduced initial lag. The release was sustained for 100 days. Conclusions The release rate of tenofovir alafenamide mechanistically varied with geometry and rate controlling membrane composition. The polyether urethane implant presented herein is modular and tunable to adjust the release rate and duration of the TAF release.

Long-acting antiretroviral implants could help protect high-risk individuals from HIV infection. We describe the design and testing of a long-acting reservoir subcutaneous implant capable of releasing cabotegravir for several months. We compressed cabotegravir and excipients into cylindrical pellets and heat-sealed them in tubing composed of hydrophilic poly(ether-urethane) -. The implants have a 47 mm lumen length, 3.6 mm outer diameter, and 200 μm wall thickness. Four cabotegravir pellets were sealed in the membrane, with a total drug loading of 274 ± 3 mg. In vivo, the implants released 348 ± 107 μg/day (median value per implant, N = 41) of cabotegravir in rhesus macaques. Five implants generated an average cabotegravir plasma concentration of 373 ng/ml in rhesus macaques. The non-human primates tolerated the implant without gross pathology or microscopic signs of histopathology compared to placebo implants. Cabotegravir plasma levels in macaques dropped below detectable levels within two weeks after the removal of the implants.

Design of intravaginal rings (IVRs) for delivery of antiretrovirals is often guided by in vitro release under sink conditions, based on the assumption that in vivo release will follow a similar release profile.

BACKGROUND:Reported vaginal and seminal fluid simulants have complex compositions with multiple preparatory steps that contribute to physical instability. We report the design and characterization of stable and simplified buffers that mimic the salient physical/chemical properties of the physiological fluids.STUDY DESIGN/METHODS:Human cervicovaginal and seminal fluid samples were collected and buffering capacity was determined. The major buffering species were identified from published compositions of reproductive tract fluids. These values were used to compute the composition of vaginal and seminal fluid simulants. Ionic strength, buffering capacities, pH and osmolalities were then calculated or experimentally determined. Finally, cytotoxicity was evaluated in HEC-1-A cells and 3D reconstructed EpiVaginal™ tissue (VEC-100-FT) using naïve cells/tissue and nonoxynol-9 as controls.RESULTS:The use of calculated amounts of conjugate acid and base for buffer development resulted in compositions that did not require endpoint pH adjustment and could be formulated as stable 10× concentrates. Furthermore, due to the absence of complex divalent salts, all our proposed simulants were stable at 4 °C for 1 month whereas precipitation and pH and osmolality changes were noted in reported buffers. Experimental determination of buffering capacities yielded similar values for undiluted cervicovaginal fluid (β4.2-5.2=35.6 ± 12.3 mM, N=7) and human seminal fluid (β7-6=37.5 ± 5 mM, N=3). All neat simulants showed significant cytotoxicity in HEC-1-A cells but were well tolerated by organotypic vaginal tissue.CONCLUSIONS:We report revised and improved compositions of buffers mimicking salient properties of vaginal and seminal fluid necessary for in vitro product evaluation.IMPLICATIONS:To support research in reproductive health and in particular drug delivery, we have designed and characterized stable new media to mimic these important fluids that can be used in a variety of in vitro studies.

ABSTRACT Intravaginal rings releasing tenofovir (TFV) or its prodrug, tenofovir disoproxil fumarate (TDF), are being evaluated for HIV and herpes simplex virus (HSV) prevention. The current studies were designed to determine the mechanisms of drug accumulation in human vaginal and immune cells. The exposure of vaginal epithelial or T cells to equimolar concentrations of radiolabeled TDF resulted in over 10-fold higher intracellular drug levels than exposure to TFV. Permeability studies demonstrated that TDF, but not TFV, entered cells by passive diffusion. TDF uptake was energy independent but its accumulation followed nonlinear kinetics, and excess unlabeled TDF inhibited radiolabeled TDF uptake in competition studies. The carboxylesterase inhibitor bis-nitrophenyl phosphate reduced TDF uptake, suggesting saturability of intracellular carboxylesterases. In contrast, although TFV uptake was energy dependent, no competition between unlabeled and radiolabeled TFV was observed, and the previously identified transporters, organic anion transporters (OATs) 1 and 3, were not expressed in human vaginal or T cells. The intracellular accumulation of TFV was reduced by the addition of endocytosis inhibitors, and this resulted in the loss of TFV antiviral activity. Kinetics of drug transport and metabolism were monitored by quantifying the parent drugs and their metabolites by high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). Results were consistent with the identified mechanisms of transport, and the exposure of vaginal epithelial cells to equimolar concentrations of TDF compared to TFV resulted in ∼40-fold higher levels of the active metabolite, tenofovir diphosphate. Together, these findings indicate that substantially lower concentrations of TDF than TFV are needed to protect cells from HIV and HSV-2.

Controlled enzyme dehydration using a new processing technique of Microglassification™ has been investigated. Aqueous solution microdroplets of lysozyme, α-chymotrypsin, catalase, and horseradish peroxidase were dehydrated in n-pentanol, n-octanol, n-decanol, triacetin, or butyl lactate, and changes in their structure and function were analyzed upon rehydration. Water solubility and microdroplet dissolution rate in each solvent decreased in the order: butyl lactate > n-pentanol > triacetin > n-octanol > n-decanol. Enzymes Microglassified™ in n-pentanol retained higher activity (93%-98%) than n-octanol (78%-85%) or n-decanol (75%-89%), whereas those Microglassified™ in triacetin (36%-75%) and butyl lactate (48%-79%) retained markedly lower activity. FTIR spectroscopy analyses showed α-helix to β-sheet transformation for all enzymes upon Microglassification™, reflecting a loss of bound water in the dried state; however, the enzymes reverted to native-like conformation upon rehydration. Accelerated stressed-storage tests using Microglassified™ lysozyme showed a significant (p < 0.01) decrease in enzymatic activity from 46,560 ± 2736 to 31,060 ± 4327 units/mg after 3 months of incubation; however, it was comparable to the activity of the lyophilized formulation throughout the test period. These results establish Microglassification™ as a viable technique for enzyme preservation without affecting its structure or function.

The Epstein-Plesset equation has recently been shown to predict accurately the dissolution of a pure liquid microdroplet into a second immiscible solvent, such as oil into water. Here, we present a series of new experiments and a modification to this equation to model the dissolution of a two-component oil-mixture microdroplet into a second immiscible solvent in which the two materials of the droplet have different solubilities. The model is based on a reduced surface area approximation and the assumption of ideal homogeneous mixing [mass flux d(m(i))/dt = A(frac(i))D(i)(c(i) - c(s)){(1/R) + (1/(πD(i)t)(1/2)}] where A(frac(i)) is the area fraction of component i, c(i) and c(s) are the initial and saturation concentrations of the droplet material in the surrounding medium, R is the radius of the droplet, t is time, and D(i) is the coefficient of diffusion of component i in the surrounding medium. This new model has been tested by the use of a two-chamber micropipet-based method, which measured the dissolution of single individual microdroplets of mutually miscible liquid mixtures (ethyl acetate/butyl acetate and butyl acetate/amyl acetate) in water. We additionally measured the diffusion coefficient of the pure materials-ethyl acetate, butyl acetate, and amyl acetate-in water at 22 °C. Diffusion coefficients for the pure acetates in water were 8.65 × 10(-6), 7.61 × 10(-6), and 9.14 × 10(-6) cm(2)/s, respectively. This model accurately predicts the dissolution of microdroplets for the ethyl acetate/butyl acetate and butyl acetate/amyl acetate systems given the solubility and diffusion coefficients of each of the individual components in water as well as the initial droplet radius. The average mean squared error was 8.96%. The dissolution of a spherical ideally mixed multicomponent droplet closely follows the modified Epstein-Plesset model presented here.