Intracellular islatravir pharmacology differs between species in an in vitro model: implications for preclinical study design.

BACKGROUND: Islatravir (4'-ethynyl-2-fluoro-2'-deoxyadenosine; EFdA) is a first-in-class nucleoside reverse transcriptase translocation inhibitor (NRTTI) being investigated for HIV treatment and prevention. EFdA is intracellularly phosphorylated to EFdA-triphosphate (EFdA-tp), a competitive substrate of deoxyadenosine-triphosphate (dATP). Thus, translating safety and efficacy findings from preclinical studies relies on the assumption that EFdA's intracellular pharmacology can be extrapolated across species. OBJECTIVES: We investigated how EFdA is phosphorylated across animal species commonly used for preclinical models in drug development to identify those that most closely matched humans. METHODS: PBMCs were isolated from whole blood of six species (human, rhesus macaque non-human primate (rmNHP), rat, minipig, dog, and rabbit) using Ficoll separation and counted on a haemocytometer by Trypan blue staining. One million live cells were cultured in media supplemented with 10 U/mL human IL-2, 10% FBS and 1% antibiotics and treated with 0, 17, 170, and 1700 nM EFdA (n = 3 replicates per concentration). After 24 h, representative cell counts were derived from untreated control wells (as above), cells were washed in PBS, and lysed with 70:30 methanol:water. EFdA-tp and dATP concentrations were quantified by HPLC-MS/MS and normalized to the representative live cell counts for each species. RESULTS: When compared to human values, EFdA-tp concentrations for each EFdA treatment concentration were lower in all species (rmNHP 1.5-2.1-fold, rat 4.5-15-fold, minipig 37-71-fold, dog and rabbit >100-fold). Additionally, rmNHP and dog PBMCs exhibited significantly higher (7-10-fold; P < 0.001) dATP when compared with human PBMCs. CONCLUSIONS: Given intracellular pharmacology differences, these preclinical models may be a conservative estimate of EFdA's intracellular pharmacokinetics and efficacy in humans.

Scalable purification of recombinant structural proteins, hagfish intermediate filament α and É£, from inclusion bodies for fiber formation.

The purpose of this study was to determine a method to purify recombinant hagfish intermediate filament proteins, alpha and gamma, in a scalable manner. The study succeeded by having an increase in protein recovery of up to 35% when comparing centrifuge purification and the developed tangential flow purification. The proteins were approximately the same purity of 70% pure but further purification increased the purity of the proteins by 16%, based on ImageJ analysis. The developed tangential flow filtration purification and final purification methods could be easily scaled up to meet industry scale purification needs. The scaled-up processes described in this study did not interfere with fiber production or formation, indicating the methods can produce usable proteins for material development.

Large scale production of synthetic spider silk proteins in Escherichia coli.

Spider silk, which has remarkable mechanical properties, is a natural protein fiber produced by spiders. Spiders cannot be farmed because of their cannibalistic and territorial nature. Hence, large amounts of spider silk cannot be produced from spiders. Genetic engineering is an alternative approach to produce large quantities of spider silk. Our group has produced synthetic spider silk proteins in E. coli to study structure/function and to produce biomaterials comparable to the silks produced by orb-weaving spiders. Here we give a detailed description of our cloning, expression, and purification methods of synthetic spider silk proteins ranging from ~30 to ~200 kDa. We have cloned the relevant genes of the spider Nephila clavipes and introduced them into bacteria to produce synthetic spider silk proteins using small and large-scale bioreactors. We have optimized the fermentation process, and we have developed protein purification methods as well. The purified proteins are spun into fibers and are used to make alternative materials like films and adhesives with various possible commercial applications.

Functional Leadership in Interteam Contexts: Understanding 'What' in the Context of Why? Where? When? and Who?

Research on team leadership has primarily focused on leadership processes targeted within teams, in support of team objectives. Yet, teams are open systems that interact with other teams to achieve proximal as well as distal goals. This review clarifies that defining 'what' constitutes functionally effective leadership in interteam contexts requires greater precision with regard to where (within teams, across teams) and why (team goals, system goals) leadership processes are enacted, as well as greater consideration of when and among whom leadership processes arise. We begin by synthesizing findings from empirical studies published over the past 30 years that shed light on questions of what, where, why, when, and who related to interteam leadership and end by providing three overarching recommendations for how research should proceed in order to provide a more comprehensive picture of leadership in interteam contexts.

CRISPR/Cas9 Initiated Transgenic Silkworms as a Natural Spinner of Spider Silk.

Using transgenic silkworms with their natural spinning apparatus has proven to be a promising way to spin spider silk-like fibers. The challenges are incorporating native-size spider silk proteins and achieving an inheritable transgenic silkworm strain. In this study, a CRISPR/Cas9 initiated fixed-point strategy was used to successfully incorporate spider silk protein genes into the Bombyx mori genome. Native-size spider silk genes (up to 10 kb) were inserted into an intron of the fibroin heavy or light chain (FibH or FibL) ensuring that any sequence changes induced by the CRISPR/Cas9 would not impact protein production. The resulting fibers are as strong as native spider silks (1.2 GPa tensile strength). The transgenic silkworms have been tracked for several generations with normal inheritance of the transgenes. This strategy demonstrates the feasibility of using silkworms as a natural spider silk spinner for industrial production of high-performance fibers.

PDGFB-based stem cell gene therapy increases bone strength in the mouse.

Substantial advances have been made in the past two decades in the management of osteoporosis. However, none of the current medications can eliminate the risk of fracture and rejuvenate the skeleton. To this end, we recently reported that transplantation of hematopoietic stem/progenitor cells (HSCs) or Sca1(+) cells engineered to overexpress FGF2 results in a significant increase in lamellar bone matrix formation at the endosteum; but this increase was attended by the development of secondary hyperparathyroidism and severe osteomalacia. Here we switch the therapeutic gene to PDGFB, another potent mitogen for mesenchymal stem cells (MSCs) but potentially safer than FGF2. We found that modest overexpression of PDGFB using a relatively weak phosphoglycerate kinase (PGK) promoter completely avoided osteomalacia and secondary hyperparathyroidism, and simultaneously increased trabecular bone formation and trabecular connectivity, and decreased cortical porosity. These effects led to a 45% increase in the bone strength. Transplantation of PGK-PDGFB-transduced Sca1(+) cells increased MSC proliferation, raising the possibility that PDGF-BB enhances expansion of MSC in the vicinity of the hematopoietic niche where the osteogenic milieu propels the differentiation of MSCs toward an osteogenic destination. Our therapy should have potential clinical applications for patients undergoing HSC transplantation, who are at high risk for osteoporosis and bone fractures after total body irradiation preconditioning. It could eventually have wider application once the therapy can be applied without the preconditioning.

Sticky Situation: An Investigation of Robust Aqueous-Based Recombinant Spider Silk Protein Coatings and Adhesives.

The mechanical properties and biocompatibility of spider silks have made them one of the most sought after and studied natural biomaterials. A biomimetic process has been developed that uses water to solvate purified recombinant spider silk proteins (rSSps) prior to material formation. The absence of harsh organic solvents increases cost effectiveness, safety, and decreases the environmental impact of these materials. This development allows for the investigation of aqueous-based rSSps as coatings and adhesives and their potential applications. In these studies it was determined that fiber-based rSSps in nonfiber formations have the capability to coat and adhere numerous substrates, whether rough, smooth, hydrophobic, or hydrophilic. Further, these materials can be functionalized for a variety of processes. Drug-eluting coatings have been made with the capacity to release a variety of compounds in addition to their inherent ability to prevent blood clotting and biofouling. Additionally, spider silk protein adhesives are strong enough to outperform some conventional glues and still display favorable tissue implantation properties. The physical properties, corresponding capabilities, and potential applications of these nonfibrous materials were characterized in this study. Mechanical properties, ease of manufacturing, biodegradability, biocompatibility, and functionality are the hallmarks of these revolutionary spider silk protein materials.

Gr1+ cells suppress T-dependent antibody responses in (NZB x NZW)F1 male mice through inhibition of T follicular helper cells and germinal center formation.

Systemic lupus erythematosus is an autoimmune disease characterized by elevated production of autoreactive Abs. The disease has a much higher prevalence in women than in men. Although testosterone has been shown to be protective in the disease, and estrogens exacerbating, the discrepancy in prevalence between men and women is still not well understood and the mechanism behind it is unknown. We have recently described that male (New Zealand black [NZB] × New Zealand white [NZW])F1 mice have higher levels of Gr1(+)CD11b(+) cells, and that these cells suppress autoantibody production in vivo. In this article, we extend our findings to show that similarly to humans, female lupus-prone (NZB × NZW)F1 mice also respond with stronger Ab responses to thymus-dependent Ag immunization than male littermates. Furthermore, the presence or absence of Gr1-expressing cells not only control Ag-specific Ab responses in male, but not female, (NZB × NZW)F1 mice, but also significantly alter the activation and differentiation of CD4(+) T cells in vitro and in vivo. In particular, we found that Gr1(+) cells from male (NZB × NZW)F1 mice suppress the differentiation and effector function of CXCR5(+)PD-1(+) T follicular helper cells, thereby controlling germinal center formation and plasma cell differentiation. This new finding strongly supports efforts to develop new drugs that target myeloid cell subsets in a number of T and B cell-mediated diseases with a female predominance.

Successful hemostasis and reversal of highly elevated PT/INR after dabigatran etexilate use in a patient with acute kidney injury.

Dabigatran etexilate is a novel oral anticoagulant indicated for anticoagulation in the management of atrial fibrillation and venous thromboembolism. Before its approval by the US Food and Drug Administration, warfarin, a vitamin K antagonist, was one of few oral anticoagulant options. The burden of therapeutic drug monitoring, dietary restrictions, and various drug interactions associated with warfarin have countered its extensive history of efficacy. Although dabigatran etexilate may alleviate some concerns encountered with warfarin therapy, there remains a paucity of evidence surrounding emergent reversal strategies in severe hemorrhage. We report here a 71-year-old man who presented to the emergency department with gastrointestinal hemorrhage precipitated by acute kidney injury while on dabigatran etexilate, with laboratory derangements highly uncharacteristic of dabigatran therapy (international normalized ratio, > 10, and activated partial thromboplastin time, 93 seconds). After admission to the intensive care unit and 7 U of fresh frozen plasma, the patient remained hemodynamically unstable due to blood loss. Other observations were made that are poorly characterized in medical literature related to dabigatran: refractory hemorrhagic shock after 7 U of fresh frozen plasma, rapid correction of coagulation parameters (international normalized ratio, 1.7, and activated partial thromboplastin time, 44 seconds) achieved 4 hours after 26 U/kg of 4-factor prothrombin complex concentrate (Kcentra; CSL Behring, King of Prussia, PA), and with subsequent achievement of hemostasis. The patient was discharged to home 7 days later without sequelae.

Lack of Association Between Dexamethasone and Long-Term Survival After Non-Small Cell Lung Cancer Surgery.

OBJECTIVE: To evaluate the association between the use of intraoperative dexamethasone with an increase in recurrence-free survival (RFS) and overall survival (OS) after non-small cell lung cancer (NSCLC) surgery. DESIGN: This was a propensity score-matched (PSM) retrospective study. SETTING: Single academic center. PARTICIPANTS: The study comprised patients with stage I through IIIa NSCLC. Patients were excluded if they were younger than 18 years, had missing data, and died within 30 days after surgery. MEASUREMENTS AND MAIN RESULTS: Primary outcomes of the study were RFS and OS. The data were PSM. RFS and OS were evaluated using univariate and multivariate Cox proportional hazards models after PSM to assess the association between intraoperative dexamethasone use and the primary outcomes. A p value of<0.05 was considered statistically significant. After PSM, 436 patients were included in each treatment group. Adjusting for significant covariates, the multivariate analysis demonstrated no association between the use of dexamethasone and RFS (hazard ratio [95% confidence interval]: 0.98 [0.78-1.24]; p = 0.915). The multivariate analysis also demonstrated no association between the administration of dexamethasone and OS (hazard ratio [95% confidence interval]: 1.08 [0.81-1.44]; p = 0.58). CONCLUSIONS: This study demonstrated that intraoperative dexamethasone administration to NSCLC patients was not associated with a significant impact on RFS and OS. The results were similar to a previous study on ovarian cancer patients. A randomized controlled study should be conducted to confirm the results of this study.

Importance of Heat and Pressure for Solubilization of Recombinant Spider Silk Proteins in Aqueous Solution.

The production of recombinant spider silk proteins continues to be a key area of interest for a number of research groups. Several key obstacles exist in their production as well as in their formulation into useable products. The original reported method to solubilize recombinant spider silk proteins (rSSp) in an aqueous solution involved using microwaves to quickly generate heat and pressure inside of a sealed vial containing rSSp and water. Fibers produced from this system are remarkable in their mechanical ability and demonstrate the ability to be stretched and recover 100 times. The microwave method dissolves the rSSPs with dissolution time increasing with higher molecular weight constructs, increasing concentration of rSSPs, protein type, and salt concentration. It has proven successful in solvating a number of different rSSPs including native-like sequences (MaSp1, MaSp2, piriform, and aggregate) as well as chimeric sequences (FlAS) in varied concentrations that have been spun into fibers and formed into films, foams, sponges, gels, coatings, macro and micro spheres and adhesives. The system is effective but inherently unpredictable and difficult to control. Provided that the materials that can be generated from this method of dissolution are impressive, an alternative means of applying heat and pressure that is controllable and predictable has been developed. Results indicate that there are combinations of heat and pressure (135 °C and 140 psi) that result in maximal dissolution without degrading the recombinant MaSp2 protein tested, and that heat and pressure are the key elements to the method of dissolution.

Secondary Structure Adopted by the Gly-Gly-X Repetitive Regions of Dragline Spider Silk.

Solid-state NMR and molecular dynamics (MD) simulations are presented to help elucidate the molecular secondary structure of poly(Gly-Gly-X), which is one of the most common structural repetitive motifs found in orb-weaving dragline spider silk proteins. The combination of NMR and computational experiments provides insight into the molecular secondary structure of poly(Gly-Gly-X) segments and provides further support that these regions are disordered and primarily non-ß-sheet. Furthermore, the combination of NMR and MD simulations illustrate the possibility for several secondary structural elements in the poly(Gly-Gly-X) regions of dragline silks, including ß-turns, 310-helicies, and coil structures with a negligible population of α-helix observed.

More than just fibers: an aqueous method for the production of innovative recombinant spider silk protein materials.

Spider silk is a striking and robust natural material that has an unrivaled combination of strength and elasticity. There are two major problems in creating materials from recombinant spider silk proteins (rSSps): expressing sufficient quantities of the large, highly repetitive proteins and solvating the naturally self-assembling proteins once produced. To address the second problem, we have developed a method to rapidly dissolve rSSps in water in lieu of traditional organic solvents and accomplish nearly 100% solvation and recovery of the protein. Our method involves generating pressure and temperature in a sealed vial by using short, repetitive bursts from a conventional microwave. The method is scalable and has been successful with all rSSps used to date. From these easily generated aqueous solutions of rSSps, a wide variety of materials have been produced. Production of fibers, films, hydrogels, lyogels, sponges, and adhesives and studies of their mechanical and structural properties are reported. To our knowledge, ours is the only method that is cost-effective and scalable for mass production. This solvation method allows a choice of the physical form of product to take advantage of spider silks' mechanical properties without using costly and problematic organic solvents.

Mechanistic studies of protein arginine deiminase 2: evidence for a substrate-assisted mechanism.

Citrullination, which is catalyzed by protein arginine deiminases (PADs 1-4 and 6), is a post-translational modification (PTM) that effectively neutralizes the positive charge of a guanidinium group by its replacement with a neutral urea. Given the sequence similarity of PAD2 across mammalian species and the genomic organization of the PAD2 gene, PAD2 is predicted to be the ancestral homologue of the PADs. Although PAD2 has long been known to play a role in myelination, it has only recently been linked to other cellular processes, including gene transcription and macrophage extracellular trap formation. For example, PAD2 deiminates histone H3 at R26, and this PTM leads to the increased transcription of more than 200 genes under the control of the estrogen receptor. Given that our understanding of PAD2 biology remains incomplete, we initiated mechanistic studies on this enzyme to aid the development of PAD2-specific inhibitors. Herein, we report that the substrate specificity and calcium dependence of PAD2 are similar to those of PADs 1, 3, and 4. However, unlike those isozymes, PAD2 appears to use a substrate-assisted mechanism of catalysis in which the positively charged substrate guanidinium depresses the pKa of the nucleophilic cysteine. By contrast, PADs 1, 3, and 4 use a reverse-protonation mechanism. These mechanistic differences will aid the development of isozyme-specific inhibitors.

A disintegrin and metalloproteinase-12 (ADAM12): function, roles in disease progression, and clinical implications.

BACKGROUND: A disintegrin and metalloproteinase-12 (ADAM12) is a member of the greater ADAM family of enzymes: these are multifunctional, generally membrane-bound, zinc proteases for which there are forty genes known (21 of these appearing in humans). ADAM12 has been implicated in the pathogenesis of various cancers, liver fibrogenesis, hypertension, and asthma, and its elevation or decrease in human serum has been linked to these and other physiological/pathological conditions. SCOPE: In this review, we begin with a brief overview of the ADAM family of enzymes and protein structure. We then discuss the role of ADAM12 in the progression and/or diagnosis of various disease conditions, and we will conclude with an exploration of currently known natural and synthetic inhibitors. MAJOR CONCLUSION: ADAM12 has potential to emerge as a successful drug target, although targeting the metalloproteinase domain with any specificity will be difficult to achieve due to structural similarity between the members of the ADAM and MMP family of enzymes. Overall, more research is required to establish ADAM12 being as a highly desirable biomarker and drug target of different diseases, and their selective inhibitors as potential therapeutic agents. GENERAL SIGNIFICANCE: Given the appearance of elevated levels of ADAM12 in various diseases, particularly breast cancer, our understanding of this enzyme both as a biomarker and a potential drug target could help make significant inroads into both early diagnosis and treatment of disease.

Mechanical and physical properties of recombinant spider silk films using organic and aqueous solvents.

Spider silk has exceptional mechanical and biocompatibility properties. The goal of this study was optimization of the mechanical properties of synthetic spider silk thin films made from synthetic forms of MaSp1 and MaSp2, which compose the dragline silk of Nephila clavipes. We increased the mechanical stress of MaSp1 and 2 films solubilized in both HFIP and water by adding glutaraldehyde and then stretching them in an alcohol based stretch bath. This resulted in stresses as high as 206 MPa and elongations up to 35%, which is 4× higher than the as-poured controls. Films were analyzed using NMR, XRD, and Raman, which showed that the secondary structure after solubilization and film formation in as-poured films is mainly a helical conformation. After the post-pour stretch in a methanol/water bath, the MaSp proteins in both the HFIP and water-based films formed aligned ß-sheets similar to those in spider silk fibers.

Inferring Population HIV Viral Load From a Single HIV Clinic's Electronic Health Record: Simulation Study With a Real-World Example.

BACKGROUND: Population viral load (VL), the most comprehensive measure of the HIV transmission potential, cannot be directly measured due to lack of complete sampling of all people with HIV. OBJECTIVE: A given HIV clinic's electronic health record (EHR), a biased sample of this population, may be used to attempt to impute this measure. METHODS: We simulated a population of 10,000 individuals with VL calibrated to surveillance data with a geometric mean of 4449 copies/mL. We sampled 3 hypothetical EHRs from (A) the source population, (B) those diagnosed, and (C) those retained in care. Our analysis imputed population VL from each EHR using sampling weights followed by Bayesian adjustment. These methods were then tested using EHR data from an HIV clinic in Delaware. RESULTS: Following weighting, the estimates moved in the direction of the population value with correspondingly wider 95% intervals as follows: clinic A: 4364 (95% interval 1963-11,132) copies/mL; clinic B: 4420 (95% interval 1913-10,199) copies/mL; and clinic C: 242 (95% interval 113-563) copies/mL. Bayesian-adjusted weighting further improved the estimate. CONCLUSIONS: These findings suggest that methodological adjustments are ineffective for estimating population VL from a single clinic's EHR without the resource-intensive elucidation of an informative prior.

How microbial community composition regulates coral disease development.

Reef coral cover is in rapid decline worldwide, in part due to bleaching (expulsion of photosynthetic symbionts) and outbreaks of infectious disease. One important factor associated with bleaching and in disease transmission is a shift in the composition of the microbial community in the mucus layer surrounding the coral: the resident microbial community-which is critical to the healthy functioning of the coral holobiont-is replaced by pathogenic microbes, often species of Vibrio. In this paper we develop computational models for microbial community dynamics in the mucus layer in order to understand how the surface microbial community responds to changes in environmental conditions, and under what circumstances it becomes vulnerable to overgrowth by pathogens. Some of our model's assumptions and parameter values are based on Vibrio spp. as a model system for other established and emerging coral pathogens. We find that the pattern of interactions in the surface microbial community facilitates the existence of alternate stable states, one dominated by antibiotic-producing beneficial microbes and the other pathogen-dominated. A shift to pathogen dominance under transient stressful conditions, such as a brief warming spell, may persist long after environmental conditions have returned to normal. This prediction is consistent with experimental findings that antibiotic properties of Acropora palmata mucus did not return to normal long after temperatures had fallen. Long-term loss of antibiotic activity eliminates a critical component in coral defense against disease, giving pathogens an extended opportunity to infect and spread within the host, elevating the risk of coral bleaching, disease, and mortality.

Nephila clavipes Flagelliform silk-like GGX motifs contribute to extensibility and spacer motifs contribute to strength in synthetic spider silk fibers.

Flagelliform spider silk is the most extensible silk fiber produced by orb weaver spiders, though not as strong as the dragline silk of the spider. The motifs found in the core of the Nephila clavipes flagelliform Flag protein are GGX, spacer, and GPGGX. Flag does not contain the polyalanine motif known to provide the strength of dragline silk. To investigate the source of flagelliform fiber strength, four recombinant proteins were produced containing variations of the three core motifs of the Nephila clavipes flagelliform Flag protein that produces this type of fiber. The as-spun fibers were processed in 80% aqueous isopropanol using a standardized process for all four fiber types, which produced improved mechanical properties. Mechanical testing of the recombinant proteins determined that the GGX motif contributes extensibility and the spacer motif contributes strength to the recombinant fibers. Recombinant protein fibers containing the spacer motif were stronger than the proteins constructed without the spacer that contained only the GGX motif or the combination of the GGX and GPGGX motifs. The mechanical and structural X-ray diffraction analysis of the recombinant fibers provide data that suggests a functional role of the spacer motif that produces tensile strength, though the spacer motif is not clearly defined structurally. These results indicate that the spacer is likely a primary contributor of strength, with the GGX motif supplying mobility to the protein network of native N. clavipes flagelliform silk fibers.