Stretching and Self-Myofascial Release in Helicopter Aircrew to Reduce Neck and Back Pain (Phase 1).

INTRODUCTION: This prospective intervention study was designed to determine the efficacy of a standardized Preflight/Postflight Stretches (PPS) protocol to reduce subjective neck and back pain scores in helicopter aircrew. Aircrew transient back and neck pain is well documented, and there is currently no standardized preflight and postflight stretching protocol for Naval Aviation. METHODS: Subjects were recruited from two carrier air wing MH-60R squadrons at Naval Air Station Jacksonville. These carrier air wing squadrons were selected to control for size (number of aircrew), age, and operational tempo (number of flight hours). Subjects consisted of both pilots and enlisted aircrew. One squadron was designated as the control group, although the second squadron served as the intervention group. Subjects from both groups filled out the questionnaire. Only the intervention group completed the PPS protocol immediately after completing the questionnaire and before departing the squadron spaces for the aircraft outside. Upon landing, the aircrew completed a postflight debrief. Only the intervention group completed the PPS protocol after debrief. Both the intervention and control groups once again completed the questionnaire. Questionnaires were matched by using a generated anonymous subject ID. The amounts of change and pain levels were then compared using the Mann-Whitney test and the Fisher's exact test, respectively. RESULTS: The Kolmogorov-Smirnov test found the data to be nonparametric. The preflight and postflight overall (P ≤ .001), cervical (P ≤ .001), thoracic (P = .006), and lumbar (P = .004) differences between the control and intervention groups were found to be statistically significant when using the Mann-Whitney test. Preflight and postflight pain differences in the sacral region and "other" section were not found to be statistically significant (sacral, P = .618; others, P = .182). When evaluating the worsening of the pain level, 50 (92%) of the control flights in which PPS was not performed reported worse pain, compared to 21 (61.8%) in the intervention group where PPS was performed. The Fisher's exact test found the association between performing PPS and the worsening in pain to be statistically significant (P = .001) in the overall, cervical, thoracic, and lumbar regions. Therefore, the hypothesis was accepted in regard to overall pain, as well as in the cervical, thoracic, and lumbar regions. CONCLUSION: Aircrew back and neck pain because of flying is well documented. However, there is no standardized stretching protocol for aircrew to perform immediately preflight or postflight in U.S. Naval Aviation. This study demonstrated that PPS, a simple 5- to 7-min stretching routine, gives aircrew structure and can reduce postflight cervical, thoracic, lumbar, and overall pain. This phase proved to be safe as no adverse events were reported. The prehabilitation aspect could reduce conventional medical intervention, costly pharmacological management of neck and back pain, and be applied to other aviation populations in military and civilian communities.

The effect of air purifiers and curtains on aerosol dispersion and removal in multi-patient hospital rooms.

Airborne transmission of disease is of concern in many indoor spaces. Here, aerosol dispersion and removal in an unoccupied 4-bed hospital room were characterized using a transient aerosol tracer experiment for 38 experiments covering 4 configurations of air purifiers and 3 configurations of curtains. NaCl particle (mass mean aerodynamic diameter ~3 µm) concentrations were measured around the room following an aerosol release. Particle transport across the room was 1.5-4 min which overlaps with the characteristic times for significant viral deactivation and gravitational settling of larger particles. Concentrations were close to spatially uniform except very near the source. Curtains resulted in a modest increase in delay and decay times, less so when combined with purifiers. The aerosol decay rate was in most cases higher than expected from the clean air delivery rate, but the reduction in steady-state concentrations resulting from air purifiers was less than suggested by the decay rates. Apparently, a substantial (and configuration-dependent) fraction of the aerosol is removed immediately, and this effect is not captured by the decay rate. Overall, the combination of curtains and purifiers is likely to reduce disease transmission in multi-patient hospital rooms.

Neuroendocrine, inflammatory, and extracellular vesicle responses during the Navy Special Warfare Screener Selection Course.

Military operational stress is known to increase adrenal hormones and inflammatory cytokines, while decreasing hormones associated with the anabolic milieu and neuroendocrine system. Less is known about the role of extracellular vesicles (EVs), a form of cell-to-cell communication, in military operational stress and their relationship to circulating hormones. The purpose of this study was to characterize the neuroendocrine, cytokine, and EV response to an intense. 24-h selection course known as the Naval Special Warfare (NSW) Screener and identify associations between EVs and cytokines. Blood samples were collected the morning of and following the NSW Screener in 29 men (18-26 yr). Samples were analyzed for concentrations of cortisol, insulin-like growth factor I (IGF-I), neuropeptide-Y (NPY), brain-derived neurotrophic factor (BDNF), α-klotho, tumor necrosis factor-α (TNFα), and interleukins (IL) -1ß, -6, and -10. EVs stained with markers associated with exosomes (CD63), microvesicles (VAMP3), and apoptotic bodies (THSD1) were characterized using imaging flow cytometry and vesicle flow cytometry. The selection event induced significant changes in circulating BDNF (-43.2%), IGF-I (-24.6%), TNFα (+17.7%), and IL-6 (+13.6%) accompanied by increases in intensities of THSD1+ and VAMP3+ EVs (all P < 0.05). Higher concentrations of IL-1ß and IL-10 were positively associated with THSD1+ EVs (P < 0.05). Military operational stress altered the EV profile. Surface markers associated with apoptotic bodies were positively correlated with an inflammatory response. Future studies should consider a multiomics assessment of EV cargo to discern canonical pathways that may be mediated by EVs during military stress.

A Selection of Macrocyclic Peptides That Bind STING From an mRNA-Display Library With Split Degenerate Codons.

Recent improvements in mRNA display have enabled the selection of peptides that incorporate non-natural amino acids, thus expanding the chemical diversity of macrocycles beyond what is accessible in nature. Such libraries have incorporated non-natural amino acids at the expense of natural amino acids by reassigning their codons. Here we report an alternative approach to expanded amino-acid diversity that preserves all 19 natural amino acids (no methionine) and adds 6 non-natural amino acids, resulting in the highest sequence complexity reported to date. We have applied mRNA display to this 25-letter library to select functional macrocycles that bind human STING, a protein involved in immunoregulation. The resulting STING-binding peptides include a 9-mer macrocycle with a dissociation constant (KD ) of 3.4 nM, which blocks binding of cGAMP to STING and induces STING dimerization. This approach is generalizable to expanding the amino-acid alphabet in a library beyond 25 building blocks.

Adnectin-drug conjugates for Glypican-3-specific delivery of a cytotoxic payload to tumors.

Tumor-specific delivery of cytotoxic agents remains a challenge in cancer therapy. Antibody-drug conjugates (ADC) deliver their payloads to tumor cells that overexpress specific tumor-associated antigens-but the multi-day half-life of ADC leads to high exposure even of normal, antigen-free, tissues and thus contributes to dose-limiting toxicity. Here, we present Adnectin-drug conjugates, an alternative platform for tumor-specific delivery of cytotoxic payloads. Due to their small size (10 kDa), renal filtration eliminates Adnectins from the bloodstream within minutes to hours, ensuring low exposure to normal tissues. We used an engineered cysteine to conjugate an Adnectin that binds Glypican-3, a membrane protein overexpressed in hepatocellular carcinoma, to a cytotoxic derivative of tubulysin, with the drug-to-Adnectin ratio of 1. We demonstrate specific, nanomolar binding of this Adnectin-drug conjugate to human and murine Glypican-3; its high thermostability; its localization to target-expressing tumor cells in vitro and in vivo, its fast clearance from normal tissues and its efficacy against Glypican-3-positive mouse xenograft models.

Synthesis and Biologic Evaluation of a Novel 18F-Labeled Adnectin as a PET Radioligand for Imaging PD-L1 Expression.

The programmed death protein (PD-1) and its ligand (PD-L1) play critical roles in a checkpoint pathway cancer cells exploit to evade the immune system. A same-day PET imaging agent for measuring PD-L1 status in primary and metastatic lesions could be important for optimizing drug therapy. Herein, we have evaluated the tumor targeting of an anti-PD-L1 adnectin after 18F-fluorine labeling. Methods: An anti-PD-L1 adnectin was labeled with 18F in 2 steps. This synthesis featured fluorination of a novel prosthetic group, followed by a copper-free click conjugation to a modified adnectin to generate 18F-BMS-986192. 18F-BMS-986192 was evaluated in tumors using in vitro autoradiography and PET with mice bearing bilateral PD-L1-negative (PD-L1(-)) and PD-L1-positive (PD-L1(+)) subcutaneous tumors. 18F-BMS-986192 was evaluated for distribution, binding, and radiation dosimetry in a healthy cynomolgus monkey. Results:18F-BMS-986192 bound to human and cynomolgus PD-L1 with a dissociation constant of less than 35 pM, as measured by surface plasmon resonance. This adnectin was labeled with 18F to yield a PET radioligand for assessing PD-L1 expression in vivo. 18F-BMS-986192 bound to tumor tissues as a function of PD-L1 expression determined by immunohistochemistry. Radioligand binding was blocked in a dose-dependent manner. In vivo PET imaging clearly visualized PD-L1 expression in mice implanted with PD-L1(+), L2987 xenograft tumors. Two hours after dosing, a 3.5-fold-higher uptake (2.41 ± 0.29 vs. 0.82 ± 0.11 percentage injected dose per gram, P < 0.0001) was observed in L2987 than in control HT-29 (PD-L1(-)) tumors. Coadministration of 3 mg/kg ADX_5322_A02 anti-PD-L1 adnectin reduced tumor uptake at 2 h after injection by approximately 70%, whereas HT-29 uptake remained unchanged, demonstrating PD-L1-specific binding. Biodistribution in a nonhuman primate showed binding in the PD-L1-rich spleen, with rapid blood clearance through the kidneys and bladder. Binding in the PD-L1(+) spleen was reduced by coadministration of BMS-986192. Dosimetry estimates indicate that the kidney is the dose-limiting organ, with an estimated human absorbed dose of 2.20E-01 mSv/MBq. Conclusion:18F-BMS-986192 demonstrated the feasibility of noninvasively imaging the PD-L1 status of tumors by small-animal PET studies. Clinical studies with 18F-BMS-986192 are under way to measure PD-L1 expression in human tumors.

Improving iSC performance through outsourcing - Considerations for using third-party service providers to increase innovation, capacity and efficiency.

Development partners and donors have encouraged and incentivized governments in developing countries to explore ways of working with third-party service suppliers to reduce costs and increase service delivery capacity. The distribution of vaccines and medicines has for a long time shown demand for outsourcing but public health systems have struggled to develop the expertise and capital assets necessary to manage such ventures. Existing transport and logistics capacity within public health systems, in particular, is well documented as being insufficient to support existing, let alone future immunization needs. Today, a number of countries are contracting party logistics providers (3PLs) to supplement the in-house distribution operations of public health systems. This commentary reflects on recent, leading examples of outsourcing initiatives to address critical gaps in transport and logistics.

Developing Adnectins that target SRC co-activator binding to PXR: a structural approach toward understanding promiscuity of PXR.

The human pregnane X receptor (PXR) is a promiscuous nuclear receptor that functions as a sensor to a wide variety of xenobiotics and regulates expression of several drug metabolizing enzymes and transporters. We have generated "Adnectins", derived from 10th fibronectin type III domain ((10)Fn3), that target the PXR ligand binding domain (LBD) interactions with the steroid receptor co-activator-1 (SRC-1) peptide, displacing SRC-1 binding. Adnectins are structurally homologous to the immunoglobulin superfamily. Three different co-crystal structures of PXR LBD with Adnectin-1 and CCR1 (CC chemokine receptor-1) antagonist Compound-1 were determined. This structural information was used to modulate PXR affinity for a related CCR1 antagonist compound that entered into clinical trials for rheumatoid arthritis. The structures of PXR with Adnectin-1 reveal specificity of Adnectin-1 in not only targeting the interface of the SRC-1 interactions but also engaging the same set of residues that are involved in binding of SRC-1 to PXR. Substituting SRC-1 with Adnectin-1 does not alter the binding conformation of Compound-1 in the ligand binding pocket. The structure also reveals the possibility of using Adnectins as crystallization chaperones to generate structures of PXR with compounds of interest.

Structures of adnectin/protein complexes reveal an expanded binding footprint.

Adnectins are targeted biologics derived from the tenth type III domain of human fibronectin (¹°Fn3), a member of the immunoglobulin superfamily. Target-specific binders are selected from libraries generated by diversifying the three ¹°Fn3 loops that are analogous to the complementarity determining regions of antibodies. The crystal structures of two Adnectins were determined, each in complex with its therapeutic target, EGFR or IL-23. Both Adnectins bind different epitopes than those bound by known monoclonal antibodies. Molecular modeling suggests that some of these epitopes might not be accessible to antibodies because of the size and concave shape of the antibody combining site. In addition to interactions from the Adnectin diversified loops, residues from the N terminus and/or the ß strands interact with the target proteins in both complexes. Alanine-scanning mutagenesis confirmed the calculated binding energies of these ß strand interactions, indicating that these nonloop residues can expand the available binding footprint.

A fibronectin scaffold approach to bispecific inhibitors of epidermal growth factor receptor and insulin-like growth factor-I receptor.

Engineered domains of human fibronectin (Adnectins™) were used to generate a bispecific Adnectin targeting epidermal growth factor receptor (EGFR) and insulin-like growth factor-I receptor (IGF-IR), two transmembrane receptors that mediate proliferative and survival cell signaling in cancer. Single-domain Adnectins that specifically bind EGFR or IGF-IR were generated using mRNA display with a library containing as many as 10 ( 13) Adnectin variants. mRNA display was also used to optimize lead Adnectin affinities, resulting in clones that inhibited EGFR phosphorylation at 7 to 38 nM compared to 2.6 µM for the parental clone. Individual, optimized, Adnectins specific for blocking either EGFR or IGF-IR signaling were engineered into a single protein (EI-Tandem Adnectin). The EI-Tandems inhibited phosphorylation of EGFR and IGF-IR, induced receptor degradation, and inhibited down-stream cell signaling and proliferation of human cancer cell lines (A431, H292, BxPC3 and RH41) with IC 50 values ranging from 0.1 to 113 nM. Although Adnectins bound to EGFR at a site distinct from those of anti-EGFR antibodies cetuximab, panitumumab and nimotuzumab, like the antibodies, the anti-EGFR Adnectins blocked the binding of EGF to EGFR. PEGylated EI-Tandem inhibited the growth of both EGFR and IGF-IR driven human tumor xenografts, induced degradation of EGFR, and reduced EGFR phosphorylation in tumors. These results demonstrate efficient engineering of bispecific Adnectins with high potency and desired specificity. The bispecificity may improve biological activity compared to monospecific biologics as tumor growth is driven by multiple growth factors. Our results illustrate a technological advancement for constructing multi-specific biologics in cancer therapy.

Anti-tumor effect of CT-322 as an adnectin inhibitor of vascular endothelial growth factor receptor-2.

CT-322 is a new anti-angiogenic therapeutic agent based on an engineered variant of the tenth type III domain of human fibronectin, i.e., an Adnectin™, designed to inhibit vascular endothelial growth factor receptor (VEGFR)-2. This PE Gylated Adnectin was developed using an mRNA display technology. CT-322 bound human VEGFR-2 with high affinity (K(D), 11 nM), but did not bind VEGFR-1 or VEGFR-3 at concentrations up to 100 nM, as determined by surface plasmon resonance studies. Western blot analysis showed that CT-322 blocked VEGF-induced phosphorylation of VEGFR-2 and mitogen-activated protein kinase in human umbilical vascular endothelial cells. CT-322 significantly inhibited the growth of human tumor xenograft models of colon carcinoma and glioblastoma at doses of 15-60 mg/kg administered 3 times/week. Anti-tumor effects of CT-322 were comparable to those of sorafenib or sunitinib, which inhibit multiple kinases, in a colon carcinoma xenograft model, although CT-322 caused less overt adverse effects than the kinase inhibitors. CT-322 also enhanced the anti-tumor activity of the chemotherapeutic agent temsirolimus in the colon carcinoma model. The high affinity and specificity of CT-322 binding to VEGFR-2 and its anti-tumor activities establish CT-322 as a promising anti-angiogenic therapeutic agent. Our results further suggest that Adnectins are an important new class of targeted biologics that can be developed as potential treatments for a wide variety of diseases.

Effectiveness of a bivalent Haemophilus influenzae type B-hepatitis B vaccine in preventing hepatitis B virus infection among children born to hepatitis B e antigen-positive carrier mothers.

BACKGROUND: This observational study evaluated a modified immunoprophylactic regimen (hepatitis B immune globulin [HBIG]) and a dose of thimerosal-free monovalent hepatitis B (HB) vaccine shortly after birth followed by doses of thimerosal-free bivalent Haemophilus influenzae type b (Hib)-HB vaccine at 2 and 4 months of age, and a booster at 12 months of age) in infants at high risk of hepatitis B virus (HBV) infection (mothers HBeAg+). METHODS: Children >or=6 months of age vaccinated in routine clinical practice were tested twice (>or=6 months apart) for HBV antigens surface antigen (HBsAg) and "e" antigen, and for antibody to HBsAg. Partial nucleotide sequence analysis was performed on HBV DNA isolated from infants identified with a breakthrough chronic HBV infection. A fully sequential statistical design was used to maximize patient safety and study efficiency. RESULTS: Four of 60 children developed chronic HBV infection despite vaccination, but at no point did the cumulative number of cases reach the boundary of statistical significance. Overall, the analysis adjusted for sequential testing yielded an estimated breakthrough rate of 6.7% (90% CI: 2.3%-14.6%). In a subset of uninfected children tested for antibody to HBsAg 1 to 4 months after the second dose of Hib-HB vaccine, 90% (9/10) had >or=10 milli-International Units per milliliter (mIU/mL). The third dose of Hib-HB vaccine induced a secondary increase in the level of antibody; 94.7% (18/19) of a second group developed >or=100 mIU/mL, with a geometric mean concentration of 771 mIU/mL (95% CI: 351.4-1692.1 mIU/mL). CONCLUSION: The tested regimen is comparably effective to historical experience with a standard one employing HBIG plus monovalent thimerosal-containing HB vaccine given at 0, 1, and 6 months of age.

Measurement of 3-D hydraulic conductivity in aquifer cores at in situ effective stresses.

An innovative and nondestructive method to measure the hydraulic conductivity of drill core samples in horizontal and vertical directions within a triaxial cell has been developed. This has been applied to characterizing anisotropy and heterogeneity of a confined consolidated limestone aquifer. Most of the cores tested were isotropic, but hydraulic conductivity varied considerably and the core samples with lowest values were also the most anisotropic. Hydraulic conductivity decreased with increasing effective stress due to closure of microfractures caused by sampling for all core samples. This demonstrates the importance of replicating in situ effective stresses when measuring hydraulic conductivity of cores of deep aquifers in the laboratory.

Identification of epitope-like consensus motifs using mRNA display.

The mRNA display approach to in vitro protein selection is based upon the puromycin-mediated formation of a covalent bond between an mRNA and its gene product. This technique can be used to identify peptide sequences involved in macromolecular recognition, including those identical or homologous to natural ligand epitopes. To demonstrate this approach, we determined the peptide sequences recognized by the trypsin active site, and by the anti-c-Myc antibody, 9E10. Here we describe the use of two peptide libraries of different diversities, one a constrained library based on the trypsin inhibitor EETI-II, where only the six residues in the first loop were randomized (6.4 x 10(7) possible sequences, 6.0 x 10(11) sequences in the library), the other a linear-peptide library with 27 randomized amino acids (1.3 x 10(35) possible sequences, 2 x 10(13) sequences in the library). The constrained library was screened against the natural target of wild-type EETI, bovine trypsin, and the linear library was screened against the anti-c-myc antibody, 9E10. The analysis of selected sequences revealed minimal consensus sequences of PR(I,L,V)L for the first loop of EETI-II and LISE for the 9E10 epitope. The wild-type sequences, PRILMR for the first loop of EETI-II and QKLISE for the 9E10 epitope, were selected with the highest frequency, and in each case the complete wild-type epitope was selected from the library.

Continuous in vitro evolution of a ribozyme that catalyzes three successive nucleotidyl addition reactions.

Variants of the class I ligase ribozyme, which catalyzes joining of the 3' end of a template bound oligonucleotide to its own 5' end, have been made to evolve in a continuous manner by a simple serial transfer procedure that can be carried out indefinitely. This process was expanded to allow the evolution of ribozymes that catalyze three successive nucleotidyl addition reactions, two template-directed mononucleotide additions followed by RNA ligation. During the development of this behavior, a population of ribozymes was maintained against an overall dilution of more than 10(406). The resulting ribozymes were capable of catalyzing the three-step reaction pathway, with nucleotide addition occurring in either a 5'-->3' or a 3'-->5' direction. This purely chemical system provides a functional model of a multi-step reaction pathway that is undergoing Darwinian evolution.

Generating addressable protein microarrays with PROfusion covalent mRNA-protein fusion technology.

An mRNA-protein fusion consists of a polypeptide covalently linked to its corresponding mRNA. These species, prepared individually or en masse by in vitro translation with a modified mRNA conjugate (the PROfusion process), link phenotype to genotype and enable powerful directed evolution schemes. We have exploited the informational content of the nucleic acid component of the mRNA-protein fusion to create an addressable protein microarray that self-assembles via hybridization to surface-bound DNA capture probes. The nucleic acid component not only directs the mRNA-protein fusion to the proper coordinate of the microarray, but also positions the protein in a uniform orientation. We demonstrate the feasibility of this protein chip concept with several mRNA-protein fusions, each possessing a unique peptide epitope sequence. These addressable proteins could be visualized on the microarray both by autoradiography and highly specific monoclonal antibody binding. The anchoring of the protein to the chip surface is surprisingly robust, and the system is sensitive enough to detect sub-attomole quantities of displayed protein without signal amplification. Such protein arrays should be useful for functional screening in massively parallel formats, as well as other applications involving immobilized peptides and proteins.