Control-Normalized Fisher Ratio Analysis of Comprehensive Two-Dimensional Gas Chromatography Time-of-Flight Mass Spectrometry Data for Enhanced Biomarker Discovery in a Metabolomic Study of Orthopedic Knee-Ligament Injury.

An innovative form of Fisher ratio (F-ratio) analysis (FRA) is developed for use with comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry (GC × GC-TOFMS) data and applied to the investigation of the changes in the metabolome in human plasma for patients with injury to their anterior cruciate ligament (ACL). Specifically, FRA provides a supervised discovery of metabolites that express a statistically significant variance in a two-sample class comparison: patients and healthy controls. The standard F-ratio utilizes the between-class variance relative to the pooled within-class variance. Because standard FRA is adversely impacted by metabolites expressed with a large within-class variance in the patient class, "control-normalized FRA" has been developed to provide complementary information, by normalizing the between-class variance to the variance of the control class only. Thirty plasma samples from patients who recently suffered from an ACL injury, along with matched controls, were subjected to GC × GC-TOFMS analysis. Following both standard and control-normalized FRA, the concentration ratio for the top 30 "hits" in each comparison was obtained and then t-tested for statistical significance. Twenty four out of 30 metabolites plus the therapeutic agent, naproxen (24/30), passed the t-test for the control-normalized FRA, which included 8/24 unique to control-normalized FRA and 16/24 in common with the standard FRA. Likewise, standard FRA provided 21/30 metabolites passing the t-test, with 5/21 undiscovered by control-normalized FRA. The complementary information obtained by both F-ratio analyses demonstrates the general utility of the new approach for a variety of applications.

Progress and Future Directions of the NCAA-DoD Concussion Assessment, Research, and Education (CARE) Consortium and Mind Matters Challenge at the US Service Academies.

Despite the significant impact that concussion has on military service members, significant gaps remain in our understanding of the optimal diagnostic, management, and return to activity/duty criteria to mitigate the consequences of concussion. In response to these significant knowledge gaps, the US Department of Defense (DoD) and the National Collegiate Athletic Association (NCAA) partnered to form the NCAA-DoD Grand Alliance in 2014. The NCAA-DoD CARE Consortium was established with the aim of creating a national multisite research network to study the clinical and neurobiological natural history of concussion in NCAA athletes and military Service Academy cadets and midshipmen. In addition to the data collected for the larger CARE Consortium effort, the service academies have pursued military-specific lines of research relevant to operational and medical readiness associated with concussion. The purpose of this article is to describe the structure of the NCAA-DoD Grand Alliance efforts at the service academies, as well as discuss military-specific research objectives and provide an overview of progress to date. A secondary objective is to discuss the challenges associated with conducting large-scale studies in the Service Academy environment and highlight future directions for concussion research endeavors across the CARE Service Academy sites.

Non-Pharmaceutical Interventions and Military Hygiene at the United States Military Academy between 1890 and 1910.

INTRODUCTION: Military installations are at increased risk for the transmission of infectious disease. Personnel who live and train on military installations live and train near one another facilitating disease transmission. An understanding of historical sanitation and hygiene can inform modern practices. This is especially pertinent considering the continuing rise of variants of infectious diseases, such as the recent pandemic of the 2019 severe acute respiratory syndrome coronavirus 2. In this article, we review the rise and decline of infectious disease at the United States Military Academy (USMA) during the period spanning 1890 through 1910, and the public health interventions used to combat disease spread. MATERIALS AND METHODS: Primary data regarding cadet illness were acquired from the historical archives of the USMA. These included annual reports, clinical admission records, casualty ledgers, and sanitation reports. Unpublished documents from the medical history of USMA provide periodic trends of health among cadets because of infectious disease. RESULTS: Between 1890 and 1910, the USMA at West Point was confronted with cases of influenza, measles, mumps, scarlet fever, smallpox, typhus, and malaria. In response, a series of non-pharmaceutical interventions (NPIs) were instituted to curb the spread of infectious disease. These interventions most likely proved effective in suppressing the transmission of communicable diseases. The most common and arguably the most effective NPI was the physical separation of the sick from the well. CONCLUSIONS: The USMA experience mirrored what was occurring in the larger U.S. Army in the early 20th century and may serve as a model for the application of NPIs in response to modern infectious diseases resulting from novel or unknown etiologies.

A Salt-Templated Synthesis Method for Porous Platinum-based Macrobeams and Macrotubes.

The synthesis of high surface area porous noble metal nanomaterials generally relies on time consuming coalescence of pre-formed nanoparticles, followed by rinsing and supercritical drying steps, often resulting in mechanically fragile materials. Here, a method to synthesize nanostructured porous platinum-based macrotubes and macrobeams with a square cross section from insoluble salt needle templates is presented. The combination of oppositely charged platinum, palladium, and copper square planar ions results in the rapid formation of insoluble salt needles. Depending on the stoichiometric ratio of metal ions present in the salt-template and the choice of chemical reducing agent, either macrotubes or macrobeams form with a porous nanostructure comprised of either fused nanoparticles or nanofibrils. Elemental composition of the macrotubes and macrobeams, determined with x-ray diffractometry and x-ray photoelectron spectroscopy, is controlled by the stoichiometric ratio of metal ions present in the salt-template. Macrotubes and macrobeams may be pressed into free standing films, and the electrochemically active surface area is determined with electrochemical impedance spectroscopy and cyclic voltammetry. This synthesis method demonstrates a simple, relatively fast approach to achieve high-surface area platinum-based macrotubes and macrobeams with tunable nanostructure and elemental composition that may be pressed into free-standing films with no required binding materials.

Uniform wet-Spinning Mechanically Automated (USMA) fiber device.

Bioengineering techniques for producing fibers from biomaterials is a growing requirement in medical device technology research and development environments. Scale-up and control of diameter, shape, and length of fibrous proteins and elastomeric polymers are essential to produce defined and consistent materials for experimentation and clinical use. Here, we developed a novel wet spinning fiber extruder and spooler system engineered to draw precipitated fibers several meters in length across five spools. By controlling both the extrusion and spooling rate, the diameter of the fiber can be controlled on the order of 10-1000 µm. Using this system, we extruded and spooled precipitated Type-1 Collagen fibers up to 7.5 m in length on a single spool with a controllable diameter range of 30-50 µm. Furthermore, this device facilitated bundling of fibers directly on the spool in order to create 1-12 cm long fiber bundles for experimentation. This system may be used in the laboratory to scale up biomaterial fiber production to produce degradable scaffolds made from synthetic or natural materials for a range of biomedical applications.

Synthesis Method for Cellulose Nanofiber Biotemplated Palladium Composite Aerogels.

Here, a method to synthesize cellulose nanofiber biotemplated palladium composite aerogels is presented. Noble metal aerogel synthesis methods often result in fragile aerogels with poor shape control. The use of carboxymethylated cellulose nanofibers (CNFs) to form a covalently bonded hydrogel allows for the reduction of metal ions such as palladium on the CNFs with control over both nanostructure and macroscopic aerogel monolith shape after supercritical drying. Crosslinking the carboxymethylated cellulose nanofibers is achieved using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) in the presence of ethylenediamine. The CNF hydrogels maintain their shape throughout synthesis steps including covalent crosslinking, equilibration with precursor ions, metal reduction with high concentration reducing agent, rinsing in water, ethanol solvent exchange, and CO2 supercritical drying. Varying the precursor palladium ion concentration allows for control over the metal content in the final aerogel composite through a direct ion chemical reduction rather than relying on the relatively slow coalescence of pre-formed nanoparticles used in other sol-gel techniques. With diffusion as the basis to introduce and remove chemical species into and out of the hydrogel, this method is suitable for smaller bulk geometries and thin films. Characterization of the cellulose nanofiber-palladium composite aerogels with scanning electron microscopy, X-ray diffractometry, thermal gravimetric analysis, nitrogen gas adsorption, electrochemical impedance spectroscopy, and cyclic voltammetry indicates a high surface area, metallized palladium porous structure.

Latent Lyme Disease Resulting in Chronic Arthritis and Early Career Termination in a United States Army Officer.

Lyme disease is a continuing threat to military personnel operating in arboriferous and mountainous environments. Here we present the case of a 24-year-old Second Lieutenant, a recent graduate from the United States Military Academy, with a history of Lyme disease who developed recurrent knee effusions following surgery to correct a hip impingement. Although gonococcal arthritis was initially suspected from preliminary laboratory results, a comprehensive evaluation contradicted this diagnosis. Despite antibiotic therapy, aspiration of the effusions, and steroid treatment to control inflammation, the condition of the patient deteriorated to the point where he was found to be unfit for duty and subsequently discharged from active military service. This case illustrates the profound effect that latent Lyme disease can have on the quality of life and the career of an active duty military member. It highlights the need for increased surveillance for Borrelia burgdorferi (B. burgdorferi) in military training areas and for the early and aggressive diagnosis and treatment of military personnel who present with the symptoms of acute Lyme disease.

Noble Metal Composite Porous Silk Fibroin Aerogel Fibers.

Nobel metal composite aerogel fibers made from flexible and porous biopolymers offer a wide range of applications, such as in catalysis and sensing, by functionalizing the nanostructure. However, producing these composite aerogels in a defined shape is challenging for many protein-based biopolymers, especially ones that are not fibrous proteins. Here, we present the synthesis of silk fibroin composite aerogel fibers up to 2 cm in length and a diameter of ~300 µm decorated with noble metal nanoparticles. Lyophilized silk fibroin dissolved in hexafluoro-2-propanol (HFIP) was cast in silicon tubes and physically crosslinked with ethanol to produce porous silk gels. Composite silk aerogel fibers with noble metals were created by equilibrating the gels in noble metal salt solutions reduced with sodium borohydride, followed by supercritical drying. These porous aerogel fibers provide a platform for incorporating noble metals into silk fibroin materials, while also providing a new method to produce porous silk fibers. Noble metal silk aerogel fibers can be used for biological sensing and energy storage applications.

Progress in the development of olfactory-based bioelectronic chemosensors.

Artificial chemosensory devices have a wide range of applications in industry, security, and medicine. The development of these devices has been inspired by the speed, sensitivity, and selectivity by which the olfactory system in animals can probe the chemical nature of the environment. In this review, we examine how molecular and cellular components of natural olfactory systems have been incorporated into artificial chemosensors, or bioelectronic sensors. We focus on the biological material that has been combined with signal transduction systems to develop artificial chemosensory devices. The strengths and limitations of different biological chemosensory material at the heart of these devices, as well as the reported overall effectiveness of the different bioelectronic sensor designs, is examined. This review also discusses future directions and challenges for continuing to advance development of bioelectronic sensors.

Cellulose Nanofiber Biotemplated Palladium Composite Aerogels.

Noble metal aerogels offer a wide range of catalytic applications due to their high surface area and tunable porosity. Control over monolith shape, pore size, and nanofiber diameter is desired in order to optimize electronic conductivity and mechanical integrity for device applications. However, common aerogel synthesis techniques such as solvent mediated aggregation, linker molecules, sol⁻gel, hydrothermal, and carbothermal reduction are limited when using noble metal salts. Here, we present the synthesis of palladium aerogels using carboxymethyl cellulose nanofiber (CNF) biotemplates that provide control over aerogel shape, pore size, and conductivity. Biotemplate hydrogels were formed via covalent cross linking using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) with a diamine linker between carboxymethylated cellulose nanofibers. Biotemplate CNF hydrogels were equilibrated in precursor palladium salt solutions, reduced with sodium borohydride, and rinsed with water followed by ethanol dehydration, and supercritical drying to produce freestanding aerogels. Scanning electron microscopy indicated three-dimensional nanowire structures, and X-ray diffractometry confirmed palladium and palladium hydride phases. Gas adsorption, impedance spectroscopy, and cyclic voltammetry were correlated to determine aerogel surface area. These self-supporting CNF-palladium aerogels demonstrate a simple synthesis scheme to control porosity, electrical conductivity, and mechanical robustness for catalytic, sensing, and energy applications.

Similar pathogen targets in Arabidopsis thaliana and homo sapiens protein networks.

We study the behavior of pathogens on host protein networks for humans and Arabidopsis - noting striking similarities. Specifically, we preform [Formula: see text]-shell decomposition analysis on these networks - which groups the proteins into various "shells" based on network structure. We observe that shells with a higher average degree are more highly targeted (with a power-law relationship) and that highly targeted nodes lie in shells closer to the inner-core of the network. Additionally, we also note that the inner core of the network is significantly under-targeted. We show that these core proteins may have a role in intra-cellular communication and hypothesize that they are less attacked to ensure survival of the host. This may explain why certain high-degree proteins are not significantly attacked.

Hyperspectral imaging of cuttlefish camouflage indicates good color match in the eyes of fish predators.

Camouflage is a widespread phenomenon throughout nature and an important antipredator tactic in natural selection. Many visual predators have keen color perception, and thus camouflage patterns should provide some degree of color matching in addition to other visual factors such as pattern, contrast, and texture. Quantifying camouflage effectiveness in the eyes of the predator is a challenge from the perspectives of both biology and optical imaging technology. Here we take advantage of hyperspectral imaging (HSI), which records full-spectrum light data, to simultaneously visualize color match and pattern match in the spectral and the spatial domains, respectively. Cuttlefish can dynamically camouflage themselves on any natural substrate and, despite their colorblindness, produce body patterns that appear to have high-fidelity color matches to the substrate when viewed directly by humans or with RGB images. Live camouflaged cuttlefish on natural backgrounds were imaged using HSI, and subsequent spectral analysis revealed that most reflectance spectra of individual cuttlefish and substrates were similar, rendering the color match possible. Modeling color vision of potential di- and trichromatic fish predators of cuttlefish corroborated the spectral match analysis and demonstrated that camouflaged cuttlefish show good color match as well as pattern match in the eyes of fish predators. These findings (i) indicate the strong potential of HSI technology to enhance studies of biological coloration and (ii) provide supporting evidence that cuttlefish can produce color-coordinated camouflage on natural substrates despite lacking color vision.

Kinetics of riboswitch regulation studied by in vitro transcription.

Riboswitches encompass messenger ribonucleic acid transcripts that sense the concentration of small molecule metabolites through binding the target compound and then control the expression of metabolite-related genes in response to the metabolite concentration. While much of the riboswitch-related research has focused on the remarkable ability of different aptamer domains to adopt the intricate structures required to bind a spectrum of biological metabolites with high affinity and specificity, less attention has been paid to the mechanism of riboswitch action. Specifically, the genetic control element of the riboswitch, known as the expression platform, must function cotranscriptionally in the case of transcription termination-controlled riboswitches. By correlating the transcriptional kinetics of the entire switch and the kinetics and thermodynamics of metabolite binding of the aptamer domain, it was found that the FMN-binding riboswitch in the 5' UTR of the Bacillus subtilis ribGBAHT operon functions as a kinetically controlled genetic switch chiefly dependent upon transcriptional pausing and the concentration of the target metabolite. This study has emphasized the importance of studying the switch in its entirety and in the context of an actively transcribing RNA polymerase. Herein I will describe the study of the kinetics of riboswitch transcription and the proposed mechanism for the transcription termination-associated riboswitch control of riboflavin-related genes.

The kinetics of ligand binding by an adenine-sensing riboswitch.

A riboswitch within the 5' untranslated region (UTR) of the Bacillus subtilis pbuE mRNA binds adenine and related analogues in the absence of protein factors; excess adenine added to bacterial growth media triggers activation of a reporter gene that carries this riboswitch. To assess whether the riboswitch reaches thermodynamic equilibrium, or is operated by the kinetics of ligand binding and RNA transcription, we examined the detailed equilibrium and kinetic parameters for the complex formation between the aptamer domain of this riboswitch and the ligands adenine, 2-aminopurine (2AP), and 2,6-diaminopurine (DAP). Using a fluorescence-based assay, we have confirmed that adenine and 2AP have nearly equal binding affinity, with KD values for 2AP ranging from 250 nM to 3 microM at temperatures ranging from 15 to 35 degrees C, while DAP binds with much higher affinity. The association rate constant, however, favors adenine over DAP and 2AP by 3- and 10-fold, respectively, at 25 degrees C. Furthermore, the rate constants for adenine association and dissociation with the aptamer suggest that the pbuE riboswitch could be either kinetically or thermodynamically controlled depending upon the time scale of transcription and external variables such as temperature. We cite data that suggest kinetic control under certain conditions and illustrate with a model calculation how the system can switch between kinetic and equilibrium control. These findings further support the hypothesis that many riboswitches rely on the kinetics of ligand binding and the speed of RNA transcription, rather than simple ligand affinity, to establish the concentration of metabolite needed to trigger riboswitch function.

The speed of RNA transcription and metabolite binding kinetics operate an FMN riboswitch.

Riboswitches are genetic control elements that usually reside in untranslated regions of messenger RNAs. These folded RNAs directly bind metabolites and undergo allosteric changes that modulate gene expression. A flavin mononucleotide (FMN)-dependent riboswitch from the ribDEAHT operon of Bacillus subtilis uses a transcription termination mechanism wherein formation of an RNA-FMN complex causes formation of an intrinsic terminator stem. We assessed the importance of RNA transcription speed and the kinetics of FMN binding to the nascent mRNA for riboswitch function. The riboswitch does not attain thermodynamic equilibrium with FMN before RNA polymerase needs to make a choice between continued transcription and transcription termination. Therefore, this riboswitch is kinetically driven, and functions more like a "molecular fuse." This reliance on the kinetics of ligand association and RNA polymerization speed might be common for riboswitches that utilize transcription termination mechanisms.

New RNA motifs suggest an expanded scope for riboswitches in bacterial genetic control.

The expression of certain genes involved in fundamental metabolism is regulated by metabolite-binding "riboswitch" elements embedded within their corresponding mRNAs. We have identified at least six additional elements within the Bacillus subtilis genome that exhibit characteristics of riboswitch function (glmS, gcvT, ydaO/yuaA, ykkC/yxkD, ykoK, and yybP/ykoY). These motifs exhibit extensive sequence and secondary-structure conservation among many bacterial species and occur upstream of related genes. The element located upstream of the glmS gene in Gram-positive organisms functions as a metabolite-dependent ribozyme that responds to glucosamine-6-phosphate. Other motifs form complex folded structures when transcribed as RNA molecules and carry intrinsic terminator structures. These findings indicate that riboswitches serve as a major genetic regulatory mechanism for the control of metabolic genes in many microbial species.

An mRNA structure in bacteria that controls gene expression by binding lysine.

Riboswitches are metabolite-responsive genetic control elements that reside in the untranslated regions (UTRs) of certain messenger RNAs. Herein, we report that the 5'-UTR of the lysC gene of Bacillus subtilis carries a conserved RNA element that serves as a lysine-responsive riboswitch. The ligand-binding domain of the riboswitch binds to L-lysine with an apparent dissociation constant (KD) of approximately 1 micro M, and exhibits a high level of molecular discrimination against closely related analogs, including D-lysine and ornithine. Furthermore, we provide evidence that this widespread class of riboswitches serves as a target for the antimetabolite S-(2-aminoethyl)-L-cysteine (AEC). These findings add support to the hypotheses that direct sensing of metabolites by messenger RNAs is a fundamental form of genetic control and that riboswitches represent a new class of antimicrobial drug targets.