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The awareness of individuals’ biological status is critical for creating interactive and adaptive environments that can actively assist the users to achieve optimal outcomes. Accordingly, specialized human–machine interfaces—equipped with bioperception and interpretation capabilities—are required. To this end, we devised a multimodal cryptographic bio-human–machine interface (CB-HMI), which seamlessly translates the user’s touch-based entries into encrypted biochemical, biophysical, and biometric indices. As its central component, the CB-HMI features thin hydrogel-coated chemical sensors and inference algorithms to noninvasively and inconspicuously acquire biochemical indices such as circulating molecules that partition onto the skin (here, ethanol and acetaminophen). Additionally, the CB-HMI hosts physical sensors and associated algorithms to simultaneously acquire the user’s heart rate, blood oxygen level, and fingerprint minutiae pattern. Supported by human subject studies, we demonstrated the CB-HMI’s capability in terms of acquiring physiologically relevant readouts of target bioindices, as well as user-identifying and biometrically encrypting/decrypting these indices in situ (leveraging the fingerprint feature). By upgrading the common surrounding objects with the CB-HMI, we created interactive solutions for driving safety and medication use. Specifically, we demonstrated a vehicle-activation system and a medication-dispensing system, where the integrated CB-HMI uniquely enabled user bioauthentication (on the basis of the user’s biological state and identity) prior to rendering the intended services. Harnessing the levels of bioperception achieved by the CB-HMI and other intelligent HMIs, we can equip our surroundings with a comprehensive and deep awareness of individuals’ psychophysiological state and needs.

Corynebacterium glutamicum has been successfully employed for the industrial production of amino acids and other bioproducts, partially due to its native ability to utilize a wide range of carbon substrates. We demonstrated C. glutamicum as an efficient microbial host for utilizing diverse carbon substrates present in biomass hydrolysates, such as glucose, arabinose, and xylose, in addition to its natural ability to assimilate lignin-derived aromatics. As a case study to demonstrate its bioproduction capabilities, L-lactate was chosen as the primary fermentation end product along with acetate and succinate. C. glutamicum was found to grow well in different aromatics (benzoic acid, cinnamic acid, vanillic acid, and p-coumaric acid) up to a concentration of 40 mM. Besides, 13 C-fingerprinting confirmed that carbon from aromatics enter the primary metabolism via TCA cycle confirming the presence of β-ketoadipate pathway in C. glutamicum . 13 C-fingerprinting in the presence of both glucose and aromatics also revealed coumarate to be the most preferred aromatic by C. glutamicum contributing 74 and 59% of its carbon for the synthesis of glutamate and aspartate respectively. 13 C-fingerprinting also confirmed the activity of ortho-cleavage pathway, anaplerotic pathway, and cataplerotic pathways. Finally, the engineered C. glutamicum strain grew well in biomass hydrolysate containing pentose and hexose sugars and produced L-lactate at a concentration of 47.9 g/L and a yield of 0.639 g/g from sugars with simultaneous utilization of aromatics. Succinate and acetate co-products were produced at concentrations of 8.9 g/L and 3.2 g/L, respectively. Our findings open the door to valorize all the major carbon components of biomass hydrolysate by using C. glutamicum as a microbial host for biomanufacturing., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Mhatre, Shinde, Jha, Rodriguez, Wardak, Jansen, Gladden, George, Davis and Varman.)

Global surgery is a medical field dedicated to the facilitation of timely access to safe, affordable, and high-quality surgical healthcare worldwide, including support for necessary surgery and anesthesia infrastructure. Standard surgical training in the US does not provide necessary exposure to the range of surgical operations and nontechnical skills critical to practice in resource-limited contexts. Therefore, academic medical institutions have sought to bridge this training gap by establishing global surgery-focused rotations, fellowships, and integrated global surgery residencies. However, the presence of trainees pursuing education for careers in resource-variable settings presents an added layer of ethical complexity that must be carefully considered on the individual, programmatic, and institutional level. This article reviews the complexities relevant to global surgery trainees across these levels and offers potential mechanisms for addressing these ethical challenges., (Copyright © 2022 by the American College of Surgeons. Published by Wolters Kluwer Health, Inc. All rights reserved.)

Background: Microalgae possess numerous advantages for use as a feedstock in producing renewable fuels and products, with techno-economic analysis (TEA) frequently used to highlight the economic potential and technical challenges of utilizing this biomass in a biorefinery context. However, many historical TEA studies have focused on the conversion of biomass with elevated levels of carbohydrates and lipids and lower levels of protein, incurring substantial burdens on the ability to achieve high cultivation productivity rates relative to nutrient-replete, high-protein biomass. Given a strong dependence of algal biomass production costs on cultivation productivity, further TEA assessment is needed to understand the economic potential for utilizing potentially lower-cost but lower-quality, high-protein microalgae for biorefinery conversion., Results: In this work, we conduct rigorous TEA modeling to assess the economic viability of two conceptual technology pathways for processing proteinaceous algae into a suite of fuels and products. One approach, termed mild oxidative treatment and upgrading (MOTU), makes use of a series of thermo-catalytic operations to upgrade solubilized proteins and carbohydrates to hydrocarbon fuels, while another alternative focuses on the biological conversion of those substrates to oxygenated fuels in the form of mixed alcohols (MA). Both pathways rely on the production of polyurethanes from unsaturated fatty acids and valorization of unconverted solids for use as a material for synthesizing bioplastics. The assessment found similar, albeit slightly higher fuel yields and lower costs for the MA pathway, translating to a residual solids selling price of $899/ton for MA versus $1033/ton for MOTU as would be required to support a $2.50/gallon gasoline equivalent (GGE) fuel selling price. A variation of the MA pathway including subsequent upgrading of the mixed alcohols to hydrocarbon fuels (MAU) reflected a required solids selling price of $975/ton., Conclusion: The slight advantages observed for the MA pathway are partially attributed to a boundary that stops at oxygenated fuels versus fungible drop-in hydrocarbon fuels through a more complex MOTU configuration, with more comparable results obtained for the MAU scenario. In either case, it was shown that an integrated algal biorefinery can be economical through optimal strategies to utilize and valorize all fractions of the biomass., (© 2022. The Author(s).)

Objectives: While lateral and forward step-down tasks are commonly used, they may have different kinematic and performance demands that could influence clinical assessment and rehabilitation. Therefore, the purpose of this study was to compare 3D lower extremity kinematics and 2D quality of movement between the tasks., Design: Cross-sectional comparative study., Setting: Research laboratory., Participants: Thirty healthy adults (18 females, age = 23.2 ± 1.4 years, BMI = 23.9 ± 2.2 kg/m 2 )., Main Outcome Measures: Participants underwent 3D and 2D motion analysis. 3D variables were peak hip, knee, and ankle angles. Dichotomous clinical criteria were used for 2D assessment. An alpha level of 0.05 was used for statistical analyses., Results: In the sagittal plane, the forward step-down averaged 7° more knee flexion (p < 0.001, d = 2.30) and 4° more ankle dorsiflexion (p < 0.001, d = 1.72), but 2° less hip flexion (p = 0.001, d = 0.64). In the frontal plane, forward step-downs averaged 1° more hip adduction (p = 0.006, d = 0.54) and 1° more ankle eversion (p < 0.001, d = 1.04). The forward step-down elicited 2D movement faults more often (p = 0.003)., Conclusions: The increased knee flexion and ankle dorsiflexion demands of the forward step-down were accompanied by increases in frontal plane aberrations. The forward step-down may be more challenging for individuals with reduced tolerance to loaded knee flexion and/or limited ankle mobility., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Lab-on-a-chip (LOC) devices capable of manipulating micro/nano-sized samples have spurred advances in biotechnology and chemistry. Designing and analyzing new and more advanced LOCs require accurate modeling and simulation of sample/particle dynamics inside such devices. In this work, we present a generalized computational physics model to simulate particle/sample trajectories under the influence of dielectrophoretic or optical forces inside LOC devices. The model takes into account time varying applied forces, Brownian motion, fluid flow, collision mechanics, and hindered diffusion caused by hydrodynamic interactions. We develop a numerical solver incorporating the aforementioned physics and use it to simulate two example cases: first, an optical trapping experiment, and second, a dielectrophoretic cell sorter device. In both cases, the numerical results are found to be consistent with experimental observations, thus proving the generality of the model. The numerical solver can simulate time evolution of the positions and velocities of an arbitrarily large number of particles simultaneously. This allows us to characterize and optimize a wide range of LOCs. The developed numerical solver is made freely available through a GitHub repository so that researchers can use it to develop and simulate new designs.

One in four myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) patients are estimated to be severely affected by the disease, and these house-bound or bedbound patients are currently understudied. Here, we report a comprehensive examination of the symptoms and clinical laboratory tests of a cohort of severely ill patients and healthy controls. The greatly reduced quality of life of the patients was negatively correlated with clinical depression. The most troublesome symptoms included fatigue (85%), pain (65%), cognitive impairment (50%), orthostatic intolerance (45%), sleep disturbance (35%), post-exertional malaise (30%), and neurosensory disturbance (30%). Sleep profiles and cognitive tests revealed distinctive impairments. Lower morning cortisol level and alterations in its diurnal rhythm were observed in the patients, and antibody and antigen measurements showed no evidence for acute infections by common viral or bacterial pathogens. These results highlight the urgent need of developing molecular diagnostic tests for ME/CFS. In addition, there was a striking similarity in symptoms between long COVID and ME/CFS, suggesting that studies on the mechanism and treatment of ME/CFS may help prevent and treat long COVID and vice versa.

Management of gases during diving is not well understood across marine mammal species. Prior to diving, phocid (true) seals generally exhale, a behaviour thought to assist with the prevention of decompression sickness. Otariid seals (fur seals and sea lions) have a greater reliance on their lung oxygen stores, and inhale prior to diving. One otariid, the Antarctic fur seal ( Arctocephalus gazella ), then exhales during the final 50-85% of the return to the surface, which may prevent another gas management issue: shallow-water blackout. Here, we compare data collected from animal-attached tags (video cameras, hydrophones and conductivity sensors) deployed on a suite of otariid seal species to examine the ubiquity of ascent exhalations for this group. We find evidence for ascent exhalations across four fur seal species, but that such exhalations are absent for three sea lion species. Fur seals and sea lions are no longer genetically separated into distinct subfamilies, but are morphologically distinguished by the thick underfur layer of fur seals. Together with their smaller size and energetic dives, we suggest their air-filled fur might underlie the need to perform these exhalations, although whether to reduce buoyancy and ascent speed, for the avoidance of shallow-water blackout or to prevent other cardiovascular management issues in their diving remains unclear. This article is part of the theme issue 'Measuring physiology in free-living animals (Part I)'.

Basal metabolic rate generally scales with body mass in mammals, and variation from predicted levels indicates adaptive metabolic remodeling. As a thermogenic adaptation for living in cool water, sea otters have a basal metabolic rate approximately three times that of the predicted rate; however, the tissue-level source of this hypermetabolism is unknown. Because skeletal muscle is a major determinant of whole-body metabolism, we characterized respiratory capacity and thermogenic leak in sea otter muscle. Compared with that of previously sampled mammals, thermogenic muscle leak capacity was elevated and could account for sea otter hypermetabolism. Muscle respiratory capacity was modestly elevated and reached adult levels in neonates. Premature metabolic development and high leak rate indicate that sea otter muscle metabolism is regulated by thermogenic demand and is the source of basal hypermetabolism., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Problem: The World Health Organization and the World Bank have identified improvement in access to surgical care as an urgent global health challenge and a cost-effective investment in public health. However, trainees in standard U.S. general surgery programs do not have adequate exposure to the procedures, technical skills, and foundational knowledge essential for providing surgical care in resource-limited settings., Approach: The Michael E. DeBakey Department of Surgery at Baylor College of Medicine (BCM) created a 7-year global surgery track within its general surgery residency in 2014. Individualized rotations equip residents with the necessary skills, knowledge, and experience to operate in regions with low surgeon density and develop sustainable surgical infrastructures. BCM provides a formal, integrated global surgery curriculum-including 2 years dedicated to global surgery-with surgical specialty rotations in domestic and international settings. Residents tailor their individual experience to the needs of their future clinical practice, region of interest, and surgical specialty., Outcomes: There have been 4 major outcomes of the BCM global surgery track: (1) increased exposure for trainees to a broad range of surgeries critical in resource-limited settings, (2) meaningful international partnerships, (3) contributions to global surgery scholarship, and (4) establishment of sustainable global surgery activities., Next Steps: To better facilitate access to safe, timely, and affordable surgical care worldwide, global surgeons should pursue expertise in topics not currently included in U.S. general surgical curricula, such as setting-specific technical skills, capacity building, and organizational collaboration. Future evaluations of the BCM global surgery track will assess the effect of individualized education on trainees' professional identities, clinical practices, academic pursuits, global surgery leadership preparedness, and comfort with technical skills not encompassed in general surgery programs. Increasing availability of quality global surgery training programs would provide a critical next step toward contributing to the delivery of safe surgical care worldwide., (Copyright © 2020 by the Association of American Medical Colleges.)

Sequential diving by wild marine mammals results in a lifetime of rapid physiological transitions between lung collapse-reinflation, bradycardia-tachycardia, vasoconstriction-vasodilation, and oxygen store depletion-restoration. The result is a cycle of normoxia and hypoxia in which blood oxygen partial pressures can decline to <20-30 mmHg during a dive, a level considered injurious to oxygen-dependent human tissues (i.e., brain, heart). Safeguards in the form of enhanced on-board oxygen stores, selective oxygen transport, and unique tissue buffering capacities enable marine-adapted mammals to maintain physiological homeostasis and energy metabolism even when breathing and pulmonary gas exchange cease. This stands in stark contrast to the vulnerability of oxygen-sensitive tissues in humans that may undergo irreversible damage within minutes of ischemia and tissue hypoxia. Recently, these differences in protection against hypoxic injury have become evident in the systemic, multi-organ physiological failure during COVID-19 infection in humans. Prolonged recoveries in some patients have led to delays in the return to normal exercise levels and cognitive function even months later. Rather than a single solution to this problem, we find that marine mammals rely on a unique, integrative assemblage of protections to avoid the deleterious impacts of hypoxia on tissues. Built across evolutionary time, these solutions provide a natural template for identifying the potential for tissue damage when oxygen is lacking, and for guiding management decisions to support oxygen-deprived tissues in other mammalian species, including humans, challenged by hypoxia., (Copyright © 2020. Published by Elsevier Inc.)

The purpose of this study is to determine the potential for an attached algae flow-way system to efficiently produce algal biomass in estuarine surface waters by utilizing dilute non-point source nutrients from local urban, industrial, and agricultural discharges into the Upper Laguna Madre, Corpus Christi, Texas. The study was conducted over the course of two years to establish seasonal base-line biomass productivity and composition for bioproducts applications, and to identify key environmental factors and flow-way cohorts impacting biomass production. For the entire cultivation period, continuous ash-free biomass production at 4 to 10 g/m 2 /day (corresponding to nutrient recovery at 300 to 500 mg of nitrogen/m 2 /day and 15 to 30 mg of phosphorus/m 2 /day) was successfully achieved without system restart. Upon start-up, a latency period was observed which indicates roles for species succession from relatively low productivity, high ash content pioneer periphytic culture composed primarily of benthic diatoms from the source waters to higher productivity, reduced ash content, and more resilient culture mainly composed of filamentous chlorophyta, Ulva lactuca. Principal Component Analysis (PCA) was used to identify environmental factors driving biomass production, and machine learning (ML) models were constructed to assess the predictive capability of the data set for system performance using the local multi-season environmental variations. Environmental datasets were segregated for ML training, validation, and testing using three methods: regression tree, ensemble regression, and Gaussian process regression (GPR). The predicted ash-free biomass productivity using ML models resulted in root-squared-mean-errors (RSME) from 1.78 to 1.86 g/m 2 /day, and R 2 values from 0.67 to 0.75 using different methods. The greatest contributor to net productivity was total solar irradiation, followed by air temperature, salinity, and pH. The results of the study should be useful as a decision-making tool to application of attached algae flow-ways for biomass production while preventing algal blooms in the environment., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021. Published by Elsevier Ltd.)

We investigated the use of termite baiting, a proven system of targeted colony elimination, in an overall area-wide control strategy against subterranean termites. At two field sites, we used microsatellite markers to estimate the total number of Reticulitermes colonies, their spatial partitioning, and breeding structure. Termite pressure was recorded for two years before and after the introduction of Trelona ® (active ingredient novaluron) to a large area of one of the sites. Roughly 70% of the colonies in the treatment site that were present at the time of baiting were not found in the site within two months after the introduction of novaluron. Feeding activity of the remaining colonies subsequently ceased over time and new invading colonies were unable to establish within this site. Our study provides novel field data on the efficacy of novaluron in colony elimination of Reticulitermes flavipes , as well as evidence that an area-wide baiting program is feasible to maintain a termite-free area within its native range., Competing Interests: The funders had no role in the collection, analyses, or interpretation of data; in the writing of the original manuscript; or in the decision to publish the results.

Investment in healthcare infrastructure in resource-limited settings is a vital and cost-effective approach for diminishing world-wide disease burden, increasing quality of life, and lengthening life expectancy. Graduate medical trainees enthusiastically express interest in supporting global health efforts that expand healthcare access and capacity in resource-limited settings. Academic institutions are responding by developing training programs to equip graduate medical trainees with the technical, interpersonal, scholastic, and ethical skillsets necessary for the pursuit of global health efforts. Drawn from real-world experience and current literature, the following twelve tips will strengthen a global health curriculum in graduate medical training programs with dedicated global health education.

Point-of-care testing (POCT) allows physicians to detect and diagnose diseases at or near the patient site, faster than conventional lab-based testing. The importance of POCT is considerably amplified in the trying times of the COVID-19 pandemic. Numerous point-of-care tests and diagnostic devices are available in the market including, but not limited to, glucose monitoring, pregnancy and infertility testing, infectious disease testing, cholesterol testing and cardiac markers. Integrating microfluidics in POCT allows fluid manipulation and detection in a singular device with minimal sample requirements. This review presents an overview of two technologies - (a.) Lateral Flow Assay (LFA) and (b.) Nucleic Acid Amplification - upon which a large chunk of microfluidic POCT diagnostics is based, some of their applications, and commercially available products. Apart from this, we also delve into other microfluidic-based diagnostics that currently dominate the in-vitro diagnostic (IVD) market, current testing landscape for COVID-19 and prospects of microfluidics in next generation diagnostics., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Sachdeva, Davis and Saha.)

The provision of intestinal stoma care is challenging in austere settings due to limitations in surgical and wound care access as well as the high cost and sparsity of ostomy supplies. As a result, many surgical patients suffer from ostomy-related complications such as peristomal wounds and are unable to find relief for these complications from standard treatments and measures. This article describes the external stoma diversion, a cost-effective palliative surgical procedure that assists in the healing of peristomal wounds in resource-limited settings., (Published by Oxford University Press and JSCR Publishing Ltd. All rights reserved. © The Author(s) 2020.)

Northern elephant seals (NES, Mirounga angustirostris ) undergo an annual molt during which they spend ∼40 days fasting on land with reduced activity and lose approximately one-quarter of their body mass. Reduced activity and muscle load in stereotypic terrestrial mammalian models results in decreased muscle mass and capacity for force production and aerobic metabolism. However, the majority of lost mass in fasting female NES is from fat while muscle mass is largely preserved. Although muscle mass is preserved, potential changes to the metabolic and contractile capacity are unknown. To assess potential changes in NES skeletal muscle during molt, we collected muscle biopsies from 6 adult female NES before the molt and after ∼30 days at the end of the molt. Skeletal muscle was assessed for respiratory capacity using high resolution respirometry, and RNA was extracted to assess changes in gene expression. Despite a month of reduced activity, fasting, and weight loss, skeletal muscle respiratory capacity was preserved with no change in OXPHOS respiratory capacity. Molt was associated with 162 upregulated genes including those favoring lipid metabolism. We identified 172 downregulated genes including those coding for ribosomal proteins and genes associated with skeletal muscle force transduction and glucose metabolism. Following ∼30 days of molt, NES skeletal muscle metabolic capacity is preserved although mechanotransduction may be compromised. In the absence of exercise stimulus, fasting-induced shifts in muscle metabolism may stimulate pathways associated with preserving the mass and metabolic capacity of slow oxidative muscle., (Copyright © 2020 Wright, Davis, Holser, Hückstädt, Danesi, Porter, Widen, Williams, Costa and Sheffield-Moore.)

To achieve the mission of personalized medicine, centering on delivering the right drug to the right patient at the right dose, therapeutic drug monitoring solutions are necessary. In that regard, wearable biosensing technologies, capable of tracking drug pharmacokinetics in noninvasively retrievable biofluids (e.g., sweat), play a critical role, because they can be deployed at a large scale to monitor the individuals' drug transcourse profiles (semi)continuously and longitudinally. To this end, voltammetry-based sensing modalities are suitable, as in principle they can detect and quantify electroactive drugs on the basis of the target's redox signature. However, the target's redox signature in complex biofluid matrices can be confounded by the immediate biofouling effects and distorted/buried by the interfering voltammetric responses of endogenous electroactive species. Here, we devise a wearable voltammetric sensor development strategy-centering on engineering the molecule-surface interactions-to simultaneously mitigate biofouling and create an "undistorted potential window" within which the target drug's voltammetric response is dominant and interference is eliminated. To inform its clinical utility, our strategy was adopted to track the temporal profile of circulating acetaminophen (a widely used analgesic and antipyretic) in saliva and sweat, using a surface-modified boron-doped diamond sensing interface (cross-validated with laboratory-based assays, R 2 ∼ 0.94). Through integration of the engineered sensing interface within a custom-developed smartwatch, and augmentation with a dedicated analytical framework (for redox peak extraction), we realized a wearable solution to seamlessly render drug readouts with minute-level temporal resolution. Leveraging this solution, we demonstrated the pharmacokinetic correlation and significance of sweat readings., Competing Interests: The authors declare no competing interest.

To prepare global surgeons, academic institutions have created training programs that provide opportunities to develop foundational clinical knowledge, pursue academic inquiry, build surgical infrastructure and capacity, and become advocates and collaborators in resource-limited settings. Academic institutions can create a short course in global surgery, global surgery rotation, global surgery fellowship, or integrated global surgery residency. Global surgery training programs must account for ethics of global surgery engagement, sources of funding, structures for professional advancement, and trainee-appropriate partnerships. Global surgery training must include the establishment of accreditation systems, development of integrated training programs, and institutional investment in global surgery education., Competing Interests: Disclosure The authors have nothing to disclose., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Perception of sound is initiated by mechanically gated ion channels at the tips of stereocilia. Mature mammalian auditory hair cells require transmembrane channel-like 1 (TMC1) for mechanotransduction, and mutations of the cognate genetic sequences result in dominant or recessive heritable deafness forms in humans and mice. In contrast, zebrafish lateral line hair cells, which detect water motion, require Tmc2a and Tmc2b. Here, we use standard and multiplex genome editing in conjunction with functional and behavioral assays to determine the reliance of zebrafish hearing and vestibular organs on Tmc proteins. Surprisingly, our approach using multiple mutant alleles demonstrates that hearing in zebrafish is not dependent on Tmc1, nor is it fully dependent on Tmc2a and Tmc2b. Hearing however is absent in triple-mutant zebrafish that lack Tmc1, Tmc2a and Tmc2b. These outcomes reveal a striking resemblance of Tmc protein reliance in the vestibular sensory epithelia of mammals to the maculae of zebrafish. Moreover, our findings disclose a logic of Tmc use where hearing depends on a complement of Tmc proteins beyond those employed to sense water motion., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Red blood cell biomechanics can provide us with a deeper understanding of macroscopic physiology and have the potential of being used for diagnostic purposes. In diseases like sickle cell anemia and malaria, reduced red blood cell deformability can be used as a biomarker, leading to further assays and diagnoses. A microfluidic system is useful for studying these biomechanical properties. We can observe detailed red blood cell mechanical behavior as they flow through microcapillaries using high-speed imaging and microscopy. Microfluidic devices are advantageous over traditional methods because they can serve as high-throughput tests. However, to rapidly analyze thousands of cells, there is a need for powerful image processing tools and software automation. We describe a workflow process using Image-Pro to identify and track red blood cells in a video, take measurements, and export the data for use in statistical analysis tools. The information in this protocol can be applied to large-scale blood studies where entire cell populations need to be analyzed from many cohorts of donors. © 2020 The Authors. Basic Protocol 1: Enhancing raw video for motion tracking Basic Protocol 2: Extracting motion tracking data from enhanced video., (© 2020 The Authors.)

There is a lack of current research to support the efficacy of a combination of equine-assisted activities (EAA) and brain building activities to influence motor skill competencies in youth with neurodevelopmental disorders (ND). The primary objective of this study was to quantify changes in motor skill proficiency before and after 8 weeks of EAA and brain-building activities in youth with ND. A secondary objective was to quantify changes in motor skill proficiency before and after 1 year of EAA and brain-building activities in youth with ND. Twenty-five youth completed the same 32-week protocol that was separated into 4, 8-week blocks, in the following order: (1) control; (2) EAA-only; (3) washout; (4) GaitWay block (EAA and brain building activities). Before and after each block, motor skills were assessed using the Short Form of the Bruininks-Oseretsky Test of Motor Proficiency-Version 2 (BOT-2). Seven youth continued with the GaitWay intervention for one additional year, and the BOT-2 Short Form was also administered following this intervention. A repeated-measures analysis-of-variance was performed to compare BOT-2 subtest and overall scores between interventions with a significance of 0.05. Manual dexterity was higher at Post-Washout [3.3 (2.4)] vs. Pre-Control [2.2 (2.1); p = 0.018] and Post-Control [2.6 (2.0); p = 0.024], and at Post-GaitWay vs. Pre-Control [3.2 (2.4) vs. 2.2 (2.1); p = 0.037]. Upper-limb coordination was higher at Post-GaitWay vs. Post-Control [6.0 (4.1) vs. 3.9 (3.8); p = 0.050]. When compared to Pre-Control [3.2 (3.0)], strength was higher at Post-EAA [4.9 (3.5); p = 0.028] and at Post-GaitWay [5.2 (2.9); p = 0.015]. Overall scores were higher at Post-GaitWay [39.1 (22.2)] when compared to Pre-Control [32.4 (21.6); p = 0.003] and Post-Control [32.5 (21.9); p = 0.009]. Additionally, motor skills were maintained for 1 year following the Post-GaitWay testing session among seven participants. This is the first known study to include and demonstrate the short-term and long-term effects of a combination of EAA and brain building activities with motor proficiency in youth with ND. Clinical Trial Registration: Motor Skill Proficiency After Equine-Assisted Activities and Brain-building Tasks; www.ClinicalTrials.gov, identifier: NCT04158960., (Copyright © 2020 Rigby, Davis, Bittner, Harwell, Leek, Johnson and Nichols.)

Shotgun assays are widely used in biotechnologies to characterize large molecules, which are hard to be measured as a whole directly. For instance, in Liquid Chromatography - Mass Spectrometry (LC-MS) shotgun experiments, proteins in biological samples are digested into peptides, and then peptides are separated and measured. However, in proteomics study, investigators are usually interested in the performance of the whole proteins instead of those peptide fragments. In light of meta-analysis, we propose an adaptive thresholding method to select informative peptides, and combine peptide-level models to protein-level analysis. The meta-analysis procedure and modeling rationale can be adapted to data analysis of other types of shotgun assays.

The use of molecular probe technology is demonstrated for routine identification and tracking of cultured and uncultured microorganisms in an activated sludge bioreactor treating domestic wastewater. A key advantage of molecular probe technology is that it can interrogate hundreds of microbial species of interest in a single measurement. In environmental niches where a single genus (such as Competibacteraceae) dominates, it can be difficult and expensive to identify microorganisms that are present at low relative abundance. With molecular probe technology, it is straightforward. Members of the Competibacteraceae family, none of which have been grown in pure culture, are abundant in an activated sludge system in the San Francisco Bay Area, California, USA. Molecular probe ensembles with and without Competibacteraceae probes were constructed. Whereas the probe ensemble with Competibacteraceae probes identified a total of ten bacteria, the molecular probe ensemble without Competibacteraceae probes identified 29 bacteria, including many at low relative abundance and including some species of public health significance., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Despite poor access to quality surgical and anesthesia care for the majority of the world's people, with greatest impact on low- and middle-income countries, surgery has only recently begun to gain acceptance as a necessary component of global health. As a leader in global surgical funding, the field of Plastic and Reconstructive Surgery is uniquely positioned to influence change in global policy and financial support. For improvements in surgical access and outcomes worldwide, investment in surgical systems, commitment to national surgery, obstetric, and anesthesia planning, and continued evaluation and improvement of care delivery should be pursued., (Copyright © 2019 The Authors. Published by Wolters Kluwer Health, Inc. on behalf of The American Society of Plastic Surgeons.)

Glial cell line-derived neurotrophic factor (GDNF) improved motor function in Parkinson's disease (PD) patients in Phase I clinical trials, and these effects persisted months after GDNF discontinuation. Conversely, phase II clinical trials reported no significant effects on motor improvement vs placebo. The disease duration and the quantity, infusion approach, and duration of GDNF delivery may affect GDNF efficacy in PD treatment. However, identifying mechanisms activated by GDNF that affect nigrostriatal function may reveal additional avenues to partially restore nigrostriatal function. In PD and aging models, GDNF affects tyrosine hydroxylase (TH) expression or phosphorylation in substantia nigra (SN), long after a single GDNF injection in striatum. In aged rats, the GDNF family receptor, GFR-α1, increases TH expression and phosphorylation in SN. To determine if GFR-α1 could be a mechanistic link in long-term GDNF impact, we conducted two studies; first to determine if a single unilateral striatal delivery of GDNF affected GFR-α1 and TH over time (1 day, 1 week, and 4 weeks) in the striatum or SN in aged rats, and second, to determine if soluble GFR-α1 could mitigate TH loss following 6-hydroxydopamine (6-OHDA) lesion. In aged rats, GDNF bilaterally increased ser31 TH phosphorylation and GFR-α1 expression in SN at 1 day and 4 weeks after GDNF, respectively. In striatum, GFR-α1 expression decreased 1 week after GDNF, only on the GDNF-injected side. In 6-OHDA-lesioned rats, recombinant soluble GFR-α1 mitigated nigral, but not striatal, TH protein loss following 6-OHDA. Together, these results show GDNF has immediate and long-term impact on dopamine regulation in the SN, which includes a gradual increase in GFR-α1 expression that may sustain TH expression and dopamine function therein.

Distiller's grains are a byproduct of corn ethanol production and provide an opportunity for increasing the economic viability and sustainability of the overall grain-to-fuels process. Typically, these grains are dried and sold as a ruminant feed adjunct. This study considers utilization of the residuals in a novel supplementary fermentation process to produce two products, enriched protein and fusel alcohols. The value-added proposition and environmental impact of this second fermentation step for distiller's grains are evaluated by considering three different processing scenarios. Techno-economic results show the minimum protein selling price, assuming fusel alcohol products are valued at $0.79 per liter gasoline equivalent, ranges between $1.65-$2.48 kg protein -1 for the different cases. Environmental impacts of the systems were evaluated through life cycle assessment. Results show a baseline emission results of 17 g CO 2-eq (MJ fuel) -1 for the fuel product and 10.3 kg CO 2-eq kg protein -1 for the protein product. Sensitivity to allocation methods show a dramatic impact with results ranging between -8 to 140 g CO 2-eq (MJ fuel) -1 for the fuel product and -0.3 to 6.4 kg CO 2-eq kg protein -1 for the protein product. The discussion is focused on the potential impact of the technology on corn ethanol production economics and sustainability.

Metal-Organic Frameworks (MOFs) that catalyze hydrogenolysis reactions are rare and there is little understanding of how the MOF, hydrogen, and substrate molecules interact. In this regard, the isoreticular IRMOF-74 series, two of which are known catalysts for hydrogenolysis of aromatic C-O bonds, provides an unusual opportunity for systematic probing of these reactions. The diameter of the 1D open channels can be varied within a common topology owing to the common secondary building unit (SBU) and controllable length of the hydroxy-carboxylate struts. We show that the first four members of the IRMOF-74(Mg) series are inherently catalytic for aromatic C-O bond hydrogenolysis and that the conversion varies non-monotonically with pore size. These catalysts are recyclable and reusable, retaining their crystallinity and framework structure after the hydrogenolysis reaction. The hydrogenolysis conversion of phenylethylphenyl ether (PPE), benzylphenyl ether (BPE), and diphenyl ether (DPE) varies as PPE > BPE > DPE, consistent with the strength of the C-O bond. Counterintuitively, however, the conversion also follows the trend IRMOF-74(III) > IRMOF-74(IV) > IRMOF-74(II) > IRMOF-74(I), with little variation in the corresponding selectivity. DFT calculations suggest the unexpected behavior is due to much stronger ether and phenol binding to the Mg(ii) open metal sites (OMS) of IRMOF-74(III), resulting from a structural distortion that moves the Mg 2+ ions toward the interior of the pore. Solid-state 25 Mg NMR data indicate that both H 2 and ether molecules interact with the Mg(ii) OMS and hydrogen-deuterium exchange reactions show that these MOFs activate dihydrogen bonds. The results suggest that both confinement and the presence of reactive metals are essential for achieving the high catalytic activity, but that subtle variations in pore structure can significantly affect the catalysis. Moreover, they challenge the notion that simply increasing MOF pore size within a constant topology will lead to higher conversions., (This journal is © The Royal Society of Chemistry 2019.)

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a debilitating noncommunicable disease brandishing an enormous worldwide disease burden with some evidence of inherited genetic risk. Absence of measurable changes in patients' standard blood work has necessitated ad hoc symptom-driven therapies and a dearth of mechanistic hypotheses regarding its etiology and possible cure. A new hypothesis, the indolamine-2,3-dioxygenase (IDO) metabolic trap, was developed and formulated as a mathematical model. The historical occurrence of ME/CFS outbreaks is a singular feature of the disease and implies that any predisposing genetic mutation must be common. A database search for common damaging mutations in human enzymes produces 208 hits, including IDO2 with four such mutations. Non-functional IDO2, combined with well-established substrate inhibition of IDO1 and kinetic asymmetry of the large neutral amino acid transporter, LAT1, yielded a mathematical model of tryptophan metabolism that displays both physiological and pathological steady-states. Escape from the pathological one requires an exogenous perturbation. This model also identifies a critical point in cytosolic tryptophan abundance beyond which descent into the pathological steady-state is inevitable. If, however, means can be discovered to return cytosolic tryptophan below the critical point, return to the normal physiological steady-state is assured. Testing this hypothesis for any cell type requires only labelled tryptophan, a means to measure cytosolic tryptophan and kynurenine, and the standard tools of tracer kinetics.

Improving the economic feasibility is necessary for algae-based processes to achieve commercial scales for biofuels and bioproducts production. A closed-loop system for fusel alcohol production from microalgae biomass with integrated nutrient recycling was developed, which enables the reuse of nitrogen and phosphorus for downstream application and thus reduces the operational requirement for external major nutrients. Mixed fusel alcohols, primarily isobutanol and isopentanol were produced from Microchloropsis salina hydrolysates by an engineered E. coli co-culture. During the process, cellular nitrogen from microalgae biomass was converted into ammonium, whereas cellular phosphorus was liberated by an osmotic shock treatment. The formation of struvite from the liberated ammonium and phosphate, and the subsequent utilization of struvite to support M. salina cultivation was demonstrated. The closed loop system established here should help overcome one of the identified economic barriers to scale-up of microalgae production, and enhance the sustainability of microalgae-based chemical commodities production., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Purpose: Improved second-tier tools are needed to reduce false-positive outcomes in newborn screening (NBS) for inborn metabolic disorders on the Recommended Universal Screening Panel (RUSP)., Methods: We designed an assay for multiplex sequencing of 72 metabolic genes (RUSPseq) from newborn dried blood spots. Analytical and clinical performance was evaluated in 60 screen-positive newborns for methylmalonic acidemia (MMA) reported by the California Department of Public Health NBS program. Additionally, we trained a Random Forest machine learning classifier on NBS data to improve prediction of true and false-positive MMA cases., Results: Of 28 MMA patients sequenced, we found two pathogenic or likely pathogenic (P/LP) variants in a MMA-related gene in 24 patients, and one pathogenic variant and a variant of unknown significance (VUS) in 1 patient. No such variant combinations were detected in MMA false positives and healthy controls. Random Forest-based analysis of the entire NBS metabolic profile correctly identified the MMA patients and reduced MMA false-positive cases by 51%. MMA screen-positive newborns were more likely of Hispanic ethnicity., Conclusion: Our two-pronged approach reduced false positives by half and provided a reportable molecular finding for 89% of MMA patients. Challenges remain in newborn metabolic screening and DNA variant interpretation in diverse multiethnic populations.

Background: Due to their high energy density and compatible physical properties, several monoterpenes have been investigated as potential renewable transportation fuels, either as blendstocks with petroleum or as drop-in replacements for use in vehicles (both heavy and light-weight) or in aviation. Sustainable microbial production of these biofuels requires the ability to utilize cheap and readily available feedstocks such as lignocellulosic biomass, which can be depolymerized into fermentable carbon sources such as glucose and xylose. However, common microbial production platforms such as the yeast Saccharomyces cerevisiae are not naturally capable of utilizing xylose, hence requiring extensive strain engineering and optimization to efficiently utilize lignocellulosic feedstocks. In contrast, the oleaginous red yeast Rhodosporidium toruloides is capable of efficiently metabolizing both xylose and glucose, suggesting that it may be a suitable host for the production of lignocellulosic bioproducts. In addition, R. toruloides naturally produces several carotenoids (C40 terpenoids), indicating that it may have a naturally high carbon flux through its mevalonate (MVA) pathway, providing pools of intermediates for the production of a wide range of heterologous terpene-based biofuels and bioproducts from lignocellulose., Results: Sixteen terpene synthases (TS) originating from plants, bacteria and fungi were evaluated for their ability to produce a total of nine different monoterpenes in R. toruloides. Eight of these TS were functional and produced several different monoterpenes, either as individual compounds or as mixtures, with 1,8-cineole, sabinene, ocimene, pinene, limonene, and carene being produced at the highest levels. The 1,8-cineole synthase HYP3 from Hypoxylon sp. E74060B produced the highest titer of 14.94 ± 1.84 mg/L 1,8-cineole in YPD medium and was selected for further optimization and fuel properties study. Production of 1,8-cineole from lignocellulose was also demonstrated in a 2L batch fermentation, and cineole production titers reached 34.6 mg/L in DMR-EH (Deacetylated, Mechanically Refined, Enzymatically Hydorlized) hydrolysate. Finally, the fuel properties of 1,8-cineole were examined, and indicate that it may be a suitable petroleum blend stock or drop-in replacement fuel for spark ignition engines., Conclusion: Our results demonstrate that Rhodosporidium toruloides is a suitable microbial platform for the production of non-native monoterpenes with biofuel applications from lignocellulosic biomass.

Multidrug resistance is highly conserved in mammalian, fungal, and bacterial cells, is characterized by resistance to several unrelated xenobiotics, and poses significant challenges to managing infections and many cancers. Eukaryotes use a highly conserved set of drug efflux transporters that confer pleiotropic drug resistance (PDR). To interrogate the regulation of this critical process, here we developed a small molecule-responsive biosensor that couples transcriptional induction of PDR genes to growth rate in the yeast Saccharomyces cerevisiae Using diverse PDR inducers and the homozygous diploid deletion collection, we applied this biosensor system to genome-wide screens for potential PDR regulators. In addition to recapitulating the activity of previously known factors, these screens identified a series of genes involved in a variety of cellular processes with significant but previously uncharacterized roles in the modulation of yeast PDR. Genes identified as down-regulators of the PDR included those encoding the MAD family of proteins involved in the mitotic spindle assembly checkpoint (SAC) complex. Of note, we demonstrated that genetic disruptions of the mitotic spindle assembly checkpoint elevate expression of PDR-mediating efflux pumps in response to exposure to a variety of compounds that themselves have no known influence on the cell cycle. These results not only establish our biosensor system as a viable tool for investigating PDR in a high-throughput fashion, but also uncover critical control mechanisms governing the PDR response and a previously uncharacterized link between PDR and cell cycle regulation in yeast., (© 2019 Li et al.)

Inflammatory cells, most especially neutrophils, can be a necessary component of the antitumor activity occurring after administration of photodynamic therapy. Generation of neutrophil responses has been suggested to be particularly important in instances when the delivered photodynamic therapy (PDT) dose is insufficient. In these cases, the release of neutrophil granules and engagement of antitumor immunity may play an important role in eliminating residual disease. Herein, we utilize in vivo imaging of luminol chemiluminescence to noninvasively monitor neutrophil activation after PDT administration. Studies were performed in the AB12 murine model of mesothelioma, treated with Photofrin-PDT. Luminol-generated chemiluminescence increased transiently 1 h after PDT, followed by a subsequent decrease at 4 h after PDT. The production of luminol signal was not associated with the influx of Ly6G + cells, but was related to oxidative burst, as an indicator of neutrophil function. Most importantly, greater levels of luminol chemiluminescence 1 h after PDT were prognostic of a complete response at 90 days after PDT. Taken together, this research supports an important role for early activity by Ly6G + cells in the generation of long-term PDT responses in mesothelioma, and it points to luminol chemiluminescence as a potentially useful approach for preclinical monitoring of neutrophil activation by PDT., (© 2018 The American Society of Photobiology.)

Background: Myalgic encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a poorly understood disease. Amongst others symptoms, the disease is associated with profound fatigue, cognitive dysfunction, sleep abnormalities, and other symptoms that are made worse by physical or mental exertion. While the etiology of the disease is still debated, evidence suggests oxidative damage to immune and hematological systems as one of the pathophysiological mechanisms of the disease. Since red blood cells (RBCs) are well-known scavengers of oxidative stress, and are critical in microvascular perfusion and tissue oxygenation, we hypothesized that RBC deformability is adversely affected in ME/CFS., Methods: We used a custom microfluidic platform and high-speed microscopy to assess the difference in deformability of RBCs obtained from ME/CFS patients and age-matched healthy controls., Results and Conclusion: We observed from various measures of deformability that the RBCs isolated from ME/CFS patients were significantly stiffer than those from healthy controls. Our observations suggest that RBC transport through microcapillaries may explain, at least in part, the ME/CFS phenotype, and promises to be a novel first-pass diagnostic test.

Linoleate triol esters are intermediates along the pathway of formation of the mammalian skin permeability barrier. In connection with the study of their involvement in barrier formation we required access to isomerically pure and defined samples of four linoleate triol esters. A common synthetic strategy was developed starting from isomeric alkynols derived from d-tartaric acid and 2-deoxy-d-ribose.

Microbial production of fuels and commodity chemicals has been performed primarily using natural or slightly modified enzymes, which inherently limits the types of molecules that can be produced. Type I modular polyketide synthases (PKSs) are multi-domain enzymes that can produce unique and diverse molecular structures by combining particular types of catalytic domains in a specific order. This catalytic mechanism offers a wealth of engineering opportunities. Here we report engineered microbes that produce various short-chain (C5-C7) ketones using hybrid PKSs. Introduction of the genes into the chromosome of Streptomyces albus enables it to produce >1 g · l -1 of C6 and C7 ethyl ketones and several hundred mg · l -1 of C5 and C6 methyl ketones from plant biomass hydrolysates. Engine tests indicate these short-chain ketones can be added to gasoline as oxygenates to increase the octane of gasoline. Together, it demonstrates the efficient and renewable microbial production of biogasolines by hybrid enzymes.

The purpose of this study was to compare acute cardiometabolic responses to 3 modes of treadmill exercise in adults diagnosed with Parkinson's disease (PD). Eight elderly adults with PD (67.9 ± 3.0 yr) completed 1 session each on a land, aquatic, and antigravity treadmill at 50% body weight. Participants walked from 1 to 3 mph in 0.5-mph increments at 0% grade for 5 min at each speed. Heart rate, energy expenditure, blood pressure, and rating of perceived exertion were measured at rest and during exercise. All variables except diastolic blood pressure increased with speed on all treadmills (p < .001). At all speeds except 1.5 mph, heart rate was higher on the land treadmill than the antigravity treadmill (p < .05). Exercising on an aquatic or antigravity treadmill elicits similar submaximal physiologic responses to exercise on a land treadmill in adults with PD.

Objective: Inflammation is a well-known consequence of surgery. Although surgical debulking of tumor is beneficial to patients, the onset of inflammation in injured tissue may impede the success of adjuvant therapies. One marker for postoperative inflammation is IL-6, which is released as a consequence of surgical injuries. IL-6 is predictive of response to many cancer therapies, and it is linked to various molecular and cellular resistance mechanisms. The purpose of this study was to establish a murine model by which therapeutic responses to photodynamic therapy (PDT) can be studied in the context of surgical inflammation., Materials and Methods: Murine models with AB12 mesothelioma tumors were treated with either surgical resection or sham surgery with tumor incision but no resection. The timing and extent of IL-6 release in the tumor and/or serum was measured using enzyme-linked immunosorbent assay (ELISA) and compared to that measured in the serum of 27 consecutive, prospectively enrolled patients with malignant pleural mesothelioma (MPM) who underwent macroscopic complete resection (MCR)., Results: MPM patients showed a significant increase in IL-6 at the time MCR was completed. Similarly, IL-6 increased in the tumor and serum of mice treated with surgical resections. However, investigations that combine resection with another therapy make it necessary to grow tumors for resection to a larger volume than those that receive secondary therapy alone. As the larger size may alter tumor biology independent of the effects of surgical injury, we assessed the tumor incision model. In this model, tumor levels of IL-6 significantly increased after tumor incision., Conclusion: The tumor incision model induces IL-6 release as is seen in the surgical setting, yet it avoids the limitations of surgical resection models. Potential mechanisms by which surgical induction of inflammation and IL-6 could alter the nature and efficacy of tumor response to PDT are reviewed. These include a wide spectrum of molecular and cellular mechanisms through which surgically-induced IL-6 could change the effectiveness of therapies that are combined with surgery. The tumor incision model can be employed for novel investigations of the effects of surgically-induced, acute inflammation on therapeutic response to PDT (or potentially other therapies). Lasers Surg. Med. 50:440-450, 2018. © 2018 Wiley Periodicals, Inc., (© 2018 Wiley Periodicals, Inc.)

Our understanding of how genotype controls phenotype is limited by the scale at which we can precisely alter the genome and assess the phenotypic consequences of each perturbation. Here we describe a CRISPR-Cas9-based method for multiplexed accurate genome editing with short, trackable, integrated cellular barcodes (MAGESTIC) in Saccharomyces cerevisiae. MAGESTIC uses array-synthesized guide-donor oligos for plasmid-based high-throughput editing and features genomic barcode integration to prevent plasmid barcode loss and to enable robust phenotyping. We demonstrate that editing efficiency can be increased more than fivefold by recruiting donor DNA to the site of breaks using the LexA-Fkh1p fusion protein. We performed saturation editing of the essential gene SEC14 and identified amino acids critical for chemical inhibition of lipid signaling. We also constructed thousands of natural genetic variants, characterized guide mismatch tolerance at the genome scale, and ascertained that cryptic Pol III termination elements substantially reduce guide efficacy. MAGESTIC will be broadly useful to uncover the genetic basis of phenotypes in yeast.

Background: Engineering strategies to create promoters that are both higher strength and tunable in the presence of inexpensive compounds are of high importance to develop metabolic engineering technologies that can be commercialized. Lignocellulosic biomass stands out as the most abundant renewable feedstock for the production of biofuels and chemicals. However, lignin a major polymeric component of the biomass is made up of aromatic units and remains as an untapped resource. Novel synthetic biology tools for the expression of heterologous proteins are critical for the effective engineering of a microbe to valorize lignin. This study demonstrates the first successful attempt in the creation of engineered promoters that can be induced by aromatics present in lignocellulosic hydrolysates to increase heterologous protein production., Results: A hybrid promoter engineering approach was utilized for the construction of phenolic-inducible promoters of higher strength. The hybrid promoters were constructed by replacing the spacer region of an endogenous promoter, P emrR present in E. coli that was naturally inducible by phenolics. In the presence of vanillin, the engineered promoters P vtac , P vtrc , and P vtic increased protein expression by 4.6-, 3.0-, and 1.5-fold, respectively, in comparison with a native promoter, P emrR . In the presence of vanillic acid, P vtac , P vtrc , and P vtic improved protein expression by 9.5-, 6.8-, and 2.1-fold, respectively, in comparison with P emrR . Among the cells induced with vanillin, the emergence of a sub-population constituting the healthy and dividing cells using flow cytometry was observed. The analysis also revealed this smaller sub-population to be the primary contributor for the increased expression that was observed with the engineered promoters., Conclusions: This study demonstrates the first successful attempt in the creation of engineered promoters that can be induced by aromatics to increase heterologous protein production. Employing promoters inducible by phenolics will provide the following advantages: (1) develop substrate inducible systems; (2) lower operating costs by replacing expensive IPTG currently used for induction; (3) develop dynamic regulatory systems; and (4) provide flexibility in operating conditions. The flow cytometry findings strongly suggest the need for novel approaches to maintain a healthy cell population in the presence of phenolics to achieve increased heterologous protein expression and, thereby, valorize lignin efficiently.

For decades, fungi have been a source of U.S. Food and Drug Administration-approved natural products such as penicillin, cyclosporine, and the statins. Recent breakthroughs in DNA sequencing suggest that millions of fungal species exist on Earth, with each genome encoding pathways capable of generating as many as dozens of natural products. However, the majority of encoded molecules are difficult or impossible to access because the organisms are uncultivable or the genes are transcriptionally silent. To overcome this bottleneck in natural product discovery, we developed the HEx (Heterologous EXpression) synthetic biology platform for rapid, scalable expression of fungal biosynthetic genes and their encoded metabolites in Saccharomyces cerevisiae . We applied this platform to 41 fungal biosynthetic gene clusters from diverse fungal species from around the world, 22 of which produced detectable compounds. These included novel compounds with unexpected biosynthetic origins, particularly from poorly studied species. This result establishes the HEx platform for rapid discovery of natural products from any fungal species, even those that are uncultivable, and opens the door to discovery of the next generation of natural products.

Lipoxygenase (LOX)-catalyzed oxidation of the essential fatty acid, linoleate, represents a vital step in construction of the mammalian epidermal permeability barrier. Analysis of epidermal lipids indicates that linoleate is converted to a trihydroxy derivative by hydrolysis of an epoxy-hydroxy precursor. We evaluated different epoxide hydrolase (EH) enzymes in the hydrolysis of skin-relevant fatty acid epoxides and compared the products to those of acid-catalyzed hydrolysis. In the absence of enzyme, exposure to pH 5 or pH 6 at 37°C for 30 min hydrolyzed fatty acid allylic epoxyalcohols to four trihydroxy products. By contrast, human soluble EH [sEH (EPHX2)] and human or murine epoxide hydrolase-3 [EH3 (EPHX3)] hydrolyzed cis or trans allylic epoxides to single diastereomers, identical to the major isomers detected in epidermis. Microsomal EH [mEH (EPHX1)] was inactive with these substrates. At low substrate concentrations (<10 μM), EPHX2 hydrolyzed 14,15-epoxyeicosatrienoic acid (EET) at twice the rate of the epidermal epoxyalcohol, 9 R ,10 R - trans -epoxy-11 E -13 R -hydroxy-octadecenoic acid, whereas human or murine EPHX3 hydrolyzed the allylic epoxyalcohol at 31-fold and 39-fold higher rates, respectively. These data implicate the activities of EPHX2 and EPHX3 in production of the linoleate triols detected as end products of the 12 R -LOX pathway in the epidermis and implicate their functioning in formation of the mammalian water permeability barrier., (Copyright © 2018 by the American Society for Biochemistry and Molecular Biology, Inc.)

There is a growing demand for sustainable methods in research and development, where instead of hazardous chemicals, an aqueous medium is chosen to perform biological reactions. In this Perspective, we examine the history and current methodology of using enzymes to generate artificial single-stranded DNA. By using traditional solid-phase phosphoramidite chemistry as a metric, we also explore criteria for the method of template-independent enzymatic oligonucleotide synthesis (TiEOS). As its key component, we delve into the biology of one of the most enigmatic enzymes, terminal deoxynucleotidyl transferase (TdT). As TdT is found to exponentially increase antigen receptor diversity in the vertebrate immune system by adding nucleotides in a template-free manner, researchers have exploited this function as an alternative to the phosphoramidite synthesis method. Though TdT is currently the preferred enzyme for TiEOS, its random nucleotide incorporation presents a barrier in synthesis automation. Taking a closer look at the TiEOS cycle, particularly the coupling step, we find it is comprised of additions > n+1 and deletions. By tapping into the physical and biochemical properties of TdT, we strive to further elucidate its mercurial behavior and offer ways to better optimize TiEOS for production-grade oligonucleotide synthesis.

Background: Viral infections are a major cause of complications and death in solid organ and hematopoietic cell transplantation., Methods: We developed a multiplex viral sequencing assay (mVseq) to simultaneously detect 20 transplant-relevant DNA viruses from small clinical samples. The assay uses a single-tube multiplex PCR to amplify highly conserved virus genomic regions without the need for previous virus enrichment or host nucleic acid subtraction. Multiplex sample sequencing was performed using Illumina MiSeq, and reads were aligned to a database of target sequences. Analytical and clinical performance was evaluated using reference viruses spiked into human plasma, as well as patient plasma and nonplasma samples, including bronchoalveolar lavage fluid, cerebrospinal fluid, urine, and tissue from immunocompromised transplant recipients., Results: For the virus spike-in samples, mVseq's analytical sensitivity and dynamic range were similar to quantitative PCR (qPCR). In clinical specimens, mVseq showed substantial agreement with single-target qPCR (92%; k statistic, 0.77; 259 of 282 viral tests); however, clinical sensitivity was reduced (81%), ranging from 62% to 100% for specific viruses. In 12 of the 47 patients tested, mVseq identified previously unknown BK virus, human herpesvirus-7, and Epstein-Barr virus infections that were confirmed by qPCR., Conclusions: Our results reveal factors that can influence clinical sensitivity, such as high levels of host DNA background and loss of detection in coinfections when 1 virus was at much higher concentration than the others. The mVseq assay is flexible and scalable to incorporate RNA viruses, emerging viruses of interest, and other pathogens important in transplant recipients.

Purpose: This study explored the effect of whole-body vibration (WBV) using accelerations of 2.56 g to 7.68 g on lower-body detraining., Methods: All participants (N = 20) were trained using a lower-body resistance-training program for 30 min twice per week from Week 0 to Week 6. At the end of the program, they were randomly assigned to a control group that performed no further training or a WBV group that performed a progressive static WBV program. Data for the 5-repetition-maximum (5RM) squat and extensors and flexors of the knee and ankle were collected at Weeks 0, 6, 8, 10, and 12 for all participants., Results: Two-way (condition vs. time) analysis of variance revealed that although the WBV group maintained strength in the 5RM from Week 6 through Week 8 and the control group had a lower 5RM in Week 8 from Week 6, no differences in the 5RM squat existed between the groups at Week 8. Two-way factorial multivariate analysis of variance revealed no differences between the groups at any of the time for torque of knee flexion, dorsiflexion, or plantar flexion., Conclusion: Static WBV of 2.56 g to 7.68 g did not attenuate detraining of the flexors and extensors of the knee and ankle.

Proximity ligation assay (PLA) is a powerful tool for quantitative detection of protein biomarkers in biological fluids and tissues. Here, we present the circular proximity ligation assay (c-PLA), a highly specific protein detection method that outperforms traditional PLA in stringency, ease of use, and compatibility with low-affinity reagents. In c-PLA, two proximity probes bind to an analyte, providing a scaffolding that positions two free oligonucleotides such that they can be ligated into a circular DNA molecule. This assay format stabilizes antigen proximity probe complexes and enhances stringency by reducing the probability of random background ligation events. Circle formation also increases selectivity, since the uncircularized DNA can be removed enzymatically. We compare this method with traditional PLA on several biomarkers and show that the higher stringency for c-PLA improves reproducibility and enhances sensitivity in both buffer and human plasma. The limit of detection ranges from femtomolar to nanomolar concentrations for both methods. Kinetic analyses using surface plasmon resonance (SPR) and biolayer interferometry (BLI) reveal that the variation in limit of detection is due to the variation in antibody affinity and that c-PLA outperforms traditional PLA for low-affinity antibodies. The lower background signal can be used to increase proximity probe concentration while maintaining a high signal-to-noise ratio, thereby enabling the use of low-affinity reagents in a homogeneous assay format. We anticipate that the advantages of c-PLA will be useful in a variety of clinical protein detection applications where high-affinity reagents are lacking., Competing Interests: Conflict of interest statement: S.F. owns stock in a company (Olink AB) with patents on the core technology described, "Proximity Ligation Assay."

To shift the world to a more sustainable future, it is necessary to phase out the use of fossil fuels and focus on the development of low-carbon alternatives. However, this transition has been slow, so there is still a large dependence on fossil-derived power, and therefore, carbon dioxide is released continuously. Owing to the potential for assimilating and utilizing carbon dioxide to generate carbon-neutral products, such as biodiesel, the application of microalgae technology to capture CO 2 from flue gases has gained significant attention over the past decade. Microalgae offer a more sustainable source of biomass, which can be converted into energy, over conventional fuel crops because they grow more quickly and do not adversely affect the food supply. This review focuses on the technical feasibility of combined carbon fixation and microalgae cultivation for carbon reuse. A range of different carbon metabolisms and the impact of flue gas compounds on microalgae are appraised. Fixation of flue gas carbon dioxide is dependent on the selected microalgae strain and on flue gas compounds/concentrations. Additionally, current pilot-scale demonstrations of microalgae technology for carbon dioxide capture are assessed and its future prospects are discussed. Practical implementation of this technology at an industrial scale still requires significant research, which necessitates multidisciplinary research and development to demonstrate its viability for carbon dioxide capture from flue gases at the commercial level., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Recent studies have revealed that caryophyllene and its stereoisomers not only exhibit multiple biological activities but also have desired properties as renewable candidates for ground transportation and jet fuel applications. This study presents the first significant production of caryophyllene and caryolan-1-ol by an engineered E. coli with heterologous expression of mevalonate pathway genes with a caryophyllene synthase and a caryolan-1-ol synthase. By optimizing metabolic flux and fermentation parameters, the engineered strains yielded 449 mg/L of total terpene, including 406 mg/L sesquiterpene with 100 mg/L caryophyllene and 10 mg/L caryolan-1-ol. Furthermore, a marine microalgae hydrolysate was used as the sole carbon source for the production of caryophyllene and other terpene compounds. Under the optimal fermentation conditions, 360 mg/L of total terpene, 322 mg/L of sesquiterpene, and 75 mg/L caryophyllene were obtained from the pretreated algae hydrolysates. The highest yields achieved on the biomass basis were 48 mg total terpene/g algae and 10 mg caryophyllene/g algae and the caryophyllene yield is approximately ten times higher than that from plant tissues by solvent extraction. The study provides a sustainable alternative for production of caryophyllene and its alcohol from microalgae biomass as potential candidates for next generation aviation fuels.