Metabolic engineering of Caldicellulosiruptor bescii for 2,3-butanediol production from unpretreated lignocellulosic biomass and metabolic strategies for improving yields and titers.

The platform chemical 2,3-butanediol (2,3-BDO) is used to derive products, such as 1,3-butadiene and methyl ethyl ketone, for the chemical and fuel production industries. Efficient microbial 2,3-BDO production at industrial scales has not been achieved yet for various reasons, including product inhibition to host organisms, mixed stereospecificity in product formation, and dependence on expensive substrates (i.e., glucose). In this study, we explore engineering of a 2,3-BDO pathway in Caldicellulosiruptor bescii, an extremely thermophilic (optimal growth temperature = 78°C) and anaerobic bacterium that can break down crystalline cellulose and hemicellulose into fermentable C5 and C6 sugars. In addition, C. bescii grows on unpretreated plant biomass, such as switchgrass. Biosynthesis of 2,3-BDO involves three steps: two molecules of pyruvate are condensed into acetolactate; acetolactate is decarboxylated to acetoin, and finally, acetoin is reduced to 2,3-BDO. C. bescii natively produces acetoin; therefore, in order to complete the 2,3-BDO biosynthetic pathway, C. bescii was engineered to produce a secondary alcohol dehydrogenase (sADH) to catalyze the final step. Two previously characterized, thermostable sADH enzymes with high affinity for acetoin, one from a bacterium and one from an archaeon, were tested independently. When either sADH was present in C. bescii, the recombinant strains were able to produce up to 2.5-mM 2,3-BDO from crystalline cellulose and xylan and 0.2-mM 2,3-BDO directly from unpretreated switchgrass. This serves as the basis for higher yields and productivities, and to this end, limiting factors and potential genetic targets for further optimization were assessed using the genome-scale metabolic model of C. bescii.IMPORTANCELignocellulosic plant biomass as the substrate for microbial synthesis of 2,3-butanediol is one of the major keys toward cost-effective bio-based production of this chemical at an industrial scale. However, deconstruction of biomass to release the sugars for microbial growth currently requires expensive thermochemical and enzymatic pretreatments. In this study, the thermo-cellulolytic bacterium Caldicellulosiruptor bescii was successfully engineered to produce 2,3-butanediol from cellulose, xylan, and directly from unpretreated switchgrass. Genome-scale metabolic modeling of C. bescii was applied to adjust carbon and redox fluxes to maximize productivity of 2,3-butanediol, thereby revealing bottlenecks that require genetic modifications.

Optimizing Strategies for Bio-Based Ethanol Production Using Genome-Scale Metabolic Modeling of the Hyperthermophilic Archaeon, Pyrococcus furiosus.

A genome-scale metabolic model, encompassing a total of 623 genes, 727 reactions, and 865 metabolites, was developed for Pyrococcus furiosus, an archaeon that grows optimally at 100°C by carbohydrate and peptide fermentation. The model uses subsystem-based genome annotation, along with extensive manual curation of 237 gene-reaction associations including those involved in central carbon metabolism, amino acid metabolism, and energy metabolism. The redox and energy balance of P. furiosus was investigated through random sampling of flux distributions in the model during growth on disaccharides. The core energy balance of the model was shown to depend on high acetate production and the coupling of a sodium-dependent ATP synthase and membrane-bound hydrogenase, which generates a sodium gradient in a ferredoxin-dependent manner, aligning with existing understanding of P. furiosus metabolism. The model was utilized to inform genetic engineering designs that favor the production of ethanol over acetate by implementing an NADPH and CO-dependent energy economy. The P. furiosus model is a powerful tool for understanding the relationship between generation of end products and redox/energy balance at a systems-level that will aid in the design of optimal engineering strategies for production of bio-based chemicals and fuels. IMPORTANCE The bio-based production of organic chemicals provides a sustainable alternative to fossil-based production in the face of today's climate challenges. In this work, we present a genome-scale metabolic reconstruction of Pyrococcus furiosus, a well-established platform organism that has been engineered to produce a variety of chemicals and fuels. The metabolic model was used to design optimal engineering strategies to produce ethanol. The redox and energy balance of P. furiosus was examined in detail, which provided useful insights that will guide future engineering designs.

Manipulating Fermentation Pathways in the Hyperthermophilic Archaeon Pyrococcus furiosus for Ethanol Production up to 95°C Driven by Carbon Monoxide Oxidation.

Genetic engineering of hyperthermophilic organisms for the production of fuels and other useful chemicals is an emerging biotechnological opportunity. In particular, for volatile organic compounds such as ethanol, fermentation at high temperatures could allow for straightforward separation by direct distillation. Currently, the upper growth temperature limit for native ethanol producers is 72°C in the bacterium Thermoanaerobacter ethanolicus JW200, and the highest temperature for heterologously-engineered bioethanol production was recently demonstrated at 85°C in the archaeon Pyrococcus furiosus. Here, we describe an engineered strain of P. furiosus that synthesizes ethanol at 95°C, utilizing a homologously-expressed native alcohol dehydrogenase, termed AdhF. Ethanol biosynthesis was compared at 75°C and 95°C with various engineered strains. At lower temperatures, the acetaldehyde substrate for AdhF is most likely produced from acetate by aldehyde ferredoxin oxidoreductase (AOR). At higher temperatures, the effect of AOR on ethanol production is negligible, suggesting that acetaldehyde is produced by pyruvate ferredoxin oxidoreductase (POR) via oxidative decarboxylation of pyruvate, a reaction known to occur only at higher temperatures. Heterologous expression of a carbon monoxide dehydrogenase complex in the AdhF overexpression strain enabled it to use CO as a source of energy, leading to increased ethanol production. A genome reconstruction model for P. furiosus was developed to guide metabolic engineering strategies and understand outcomes. This work opens the door to the potential for 'bioreactive distillation' since fermentation can be performed well above the normal boiling point of ethanol. IMPORTANCE Previously, the highest temperature for biological ethanol production was 85°C. Here, we have engineered ethanol production at 95°C by the hyperthermophilic archaeon Pyrococcus furiosus. Using mutant strains, we showed that ethanol production occurs by different pathways at 75°C and 95°C. In addition, by heterologous expression of a carbon monoxide dehydrogenase complex, ethanol production by this organism was driven by the oxidation of carbon monoxide. A genome reconstruction model for P. furiosus was developed to guide metabolic engineering strategies and understand outcomes.

"Reduced to My Race Once Again": Perceptions about Underrepresented Minority Medical School Applicants in Canada and the United States.

Phenomenon: To increase racial diversity in medical school classes, many institutions have created underrepresented minority (URM) application streams. However, many URM students experience overt and passive marginalization throughout their training and this may be related to how matriculants from URM streams are perceived by their peers. Approach: We conducted a discourse analysis of online discussion forums to explore how URM streams across Canada and the United States are perceived. We analyzed 850 posts from 13 discussion threads published between 2015 and 2020. We used inductive content analysis to develop a data-driven coding scheme from which we identified common themes. Findings: Despite an overall appreciation of the benefits of a diverse workforce, participants engaged in prominent discussions surrounding the merits of URM streams. We identified perceptions that students admitted from URM streams are less academically and clinically competent, with URM applicants reporting feeling unworthy for admission in the eyes of non-URM applicants. Users felt that the influence of socioeconomic status was under-appreciated, and that admissions officers inadequately addressed this barrier. There were some applicants who perceived the admissions process as "broken" with non-URMs displaying a fear of social change, and URMs fearing that the system defines them by their racialized status. Insights: Online discussion forums provide unique insight into perceptions surrounding URM streams. We identified potentially harmful misconceptions about URM students applying to these streams and highlight that actionable measures to reduce marginalization against URM matriculants must begin before medical school.

An ultraluminous X-ray source powered by an accreting neutron star.

The majority of ultraluminous X-ray sources are point sources that are spatially offset from the nuclei of nearby galaxies and whose X-ray luminosities exceed the theoretical maximum for spherical infall (the Eddington limit) onto stellar-mass black holes. Their X-ray luminosities in the 0.5-10 kiloelectronvolt energy band range from 10(39) to 10(41) ergs per second. Because higher masses imply less extreme ratios of the luminosity to the isotropic Eddington limit, theoretical models have focused on black hole rather than neutron star systems. The most challenging sources to explain are those at the luminous end of the range (more than 10(40) ergs per second), which require black hole masses of 50-100 times the solar value or significant departures from the standard thin disk accretion that powers bright Galactic X-ray binaries, or both. Here we report broadband X-ray observations of the nuclear region of the galaxy M82 that reveal pulsations with an average period of 1.37 seconds and a 2.5-day sinusoidal modulation. The pulsations result from the rotation of a magnetized neutron star, and the modulation arises from its binary orbit. The pulsed flux alone corresponds to an X-ray luminosity in the 3-30 kiloelectronvolt range of 4.9 × 10(39) ergs per second. The pulsating source is spatially coincident with a variable source that can reach an X-ray luminosity in the 0.3-10 kiloelectronvolt range of 1.8 × 10(40) ergs per second. This association implies a luminosity of about 100 times the Eddington limit for a 1.4-solar-mass object, or more than ten times brighter than any known accreting pulsar. This implies that neutron stars may not be rare in the ultraluminous X-ray population, and it challenges physical models for the accretion of matter onto magnetized compact objects.

Asymmetries in core-collapse supernovae from maps of radioactive 44Ti in Cassiopeia A.

Asymmetry is required by most numerical simulations of stellar core-collapse explosions, but the form it takes differs significantly among models. The spatial distribution of radioactive (44)Ti, synthesized in an exploding star near the boundary between material falling back onto the collapsing core and that ejected into the surrounding medium, directly probes the explosion asymmetries. Cassiopeia A is a young, nearby, core-collapse remnant from which (44)Ti emission has previously been detected but not imaged. Asymmetries in the explosion have been indirectly inferred from a high ratio of observed (44)Ti emission to estimated (56)Ni emission, from optical light echoes, and from jet-like features seen in the X-ray and optical ejecta. Here we report spatial maps and spectral properties of the (44)Ti in Cassiopeia A. This may explain the unexpected lack of correlation between the (44)Ti and iron X-ray emission, the latter being visible only in shock-heated material. The observed spatial distribution rules out symmetric explosions even with a high level of convective mixing, as well as highly asymmetric bipolar explosions resulting from a fast-rotating progenitor. Instead, these observations provide strong evidence for the development of low-mode convective instabilities in core-collapse supernovae.

Cosmic-ray antinuclei as messengers of new physics: status and outlook for the new decade.

The precise measurement of cosmic-ray antinuclei serves as an important means for identifying the nature of dark matter and other new astrophysical phenomena, and could be used with other cosmic-ray species to understand cosmic-ray production and propagation in the Galaxy. For instance, low-energy antideuterons would provide a "smoking gun" signature of dark matter annihilation or decay, essentially free of astrophysical background. Studies in recent years have emphasized that models for cosmic-ray antideuterons must be considered together with the abundant cosmic antiprotons and any potential observation of antihelium. Therefore, a second dedicated Antideuteron Workshop was organized at UCLA in March 2019, bringing together a community of theorists and experimentalists to review the status of current observations of cosmic-ray antinuclei, the theoretical work towards understanding these signatures, and the potential of upcoming measurements to illuminate ongoing controversies. This review aims to synthesize this recent work and present implications for the upcoming decade of antinuclei observations and searches. This includes discussion of a possible dark matter signature in the AMS-02 antiproton spectrum, the most recent limits from BESS Polar-II on the cosmic antideuteron flux, and reports of candidate antihelium events by AMS-02; recent collider and cosmic-ray measurements relevant for antinuclei production models; the state of cosmic-ray transport models in light of AMS-02 and Voyager data; and the prospects for upcoming experiments, such as GAPS. This provides a roadmap for progress on cosmic antinuclei signatures of dark matter in the coming years.

A rapidly spinning supermassive black hole at the centre of NGC 1365.

Broad X-ray emission lines from neutral and partially ionized iron observed in active galaxies have been interpreted as fluorescence produced by the reflection of hard X-rays off the inner edge of an accretion disk. In this model, line broadening and distortion result from rapid rotation and relativistic effects near the black hole, the line shape being sensitive to its spin. Alternative models in which the distortions result from absorption by intervening structures provide an equally good description of the data, and there has been no general agreement on which is correct. Recent claims that the black hole (2 × 10(6) solar masses) at the centre of the galaxy NGC 1365 is rotating at close to its maximum possible speed rest on the assumption of relativistic reflection. Here we report X-ray observations of NGC 1365 that reveal the relativistic disk features through broadened Fe-line emission and an associated Compton scattering excess of 10-30 kiloelectronvolts. Using temporal and spectral analyses, we disentangle continuum changes due to time-variable absorption from reflection, which we find arises from a region within 2.5 gravitational radii of the rapidly spinning black hole. Absorption-dominated models that do not include relativistic disk reflection can be ruled out both statistically and on physical grounds.

Steady-state cerebral blood flow regulation at altitude: interaction between oxygen and carbon dioxide.

High-altitude ascent imposes a unique cerebrovascular challenge due to two opposing blood gas chemostimuli. Specifically, hypoxia causes cerebral vasodilation, whereas respiratory-induced hypocapnia causes vasoconstriction. The conflicting nature of these two superimposed chemostimuli presents a challenge in quantifying cerebrovascular reactivity (CVR) in chronic hypoxia. During incremental ascent to 4240 m over 7 days in the Nepal Himalaya, we aimed to (a) characterize the relationship between arterial blood gas stimuli and anterior, posterior and global (g)CBF, (b) develop a novel index to quantify cerebral blood flow (CBF) in relation to conflicting steady-state chemostimuli, and (c) assess these relationships with cerebral oxygenation (rSO2). On rest days during ascent, participants underwent supine resting measures at 1045 m (baseline), 3440 m (day 3) and 4240 m (day 7). These measures included pressure of arterial (Pa)CO2, PaO2, arterial O2 saturation (SaO2; arterial blood draws), unilateral anterior, posterior and gCBF (duplex ultrasound; internal carotid artery [ICA] and vertebral artery [VA], gCBF [{ICA + VA} × 2], respectively) and rSO2 (near-infrared spectroscopy). We developed a novel stimulus index (SI), taking into account both chemostimuli (PaCO2/SaO2). Subsequently, CBF was indexed against the SI to assess steady-state cerebrovascular responsiveness (SS-CVR). When both competing chemostimuli are taken into account, (a) SS-CVR was significantly higher in ICA, VA and gCBF at 4240 m compared to lower altitudes, (b) delta SS-CVR with ascent (1045 m vs. 4240 m) was higher in ICA vs. VA, suggesting regional differences in CBF regulation, and (c) ICA SS-CVR was strongly and positively correlated (r = 0.79) with rSO2 at 4240 m.

Renal reactivity: acid-base compensation during incremental ascent to high altitude.

KEY POINTS: Ascent to high altitude imposes an acid-base challenge in which renal compensation is integral for maintaining pH homeostasis, facilitating acclimatization and helping prevent mountain sicknesses. The time-course and extent of plasticity of this important renal response during incremental ascent to altitude is unclear. We created a novel index that accurately quantifies renal acid-base compensation, which may have laboratory, fieldwork and clinical applications. Using this index, we found that renal compensation increased and plateaued after 5 days of incremental altitude exposure, suggesting plasticity in renal acid-base compensation mechanisms. The time-course and extent of plasticity in renal responsiveness may predict severity of altitude illness or acclimatization at higher or more prolonged stays at altitude. ABSTRACT: Ascent to high altitude, and the associated hypoxic ventilatory response, imposes an acid-base challenge, namely chronic hypocapnia and respiratory alkalosis. The kidneys impart a relative compensatory metabolic acidosis through the elimination of bicarbonate (HCO3- ) in urine. The time-course and extent of plasticity of the renal response during incremental ascent is unclear. We developed an index of renal reactivity (RR), indexing the relative change in arterial bicarbonate concentration ([HCO3- ]a ) (i.e. renal response) against the relative change in arterial pressure of CO2 ( PaCO2 ) (i.e. renal stimulus) during incremental ascent to altitude ( Δ[HCO3-]a/ΔPaCO2 ). We aimed to assess whether: (i) RR magnitude was inversely correlated with relative changes in arterial pH (ΔpHa ) with ascent and (ii) RR increased over time and altitude exposure (i.e. plasticity). During ascent to 5160 m over 10 days in the Nepal Himalaya, arterial blood was drawn from the radial artery for measurement of blood gas/acid-base variables in lowlanders at 1045/1400 m and after 1 night of sleep at 3440 m (day 3), 3820 m (day 5), 4240 m (day 7) and 5160 m (day 10) during ascent. At 3820 m and higher, RR significantly increased and plateaued compared to 3440 m (P < 0.04), suggesting plasticity in renal acid-base compensations. At all altitudes, we observed a strong negative correlation (r ≤ -0.71; P < 0.001) between RR and ΔpHa from baseline. Renal compensation plateaued after 5 days of altitude exposure, despite subsequent exposure to higher altitudes. The time-course, extent of plasticity and plateau in renal responsiveness may predict severity of altitude illness or acclimatization at higher or more prolonged stays at altitude.

Age-dependent bone loss and recovery during hindlimb unloading and subsequent reloading in rats.

BACKGROUND: Bone structure and strength are rapidly lost during conditions of decreased mechanical loading, and aged bones have a diminished ability to adapt to increased mechanical loading. This is a concern for older patients that experience periods of limited mobility or bed rest, but the acute effects of disuse on the bones of aged patients have not been thoroughly described. Previous animal studies have primarily examined the effect of mechanical unloading on young animals. Those that have studied aged animals have exclusively focused on bone loss during unloading and not bone recovery during subsequent reloading. In this study, we investigated the effect of decreased mechanical loading and subsequent reloading on bone using a hindlimb unloading model in Adult (9 month old) and Aged (28 month old) male rats. METHODS: Animals from both age groups were subjected to 14 days of hindlimb unloading followed by up to 7 days of reloading. Additional Aged rats were subjected to 7 days of forced treadmill exercise during reloading or a total of 28 days of reloading. Trabecular and cortical bone structure of the femur were quantified using ex vivo micro-computed tomography (µCT), and mechanical properties were quantified with mechanical testing. RESULTS: We found that Adult rats had substantially decreased trabecular bone volume fraction (BV/TV) following unloading (- 27%) while Aged animals did not exhibit significant bone loss following unloading. However, Aged animals had lower trabecular BV/TV after 3 days of reloading (- 20% compared to baseline), while trabecular BV/TV of Adult rats was not different from baseline values after 3 days of reloading. Trabecular BV/TV of Aged animals remained lower than control animals even with exercise during 7 days of reloading and after 28 days of reloading. CONCLUSIONS: These data suggest that aged bone is less responsive to both increased and decreased mechanical loading, and that acute periods of disuse may leave older subjects with a long-term deficit in trabecular bone mass. These finding indicate the need for therapeutic strategies to improve the skeletal health of elderly patients during periods of disuse.

Hispanic ethnicity and complication profile following laparoscopic and open cholecystectomy.

BACKGROUND: Hispanic ethnicity is associated with increased incidence of gallbladder disease. Additionally, ethnicity has been shown to be an outcome determinant in several conditions and procedures but has never been studied as a potential determinant of morbidity or mortality after laparoscopic or open cholecystectomy. METHODS: Using the American College of Surgeons National Surgical Quality Improvement Program database, patients who underwent cholecystectomy and related procedures were studied in Hispanic and non-Hispanic cohorts. Mortality and postoperative complication rates were calculated and adjusted for patient demographics and comorbidities through multivariate analysis. RESULTS: Hispanics had decreased morbidity following cholecystectomy as compared to non-Hispanics on univariate analysis in combined, laparoscopic, and open cohorts (RR 0.64, P value < 0.001; 0.68, <0.001; 0.77, <0.001, respectively). The reduction was not found to be statistically significant in multivariate analysis. A similar reduction was seen for mortality (RR 0.30, <0.001; 0.39, <0.001; 0.28, <0.001, respectively) which remained on multivariate analysis in both combined and open cohorts (RR 0.63, 0.008 and 0.48, 0.021, respectively). Additionally, the rates of several postoperative complications were found to be reduced in Hispanic patients. Though our study demonstrates a lower rate of established comorbidities for poor outcomes in Hispanics, after adjustment in multivariate analysis, the entirety of the reduced risk could not be accounted for. CONCLUSIONS: While the Hispanic cohort has an increased incidence in gallbladder disease as compared to non-Hispanics, the complication, morbidity, and mortality rates are lower in unadjusted analysis. With adjustment, morbidity was not statistically significant and mortality was only significant in combined and open cohorts. This suggests that increased incidence rates do not equate with worse outcomes, but Hispanic ethnicity may be associated with better outcomes.

Ethnic disparities in traumatic brain injury care referral in a Hispanic-majority population.

BACKGROUND: Functional outcomes after traumatic brain injury (TBI) can be significantly improved by discharge to posthospitalization care facilities. Many variables influence the discharge disposition of the TBI patient, including insurance status, patient condition, and patient prognosis. The literature has demonstrated an ethnic disparity in posthospitalization care referral, with Hispanics being discharged to rehabilitation and nursing facilities less often than non-Hispanics. However, this relationship has not been studied in a Hispanic-majority population, and thus, this study seeks to determine if differences in neurorehabilitation referrals exist among ethnic groups in a predominately Hispanic region. METHODS: This study is a retrospective cohort that includes 1128 TBI patients who presented to University Medical Center El Paso, Texas, between the years 2005 and 2015. The patients' age, sex, race, residence, admission Glasgow Coma Scale (GCS), GCS motor, Injury Severity Score (ISS), hospital and intensive care unit length of stay (LOS), mechanism of injury, and discharge disposition were analyzed in univariate and multivariate models. RESULTS: Our study population had an insurance rate of 55.5%. Insurance status and markers of injury severity (hospital LOS, intensive care unit LOS, ISS, GCS, and GCS motor) were predictive of discharge disposition to rehabilitation facilities. The study population was 70% Hispanic, yet Hispanics were discharged to rehabilitation facilities (relative risk: 0.56, P: 0.001) and to long-term acute care/nursing facilities (relative risk: 0.35, P < 0.0001) less than non-Hispanics even after LOS, ISS, ethnicity, insurance status, and residence were adjusted for in multivariate analysis. CONCLUSIONS: This study suggests that patients of different ethnicities but comparable traumatic severity and insurance status receive different discharge dispositions post-TBI even in regions in which Hispanics are the demographic majority.

Performance of three techniques for diagnosing equine tapeworm infection.

Tapeworm infection in horses can cause serious health concerns, and recent data have documented treatment failures in the most common species, Anoplocephala perfoliata. The threat of anthelmintic resistance in A. perfoliata is of particular concern because of poor diagnostic performance of standard egg counting techniques for detecting this parasite. This study compared the performance of three diagnostic techniques 1) Mini-FLOTAC, 2) Cornell-Wisconsin, and 3) Proudman and Edwards used to detect and quantify A. perfoliata eggs in naturally infected horses. Eighteen adult female horses from the University of Kentucky's historic parasitology herd were included in this study. Fecal samples were collected from all horses at five collection time points two weeks apart and analyzed with the three techniques. A total of 90 samples were collected and 270 counts determined in the study. The proportions of positive samples determined by the three techniques were significantly different from each other (p<0.05): Mini-FLOTAC (16%), Cornell-Wisconsin (47%), and Proudman and Edwards (70%). The Proudman and Edwards technique counted consistently higher numbers of tapeworm eggs compared to the other two techniques throughout the study [p < 0.05]. Total raw counts of tapeworm eggs across the study for each technique were 16, 88, and 410 for the Mini-FLOTAC, Cornell-Wisconsin, and Proudman and Edwards, respectively. This study demonstrated that the Proudman and Edwards technique was superior in diagnosing A. perfoliata infection. Future work needs to assess this technique's potential for Fecal Egg Count Reduction Testing (FECRT).

Susceptibility-Weighted MRI Approximates Intraoperative Microelectrode Recording During Deep Brain Stimulation of the Subthalamic Nucleus for Parkinson's Disease.

BACKGROUND: Deep brain stimulation of the subthalamic nucleus (STN-DBS) for Parkinson's disease can be performed with intraoperative neurophysiological and radiographic guidance. Conventional T2-weighted magnetic resonance imaging sequences, however, often fail to provide definitive borders of the STN. Novel magnetic resonance imaging sequences, such as susceptibility-weighted imaging (SWI), might better localize the STN borders and facilitate radiographic targeting. We compared the radiographic location of the dorsal and ventral borders of the STN using SWI with intraoperative microelectrode recording (MER) during awake STN-DBS for Parkinson's disease. METHODS: Thirteen consecutive patients who underwent placement of 24 STN-DBS leads for Parkinson's disease were analyzed retrospectively. Preoperative targeting was performed with SWI, and MER data were obtained from intraoperative electrophysiology records. The boundaries of the STN on SWI were identified by a blinded investigator. RESULTS: The final electrode position differed significantly from the planned coordinates in depth but not in length or width, indicating that MER guided the final electrode depth. When we compared the boundaries of the STN by MER and SWI, SWI accurately predicted the entry into the STN but underestimated the length and ventral boundary of the STN by 1.2 mm. This extent of error approximates the span of a DBS contact and could affect the placement of directional contacts within the STN. CONCLUSIONS: MER might continue to have a role in STN-DBS. This could potentially be mitigated by further refinement of imaging protocols to better image the ventral boundary of the STN.

Chemobiosis reveals tardigrade tun formation is dependent on reversible cysteine oxidation.

Tardigrades, commonly known as 'waterbears', are eight-legged microscopic invertebrates renowned for their ability to withstand extreme stressors, including high osmotic pressure, freezing temperatures, and complete desiccation. Limb retraction and substantial decreases to their internal water stores results in the tun state, greatly increasing their ability to survive. Emergence from the tun state and/or activity regain follows stress removal, where resumption of life cycle occurs as if stasis never occurred. However, the mechanism(s) through which tardigrades initiate tun formation is yet to be uncovered. Herein, we use chemobiosis to demonstrate that tardigrade tun formation is mediated by reactive oxygen species (ROS). We further reveal that tuns are dependent on reversible cysteine oxidation, and that this reversible cysteine oxidation is facilitated by the release of intracellular reactive oxygen species (ROS). We provide the first empirical evidence of chemobiosis and map the initiation and survival of tardigrades via osmobiosis, chemobiosis, and cryobiosis. In vivo electron paramagnetic spectrometry suggests an intracellular release of reactive oxygen species following stress induction; when this release is quenched through the application of exogenous antioxidants, the tardigrades can no longer survive osmotic stress. Together, this work suggests a conserved dependence of reversible cysteine oxidation across distinct tardigrade cryptobioses.

Differential bone adaptation to mechanical unloading and reloading in young, old, and osteocyte deficient mice.

Mechanical unloading causes rapid loss of bone structure and strength, which gradually recovers after resuming normal loading. However, it is not well established how this adaptation to unloading and reloading changes with age. Clinically, elderly patients are more prone to musculoskeletal injury and longer periods of bedrest, therefore it is important to understand how periods of disuse will affect overall skeletal health of aged subjects. Bone also undergoes an age-related decrease in osteocyte density, which may impair mechanoresponsiveness. In this study, we examined bone adaptation during unloading and subsequent reloading in mice. Specifically, we examined the differences in bone adaptation between young mice (3-month-old), old mice (18-month-old), and transgenic mice that exhibit diminished osteocyte density at a young age (3-month-old BCL-2 transgenic mice). Mice underwent 14 days of hindlimb unloading followed by up to 14 days of reloading. We analyzed trabecular and cortical bone structure in the femur, mechanical properties of the femoral cortical diaphysis, osteocyte density and cell death in cortical bone, and serum levels of inflammatory cytokines. We found that young mice lost ~10% cortical bone volume and 27-42% trabecular bone volume during unloading and early reloading, with modest recovery of metaphyseal trabecular bone and near total recovery of epiphyseal trabecular bone, but no recovery of cortical bone after 14 days of reloading. Old mice lost 12-14% cortical bone volume and 35-50% trabecular bone volume during unloading and early reloading but had diminished recovery of trabecular bone during reloading and no recovery of cortical bone. In BCL-2 transgenic mice, no cortical bone loss was observed during unloading or reloading, but 28-31% trabecular bone loss occurred during unloading and early reloading, with little to no recovery during reloading. No significant differences in circulating inflammatory cytokine levels were observed due to unloading and reloading in any of the experimental groups. These results illustrate important differences in bone adaptation in older and osteocyte deficient mice, suggesting a possible period of vulnerability in skeletal health in older subjects during and following a period of disuse that may affect skeletal health in elderly patients.