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BackgroundCollecting lymphatic vessel (CLV) dysfunction has been implicated in various diseases, including rheumatoid arthritis (RA). Previous studies in the tumor necrosis factor-transgenic (TNF-Tg) mouse model of inflammatory-erosive arthritis have demonstrated reduced joint-draining CLV contractility that correlates with arthritic severity. Clinically, RA patients with active hand arthritis exhibit significantly reduced lymphatic clearance of the web spaces adjacent to the metacarpophalangeal (MCP) joints and a reduction in total and basilic-associated CLVs on the dorsal surface of the hand by dynamic near-infrared (NIR) imaging of indocyanine green (ICG). In this pilot study, we assessed direct lymphatic drainage from MCP joints, and aimed to visualize the total lymphatic anatomy using novel dual-agent relaxation contrast magnetic resonance lymphography (DARC-MRL) in the upper extremity of healthy human subjects.MethodsTwo healthy male subjects participated in the study. We performed NIR imaging following intra-dermal web space and intra-articular MCP joint injections of ICG to visualize the CLV anatomy on the dorsal surface of the hand and antecubital fossa. Subsequently, we performed conventional or DARC-MRL after intra-dermal web space and intra-articular MCP joint injections of gadolinium to evaluate the total lymphatic anatomy of the upper extremity and compare with NIR-ICG imaging.ResultsNIR-ICG imaging demonstrated that web space and MCP lymphatics drain via distinct CLV tributaries. Web space draining CLVs tended to be associated with the cephalic side of the antecubital fossa, while MCP draining CLVs were localized to the basilic side of the forearm. The DARC-MRL methods used in this study did not adequately nullify the contrast in the blood vessels, and limited gadolinium-filled CLVs could be identified.ConclusionThrough the use of NIR-ICG imaging, we found that MCP joints predominantly drain into basilic CLVs in the forearm, which may explain the reduction in basilic-associated CLVs in the hands of RA patients. In healthy subjects, current DARC-MRL techniques have limited ability in identifying lymphatic structures and further refinement in this technique is necessary.

Methanobactins (Mbns) are ribosomally produced, post-translationally modified peptidic natural products that bind copper with high affinity. Methanotrophic bacteria use Mbns to acquire copper needed for enzymatic methane oxidation. Despite the presence of Mbn operons in a range of methanotroph and other bacterial genomes, few Mbns have been isolated and structurally characterized. Here we report the isolation of a novel Mbn from the methanotroph Methylosinus (Ms.) sp. LW3. Mass spectrometric and nuclear magnetic resonance spectroscopic data indicate that this Mbn, the largest characterized to date, consists of a 13-amino acid backbone modified to include pyrazinedione/oxazolone rings and neighboring thioamide groups derived from cysteine residues. The pyrazinedione ring is more stable to acid hydrolysis than the oxazolone ring and likely protects the Mbn from degradation. The structure corresponds exactly to that predicted on the basis of the Ms. sp. LW3 Mbn operon content, providing support for the proposed role of an uncharacterized biosynthetic enzyme, MbnF, and expanding the diversity of known Mbns.

Analogous to 2D layered transition-metal dichalcogenides, the TlSe family of quasi-one dimensional chain materials with the Zintl-type structure exhibits novel phenomena under high pressure. In the present work, we have systematically investigated the high-pressure behavior of TlInTe2 using Raman spectroscopy, synchrotron X-ray diffraction (XRD), and transport measurements, in combination with first principles crystal structure prediction (CSP) based on evolutionary approach. We found that TlInTe2 undergoes a pressure-induced semiconductor-to-semimetal transition at 4 GPa, followed by a superconducting transition at 5.7 GPa (with Tc = 3.8 K). An unusual giant phonon mode (Ag) softening appears at ∼10-12 GPa as a result of the interaction of optical phonons with the conduction electrons. The high-pressure XRD and Raman spectroscopy studies reveal that there is no structural phase transitions observed up to the maximum pressure achieved (33.5 GPa), which is in agreement with our CSP calculations. In addition, our calculations predict two high-pressure phases above 35 GPa following the phase transition sequence as I4/mcm (B37) → Pbcm → Pm3m (B2). Electronic structure calculations suggest Lifshitz (L1 & L2-type) transitions near the superconducting transition pressure. Our findings on TlInTe2 open up a new avenue to study unexplored high-pressure novel phenomena in TlSe family induced by Lifshitz transition (electronic driven), giant phonon softening, and electron-phonon coupling.

Cigarette smoking is a risk factor for developing nonmuscle invasive bladder cancer, and continued smoking exposure after diagnosis may increase the likelihood of adverse clinical outcomes. We compare self-reported vs biochemically verified nicotine exposure to determine the accuracy of self-report among recently diagnosed nonmuscle invasive bladder cancer patients.This cross-sectional analysis consisted of 517 nonmuscle invasive bladder cancer patients who contributed a urine or saliva specimen the same day as self-reporting their smoking, use of e-cigarettes, nicotine replacement therapy and whether they lived with a smoker. Cotinine, the primary metabolite of nicotine, was used as an objective biomarker of recent nicotine exposure.The prevalence of high, low and no cotinine exposure was 13%, 54% and 33%, respectively. Overall, 7.3% of patients (38/517) reported being a current cigarette smoker, while 13% (65/517) had cotinine levels consistent with active smoking exposure. Of these 65 patients 27 denied current smoking, resulting in a sensitivity of self-reported current smoking of 58%. After considering other sources of nicotine exposure such as e-cigarettes, cigars, nicotine replacement therapy and living with a smoker, the sensitivity was higher, at 82%. Nearly all patients with low cotinine denied any smoking-related exposure.Our findings suggest either biochemical verification with cotinine or additional questions about other sources of nicotine are needed to accurately identify nonmuscle invasive bladder cancer patients who have smoking-related exposures. Accurate classification of active and passive smoking exposure is essential to allow clinicians to advise cessation and help researchers estimate the association between post-diagnosis smoking-related exposure and nonmuscle invasive bladder cancer recurrence risk.

Summary Partially hydrolyzed polyacrylamide (PHPA) friction reducer was investigated in produced water from hydraulically fractured wells in the Duvernay and Montney Formations of western Canada. Produced water from systems that used nonencapsulated breaker had little residual solids ( NOTE: An erratum has been issued for this paper and added to the PDF. A copy is also available under the Supplementary Data section.

Purpose: 7-ketocholesterol (7kCh), a natural byproduct of oxidation in lipoprotein deposits is implicated in the pathogenesis of diabetic retinopathy and age-related macular degeneration (AMD). This study was performed to investigate whether several clinical drugs can inhibit 7kCh-induced caspase activation and mitigate its apoptotic effects on retinal cells in vitro. Method: Two populations of retinal cells, human retinal pigment epithelial cells (ARPE-19) and rat neuroretinal cells (R28) were exposed to 7kCh in the presence of the following inhibitors: Z-VAD-FMK (pan-caspase inhibitor), simvastatin, memantine, epicatechin, and Z-IETD-FMK (caspase-8 inhibitor) or Z-ATAD-FMK (caspase-12 inhibitor). Caspase-3/7, -8, and -12 activity levels were measured by fluorochrome caspase assays to quantify cell death. IncuCyte live-cell microscopic images were obtained to quantify cell counts. Results: Exposure to 7kCh for 24 hours significantly increased caspase activities for both ARPE-19 and R28 cells (P < 0.05). In ARPE cells, pretreatment with various drugs had significantly lower caspase-3/7, -8, and -12 activities, reported in % change in mean signal intensity (msi): Z-VAD-FMK (48% decrease, P < 0.01), memantine (decreased 47.8% at 1 µM, P = 0.0039 and 81.9% at 1 mM, P < 0.001), simvastatin (decreased 85.3% at 0.01 µM, P < 0.001 and 84.8% at 0.05 µM, P < 0.001) or epicatechin (83.6% decrease, P < 0.05), Z-IETD-FMK (68.1% decrease, P < 0.01), and Z-ATAD-FMK (47.7% decrease, P = 0.0017). In contrast, R28 cells exposed to 7kCh continued to have elevated caspase-3/7, -8, and -12 activities (between 25.7% decrease and 17.5% increase in msi, P > 0.05) regardless of the pretreatment. Conclusion: Several current drugs protect ARPE-19 cells but not R28 cells from 7kCh-induced apoptosis, suggesting that a multiple-drug approach is needed to protect both cells types in various retinal diseases.

OBJECTIVE To assess differences between lymphatic function in the affected hands of rheumatoid arthritis (RA) patients with active synovitis and that of healthy controls, using indocyanine green (ICG) dye and near-infrared (NIR) imaging. METHODS NIR imaging of the hands of 8 patients with active RA and 13 healthy controls was performed following web space injection of 0.1 ml of 100 μM ICG. The percentage of ICG retention in the web spaces was determined by NIR imaging at baseline and at 7 days (±1 day) after the initial injections; image analysis provided contraction frequency. ICG+ lymphatic vessel (LV) length and branching architecture were assessed. RESULTS Retention of ICG in RA hands was higher compared to controls (P < 0.01). The average contraction frequency of ICG+ LVs in RA patients and in controls did not differ (mean ± SD 0.53 ± 0.39 contractions/minute versus 0.51 ± 0.35 contractions/minute). Total ICG+ LV length in RA hands was lower compared to healthy controls (58.3 ± 15.0 cm versus 71.4 ± 16.1 cm; P < 0.001), concomitant with a decrease in the number of ICG+ basilic LVs in the hands of RA patients (P < 0.05). CONCLUSION Lymphatic drainage in the hands of RA patients with active disease was reduced compared to controls. This reduction was associated with a decrease in total length of ICG+ LVs on the dorsal surface of the hands, which continued to contract at a similar rate to that observed in controls. These findings provide a plausible mechanism for exacerbation of synovitis and joint damage, specifically the accumulation and retention of inflammatory cells and catabolic factors in RA joints due to impaired efferent lymphatic flow. NIR/ICG imaging of RA hands is feasible and warrants formal investigation as a primary outcome measure for arthritis disease severity and/or persistence in future clinical trials.

BACKGROUND Despite current neurological guidelines that a single brain death examination (SBDE) is sufficient to determine brain death, a vast majority of hospitals still use a two brain death examination (TBDE) policy based on historical practice. The purpose of this study was to analyze the outcomes and implications of an SBDE policy compared with a TBDE policy with respect to organ donation outcomes. METHODS We retrospectively reviewed all adult patients declared dead by neurological criteria between 2010 and 2018 at a high-volume trauma center. The study population was divided into SBDE and TBDE cohorts. Primary outcomes included time to organ donation, terminal donor creatinine and bilirubin, and number of procured and transplanted organs. RESULTS A total of 327 patients comprised the study population: 66.7% SBDE (n = 218 of 327 patients) and 33.3% TBDE (n = 109 of 327 patients). The SBDE group had a shorter median time from examination to procurement (38 vs. 44 hours, p = 0.02) as well as lower terminal donor creatinine (1.1 vs. 1.35 mg/dL, p = 0.004) and bilirubin (0.8 vs. 1.1 mg/dL, p = 0.04). Furthermore, the SBDE group had a significantly greater proportion of kidneys (90.6% vs. 81.6%, p = 0.02), lungs (11.8% vs. 4.6%, p = 0.02), and total organs (58.2% vs. 46.6%; p = 0.0001) procured with intent to transplant and a greater proportion of total organs transplanted (53.1% vs. 42.4%, p = 0.0004). Multivariable regression analysis confirmed that SBDE was independently associated with a shorter time to procurement, lower terminal creatinine, and increased number of procured organs. CONCLUSION These data highlight the potential benefit of an SBDE policy with regards to organ donation outcomes at a high-volume trauma center and should facilitate future randomized prospective studies to more rigorously test this hypothesis. LEVEL OF EVIDENCE Therapeutic/Care Management, level IV.

TRIM family proteins play integral roles in the innate immune response to virus infection. MG53 (TRIM72) is essential for cell membrane repair and is believed to be a muscle-specific TRIM protein. Here we show human macrophages express MG53, and MG53 protein expression is reduced following virus infection. Knockdown of MG53 in macrophages leads to increases in type I interferon (IFN) upon infection. MG53 knockout mice infected with influenza virus show comparable influenza virus titres to wild type mice, but display increased morbidity accompanied by more accumulation of CD45+ cells and elevation of IFNβ in the lung. We find that MG53 knockdown results in activation of NFκB signalling, which is linked to an increase in intracellular calcium oscillation mediated by ryanodine receptor (RyR). MG53 inhibits IFNβ induction in an RyR-dependent manner. This study establishes MG53 as a new target for control of virus-induced morbidity and tissue injury., TRIM proteins are known to play critical roles in the context of viral infection. Here the authors establish MG53 (TRIM72) suppresses IFN and inflammation by modulation of ryanodine receptor related intracellular calcium induction.

The alkylating warhead of the pancreatic cancer drug streptozotocin (SZN) contains an N-nitrosourea moiety constructed from Nω-methyl-l-arginine (l-NMA) by the multi-domain metalloenzyme SznF. The enzyme's central heme-oxygenase-like (HO-like) domain sequentially hydroxylates Nδ and Nω' of l-NMA. Its C-terminal cupin domain then rearranges the triply modified arginine to Nδ-hydroxy-Nω-methyl-Nω-nitroso-l-citrulline, the proposed donor of the functional pharmacophore. Here we show that the HO-like domain of SznF can bind Fe(II) and use it to capture O2, forming a peroxo-Fe2(III/III) intermediate. This intermediate has absorption- and Mossbauer-spectroscopic features similar to those of complexes previously trapped in f erritin-like d iiron o xidases and oxygenases (FDOs) and, more recently, the HO-like fatty acid oxidase UndA. The SznF peroxo-Fe2(III/III) complex is an intermediate in both hydroxylation steps, as shown by the concentration-dependent acceleration of its decay upon exposure to either l-NMA or Nδ-hydroxy-Nω-methyl-l-Arg (l-HMA). The Fe2(III/III) cluster produced upon decay of the intermediate has a small Mossbauer quadrupole splitting parameter, implying that, unlike the corresponding product states of many FDOs, it lacks an oxo-bridge. The subsequent decomposition of the product cluster to one or more paramagnetic Fe(III) species over several hours explains why SznF was previously purified and crystallographically characterized without its cofactor. Programmed instability of the oxidized form of the cofactor appears to be a unifying characteristic of the emerging superfamily of H O-like d iiron o xidases and oxygenases (HDOs).

Citicoline is the exogenous form of the nootropic, Cytidine 5'-diphosphate-choline that exerts its neuroprotective effects in the brain as well as in the eye. The current study characterized the cytoprotective effects of purified Citicoline in transmitochondrial AMD (Age-related Macular Degeneration) RPE cybrid cells which carry diseased mitochondria from clinically characterized AMD patients. The effects of Citicoline were examined via flow cytometry analysis of AnnexinV/ PI-stained cells, IncuCyte live-cell imaging analysis to quantify cells undergoing caspase-3/7-mediated apoptosis, analyses of gene expression profiles of apoptosis, hypoxia, and angiogenesis markers, and measurement of ROS levels and cell viability. Our results demonstrated that Citicoline when added exogenously alleviates apoptotic effects as evidenced by diminished AnnexinV/PI and Caspase-3/7 staining, downregulation of apoptosis genes, enhanced cell viability, and reduced oxidative stress in AMD RPE cybrid cells. In conclusion, our study identified Citicoline as a protector in AMD RPE cybrid cells in vitro. However, further studies are required to establish the merit of Citicoline as a cytoprotective molecule in AMD and to decipher the molecular underpinnings of its mechanism of action in AMD.

ObjectiveTo measure exosomal and plasma levels of candidate blood biomarkers in veterans with history of mild traumatic brain injury (mTBI) and test their relationship with chronic symptoms.MethodsExosomal and plasma levels of neurofilament light (NfL) chain, tumor necrosis factor (TNF)–α, interleukin (IL)–6, IL-10, and vascular endothelial growth factor (VEGF) were measured using an ultrasensitive assay in a cohort of 195 veterans, enrolled in the Chronic Effects of Neurotrauma Consortium Longitudinal Study. We examined relationships between candidate biomarkers and symptoms of postconcussive syndrome (PCS), posttraumatic stress disorder (PTSD), and depression. Biomarker levels were compared among those with no traumatic brain injury (TBI) (controls), 1–2 mTBIs, and repetitive (3 or more) mTBIs.ResultsElevated exosomal and plasma levels of NfL were associated with repetitive mTBIs and with chronic PCS, PTSD, and depression symptoms. Plasma TNF-α levels correlated with PCS and PTSD symptoms. The total number of mTBIs correlated with exosomal and plasma NfL levels and plasma IL-6. Increased number of years since the most recent TBI correlated with higher exosomal NfL and lower plasma IL-6 levels, while increased number of years since first TBI correlated with higher levels of exosomal and plasma NfL, as well as plasma TNF-α and VEGF.ConclusionRepetitive mTBIs are associated with elevated exosomal and plasma levels of NfL, even years following these injuries, with the greatest elevations in those with chronic PCS, PTSD, and depression symptoms. Our results suggest a possible neuroinflammatory and axonal disruptive basis for symptoms that persist years after mTBI, especially repetitive.

The low magnetic saturation of iron oxide nanoparticles, which are developed primarily as contrast agents for magnetic resonance imaging, limits the sensitivity of their detection using magnetic particle imaging (MPI). Here, we show that FeCo nanoparticles that have a core diameter of 10 nm and bear a graphitic carbon shell decorated with poly(ethylene glycol) provide an MPI signal intensity that is sixfold and fifteenfold higher than the signals from the superparamagnetic iron oxide tracers VivoTrax and Feraheme, respectively, at the same molar concentration of iron. We also show that the nanoparticles have photothermal and magnetothermal properties and can therefore be used for tumour ablation in mice, and that they have high optical absorbance in a broad near-infrared region spectral range (wavelength, 700–1,200 nm), making them suitable as tracers for photoacoustic imaging. As sensitive multifunctional and multimodal imaging tracers, carbon-coated FeCo nanoparticles may confer advantages in cancer imaging and hyperthermia therapy. FeCo nanoparticles with a graphitic carbon shell decorated with poly(ethylene glycol) have photothermal and magnetothermal properties and are sensitive tracers for magnetic particle imaging, magnetic resonance imaging and photoacoustic imaging.

Oxygen is a transcriptional regulator responsible for tissue homeostasis and maintenance. Studies relating cellular phenotype with oxygen tension often use hypoxia chambers, which expose cells to a single, static oxygen tension. Despite their ease of use, these chambers are unable to replicate the oxygen gradients found in healthy and diseased tissues. Microfabricated devices capable of imposing an oxygen gradient across tissue-like structures are a promising tool for these studies, as they can provide a high density of information in a single experimental setup. We describe the fabrication and characterization of a modular device, which leverages the gas-permeability of silicone to impose gradients of oxygen across cell-containing regions, assembled by layering sheets of laser cut acrylic and silicone rubber. The silicone also acts as a barrier, separating the flowing gases from the cell culture medium, preventing evaporation or bubble formation in experiments that require prolonged periods of incubation. The acrylic components provide a rigid framework to provide a sterile culture environment. Using oxygen-sensing films, we show the device can support gradients of different ranges and steepness by simply changing the composition of the gases flowing through the silicone components of the BLOCC. Using a cell-based reporter assay, we demonstrate that cellular responses to hypoxia are proportional to oxygen tension.

Dehydration ≥2% loss of body mass is associated with reductions in performance capacity, and carbohydrate (CHO)-electrolyte solutions (CES) are often recommended to prevent dehydration and provide a source of exogenous carbohydrate during exercise. It is also well established that performance capacity in the heat is diminished compared to cooler conditions, a response attributable to greater cardiovascular strain caused by high skin and core temperatures. Because hydration status, environmental conditions, and carbohydrate availability interact to influence performance capacity, we sought to determine how these factors affect push-to-the-finish cycling performance. Ten young trained cyclists exercised at a moderate intensity (2.5 W·kg−1) in a hot-dry condition [40°C, 20% relative humidity (RH)] until dehydration of ~2% body mass. Subjects then consumed either no fluid (NF) or enough fluid (water, WAT; Gatorade®, GAT; or GoodSport™, GS) to replace 75% of lost body mass over 30 min. After a 30-min light-intensity warm-up (1.5 W·kg−1) in a 35°C, 20% RH environment, subjects then completed a 120-kJ time trial (TT). TT time-to-completion, absolute power, and relative power were significantly improved in WAT (535 ± 214 s, 259 ± 99 W, 3.3 ± 0.9 W·kg−1), GAT (539 ± 226 s, 260 ± 110 W, 3.3 ± 1.0 W·kg−1), and GS (534 ± 238 s, 262 ± 105 W, 3.4 ± 1.0 W·kg−1) compared to NF (631 ± 310 s, 229 ± 96 W, 3.0 ± 0.9 W·kg−1) all (p < 0.01) with no differences between WAT, GAT, and GS, suggesting that hydration is more important than carbohydrate availability during exercise in the heat. A subset of four subjects returned to the laboratory to repeat the WAT, GAT, and GS treatments to determine if between-beverage differences in time-trial performance were evident with a longer TT in thermoneutral conditions. Following dehydration, the ambient conditions in the environmental chamber were reduced to 21°C and 20% RH and subjects completed a 250-kJ TT. All four subjects improved TT performance in the GS trial (919 ± 353 s, 300 ± 100 W, 3.61 ± 0.86 W·kg−1) compared to WAT (960 ± 376 s, 283 ± 91 W, 3.43 ± 0.83 W·kg−1), while three subjects improved TT performance in the GAT trial (946 ± 365 s, 293 ± 103 W, 3.60 ± 0.97 W·kg−1) compared to WAT, highlighting the importance of carbohydrate availability in cooler conditions as the length of a push-to-the-finish cycling task increases.

Symptoms of post-traumatic stress disorder (PTSD) are common in military populations, and frequently associated with a history of combat-related mild traumatic brain injury (mTBI). In this study, we examined relationships between severity of PTSD symptoms and levels of extracellular vesicle (EV) proteins and miRNAs measured in the peripheral blood in a cohort of military service members and Veterans (SMs/Vs) with chronic mTBI(s). Participants (n = 144) were divided into groups according to mTBI history and severity of PTSD symptoms on the PTSD Checklist for DSM-5 (PCL-5). We analyzed EV levels of 798 miRNAs (miRNAs) as well as EV and plasma levels of neurofilament light chain (NfL), Tau, Amyloid beta (Aβ) 42, Aβ40, interleukin (IL)-10, IL-6, tumor necrosis factor-alpha (TNFα), and vascular endothelial growth factor (VEGF). We observed that EV levels of neurofilament light chain (NfL) were elevated in participants with more severe PTSD symptoms (PCL-5 ≥ 38) and positive mTBI history, when compared to TBI negative controls (p = 0.024) and mTBI participants with less severe PTSD symptoms (p = 0.006). Levels of EV NfL, plasma NfL, and hsa-miR-139–5p were linked to PCL-5 scores in regression models. Our results suggest that levels of NfL, a marker of axonal damage, are associated with PTSD symptom severity in participants with remote mTBI. Specific miRNAs previously linked to neurodegenerative and inflammatory processes, and glucocorticoid receptor signaling pathways, among others, were also associated with the severity of PTSD symptoms. Our findings provide insights into possible signaling pathways linked to the development of persistent PTSD symptoms after TBI and biological mechanisms underlying susceptibility to PTSD.

Lead halide perovskites have a rich landscape of structural and optical properties, which can be explored and possibly controlled by applying high pressure. Despite several reports on high-pressure...

When Mentos candies are dropped into a bottle of Diet Coke, a foamy fountain jets out of the beverage container. It has previously been argued that ingredients found in Mentos candies significantly...

Methanobactins (Mbns) are ribosomally-produced, post-translationally modified peptidic copper-binding natural products produced under conditions of copper limitation. Genes encoding Mbn biosynthetic and transport proteins have been identified in a wide variety of bacteria, indicating a broader role for Mbns in bacterial metal homeostasis. Many of the genes in the Mbn operons have been assigned functions, but two genes usually present, mbnP and mbnH, encode uncharacterized proteins predicted to reside in the periplasm. MbnH belongs to the bacterial diheme cytochrome c peroxidase (bCcP)/MauG protein family, and MbnP contains no domains of known function. Here, we performed a detailed bioinformatic analysis of both proteins and have biochemically characterized MbnH from Methylosinus (Ms.) trichosporium OB3b. We note that the mbnH and mbnP genes typically co-occur and are located proximal to genes associated with microbial copper homeostasis. Our bioinformatics analysis also revealed that the bCcP/MauG family is significantly more diverse than originally appreciated, and that MbnH is most closely related to the MauG subfamily. A 2.6 Å resolution structure of Ms. trichosporium OB3b MbnH combined with spectroscopic data and peroxidase activity assays provided evidence that MbnH indeed more closely resembles MauG than bCcPs, although its redox properties are significantly different from those of MauG. The overall similarity of MbnH to MauG suggests that MbnH could post-translationally modify a macromolecule, such as internalized CuMbn or its uncharacterized partner protein, MbnP. Our results indicate that MbnH is a MauG-like diheme protein that is likely involved in microbial copper homeostasis and represents a new family within the bCcP/MauG superfamily.

Short sediment cores (80–100 cm) from three Florida (USA) lakes (Sheelar, Wauberg, and Apopka) that range in trophic status, were analyzed for total organic carbon (TOC), total nitrogen, stable isotopes of organic carbon (δ13CTOC) and biomarkers (n-alkanes and fatty acids), to identify the sources of organic matter in the lake deposits, and to link changes in primary productivity to anthropogenic activities during the last ~ 150 years. Relatively small (0.07 km2), ultra-oligotrophic Lake Sheelar is located in a state park, and sediment analyses indicate stable trophic status in the water body since at least the middle of the nineteenth century. Algal biomarkers are in low abundance throughout the core and terrestrial lipids and δ13CTOC values suggest that vascular plants were the primary source of TOC to the lake sediments during the period of record. In larger, eutrophic Lakes Wauberg (1.5 km2) and Apopka (125 km2), algal-derived biomarkers increase in recent sediments, whereas δ13CTOC values and concentrations of terrestrial biomarkers decrease toward the sediment surface. Increasing dominance through time of autochthonous carbon sources in the Lake Wauberg and Apopka sediment records coincides with specific anthropogenic activities in the respective watersheds. Submersed macrophytes in Lake Wauberg were replaced by algal communities in the mid-1980s, following expansion of residential development in the watershed. Biomarker data from the Lake Apopka core show there was an abrupt transition in the lake in the late 1940s, from domination by vascular plants to domination by algae, which has been documented by other paleolimnological studies that used alternative trophic state indicators. The trophic state shift in Lake Apopka corresponds to a period of increased nutrient input to the lake, associated with extensive farming along the north shore. Florida lakes have experienced different trophic state trajectories over the last ~ 150 years, driven by specific human activities in their respective watersheds.

Hypoxia is a common feature in solid tumors. Clinical samples show a positive correlation between the expression of the hypoxia-inducible factor HIF-1α and estrogen receptor alpha (ERα) and a negative correlation between HIF-1α and hormone sensitivity. Results from monolayer cultures are in contention with clinical observations, showing that ER (+) cell lines no longer express ERα under hypoxic conditions (1% O2). Here, we compared the impact of hypoxia on the ERα signaling pathway for T47D cells in a 2D and 3D culture format. In the 2D format, the cells were cultured as monolayers. In the 3D format, paper-based scaffolds supported cells suspended in a collagen matrix. Using ELISA, Western blot, and immunofluorescence measurements, we show that hypoxia differentially regulates ERα protein levels in a culture environment-dependent manner. In the 2D format, the protein levels are significantly decreased in hypoxia. In the 3D format, the protein levels are maintained in hypoxia. Hypoxia reduced ERα transcriptional activation in both culture formats. These results highlight the importance of considering tissue dimensionality for in vitro studies. They also show that ERα protein levels in hypoxia are not an accurate indicator of ERα transcriptional activity, and confirm that a positive stain for ERα in a clinical sample may not necessarily indicate hormone sensitivity.

Epstein-Barr virus (EBV) is a human herpesvirus that infects over 90% of the world's population that persists as a latent infection in various lymphoid and epithelial malignancies. The total number of EBV associated malignancies is estimated to exceed 200,000 new cancers per year. Current chemotherapeutic treatments of EBV-positive cancers include broad-spectrum cytotoxic drugs that ignore the EBV positive status of tumors and have limited safety and selectivity. In an effort to develop new and more efficacious molecules for inducing EBV reactivation, we have developed high-throughput screening assays to identify a class of small molecules (referred to as the C60 series) that efficiently activate the EBV lytic cycle in multiple latency types, including lymphoblastoid and nasopharyngeal carcinoma cell lines. In this paper we report our preliminary structure activity relationship studies and demonstrate reactivation of EBV in the SNU719 gastric carcinoma mouse model and the AGS-Akata gastric carcinoma mouse model.

Aerobic methane oxidation is catalyzed by particulate methane monooxygenase (pMMO), a copper-dependent, membrane metalloenzyme composed of subunits PmoA, PmoB, and PmoC. Characterization of the copper active site has been limited by challenges in spectroscopic analysis stemming from the presence of multiple copper binding sites, effects of detergent solubilization on activity and crystal structures, and the lack of a heterologous expression system. Here we utilize nanodiscs coupled with native top-down mass spectrometry (nTDMS) to determine the copper stoichiometry in each pMMO subunit and to detect post-translational modifications (PTMs). These results indicate the presence of a mononuclear copper center in both PmoB and PmoC. pMMO-nanodisc complexes with a higher stoichiometry of copper-bound PmoC exhibit increased activity, suggesting that the PmoC copper site plays a role in methane oxidation activity. These results provide key insights into the pMMO copper centers and demonstrate the ability of nTDMS to characterize complex membrane-bound metalloenzymes., The activity of the membrane-bound enzyme pMMO depends on copper but the location of the copper centers is still under debate. Here, the authors reconstitute pMMO in nanodiscs and use native top-down MS to localize its copper centers, providing insights into which sites are essential for activity.

Neutral water splitting is attractive for its use of non-corrosive and environmentally friendly electrolytes. However, catalyst development for hydrogen and oxygen evolution remains a challenge under neutral conditions. Here we report a simple electrodeposition and reductive annealing procedure to produce a highly active Ni-Co-Cr metal/metal oxide heterostructured catalyst directly on Ni foam. The resulting electrocatalyst for hydrogen evolution reaction (HER) requires only 198 mV of overpotential to reach 100 mA/cm2 in 1 M potassium phosphate (pH = 7.4) and can operate for at least two days without significant performance decay. Scanning transmission electron microscopy coupled with electron energy loss spectroscopy (STEM-EELS) imaging reveals a Ni-Co alloy core decorated with blended oxides layers of NiO, CoO and Cr2O3. The metal/metal oxide interfaces are suggested to be responsible for the high HER activity.

Active silicon detectors built on p-type substrate are a promising technological solution for large area silicon trackers such as those at the High Luminosity LHC, but the radiation hardness of this novel approach has to be evaluated. Active n-in-p strip detector prototypes CHESS2 for ATLAS with different substrate resistivities in the range of 20–1000 Ω cm were irradiated with neutrons and protons up to a fluence of 2 × 1 0 15 n eq cm − 2 and 3 . 6 × 1 0 15 n eq cm − 2 . Charge collection in passive test structures on the chip was evaluated using Edge-TCT and minimum ionising electrons from 90Sr. Results were used to assess radiation hardness of the detector in the given fluence range and to determine parameters of initial acceptor removal in different substrates.

Adrenoleukodystrophy (ALD) is caused by mutations within the X-linked ABCD1 gene, resulting in the inability to transport acylated very long chain fatty acids (VLCFAs) into the peroxisome for degradation. VLCFAs subsequently accumulate in tissues, including the central nervous system. Up to 40% of boys develop a severe progressive demyelinating form of ALD, cerebral ALD, resulting in regions of demyelination observed on brain magnetic resonance imaging that are associated with a “garland ring” of gadolinium contrast enhancement. Gadolinium enhancement indicates blood-brain barrier (BBB) disruption and an active inflammatory disease process. Only hematopoietic cell transplant (HCT) has been shown to halt neurologic progression, although the mechanism of disease arrest is unknown. We evaluated imaging- and transplant-related biomarkers in 66 males who underwent HCT. In 77% of patients, gadolinium contrast resolved by 60 days post-HCT. We determined that time to neutrophil recovery and extent of donor chimerism correlated significantly with time to contrast resolution post-HCT. Graft failure was associated with a significantly slower rate of contrast resolution (P < .0001). Time to neutrophil recovery remained significant in multivariate analysis with other biomarkers (P = .03). Our data suggest that robust donor myeloid recovery is necessary for timely repair of the BBB.

Author(s): Atilano, Shari R; Lee, Daniel H; Fukuhara, Paula S; Chwa, Marilyn; Nesburn, Anthony B; Udar, Nitin; Kenney, M Cristina | Abstract: PurposeTo compare the levels of gene expression for enzymes involved in production and elimination of reactive oxygen/nitrogen species (ROS/RNS) in normal human corneal cells (NL cells) with those in human corneal cells with keratoconus (KC cells) in vitro.MethodsPrimary NL and KC stromal fibroblast cultures were incubated with apocynin (an inhibitor of NADPH oxidase) or N-nitro-L-arginine (N-LLA; an inhibitor of nitric oxide synthase). ROS/RNS levels were measured using an H2 DCFDA fluorescent assay. The RT2 Profiler™ PCR Array for Oxidative Stress and Antioxidant Defense was used for initial screening of the NL and KC cultures. Transcription levels for genes related to production or elimination of ROS/RNS were analyzed using quantitative PCR. Immunohistochemistry was performed on 10 intact human corneas using antibodies against SCARA3 and CPSF3.ResultsArray screening of 84 antioxidant-related genes identified 12 genes that were differentially expressed between NL and KC cultures. Compared with NL cells, quantitative PCR showed that KC cells had decreased expression of antioxidant genes SCARA3 isoform 2 (0.59-fold, P = 0.02) and FOXM1 isoform 1 (0.61-fold, P = 0.03). KC cells also had downregulation of the antioxidant genes SOD1 (0.4-fold, P = 0.0001) and SOD3 (0.37-fold, P = 0.02) but increased expression of SOD2 (3.3-fold, P l 0.0001), PRDX6 (1.47-fold, P = 0.01), and CPSF3 (1.44-fold, P = 0.02).ConclusionThe difference in expression of antioxidant enzymes between KC and NL suggests that the oxidative stress imbalances found in KC are caused by defects in ROS/RNS removal rather than increased ROS/RNS production.

Cellular movement is essential in the formation and maintenance of healthy tissues as well as in disease progression such as tumor metastasis. In this work, we describe a paper-based Transwell assay capable of quantifying cellular invasion through an extracellular matrix. The paper-based Transwell assays generate similar datasets, with equivalent reproducibility, to commercially available Transwell assays. With different culture configurations, we quantify invasion: upon addition of an exogenous factor or in the presence of medium obtained from other cell types, in an indirect or direct co-culture format whose medium composition is dynamically changing, and in a single-zone or parallel (96-zone) format.

The Alternative Stable States Model describes the two contrasting conditions of shallow lakes that occur in response to external phosphorus (P) loading: 1) a clear-water, macrophyte-dominated state during times of lesser external P loading, and 2) a turbid, phytoplankton-dominated state during periods of greater P loading. Determination of the P loading rate that distinguishes the two stable states, i.e. the threshold phosphorus loading rate (TPL), is fundamental to shallow-lake science and management. We used data from nine shallow subtropical lakes in Florida (USA) to generate a parsimonious model that predicts the TPL from the hydraulic detention time (HDT). The model reveals that the TPL is more sensitive to changes in HDT in lakes with short (10 years) HDTs. We compared our results with published data from 54 geographically distributed shallow aquatic systems and found that the Florida-based HDT-TPL model has general applicability. Given many lakes worldwide are shallow, our findings have the potential to improve understanding and management of numerous aquatic ecosystems around the globe.

Introduction The human placenta performs multiple functions necessary for successful pregnancy, but the metabolic pathways and molecular mechanisms responsible for regulating placental development and functions remain incompletely understood. Catabolism of the essential amino acid tryptophan has numerous critical roles in normal physiology, including inflammation. The kynurenine pathway, which accounts for ∼90% of tryptophan breakdown, is mediated by indoleamine 2,3 dioxygenase 1 (IDO1) in the placenta. In pregnant mice, alterations of IDO1 activity or expression result in fetal resorption and a preeclampsia-like phenotype. Decreased IDO1 expression at the maternal-fetal interface has also been linked to preeclampsia, in utero growth restriction and recurrent miscarriage in humans. These collective observations suggest essential role(s) for IDO1 in maintaining healthy pregnancy. Despite these important roles, the precise temporal, cell-specific and inflammatory cytokine-mediated patterns of IDO1 expression in the human placenta have not been thoroughly characterized across gestation. Methods Western blot and whole mount immunofluorescence (WMIF) were utilized to characterize and quantify basal and interferon (IFN)-inducible IDO1 expression in 1st trimester (7–13 weeks), 2nd trimester (14–22 weeks) and term (39–41 weeks) placental villi. Results IDO1 expression is activated in the human placenta between the 13th and 14th weeks of pregnancy, increases through the 2nd trimester and remains elevated at term. Constitutive IDO1 expression is restricted to placental endothelial cells. Interestingly, different types of IFNs have distinct effects on IDO1 expression in the human placenta. Discussion Our collective results are consistent with potential role(s) for IDO1 in the regulation of vascular functions in placental villi.

Type three secretion systems enable bacterial pathogens to inject effectors into the cytosol of eukaryotic hosts to reprogram cellular functions. It is technically challenging to label effectors and the secretion machinery without disrupting their structure/function. Herein, we present a new approach for labeling and visualization of previously intractable targets. Using genetic code expansion, we site-specifically labeled SsaP, the substrate specificity switch, and SifA, a here-to-fore unlabeled secreted effector. SsaP was secreted at later infection times; SsaP labeling demonstrated the stochasticity of injectisome and effector expression. SifA was labeled after secretion into host cells via fluorescent unnatural amino acids or non-fluorescent labels and a subsequent click reaction. We demonstrate the superiority of imaging after genetic code expansion compared to small molecule tags. It provides an alternative for labeling proteins that do not tolerate N- or C-terminal tags or fluorophores and thus is widely applicable to other secreted effectors and small proteins.

SummarySevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection leads to NF-κB activation and induction of pro-inflammatory cytokines, though the underlying mechanism for this activation is not fully understood. Our results reveal that the SARS-CoV-2 Nsp14 protein contributes to the viral activation of NF-κB signaling. Nsp14 caused the nuclear translocation of NF-κB p65. Nsp14 induced the upregulation of IL-6 and IL-8, which also occurred in SARS-CoV-2 infected cells. IL-8 upregulation was further confirmed in lung tissue samples from COVID-19 patients. A previous proteomic screen identified the putative interaction of Nsp14 with host Inosine-5’-monophosphate dehydrogenase 2 (IMPDH2) protein, which is known to regulate NF-κB signaling. We confirmed the Nsp14-IMPDH2 protein interaction and found that IMPDH2 knockdown or chemical inhibition using ribavirin (RIB) and mycophenolic acid (MPA) abolishes Nsp14-mediated NF-κB activation and cytokine induction. Furthermore, IMDPH2 inhibitors (RIB, MPA) efficiently blocked SARS-CoV-2 infection, indicating that IMDPH2, and possibly NF-κB signaling, is beneficial to viral replication. Overall, our results identify a novel role of SARS-CoV-2 Nsp14 in causing the activation of NF-κB.

ObjectiveTo characterize the relationship between persistent post-traumatic headache (pPTH) and traumatic cerebrovascular injury (TCVI) in chronic traumatic brain injury (TBI). Cerebrovascular reactivity (CVR), a measure of the cerebral microvasculature and endothelial cell function, is altered both in individuals with chronic TBI and migraine headache disorder (Amyot et al., 2017; Lee et al., 2019b). The pathophysiologies of pPTH and migraine are believed to be associated with chronic microvascular dysfunction. We therefore hypothesize that TCVI may contribute to the underlying migraine-like mechanism(s) of pPTH.Materials and Methods22 moderate/severe TBI participants in the chronic stage (>6 months) underwent anatomic and functional magnetic resonance imaging (fMRI) scanning with hypercapnia gas challenge to measure CVR as well as the change in CVR (ΔCVR) after single-dose treatment of a specific phosphodiesterase-5 (PDE-5) inhibitor, sildenafil, which potentiates vasodilation in response to hypercapnia in impaired endothelium, as part of a Phase2a RCT of sildenafil in chronic TBI (NCT01762475). CVR and ΔCVR measures of each participant were compared with the individual’s pPTH severity measured by the headache impact test-6 (HIT-6) survey.ResultsThere was a moderate correlation between HIT-6 and both CVR and ΔCVR scores [Spearman’s correlation = –0.50 (p = 0.018) and = 0.46 (p = 0.03), respectively], indicating that a higher headache burden is associated with decreased endothelial function in our chronic TBI population.ConclusionThere is a correlation between PTH and CVR in chronic moderate-severe TBI. This relationship suggests that chronic TCVI may underlie the pathobiology of pPTH. Further, our results suggest that novel treatment strategies that target endothelial function and vascular health may be beneficial in refractory pPTH.

The co-contamination of the environment by metals and organic pollutants is a significant concern, and one such example is lead (Pb) and methyl tert-butyl ether (MTBE) due to their historic use as fuel additives. Clinoptilolite is an abundant and efficient zeolite for metal removal, but the potential interference of co-existing organic pollutants on metal removal, such as MTBE, have rarely been discussed. In this study, a combination of batch sorption tests and synchrotron-based X-ray absorption spectroscopic analyses were employed to investigate Pb sorption mechanism(s) onto clinoptilolite in the presence and absence of MTBE. A comparison was made to synthetic ZSM-5 zeolite to gain insights into differences in Pb binding mechanisms between hydrophilic (clinoptilolite) and hydrophobic (ZSM-5) zeolites. Site occupancy and surface precipitation contributed equally to Pb removal by clinoptilolite, while surface precipitation was the main Pb removal mechanism for ZSM-5 followed by site occupancy. Despite the negligible effect of 100 mg/L MTBE on observed Pb removal from solution by both zeolites, a surface-embedded Pb removal mechanism, through the Mg site on clinoptilolite surface, arises when MTBE is present. This study provides an understanding of atomic-level Pb uptake mechanisms on zeolites, with and without co-contaminating MTBE, which aids in their application in water treatment at co-contaminated sites.

BackgroundMedulloblastoma (MB) is the most common malignant pediatric brain tumor that originates in the cerebellum and brainstem. Frequent somatic mutations and deregulated expression of epigenetic regulators in MB highlight the substantial role of epigenetic alterations. 5-hydroxymethylcytosine (5hmC) is a highly abundant cytosine modification in the developing cerebellum and is regulated by ten-eleven translocation (TET) enzymes.ResultsWe investigate the alterations of 5hmC and TET enzymes in MB and their significance to cerebellar cancer formation. We show total abundance of 5hmC is reduced in MB, but identify significant enrichment of MB-specific 5hmC marks at regulatory regions of genes implicated in stem-like properties and Nanog-binding motifs. While TET1 and TET2 levels are high in MBs, only knockout ofTet1in the smoothened (SmoA1)mouse model attenuates uncontrolled proliferation, leading to a favorable prognosis. The pharmacologicalTet1inhibition reduces cell viability andplatelet-derived growth factorsignaling pathway-associated genes.ConclusionsThese results together suggest a potential key role of 5hmC and indicate an oncogenic nature for TET1 in MB tumorigenesis, suggesting it as a potential therapeutic target for MBs.

Infection by SARS-CoV-2 generally causes mild symptoms but can lead to severe disease and death in certain populations, including the immunocompromised. Drug repurposing efforts are underway to identify compounds that interfere with SARS-CoV-2 replication or the immunopathology it can elicit. Rapamycin is among those being currently tested in clinical trials for impacts on COVID-19 severity. While rapamycin and rapamycin analogs (rapalogs) are FDA-approved for use as mTOR inhibitors in multiple clinical settings, including cancer, we previously found that rapamycin can increase the susceptibility of cells to infection by Influenza A virus. In this study, we tested the impact of rapalogs on cellular susceptibility to SARS-CoV-2 infection. We report that rapamycin and rapalogs increased SARS-CoV-2 titers in human cervical epithelial and lung epithelial cell lines to different extents, and a similar pattern of enhancement was observed using pseudovirus incorporating viral fusion proteins from SARS-CoV-2, SARS-CoV, MERS, and Influenza A Virus. Rapalogs also promoted cell entry driven by SARS-CoV-2 Spike in nasal cells and primary small airway cells, representing proximal and distal ends of the human respiratory tract, respectively. Interestingly, cell entry enhancement by the rapalog ridaforolimus was cell type-dependent, revealing a previously unrecognized functional divergence between rapalogs. The differential activity of rapalogs was associated with their capacity to induce the degradation of interferon-inducible transmembrane (IFITM) proteins, restriction factors that broadly inhibit virus infection. Our findings will spur the development of mTOR inhibitors that do not suppress the cells first line of antiviral defense. SignificanceDue to a lack of effective antivirals available to combat SARS-CoV-2 infection and the disease it causes (COVID-19), existing drugs that are clinically-approved for use in humans are being considered for deployment during this protracted pandemic. Rapamycin is an immunosuppressant that is administered to humans suffering from cancer, autoimmunity, atherosclerosis, and organ transplant rejection. While generally recognized for its inhibitory effects on the adaptive immune response, we previously showed that rapamycin can also impair intrinsic immunity, the immune system present within all cells of the body. Here, we show that rapamycin and rapamycin analogs (rapalogs) promote the first step of the SARS-CoV-2 infection cycle--entry into cells--by inducing the destruction of antiviral proteins that stand guard during virus invasion.

Our study assesses the effects of anti-VEGF (Vascular Endothelial Growth Factor) drugs and Trichostatin A (TSA), an inhibitor of histone deacetylase (HDAC) activity, on cultured ARPE-19 (Adult Retinal Pigment Epithelial-19) cells that are immortalized human retinal pigment epithelial cells. ARPE-19 cells were treated with the following anti-VEGF drugs: aflibercept, ranibizumab, or bevacizumab at 1× and 2× concentrations of the clinical intravitreal dose (12.5 μL/mL and 25 μL/mL, respectively) and analyzed for transcription profiles of genes associated with the pathogenesis age-related macular degeneration (AMD). HDAC activity was measured using the Fluorometric Histone Deacetylase assay. TSA downregulated HIF-1α and IL-1β genes, and upregulated BCL2L13, CASPASE-9, and IL-18 genes. TSA alone or bevacizumab plus TSA showed a significant reduction of HDAC activity compared to untreated ARPE-19 cells. Bevacizumab alone did not significantly alter HDAC activity, but increased gene expression of SOD2, BCL2L13, CASPASE-3, and IL-18 and caused downregulation of HIF-1α and IL-18. Combination of bevacizumab plus TSA increased gene expression of SOD2, HIF-1α, GPX3A, BCL2L13, and CASPASE-3, and reduced CASPASE-9 and IL-β. In conclusion, we demonstrated that anti-VEGF drugs can: (1) alter expression of genes involved in oxidative stress (GPX3A and SOD2), inflammation (IL-18 and IL-1β) and apoptosis (BCL2L13, CASPASE-3, and CASPASE-9), and (2) TSA-induced deacetylation altered transcription for angiogenesis (HIF-1α), apoptosis, and inflammation genes.