Acetate and Acetyl-CoA Metabolism of ANME-2 Anaerobic Archaeal Methanotrophs.

Acetyl-CoA synthetase (ACS) and acetate ligase (ACD) are widespread among microorganisms, including archaea, and play an important role in their carbon metabolism, although only a few of these enzymes have been characterized. Anaerobic methanotrophs (ANMEs) have been reported to convert methane anaerobically into CO2, polyhydroxyalkanoate, and acetate. Furthermore, it has been suggested that they might be able to use acetate for anabolism or aceticlastic methanogenesis. To better understand the potential acetate metabolism of ANMEs, we characterized an ACS from ANME-2a as well as an ACS and an ACD from ANME-2d. The conversion of acetate into acetyl-CoA (Vmax of 8.4 µmol mg-1 min-1 and Km of 0.7 mM acetate) by the monomeric 73.8-kDa ACS enzyme from ANME-2a was more favorable than the formation of acetate from acetyl-CoA (Vmax of 0.4 µmol mg-1 min-1 and Km of 0.2 mM acetyl-CoA). The monomeric 73.4-kDa ACS enzyme from ANME-2d had similar Vmax values for both directions (Vmax,acetate of 0.9 µmol mg-1 min-1 versus Vmax,acetyl-CoA of 0.3 µmol mg-1 min-1). The heterotetrameric ACD enzyme from ANME-2d was active solely in the acetate-producing direction. Batch incubations of an enrichment culture dominated by ANME-2d fed with 13C2-labeled acetate produced 3 µmol of [13C]methane in 7 days, suggesting that this anaerobic methanotroph might have the potential to reverse its metabolism and perform aceticlastic methanogenesis using ACS to activate acetate albeit at low rates (2 nmol g [dry weight]-1 min-1). Together, these results show that ANMEs may have the potential to use acetate for assimilation as well as to use part of the surplus acetate for methane production. IMPORTANCE Acetyl-CoA plays a key role in carbon metabolism and is found at the junction of many anabolic and catabolic reactions. This work describes the biochemical properties of ACS and ACD enzymes from ANME-2 archaea. This adds to our knowledge of archaeal ACS and ACD enzymes, only a few of which have been characterized to date. Furthermore, we validated the in situ activity of ACS in ANME-2d, showing the conversion of acetate into methane by an enrichment culture dominated by ANME-2d.

Varying magnetic field strength as an effective approach to boost up the plasma signal in laser-induced breakdown spectroscopy.

Externally variable magnetic field was incorporated with the combination of laser induced breakdown spectroscopy (LIBS) to enhance the emission characteristics of aluminum (Al) plasma. Significant emission enhancement of laser induced plasma (LIP) was obtained at different magnetic field strengths, for instance, enhancement factors of about 1.2, 1.3 and 1.4 times were observed at field-strength of 0.4, 0.5 and 0.6 T, respectively. The electron-impact excitation rates and recombination process were increased at higher field-strengths, which led to the higher emission signal due the stronger plasma confinement by the field. The electron number density and electron temperature were measured using the spectroscopic techniques at several delay times. At higher field strengths, both electron density and electron excitation temperature showed an increased trend as compared to the case when No-field was applied. Hence, the research has significance for enhancing the plasma signal which led to improve the LIBS sensitivity.

Unraveling Nitrogen, Sulfur, and Carbon Metabolic Pathways and Microbial Community Transcriptional Responses to Substrate Deprivation and Toxicity Stresses in a Bioreactor Mimicking Anoxic Brackish Coastal Sediment Conditions.

Microbial communities are key drivers of carbon, sulfur, and nitrogen cycling in coastal ecosystems, where they are subjected to dynamic shifts in substrate availability and exposure to toxic compounds. However, how these shifts affect microbial interactions and function is poorly understood. Unraveling such microbial community responses is key to understand their environmental distribution and resilience under current and future disturbances. Here, we used metagenomics and metatranscriptomics to investigate microbial community structure and transcriptional responses to prolonged ammonium deprivation, and sulfide and nitric oxide toxicity stresses in a controlled bioreactor system mimicking coastal sediment conditions. Ca. Nitrobium versatile, identified in this study as a sulfide-oxidizing denitrifier, became a rare community member upon ammonium removal. The ANaerobic Methanotroph (ANME) Ca. Methanoperedens nitroreducens showed remarkable resilience to both experimental conditions, dominating transcriptional activity of dissimilatory nitrate reduction to ammonium (DNRA). During the ammonium removal experiment, increased DNRA was unable to sustain anaerobic ammonium oxidation (anammox) activity. After ammonium was reintroduced, a novel anaerobic bacterial methanotroph species that we have named Ca. Methylomirabilis tolerans outcompeted Ca. Methylomirabilis lanthanidiphila, while the anammox Ca. Kuenenia stuttgartiensis outcompeted Ca. Scalindua rubra. At the end of the sulfide and nitric oxide experiment, a gammaproteobacterium affiliated to the family Thiohalobacteraceae was enriched and dominated transcriptional activity of sulfide:quinone oxidoreductases. Our results indicate that some community members could be more resilient to the tested experimental conditions than others, and that some community functions such as methane and sulfur oxidation coupled to denitrification can remain stable despite large shifts in microbial community structure. Further studies on complex bioreactor enrichments are required to elucidate coastal ecosystem responses to future disturbances.

Orthopaedic Trauma Theatre Efficiency in the COVID-19 Pandemic: Are We Returning to Normality?

Background Recent studies have shown a decline in theatre efficiency and productivity coinciding with the coronavirus disease 2019 (COVID-19) pandemic. In this study, we evaluate trauma theatre task efficiency in three different time periods (April 2019, April 2020, and November 2020), and analyse if productivity has altered since the start of the pandemic. Methods The records of a total of 320 patients who underwent orthopaedic trauma surgery at a large district general hospital in April 2019, April 2020 (during the first wave of the pandemic) and November 2020 (during the second wave of the pandemic) were analysed. Primary outcomes measured include time to get to the theatre, anaesthetic preparation time, the sum of time of anaesthesia and surgical preparation time, duration of surgery and time to transfer to recovery. Patient demographics as well as the type of surgery were also analysed. Results The time to get to the theatre and anaesthetic preparation time significantly increased in April 2020 (p<0.05) but fell in November 2020 with no significant difference in comparison to before the pandemic in April 2019 (p>0.05). The duration of surgery and time to transfer to recovery significantly increased in April 2020 (p<0.05) and though reduced in November 2020, was still significantly greater in comparison to April 2019 (p<0.05). In April 2020, the proportion of patients aged 18-65 was just 26% as compared to 35% in April 2019. This figure rose again to 45% in November 2020. The number of hip fracture procedures remained similar during the three time periods, with 32, 32 and 36 hip fracture operations in April 2019, April 2020 and November 2020, respectively. Conclusion While operating theatres' efficiency decreased during the first wave of the COVID-19 pandemic, it increased again in the second wave, coming close to the 'normal' levels before the pandemic struck.

Excitation-scanning hyperspectral video endoscopy: enhancing the light at the end of the tunnel.

Colorectal cancer is the 3rd leading cancer for incidence and mortality rates. Positive treatment outcomes have been associated with early detection; however, early stage lesions have limited contrast to surrounding mucosa. A potential technology to enhance early stagise detection is hyperspectral imaging (HSI). While HSI technologies have been previously utilized to detect colorectal cancer ex vivo or post-operation, they have been difficult to employ in real-time endoscopy scenarios. Here, we describe an LED-based multifurcated light guide and spectral light source that can provide illumination for spectral imaging at frame rates necessary for video-rate endoscopy. We also present an updated light source optical ray-tracing model that resulted in further optimization and provided a ∼10X light transmission increase compared to the initial prototype. Future work will iterate simulation and benchtop testing of the hyperspectral endoscopic system to achieve the goal of video-rate spectral endoscopy.

Sensitivity analysis of a multibranched light guide for real time hyperspectral imaging systems.

Hyperspectral imaging (HSI) is a spectroscopic technique which captures images at a high contrast over a wide range of wavelengths to show pixel specific composition. Traditional uses of HSI include: satellite imagery, food distribution quality control and digital archaeological reconstruction. Our lab has focused on developing applications of HSI fluorescence imaging systems to study molecule-specific detection for rapid cell signaling events or real-time endoscopic screening. Previously, we have developed a prototype spectral light source, using our modified imaging technique, excitation-scanning hyperspectral imaging (HIFEX), coupled to a commercial colonoscope for feasibility testing. The 16 wavelength LED array was combined, using a multi-branched solid light guide, to couple to the scope's optical input. The prototype acquired a spectral scan at near video-rate speeds (∼8 fps). The prototype could operate at very rapid wavelength switch speeds, limited to the on/off rates of the LEDs (∼10 µs), but imaging speed was limited due to optical transmission losses (∼98%) through the solid light guide. Here we present a continuation of our previous work in performing an in-depth analysis of the solid light guide to optimize the optical intensity throughput. The parameters evaluated include: LED intensity input, geometry (branch curvature and combination) and light propagation using outer claddings. Simulations were conducted using a Monte Carlo ray tracing software (TracePro). Results show that transmission within the branched light guide may be optimized through LED focusing lenses, bend radii and smooth tangential branch merges. Future work will test a new fabricated light guide from the optimized model framework.

Mimicking microbial interactions under nitrate-reducing conditions in an anoxic bioreactor: enrichment of novel Nitrospirae bacteria distantly related to Thermodesulfovibrio.

Microorganisms are main drivers of the sulfur, nitrogen and carbon biogeochemical cycles. These elemental cycles are interconnected by the activity of different guilds in sediments or wastewater treatment systems. Here, we investigated a nitrate-reducing microbial community in a laboratory-scale bioreactor model that closely mimicked estuary or brackish sediment conditions. The bioreactor simultaneously consumed sulfide, methane and ammonium at the expense of nitrate. Ammonium oxidation occurred solely by the activity of anammox bacteria identified as Candidatus Scalindua brodae and Ca. Kuenenia stuttgartiensis. Fifty-three percent of methane oxidation was catalyzed by archaea affiliated to Ca. Methanoperedens and 47% by Ca. Methylomirabilis bacteria. Sulfide oxidation was mainly shared between two proteobacterial groups. Interestingly, competition for nitrate did not lead to exclusion of one particular group. Metagenomic analysis showed that the most abundant taxonomic group was distantly related to Thermodesulfovibrio sp. (87-89% 16S rRNA gene identity, 52-54% average amino acid identity), representing a new family within the Nitrospirae phylum. A high quality draft genome of the new species was recovered, and analysis showed high metabolic versatility. Related microbial groups are found in diverse environments with sulfur, nitrogen and methane cycling, indicating that these novel Nitrospirae bacteria might contribute to biogeochemical cycling in natural habitats.

Nitrate- and nitrite-dependent anaerobic oxidation of methane.

Microbial methane oxidation is an important process to reduce the emission of the greenhouse gas methane. Anaerobic microorganisms couple the oxidation of methane to the reduction of sulfate, nitrate and nitrite, and possibly oxidized iron and manganese minerals. In this article, we review the recent finding of the intriguing nitrate- and nitrite-dependent anaerobic oxidation of methane (AOM). Nitrate-dependent AOM is catalyzed by anaerobic archaea belonging to the ANME-2d clade closely related to Methanosarcina methanogens. They were named 'Candidatus Methanoperedens nitroreducens' and use reverse methanogenesis with the key enzyme methyl-coenzyme M (methyl-CoM) reductase for methane activation. Their major end product is nitrite which can be taken up by nitrite-dependent methanotrophs. Nitrite-dependent AOM is performed by the NC10 bacterium 'Candidatus Methylomirabilis oxyfera' that probably utilizes an intra-aerobic pathway through the dismutation of NO to N2 and O2 for aerobic methane activation by methane monooxygenase, yet being a strictly anaerobic microbe. Environmental distribution, physiological and biochemical aspects are discussed in this article as well as the cooperation of the microorganisms involved.

Branchial nitrogen cycle symbionts can remove ammonia in fish gills.

Knowledge of the mechanisms by which fish excrete their metabolic nitrogenous waste and insights into nitrogen cycling in aquaculture systems is of utmost importance to improve the sustainable commercial production of fish. In fish, most nitrogenous waste is excreted via the gills as ammonia, a potentially toxic nitrogenous compound. In this study; activity assays, physiological experiments, molecular analysis and microscopy were used to show that the gills of fish harbor a unique combination of hitherto overlooked nitrogen-cycle microorganisms that can theoretically detoxify excreted ammonia by converting it into inert dinitrogen gas. By doing so, these microorganisms may benefit from the ammonia supply by the host and prevent the build-up of this compound to toxic concentrations. This novel relationship between vertebrates and microorganisms may shed new light on nitrogen handling by ammonotelic fish species.

Hyaluronidase and Hyaluronan Oligosaccharides Promote Neurological Recovery after Intraventricular Hemorrhage.

Intraventricular hemorrhage (IVH) in premature infants results in inflammation, arrested oligodendrocyte progenitor cell (OPC) maturation, and reduced myelination of the white matter. Hyaluronan (HA) inhibits OPC maturation and complexes with the heavy chain (HC) of glycoprotein inter-α-inhibitor to form pathological HA (HC-HA complex), which exacerbates inflammation. Therefore, we hypothesized that IVH would result in accumulation of HA, and that either degradation of HA by hyaluronidase treatment or elimination of HCs from pathological HA by HA oligosaccharide administration would restore OPC maturation, myelination, and neurological function in survivors with IVH. To test these hypotheses, we used the preterm rabbit model of glycerol-induced IVH and analyzed autopsy samples from premature infants. We found that total HA levels were comparable in both preterm rabbit pups and human infants with and without IVH, but HA receptors--CD44, TLR2, TLR4--were elevated in the forebrain of both humans and rabbits with IVH. Hyaluronidase treatment of rabbits with IVH reduced CD44 and TLR4 expression, proinflammatory cytokine levels, and microglia infiltration. It also promoted OPC maturation, myelination, and neurological recovery. HC-HA and tumor necrosis factor-stimulated gene-6 were elevated in newborns with IVH; and depletion of HC-HA levels by HA oligosaccharide treatment reduced inflammation and enhanced myelination and neurological recovery in rabbits with IVH. Hence, hyaluronidase or HA oligosaccharide treatment represses inflammation, promotes OPC maturation, and restores myelination and neurological function in rabbits with IVH. These therapeutic strategies might improve the neurological outcome of premature infants with IVH. Significance statement: Approximately 12,000 premature infants develop IVH every year in the United States, and a large number of survivors with IVH develop cerebral palsy and cognitive deficits. The onset of IVH induces inflammation of the periventricular white matter, which results in arrested maturation of OPCs and myelination failure. HA is a major component of the extracellular matrix of the brain, which regulates inflammation through CD44 and TLR2/4 receptors. Here, we show two mechanism-based strategies that effectively enhanced myelination and neurological recovery in preterm rabbit model of IVH. First, degrading HA by hyaluronidase treatment reduced CD44 and TLR4 expression, proinflammatory cytokines, and microglial infiltration, as well as promoted oligodendrocyte maturation and myelination. Second, intraventricular injection of HA oligosaccharide reduced inflammation and enhanced myelination, conceivably by depleting HC-HA levels.

Extended Production of Cortical Interneurons into the Third Trimester of Human Gestation.

In humans, the developmental origins of interneurons in the third trimester of pregnancy and the timing of completion of interneuron neurogenesis have remained unknown. Here, we show that the total and cycling Nkx2.1(+)and Dlx2(+)interneuron progenitors as well as Sox2(+)precursor cells were higher in density in the medial ganglionic eminence (MGE) compared with the lateral ganglionic eminence and cortical ventricular/subventricular zone (VZ/SVZ) of 16-35 gw subjects. The proliferation of these progenitors reduced as a function of gestational age, almost terminating by 35 gw. Proliferating Dlx2(+)cells were higher in density in the caudal ganglionic eminence (CGE) compared with the MGE, and persisted beyond 35 gw. Consistent with these findings, Sox2, Nkx2.1, Dlx2, and Mash1 protein levels were higher in the ganglionic eminences relative to the cortical VZ/SVZ. The density of gamma-aminobutyric acid-positive (GABA(+)) interneurons was higher in the cortical VZ/SVZ relative to MGE, but Nkx2.1 or Dlx2-expressing GABA(+)cells were more dense in the MGE compared with the cortical VZ/SVZ. The data suggest that the MGE and CGE are the primary source of cortical interneurons. Moreover, their generation continues nearly to the end of pregnancy, which may predispose premature infants to neurobehavioral disorders.

A Metagenomics-Based Metabolic Model of Nitrate-Dependent Anaerobic Oxidation of Methane by Methanoperedens-Like Archaea.

Methane oxidation is an important process to mitigate the emission of the greenhouse gas methane and further exacerbating of climate forcing. Both aerobic and anaerobic microorganisms have been reported to catalyze methane oxidation with only a few possible electron acceptors. Recently, new microorganisms were identified that could couple the oxidation of methane to nitrate or nitrite reduction. Here we investigated such an enrichment culture at the (meta) genomic level to establish a metabolic model of nitrate-driven anaerobic oxidation of methane (nitrate-AOM). Nitrate-AOM is catalyzed by an archaeon closely related to (reverse) methanogens that belongs to the ANME-2d clade, tentatively named Methanoperedens nitroreducens. Methane may be activated by methyl-CoM reductase and subsequently undergo full oxidation to carbon dioxide via reverse methanogenesis. All enzymes of this pathway were present and expressed in the investigated culture. The genome of the archaeal enrichment culture encoded a variety of enzymes involved in an electron transport chain similar to those found in Methanosarcina species with additional features not previously found in methane-converting archaea. Nitrate reduction to nitrite seems to be located in the pseudoperiplasm and may be catalyzed by an unusual Nar-like protein complex. A small part of the resulting nitrite is reduced to ammonium which may be catalyzed by a Nrf-type nitrite reductase. One of the key questions is how electrons from cytoplasmically located reverse methanogenesis reach the nitrate reductase in the pseudoperiplasm. Electron transport in M. nitroreducens probably involves cofactor F420 in the cytoplasm, quinones in the cytoplasmic membrane and cytochrome c in the pseudoperiplasm. The membrane-bound electron transport chain includes F420H2 dehydrogenase and an unusual Rieske/cytochrome b complex. Based on genome and transcriptome studies a tentative model of how central energy metabolism of nitrate-AOM could work is presented and discussed.

Treatment with thyroxine restores myelination and clinical recovery after intraventricular hemorrhage.

Intraventricular hemorrhage (IVH) remains a major cause of white matter injury in preterm infants with no viable therapeutic strategy to restore myelination. Maturation of oligodendrocytes and myelination is influenced by thyroid hormone (TH) signaling, which is mediated by TH receptor α (TRα) and TRß. In the brain, cellular levels of TH are regulated by deiodinases, with deiodinase-2 mediating TH activation and deiodinase-3 TH inactivation. Therefore, we hypothesized that IVH would decrease TH signaling via changes in the expression of deiodinases and/or TRs, and normalization of TH signaling would enhance maturation of oligodendrocytes and myelination in preterm infants with IVH. These hypotheses were tested using both autopsy materials from human preterm infants and a rabbit model of IVH. We found that deiodinase-2 levels were reduced, whereas deiodinase-3 levels were increased in brain samples of both humans and rabbits with IVH compared with controls without IVH. TRα expression was also increased in human infants with IVH. Importantly, treatment with TH accelerated the proliferation and maturation of oligodendrocytes, increased transcription of Olig2 and Sox10 genes, augmented myelination, and restored neurological function in pups with IVH. Consistent with these findings, the density of myelinating oligodendrocytes was almost doubled in TH-treated human preterm infants compared with controls. Thus, in infants with IVH the combined elevation in deiodinase-3 and reduction in deiodinase-2 decreases TH signaling that can be worsened by an increase in unliganded TRα. Given that TH promotes neurological recovery in IVH, TH treatment might improve the neurodevelopmental outcome of preterm infants with IVH.

Victims of bullying in the emergency department with behavioral issues.

BACKGROUND: Bullying has become one of the most significant school problems experienced by our children. Victims of bullying are prone to a variety of psychological and behavioral symptoms. We noted that many children referred to the Emergency Department (ED) with behavioral symptoms provided a history of bullying. OBJECTIVES: To measure the prevalence of bullying in children referred to the ED for behavioral symptoms and to determine its association with psychiatric disorders. METHODS: A retrospective cohort study was conducted in an urban hospital, identifying children from 8 to 19 years of age who presented to the ED with behavioral symptoms. We reviewed the ED psychiatry notes to retrieve the report indicating whether these children were bullied and had previous psychiatric diagnoses. These children were classified into bullied and non-bullied groups. RESULTS: Over the study period, 591 children visited the ED with behavioral issues. Out of 591, 143 (24%) children reported bullying. More boys (100) than girls (43) reported bullying (p = 0.034). The mean age of children in the bullied group was 10.6 years (95% confidence interval 10.1-11.2). One hundred eleven (77.6%) children in the bullied group had a prior psychiatric diagnosis. Children in the bullied group were hospitalized significantly less than children in the non-bullied group (10/143 [7%] vs. 80/368 [18%]; p = 0.002). CONCLUSION: The prevalence of bullying among the ED children with behavioral symptoms is substantial. Every fourth child with behavioral symptoms reported bullying. Four in five children who reported bullying had a prior diagnosis of "disorder of behavior."