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Objectives: This study analyzed and identified factors related to the changes of the daily life, such as the health behavior, the number of meetings, and the use of public transportation during the COVID-19. Methods: A total of 133,707 people out of 229,269 adults aged 19 years or older in Community Health Survey 2020 were analyzed in terms of the daily life changes, including the demographic characteristics, health status, health behavior, mental health, and chronic disease. The multi-variable linear regression analysis was conducted with the considerable variables extracted from the stepwise selection. Results: It was found that age, sex, household type, job, household income, education level, subjective health status, breakfast, stress level, depression, hypertension, and diabetes are related to the changes of the daily life during the COVID-19. In particular, the age of 30s and 40s, women, second generation, white-collar worker, high income, well educated, worse subjective health status, skipping breakfast, high stress and depression factors were closely related to the negative changes of daily life during the COVID-19. Conclusions: The negative changes of daily life during the COVID-19 were more likely to occur in the group of the age of 30s and 40s, women, with high socioeconomic status, with worse health status, with worse health behaviors and with worse mental health.

Climate-smart land management practices that replace shallow-rooted annual crop systems with deeply-rooted perennial plants can contribute to soil carbon sequestration. However, deep soil carbon accrual may be influenced by active microbial biomass and their capacity to assimilate fresh carbon at depth. Incorporating active microbial biomass, dormancy, and growth in microbially-explicit models can improve our ability to predict soil’s capacity to store carbon. But, so far, the microbial parameters that are needed for such modeling are poorly constrained, especially in deep soil layers. Here, we used a lab incubation experiment and growth kinetics model to estimate how microbial parameters vary along 240 cm of soil depth in profiles under shallow- (soy) and deeply-rooted (switchgrass) plants 11 years after plant cover conversion. We also assessed resource origin and availability (total organic carbon, 14C, extractable organic carbon, specific UV absorbance of K2SO4 extractable organic C, total nitrogen, total dissolved nitrogen) along the soil profiles to examine associations between soil chemical and biological parameters. Even though root biomass was greater and rooting depth was deeper under switchgrass than soy, resource availability and microbial growth parameters were generally similar between vegetation types. Instead, depth significantly influenced soil chemical and biological parameters. For example, resource availability and total and relative active microbial biomass decreased with soil depth. Decreases in the relative active microbial biomass coincided with increased lag time (response time to external carbon inputs) along the soil profiles. Even at a depth of 210–240 cm, microbial communities were activated to grow by added resources within a day. Maximum specific growth rate decreased to a depth of 90 cm and then remained consistent in deeper layers. Our findings show that >10 years of vegetation and rooting depth changes may not be long enough to alter microbial growth parameters, and suggest that at least a portion of the microbial community in deep soils can grow rapidly in response to added resources. Our study determined microbial growth parameters that can be used in microbially-explicit models to simulate carbon dynamics in deep soil layers.

Chemokine receptors constitute an important subfamily of G protein-coupled receptors (GPCRs), and they are critically involved in a broad range of immune response mechanisms. Ligand promiscuity among these receptors makes them an interesting target to explore novel aspects of biased agonism. Here, we comprehensively characterize two chemokine receptors namely, CXCR4 and CXCR7, which share a common chemokine agonist (CXCL12), in terms of their G-protein coupling, β-arrestin (βarr) recruitment, contribution of GRKs, and ERK1/2 MAP kinase activation. We observe that CXCR7 lacks G-protein coupling while maintaining robust βarr recruitment with a major contribution of GRK5/6. On the other hand, CXCR4 displays robust G-protein activation as expected, however, it exhibits significantly reduced βarr-coupling compared to CXCR7 in response to their shared natural agonist, CXCL12. These two receptors induce distinct βarr conformations even when activated by the same agonist, and CXCR7, unlike CXCR4, fails to activate ERK1/2 MAP kinase. We further determine the crystal structure of βarr2 in complex with a carboxyl-terminal phosphopeptide derived from CXCR7, which reveals a smaller interdomain rotation than observed previously for activated βarrs. Importantly, structure-guided cellular experiments reveal a key contribution of a single phosphorylation site in CXCR7 on βarr recruitment and endosomal trafficking. Taken together, our study provides molecular insights into intrinsic bias encoded in the CXCR4-CXCR7 system, and it has broad implications for therapeutically important framework of biased agonism.

Rising antibiotic resistance urgently begs for novel targets and strategies for antibiotic discovery. Here, we report that over-activation of the periplasmic DegP protease, a member of the highly conserved HtrA family, can be a viable strategy for antibiotic development. We demonstrate that tripodal peptidyl compounds that mimic DegP-activating lipoprotein variants allosterically activate DegP and inhibit the growth of an Escherichia coli strain with a permeable outer membrane in a DegP-dependent fashion. Interestingly, these compounds inhibit bacterial growth at a temperature at which DegP is not essential for cell viability, mainly by over-proteolysis of newly synthesized proteins. Co-crystal structures show that the peptidyl arms of the compounds bind to the substrate-binding sites of DegP. Overall, our results represent an intriguing example of killing bacteria by activating a non-essential enzyme, and thus expand the scope of antibiotic targets beyond the traditional essential proteins or pathways., Hyunjin Cho et al. show that peptidyl compounds activating the periplasmic DegP protease inhibit the growth of Escherichia coli by promoting the proteolysis of newly synthesized proteins. This study presents an intriguing strategy to combat antibiotic resistance by activating a non-essential bacterial enzyme, thus expanding the scope of traditional antibiotic targets.

Production and reduction of nitrous oxide (N2O) by soil denitrifiers influence atmospheric concentrations of this potent greenhouse gas. Accurate projections of the net N2O flux have three key uncertainties: (1) short- vs. long-term responses to warming, (2) interactions among soil horizons, and (3) temperature responses of different steps in the denitrification pathway. We addressed these uncertainties by sampling soil from a boreal forest climate transect encompassing a 5.2 ∘C difference in the mean annual temperature and incubating the soil horizons in isolation and together at three ecologically relevant temperatures in conditions that promote denitrification. Both short-term exposure to warmer temperatures and long-term exposure to a warmer climate increased N2O emissions from organic and mineral soils; an isotopic tracer suggested that an increase in N2O production was more important than a decline in N2O reduction. Short-term warming promoted the reduction of organic horizon-derived N2O by mineral soil when these horizons were incubated together. The abundance of nirS (a precursor gene for N2O production) was not sensitive to temperature, whereas that of nosZ clade I (a gene for N2O reduction) decreased with short-term warming in both horizons and was higher from a warmer climate. These results suggest a decoupling of gene abundance and process rates in these soils that differs across horizons and timescales. In spite of these variations, our results suggest a consistent, positive response of denitrifier-mediated net N2O efflux rates to temperature across timescales in these boreal forests. Our work also highlights the importance of understanding cross-horizon N2O fluxes for developing a predictive understanding of net N2O efflux from soils.

Microbial extracellular enzymes decompose distinct components of soil organic matter (SOM), thus influencing its stability. However, we lack the knowledge about how the kinetics of individual enzymes vary when multiple substrates change simultaneously. Here we used Japanese knotweed (Polygonum cuspidatum) invasion as a model system to explore how the Michaelis–Menten kinetics (Vmax and km) of microbial extracellular enzymes vary with corresponding SOM components across soil depth (0–5, 5–10, and 10–15 cm). We hypothesized that invasion will increase the Vmax (maximum enzyme activity) and km (substrate concentration at half Vmax) of oxidative enzymes but decrease the Vmax and km of hydrolytic enzymes, and that increasing soil depth will alleviate the invasion effects on the enzyme kinetics. The invasion of knotweed, which input litter rich in recalcitrant compounds, altered soil chemistry including an increase in lignin and fungal biomass compared to the adjacent non-invaded soils. The Vmax of peroxidase, the oxidative enzyme that degrades lignin, increased in the invaded soils (0–5 cm) compared to the non-invaded soils. Among the hydrolytic enzymes, the Vmax of N-acetyl-glucosaminidase which degrades chitin from fungal cell walls increased in the invaded soils (0–5 cm). However, there was no associated change in the km of peroxidase and N-acetyl-glucosaminidase under invasion, suggesting that microbes modified the enzyme production rates, not the types (isozyme) of enzymes under invasion. The Vmax of all enzymes decreased with depth, due to the reduced substrate availability. These results highlight that the addition of relatively recalcitrant substrates due to plant invasion altered the kinetics of microbial extracellular enzymes with implications for SOM chemistry in the invaded soils.

Soil stores over 2500 Pg carbon (C), with the majority of C stored in deep soil layers (> 30 cm). Soil C can be lost to the atmosphere when organic compounds are mineralized to carbon dioxide (CO2, via oxidative decay or respiration) and moved upward through the soil profile (via diffusion). Soil moisture status can influence the balance between respiration and diffusion, thereby altering the soil CO2 concentration and flux. However, it is unclear how wetting and drying influence soil CO2 dynamics in surface vs. deep soil layers. Thus, we irrigated three soil profiles in Mediterranean arable land and continuously monitored soil CO2 concentration at 15, 30, 50, 70 and 90 cm during wetting and drying phases under ambient temperature conditions. We estimated gas diffusivity, CO2 flux, and temperature responses of soil CO2 concentration during the experiment. Decreases in gas diffusivity during the wetting period coincided with increases in soil CO2 concentrations. However, the negative gas diffusivity-soil CO2 concentration relationship did not hold true all the time, implying that CO2 production was the driving factor for the apparent soil CO2 concentration. We observed hysteretic responses of soil CO2 concentration to temperature as soil moisture varied, with deeper soil CO2 concentration being more sensitive to temperature than surface soil CO2 concentration, especially during the drying phase. The movement of CO2 was upward at all depths during the ambient phase, but the direction and the magnitude of CO2 fluxes varied across the depth gradient during the wetting and drying phases. This study highlights that the relative contribution of gas diffusivity vs. CO2 production to soil CO2 concentration changes with wetting and drying, and that the responses of soil CO2 concentration to temperature are dependent on the antecedent environmental conditions. Also, the downward movement of CO2 during the wetting and drying phases suggests that quantifying surface soil CO2 efflux may underestimate dynamic C processes in deeper soils.

Endangered cranes migrate to the Demilitarized Zone in Korea in search for habitat and food during winter. While cranes have the potential to influence soil biogeochemical processes via dropping, foraging, and walking, few studies have investigated ecological roles of migrating birds in the new habitat. Here, we explored how cranes alter resource landscape (the amount and quality of carbon) and microbial community in soil. We set up control (fenced, no crane access) and treatment (free crane activities) plots (n = 6, respectively) in a rice paddy, and collected soils at 0–15 cm three months after the crane migration. Soils were tested for total carbon, total nitrogen, water extractable organic carbon, and Diffuse Reflectance Infrared Fourier Transform Spectroscopy, along with microbial parameters (biomass, respiration, community composition). The wintering crane activity significantly increased total carbon and nitrogen contents, but decreased the ratio of CH (aliphatic) to COO (carboxylic) in soil. Also, both microbial biomass and respiration was greater in soils under crane activities. Bacterial and fungal community composition differed with or without crane activities, with treatment soils harboring more diverse microbial communities. Our results demonstrate that crane migration created a distinct system with altered resource landscape and microbial community, highlighting beneficial effects of migratory cranes on the soil biogeochemical processes in rice paddies. This study may help encourage more farmers, local governments, and the public to participate in crane conservation campaigns targeted at rice fields.

Accurate representation of temperature sensitivity (Q10 ) of soil microbial activity across time is critical for projecting soil CO2 efflux. As microorganisms mediate soil carbon (C) loss via exo-enzyme activity and respiration, we explore temperature sensitivities of microbial exo-enzyme activity and respiratory CO2 loss across time and assess mechanisms associated with these potential changes in microbial temperature responses. We collected soils along a latitudinal boreal forest transect with different temperature regimes (long-term timescale) and exposed these soils to laboratory temperature manipulations at 5, 15, and 25°C for 84 days (short-term timescale). We quantified temperature sensitivity of microbial activity per g soil and per g microbial biomass at days 9, 34, 55, and 84, and determined bacterial and fungal community structure before the incubation and at days 9 and 84. All biomass-specific rates exhibited temperature sensitivities resistant to change across short- and long-term timescales (mean Q10 = 2.77 ± 0.25, 2.63 ± 0.26, 1.78 ± 0.26, 2.27 ± 0.25, 3.28 ± 0.44, 2.89 ± 0.55 for β-glucosidase, N-acetyl-β-d-glucosaminidase, leucine amino peptidase, acid phosphatase, cellobiohydrolase, and CO2 efflux, respectively). In contrast, temperature sensitivity of soil mass-specific rates exhibited either resilience (the Q10 value changed and returned to the original value over time) or resistance to change. Regardless of the microbial flux responses, bacterial and fungal community structure was susceptible to change with temperature, significantly differing with short- and long-term exposure to different temperature regimes. Our results highlight that temperature responses of microbial resource allocation to exo-enzyme production and associated respiratory CO2 loss per unit biomass can remain invariant across time, and thus, that vulnerability of soil organic C stocks to rising temperatures may persist in the long term. Furthermore, resistant temperature sensitivities of biomass-specific rates in spite of different community structures imply decoupling of community constituents and the temperature responses of soil microbial activities.

A new vehicle is designed for the intracellular delivery of antibodies at nanomolar concentrations by combination of domain Z, a small affibody with strong binding affinity to Fc regions of immunoglobulin G (IgG), and the multimers of LK sequences, α-helical cell penetrating peptides (CPP) with powerful cell penetrating activities. Domain Z and multimeric LK are fused together to form LK-domain Z proteins. The LK-domain Z can bind with IgG at a specific ratio at nanomolar concentrations by simple mixing. The IgG/LK-domain Z complexes can successfully penetrate live cells at nanomolar concentration and the delivery efficiency is strongly dependent upon the concentrations of IgG/LK-domain Z complex as well as the species and subclasses of IgGs. The IgG/LK-domain Z complexes penetrate cells via ATP-dependent endocytosis pathway and the majority of delivered IgG seems to escape endosome to cytosol. Remarkably, the delivered IgGs are able to control the targeted intracellular signaling pathway as shown in the down-regulation of pro-survival genes by the delivery of anti-NF-κB using an LK-domain Z vehicle with a cathepsin B-cleavable linker between the LK sequence and domain Z. The simple but very efficient intracellular delivery method of antibodies at nanomolar concentrations is expected to facilitate profound understanding of cell mechanisms and development of new future therapeutics on the basis of intracellular antibodies.

Climate-smart land management practices that replace shallow-rooted annual crop systems with deeply-rooted perennial plants can contribute to soil carbon sequestration. However, deep soil carbon accrual may be influenced by active microbial biomass and their capacity to assimilate fresh carbon at depth. Incorporating active microbial biomass, dormancy, and growth in microbially-explicit models can improve our ability to predict soil's capacity to store carbon. But, so far, the microbial parameters that are needed for such modeling are poorly constrained, especially in deep soil layers. Here, we used a lab incubation experiment and growth kinetics model to estimate how microbial parameters vary along 240 cm of soil depth in profiles under shallow- (soy) and deeply-rooted (switchgrass) plants 11 years after plant cover conversion. We also assessed resource origin and availability (total organic carbon, 14C, extractable organic carbon, specific UV absorbance of K2SO4 extractable organic C, total nitrogen, total dissolved nitrogen) along the soil profiles to examine associations between soil chemical and biological parameters. Even though root biomass was greater and rooting depth was deeper under switchgrass than soy, resource availability and microbial growth parameters were generally similar between vegetation types. Instead, depth significantly influenced soil chemical and biological parameters. For example, resource availability and total and relative active microbial biomass decreased with soil depth. Decreases in the relative active microbial biomass coincided with increased lag time (response time to external carbon inputs) along the soil profiles. Even at a depth of 210–240 cm, microbial communities were activated to grow by added resources within a day. Maximum specific growth rate decreased to a depth of 90 cm and then remained consistent in deeper layers. Our findings show that >10 years of vegetation and rooting depth changes may not be long enough to alter microbial growth parameters, and suggest that at least a portion of the microbial community in deep soils can grow rapidly in response to added resources. Our study determined microbial growth parameters that can be used in microbially-explicit models to simulate carbon dynamics in deep soil layers.

β-Arrestins (βarrs) critically regulate G-protein-coupled receptor (GPCR) signaling and trafficking. βarrs have two isoforms, βarr1 and βarr2. Receptor phosphorylation is a key determinant for the binding of βarrs, and understanding the intricate details of receptor-βarr interaction is the next frontier in GPCR structural biology. The high-resolution structure of active βarr1 in complex with a phosphopeptide derived from GPCR has been revealed, but that of βarr2 remains elusive. Here, we present a 2.3-A crystal structure of βarr2 in complex with a phosphopeptide (C7pp) derived from the carboxyl terminus of CXCR7. The structural analysis of C7pp-bound βarr2 reveals key differences from the previously determined active conformation of βarr1. One of the key differences is that C7pp-bound βarr2 shows a relatively small inter-domain rotation. Antibody-fragment-based conformational sensor and hydrogen/deuterium exchange experiments further corroborated the structural features of βarr2 and suggested that βarr2 adopts a range of inter-domain rotations.

β-arrestins (βarrs) critically regulate signaling and trafficking of G protein-coupled receptors (GPCRs), the largest family of drug targets in the human genome, and there are two isoforms of βarrs: βarr1 and βarr2. Most GPCRs interact with both the heterotrimeric G-proteins and βarrs, inducing distinct downstream signal transduction. However, certain chemokine receptors lack functional G-protein coupling, but they can efficiently recruit βarrs upon agonist-stimulation, and they are referred to as atypical chemokine receptors (ACKRs). Receptor phosphorylation is a key determinant for the binding of βarrs, and understanding the intricate details of receptor-βarr interaction is the next frontier in GPCR structural biology. To date, the high-resolution structures of active βarr1 have been revealed, but the activation mechanism of βarr2 by a phosphorylated GPCR remains elusive. Here, we present a 1.95 Å crystal structure of βarr2 in complex with a phosphopeptide (C7pp) derived from the carboxyl-terminus of ACKR3, also known as CXCR7. The structure of C7pp-bound βarr2 reveals key differences from the previously determined active conformation of βarr1. One of the key differences is that C7pp-bound βarr2 shows a relatively small inter-domain rotation. An antibody-fragment-based conformational sensor and hydrogen/deuterium exchange experiments further corroborate structural features and suggest that the determined structure is an alternative active conformation of βarr2.

Production and reduction of nitrous oxide (N2O) by soil denitrifiers influences atmospheric concentrations of this potent greenhouse gas. Accurate climate projections of net N2O flux have three key uncertainties: (1) short- vs. long-term responses to warming; (2) interactions among soil horizons; and (3) temperature responses of different steps in the denitrification pathway. We addressed these uncertainties by sampling soil from a boreal forest climate transect encompassing a 5.2 °C difference in mean annual temperature, and incubating the soil horizons in isolation and together at three ecologically relevant temperatures in conditions that promote denitrification. Both short-term exposure to warmer temperatures and long-term exposure to a warmer climate increased N2O emissions from organic and mineral soils; an isotopic tracer suggested an increase in N2O production was more important than a decline in N2O reduction. Short-term warming promoted reduction of organic horizon-derived N2O by mineral soil when these horizons were incubated together. The abundance of nirS (a precursor gene for N2O production) was not sensitive to temperature, while that of nosZ clade I (a gene for N2O reduction) decreased with short-term warming in both horizons and was higher from a warmer climate. These results suggest a decoupling of gene abundance and process rates in these soils that differs across horizons and timescales. In spite of these variations, our results suggest a consistent, positive response of denitrifier-mediated, net N2O efflux rates to temperature across timescales in these boreal forests. Our work also highlights the importance of understanding cross-horizon N2O fluxes for developing a predictive understanding of net N2O efflux from soils.

FtsZ, a tubulin homologue, is an essential protein of the Z-ring assembly in bacterial cell division. It consists of two domains, the N-terminal and C-terminal core domains, and has a conserved C-terminal tail region. Lateral interactions between FtsZ protofilaments and several Z-ring associated proteins (Zaps) are necessary for modulating Z-ring formation. ZapD, one of the positive regulators of Z-ring assembly, directly binds to the C-terminal tail of FtsZ and promotes stable Z-ring formation during cytokinesis. To gain structural and functional insights into how ZapD interacts with the C-terminal tail of FtsZ, we solved two crystal structures of ZapD proteins from Salmonella typhimurium (StZapD) and Escherichia coli (EcZapD) at a 2.6 and 3.1 Å resolution, respectively. Several conserved residues are clustered on the concave sides of the StZapD and EcZapD dimers, the suggested FtsZ binding site. Modeled structures of EcZapD-EcFtsZ and subsequent binding studies using bio-layer interferometry also identified the EcFtsZ binding site on EcZapD. The structural insights and the results of bio-layer interferometry assays suggest that the two FtsZ binding sites of ZapD dimer might be responsible for the binding of ZapD dimer to two protofilaments to hold them together.

The rate at which catalytic capacity of microbial exo-enzymes degrades post-exudation will influence the time during which return on microbes’ investment in exo-enzyme production can be realized. Further, if exo-enzyme degradation rates vary across exo-enzymes, microbial investment returns may vary by element across time. We quantify how aging of two soil organic matter (SOM)-decaying enzymes (β-D-cellobioside, BGase; and N-acetyl-β-D-glucosaminide, NAGase) influences enzyme-substrate V max at multiple temperatures (5, 15, 25 °C), and compute how enzyme age influences relative availabilities of C and N. Both BGase and NAGase exhibited similar, exponential declines in catalytic rate with age at 25 °C (0.22 ± 0.02 and 0.36 ± 0.14 d−1, respectively). At 15 °C, NAGase exhibited exponential declines in catalytic rates with age (0.79 ± 0.31 d−1), but BGase exhibited no decline. Neither enzyme exhibited a decline in catalytic rate over 72 h at 5 °C. At 15 °C, the amount of C liberated from cellulose and chitin analogues relative to N increased, on average, by more than one order of magnitude. The ratio of C:N liberated from the two substrates remained constant across enzyme age at 25 and 5 °C, but for different reasons: no differences in decay rate across enzymes at 25 °C, and no observed decay at 5 °C. Thus, temperature-dependent decreases of catalytic activity over time may influence microbial resource allocation strategies and rates of SOM decomposition. Because the enzyme decay rates we observed differ considerably from values assumed in most models, such assumptions should be revisited when parameterizing microbial process models.

Understanding how carbon dioxide (CO2) flux from ecosystems feeds back to climate warming depends in part on our ability to quantify the efficiency with which microorganisms convert organic carbon (C) into either biomass or CO2. Quantifying ecosystem-level respiratory CO2 losses often also requires assumptions about stable C isotope fractionations associated with the microbial transformation of organic substrates. However, the diversity of organic substrates' δ13C and the challenges of measuring microbial C use efficiency (CUE) in their natural environment fundamentally limit our ability to project ecosystem C budgets in a warming climate. Here, we quantify the effect of temperature on C fluxes during metabolic transformations of cellobiose, a common microbial substrate, by a cosmopolitan microorganism growing at a constant rate. Biomass C specific respiration rate increased by 250 % between 13 and 26.5 °C, decreasing CUE from 77 to 56 %. Biomass C specific respiration rate was positively correlated with an increase in respiratory 13C discrimination from 4.4 to 6.7 ‰ across the same temperature range. This first demonstration of a direct link between temperature, microbial CUE, and associated isotope fluxes provides a critical step towards understanding δ13C of respired CO2 at multiple scales, and towards a framework for predicting future ecosystem C fluxes.

Investigators of soil organic matter (SOM) transformations struggle with a deceptively simple-sounding question: "Why does some SOM leave the soil profile relatively quickly, while other compounds, especially those at depth, appear to be retained on timescales ranging from the decadal to the millennial?" This question is important on both practical and academic levels, but addressing it is challenging for a multitude of reasons. Simultaneous with soil-specific advances, multiple other disciplines have enhanced their knowledge bases in ways potentially useful for future investigations of SOM decay. In this article, we highlight observations highly relevant for those investigating SOM decay and retention but often emanating from disparate fields and residing in literature seldom cited in SOM research. We focus on recent work in two key areas. First, we turn to experimental approaches using natural and artificial aquatic environments to investigate patterns of microbially-mediated OM transformations as environmental conditions change, and highlight how aquatic microbial responses to environmental change can reveal processes likely important to OM decay and retention in soils. Second, we emphasize the importance of establishing intrinsic patterns of decay kinetics for purified substrates commonly found in soils to develop baseline rates. These decay kinetics – which represent the upper limit of the reaction rates – can then be compared to substrate decay kinetics observed in natural samples, which integrate intrinsic decay reaction rates and edaphic factors essential to the site under study but absent in purified systems. That comparison permits the site-specific factors to be parsed from the fundamental decay kinetics, an important advance in our understanding of SOM decay (and thus persistence) in natural systems. We then suggest ways in which empirical observations from aquatic systems and purified enzyme-substrate reaction kinetics can be used to advance recent theoretical efforts in SOM-focused research. Finally, we suggest how the observations in aquatic and purified enzyme-substrate systems could be used to help unravel the puzzles presented by oft-observed patterns of SOM characteristics with depth, as one example of the many perplexing SOM-related problems.

Accurate representation of temperature sensitivity (Q

Clustered regularly interspaced short palindromic repeats (CRISPRs) from Prevotella and Francisella 1 (Cpf1) are RNA-guided endonucleases that produce cohesive double-stranded breaks in DNA by specifically recognizing thymidine-rich protospacer-adjacent motif (PAM) sequences. Cpf1 is emerging as a powerful genome-editing tool. Despite previous structural studies on various Cpf1 proteins, the apo-structure of Cpf1 remains unknown. In the present study, we determined the solution structure of the Cpf1 protein from Francisella novicida (FnCpf1) with and without CRISPR RNA (crRNA) using small-angle X-ray scattering, providing the insights into the apo-structure of FnCpf1. The apo-structure of FnCpf1 was also visualized using negative staining electron microcopy. When we compared the apo-structure of FnCpf1 with crRNA-bound structure, their overall shapes (a closed form) were similar, suggesting that conformational change upon crRNA binding to FnCpf1 is not drastic, but a local induced fit might occur to recognize PAM sequences. In contrast, the apo Cpf1 from Moraxella bovoculi 237 (MbCpf1) was analyzed as an open form, implying that a large conformational change from an open to a closed form might be required for crRNA binding to MbCpf1. These results suggested that the crRNA-induced conformational changes in Cpf1 differ among species.

Phenolics are the most abundant plant metabolites and are believed to decompose slowly in soils compared to other soil organic matter (SOM). Thus, they have often been considered as a slow carbon (C) pool in soil dynamics models. Here, however, we review changes in our concept about the turnover rate of phenolics and quantification of different types of phenolics in soils. Also, we synthesize current research on the degradation of phenolics and their regulatory effects on decomposition. Environmental changes, such as elevated CO2, warming, nitrogen (N) deposition, and drought, could influence the production and form of phenolics, leading to a change in SOM dynamics, and thus we also review the fate of phenolics under environmental disturbances. Finally, we propose the use of phenolics as a tool to control rates of SOM decomposition to stabilize organic carbon in ecosystems. Further studies to clarify the role of phenolics in SOM dynamics should include improving quantification methods, elucidating the relationship between phenolics and soil microorganisms, and determining the interactive effects of combinations of environmental changes on the phenolics production and degradation and subsequent impact on SOM processing.

Soil microorganisms release extracellular enzymes into the soil matrix to access carbon (C) and nitrogen (N) from soil organic matter (SOM). Temperature and pH are major factors governing the rates at which these enzymes decay SOM, hence influencing the availability of C and N for microbial assimilation. As temperature increases, the rate of decomposition is also expected to increase. Recent advances provide estimates of intrinsic temperature sensitivities of key decay reactions at one, circum-neutral pH, but how temperature sensitivity of enzymatic SOM degradation is influenced by pH remains unclear. Here we expand on recent work by determining specific activities of C-acquiring (β-glucosidase; BGase) and C- and N-acquiring (N-Acetyl-Glucosaminidase; NAGase) enzymes with purified, fluorescently labeled organic substrate at temperatures from 5 to 25 °C (5 °C steps) and at pH values from 3.5 to 8.5 (1 pH unit steps). Using specific activity data, we quantified temperature sensitivities of the reactions with estimates of activation energy (Ea) at each pH value. We then used Ea estimates to compute temperature-induced changes in the C:N flow ratio, which is defined as the ratio of enzymatic liberation rates of C to N from the substrates. Across all temperatures, BGase activity was generally high in the pH range of 5.5–8.5, while NAGase exhibited a relatively narrow optimum between pH 5.5–6.5. Temperature sensitivity of BGase differed significantly among pH values; the strongest temperature responses were observed at pH 4.5. NAGase, in contrast, did not exhibit any significant pH-dependent changes in temperature sensitivity. The temperature increase from 5 to 25 °C induced changes in the C:N flow ratio, with direction and magnitude strongly dependent on the pH. We observed a large, temperature-induced increase in C:N flow ratio at pH 4.5 and decreases in C:N flow ratio at pH > 5.5 that were most pronounced at pH 7.5. Our data show that pH can induce differential effects on reaction rates and temperature sensitivity of organic C and N liberation, with consequences for changes in the relative availabilities of C and N for microbial assimilation.

8-Halogenated guanine (haloG), a major DNA adduct formed by reactive halogen species during inflammation, is a promutagenic lesion that promotes misincorporation of G opposite the lesion by various DNA polymerases. Currently, the structural basis for such misincorporation is unknown. To gain insights into the mechanism of misincorporation across haloG by polymerase, we determined seven x-ray structures of human DNA polymerase β (polβ) bound to DNA bearing 8-bromoguanine (BrG). We determined two pre-catalytic ternary complex structures of polβ with an incoming nonhydrolyzable dGTP or dCTP analog paired with templating BrG. We also determined five binary complex structures of polβ in complex with DNA containing BrG·C/T at post-insertion and post-extension sites. In the BrG·dGTP ternary structure, BrG adopts syn conformation and forms Hoogsteen base pairing with the incoming dGTP analog. In the BrG·dCTP ternary structure, BrG adopts anti conformation and forms Watson-Crick base pairing with the incoming dCTP analog. In addition, our polβ binary post-extension structures show Hoogsteen BrG·G base pair and Watson-Crick BrG·C base pair. Taken together, the first structures of haloG-containing DNA bound to a protein indicate that both BrG·G and BrG·C base pairs are accommodated in the active site of polβ. Our structures suggest that Hoogsteen-type base pairing between G and C8-modified G could be accommodated in the active site of a DNA polymerase, promoting G to C mutation.

The inflammation that accompanies acute injury has dual functions: bactericidal action and repair. Bactericidal functions protect damaged tissue from infection, and repair functions are initiated to aid in the recovery of damaged tissue. Brain injury is somewhat different from injuries in other tissues in two respects. First, many cases of brain injury are not accompanied by infection: there is no chance of pathogens to enter in ischemia or even in traumatic injury if the skull is intact. Second, neurons are rarely regenerated once damaged. This raises the question of whether bactericidal inflammation really occurs in the injured brain; if so, how is this type of inflammation controlled? Many brain inflammation studies have been conducted using cultured microglia (brain macrophages). Even where animal models have been used, the behavior of microglia and neurons has typically been analyzed at or after the time of neuronal death, a time window that excludes the inflammatory response, which begins immediately after the injury. Therefore, to understand the patterns and roles of brain inflammation in the injured brain, it is necessary to analyze the behavior of all cell types in the injured brain immediately after the onset of injury. Based on our experience with both in vitro and in vivo experimental models of brain inflammation, we concluded that not only microglia, but also astrocytes, blood inflammatory cells, and even neurons participate and/or regulate brain inflammation in the injured brain. Furthermore, brain inflammation played by these cells protects neurons and repairs damaged microenvironment but not induces neuronal damage.

Soil microorganisms produce exoenzymes to access resources stored in soil organic matter. Knowledge about the effect of temperature on the rates at which exoenzymes degrade substrates is particularly important for understanding carbon and nitrogen cycling with warming, and possible feedbacks to climate change. Here, we studied reaction rates of purified enzymes and substrates in controlled laboratory conditions at temperatures ranging from 5 °C to 27 °C. We employed three enzyme-substrate pairings representative of reactions common to soil profiles: β-glucosidase and β- d -cellobioside (BGase/BG), β-N-acetyl glucosaminidase and N-acetyl-β- d -glucosaminide (NAGase/NAG), and peroxidase and 3,4-Dihydroxy- l -phenylalanine (peroxidase/ l -Dopa). Across the entire temperature range studied, BGase showed the highest specific activity (Vmax 27 °C = 1338 μmol h−1 mgBGase−1), followed by NAGase (Vmax 25 °C = 260 μmol h−1 mgNAGase−1) and peroxidase (Vmax 25 °C = 36 μmol h−1 mgperoxidase−1). From 7.5 °C to 25 °C, the specific activities of BGase, NAGase and peroxidase increased by 103%, 111% and 835%, respectively. The activation energy (Ea) required for a reaction to proceed thus was highest for peroxidase/ l -Dopa (99.8 kJ mol l -Dopa−1), followed by NAGase/NAG (41.3 kJ molNAG−1) and BGase/BG (31.4 kJ molBG−1). We use a simple model, parameterized with empirical data from these reactions in three different ways, to illustrate how the flow of carbon relative to nitrogen can change with temperature as these resources are liberated from their organic precursors. The results highlight the importance of relative temperature sensitivities among reactions and the substrates' carbon to nitrogen ratio as key determinants of temperature-mediated changes in relative availabilities of carbon and nitrogen to microorganisms.

Microbial transformations of organic carbon (OC) generate a large flux of CO2 into the atmosphere and influence the C balance of terrestrial and aquatic ecosystems. Yet, inherent heterogeneity in natural environments precludes direct quantification of multiple microbial C fluxes that underlie CO2 production. Here we used a continuous flow bioreactor coupled with a stable C isotope analyzer to determine the effects of temperature and C availability (cellobiose concentration) on C fluxes and 13C discrimination of a microbial population growing at steady-state in a homogeneous, well-mixed environment. We estimated C uptake affinity and C use efficiency (CUE) to characterize the physiological responses of microbes to changing environmental conditions. Temperature increased biomass-C specific respiration rate and C uptake affinity at lower C availability, but did not influence those parameters at higher C availability. CUE decreased non-linearly with increasing temperature. The non-linear, negative relationship between CUE and temperature was more pronounced under lower C availability than under relatively high C availability. We observed stable isotope fractionation between C substrate and microbial biomass C (7~12‰ depletion), and between microbial biomass and respired CO2 (4~10‰ depletion). Microbial discrimination against 13C-containing cellobiose during C uptake was influenced by temperature and C availability, while discrimination during respiration was only influenced by C availability. Shifts in C uptake affinity with temperature and C availability may have modified uptake-induced 13C fractionation. By stressing the importance of C availability on temperature responses of microbial C fluxes, C uptake affinity, CUE, and isotopic fractionation, this study contributes to a fundamental understanding of C flow through microbes. This will help guide parameterization of microbial responses to varying temperature and C availability within Earth-system models.

Wetlands have been recognized as a soil carbon (C) sink due to low decomposition. As decomposition is largely controlled by the availability of soil nitrogen (N), an elevated anthropogenic N input could influence the C balance in wetlands. However, the effects of the form of N on decomposition are poorly understood. Here, a 54-day laboratory incubation experiment was conducted, with a diel cycle (day: 22 °C for 13 h; night: 17 °C for 11 h) in order to determine how the dominant N form influences the mineralization of soil C in two adjacent wetland soils, with distinct physicochemical characteristics. Three combinations of N compounds were added at three different rates (0, 30, 60 kg N ha −1 yr −1 ): Ammonium dominant (NH 4 Cl + NH 4 NO 3 ); nitrate dominant (NH 4 NO 3 + NaNO 3 ); and ammonium nitrate treatments (NH 4 NO 3 ). In the acidic soil, the CO 2 efflux was reduced with N additions, especially with NH 4 NO 3 treatment. In addition, decreases in the microbial enzyme activities ( β -glucosidase, N-acetyl-glucosaminidase, phosphatase, and phenol oxidase) and soil pH were observed with NH 4 NO 3 and NH 4 + -dominant treatment. Under alkaline conditions, marginal changes in response to N additions were observed in the soil CO 2 efflux, extractable DOC, simple substrate utilization, enzyme activities and pH. A regression analysis revealed that the changes in pH and enzyme activities after fertilization significantly influenced the soil CO 2 efflux. Our findings suggest that the form of N additions could influence the rate of C cycling in wetland soils via biological (enzyme activities) and chemical (pH) changes.

Hypoxia-inducible factor 1 (HIF-1) is a master transcription factor that mediates cellular and systemic homeostatic responses to reduce O2 availability, such as erythropoiesis, angiogenesis, and glycolysis. Using the yeast two-hybrid screening system, we found that the oxygen dependent degradation (ODD) domain of HIF-1alpha interacts with necdin, a growth suppressor. The interaction of necdin with HIF-1alpha was confirmed using coimmunoprecipitation with the overexpressed HIF-1alpha. Biological effect of necdin on HIF-1alpha showed that necdin reduces the transcriptional activity of HIF-1 under hypoxia. Moreover, necdin decreased the level of the HIF-1alpha protein, but not that of mRNA, implying a possibility of necdin-mediated HIF-1alpha degradation. Furthermore, necdin has an anti-angiogenic activity in the tube formation assay and CAM assay, which might be due to the downregulation of HIF-1alpha. Collectively, these results suggest that necdin can be a novel negative regulator of HIF-1alpha stability via the direct interaction.

O6-methylguanine (O6MeG) is a minor, yet highly genotoxic lesion that promotes misincorporation of T opposite O6MeG by various DNA polymerases in vitro. Human DNA polymerase β (polβ) has been shown...

We have synthesized 16,22-diketocholesterol, a novel ligand for oxysterol-binding protein Osh4, and determined X-ray structure of the diketocholesterol in complex with Osh4. The X-ray structure shows that α7 helix of Osh4 assumes open conformation while the rest of Osh4, closed conformation, implying this diketocholesterol–bound Osh4 structure may represent a structural intermediate between the two conformations.

Alterations in metabolic pathways are gaining attention as important environmental factors affecting life span, but the determination of specific metabolic pathways and enzymes involved in life span remains largely unexplored. By applying an NMR-based metabolomics approach to a calorie-restricted yeast (Saccharomyces cerevisiae) model, we found that alanine level is inversely correlated with yeast chronological life span. The involvement of the alanine-metabolizing pathway in the life span was tested using a deletion mutant of ALT1, the gene for a key alanine-metabolizing enzyme. The mutant exhibited increased endogenous alanine level and much shorter life span, demonstrating the importance of ALT1 and alanine metabolic pathways in the life span. ALT1's effect on life span was independent of the TOR pathway, as revealed by a tor1 deletion mutant. Further mechanistic studies showed that alt1 deletion suppresses cytochrome c oxidase subunit 2 expression, ultimately generating reactive oxygen species. Overall, ALT1 seems critical in determining yeast life span, and our approach should be useful for the mechanistic studies of life span determinations.

With MOSFET scaling, increased design complexity, and multiple system power domains, ESD failures occur in internal core areas which are not connected to external package pins. A review of the various internal core device failure mechanisms and design recommendations are presented.

This report examines three aspects of adipokinetic hormone (AKH) involvement in migratory flight behavior in the grasshopper, Melanoplus sanguinipes. The titer of hemolymph AKH I during long-duration tethered flight was examined using radioimmunoassay (RIA) after narrow bore RP-HPLC. The hemolymph fraction containing AKH I was assayed using commercially available anti-Tyr1-AKH I serum. Titer determinations of hemolymph AKH were done at rest and after various periods of flight. The amount of AKH I released from the corpora cardiaca during flight was estimated. When resting levels of AKH I and II in corpora cardiaca (CC) of migrants and non-migrants were examined with HPLC, no significant differences in AKH levels were detected between non-migrants, animals that had flown for 1 h to identify them as migrants, and animals that had flown to exhaustion (i.e., voluntary cessation). CC levels of both AKH I and II were less in this species than in locusts. When the lipid mobilization in response to AKH I and II was compared in migrants (animals that had self-identified as migrants in a 1-h tethered flight test) and non-migrants (animals that would not perform a 1-h flight in a tethered flight test), the adipokinetic response to AKH I was greater in migrants than in non-migrants, possibly indicating differences in level of sensitivity or number of receptors in the target tissues. AKH II had little effect on hemolymph lipid levels in either flight group, and may not play a significant role in lipid mobilization in this species.

Transient poly-Si etching behavior in CF4+D2 electron cyclotron resonance plasmas containing different D2 proportions was investigated. Higher D2 proportions resulted in lower atomic F and higher CF2 concentration in the plasma, as evidenced by optical emission spectroscopy (OES), and in greater oxide-to-Si etch selectivity. A high initial poly-Si etch rate that declined very rapidly to a finite-steady-state value was observed for plasma etching under conditions giving low (3:1) oxide-to-Si etch selectivity. In contrast, a lower initial etch rate that declined to approximately zero over a longer (∼45 s) period was observed for poly-Si etching under plasma conditions giving (∼15:1) selectivity. In the latter case, Si consumption during overetching would be significantly underestimated if calculated on the basis of the conventional 60 s selectivity ration. X-ray photoelectron spectroscopy analysis indicated that a thick, more F-deficient fluorocarbon film was deposited on Si under the high-selectivity etchi...