A Robust and Efficient Method to Purify DNA-Scaffolded Nanostructures by Gravity-Driven Size Exclusion Chromatography.

In recent decades, nucleic acid self-assemblies have emerged as popular nanomaterials due to their programmable and robust assembly, prescribed geometry, and versatile functionality. However, it remains a challenge to purify large quantities of DNA nanostructures or DNA-templated nanocomplexes for various applications. Commonly used purification methods are either limited by a small scale or incompatible with functionalized structures. To address this unmet need, we present a robust and scalable method of purifying DNA nanostructures by Sepharose resin-based size exclusion. The resin column can be manually packed in-house with reusability. The separation is driven by a low-pressure gravity flow in which large DNA nanostructures are eluted first followed by smaller impurities of ssDNA and proteins. We demonstrated the efficiency of the method for purifying DNA origami assemblies and protein-immobilized DNA nanostructures. Compared to routine agarose gel electrophoresis that yields 1 µg or less of purified products, this method can purify ∼100-1000 µg of DNA nanostructures in less than 30 min, with the overall collection yield of 50-70% of crude preparation mixture. The purified nanocomplexes showed more precise activity in evaluating enzyme functions and antibody-triggered activation of complement protein reactions.

Rapid measurement of hemoglobin-oxygen dissociation by leveraging Bohr effect and Soret band bathochromic shift.

Oxygen (O2) binds to hemoglobin (Hb) in the lungs and is then released (dissociated) in the tissues. The Bohr effect is a physiological mechanism that governs the affinity of Hb for O2 based on pH, where a lower pH results in a lower Hb-O2 affinity and higher Hb-O2 dissociation. Hb-O2 affinity and dissociation are crucial for maintaining aerobic metabolism in cells and tissues. Despite its vital role in human physiology, Hb-O2 dissociation measurement is underutilized in basic research and in clinical laboratories, primarily due to the technical complexity and limited throughput of existing methods. We present a rapid Hb-O2 dissociation measurement approach by leveraging the Bohr effect and detecting the optical shift in the Soret band that corresponds to the light absorption by the heme group in Hb. This new method reduces Hb-O2 dissociation measurement time from hours to minutes. We show that Hb deoxygenation can be accelerated chemically at the optimal pH of 6.9. We show that time and pH-controlled deoxygenation of Hb results in rapid and distinct conformational changes in its tertiary structure. These molecular conformational changes are manifested as significant, detectable shifts in Hb's optical absorption spectrum, particularly in the characteristic Soret band (414 nm). We extensively validated the method by testing human blood samples containing normal Hb and Hb variants. We show that rapid Hb-O2 dissociation can be used to screen for and detect Hb-O2 affinity disorders and to evaluate the function and efficacy of Hb-modifying therapies. The ubiquity of optical absorption spectrophotometers positions this approach as an accessible, rapid, and accurate Hb-O2 dissociation measurement method for basic research and clinical use. We anticipate this method's broad adoption will democratize the diagnosis and prognosis of Hb disorders, such as sickle cell disease. Further, this method has the potential to transform the research and development of new targeted and genome-editing-based therapies that aim to modify or improve Hb-O2 affinity.

Trait anxiety predicting the developmental trajectories of depression symptoms in children: The mediating role of attentional control.

Trait anxiety and attentional control are important factors related to depression symptoms. The study investigated how trait anxiety and attentional control predicted the trajectories of depression symptoms during the transition into early adolescence. The mediating effect of attentional control on the relationship of trait anxiety to the trajectories of depression symptoms was also examined. Children of 9 to 10 years were recruited at Time 1. Trait anxiety, attentional control, and depression symptoms were assessed at Time 1. Depression symptoms were measured at three follow-up assessments across 18 months. Latent class growth modeling revealed high (14.4%) and low (85.6%) trajectories of depression symptoms. Higher trait anxiety and lower attentional control predicted a higher likelihood of showing the trajectory of high depressive symptoms. Attentional control mediated the relationship of trait anxiety to the trajectory membership of depression symptoms. The findings had important implications for the association of trait anxiety with the trajectory membership of depression symptoms and highlighted the importance of attentional control in the development of depression symptoms for children with high trait anxiety.

Comparison of Treatment Methods of Thumb-Tip Defects With Phalangeal Bone Exposure: Modified Heterodigital Neurovascular Island Flap Versus Free Lateral Great Toe Flap.

PURPOSE: Modified heterodigital neurovascular island flaps and free lateral great toe flaps are dependable methods for treating thumb-tip defects with phalangeal bone exposure. We retrospectively analyzed and compared the details and results of the two methods. METHODS: This retrospective study included 25 patients with thumb injuries with phalangeal bone exposure treated between 2018 and 2021. Patients were categorized as per the following surgical methods: (1) modified heterodigital neurovascular island flap (12 patients, finger flap group) and (2) free lateral great toe flap (13 patients, toe flap group). The Michigan Hand Outcome Questionnaire, aesthetic appearance, Vancouver Scar Scale, Cold Intolerance Severity Score, static 2-point discrimination, Semmes-Weinstein monofilament, and range of motion of the metacarpophalangeal joint of the injured thumb were evaluated and compared. In addition, operation time, hospital stay, return-to-work time, and complications were recorded and compared. RESULTS: In both groups, the defect was successfully repaired, with no cases of complete necrosis. The 2 groups had similar mean scores in static 2-point discrimination, Semmes-Weinstein monofilament, range of motion, and Michigan Hand Outcome Questionnaire scores. The aesthetic appearance, scarring, and cold tolerance of the toe flap group were better than the finger flap group. The operation time, hospital stay, and return-to-work time in the finger flap group were shorter than the toe flap group. The finger flap group had 2 complications-a superficial infection and 1 case of partial flap necrosis. The toe flap group had 3 complications-a superficial infection, 1 case each of partial flap necrosis, and partial skin graft loss. CONCLUSION: Both treatments can achieve satisfactory results; however, they each have advantages and disadvantages. TYPE OF STUDY/LEVEL OF EVIDENCE: Therapeutic IV.

Exosomes derived from placental trophoblast cells regulate endometrial epithelial receptivity in dairy cows during pregnancy.

Inadequate fetomaternal interactions could directly lead to pregnancy failure in dairy cows. Exosomes are widely involved in endometrial matrix remodeling, immune function changes, placental development, and other processes of embryo implantation and pregnancy in dairy cows. However, the role of exosomes derived from placental trophoblast cells in regulating the receptivity of endometrial cells and facilitating fetomaternal interaction remains unclear. In this study, bovine trophoblast cells (BTCs) were obtained from bovine placenta and immortalized by transfection with telomerase reverse transcriptase (TERT). Immortalized BTCs still possess the basic and key properties of primary BTCs without exhibiting any neoplastic transformation signs. Subsequently, the effect of trophoblast-derived exosomes (TDEs) on endometrial receptivity in endometrial epithelial cells (EECs) was determined, and the mechanism whereby TDEs and their proteins participate in the fetomaternal interaction during bovine pregnancy were explored. EECs were co-cultured with the exosomes derived from BTCs treated with progesterone (P4). Such treatment enhanced the expression of the endometrial receptivity factors, integrin αv, ß3, Wnt7a, and MUC1 by changing the extracellular environment, metabolism, and redox balance in EECs via proteome alignment, compared with no treatment according to the DIA quantitation analysis. Our study demonstrated that trophoblast-derived exosome proteins are one of the most critical elements in fetomaternal interaction, and their changes may act as a key signal in altering endometrial receptivity and provide a potential target for improving fertility.

Morphological and immunohistochemical characteristics associated with metastatic and recurrent progression in pheochromocytoma/paraganglioma: A cohort study.

This study aimed to evaluate immunohistochemical markers of pheochromocytoma/paraganglioma (PPGL) and the relationships between the grading system for adrenal pheochromocytoma and paraganglioma (GAPP) and the prognosis of PPGL in a Chinese population. A retrospective analysis was conducted on a cohort of 102 PPGL cases, from January 2012 to December 2019, with complete clinicopathological and follow-up data. Surgical pathology slides were re-reviewed. All histological parameters involved in GAPP were summarized. The relationship between clinical characteristics, expression of SDHB (succinate dehydrogenase), S-100 and Ki-67 as well as GAPP classifications and prognosis of PPGL was statistically analyzed. The 102 cases included 51 males (50%) and 51 females (50%) with a median age of 48.7 years. The median tumor size was 6.8 cm. Metastases or relapse developed in 23 (22.5%) cases. Larger tumor size, extra-adrenal location, and poorly differentiated PPGL according to GAPP were associated with metastases or relapse (P < 0.05). Histological parameters, including the appearance of large or fused cell nests, necrosis, vascular invasion, and capsular invasion, were more common in the cases with metastases or relapse (P < 0.01). Loss of SDHB or S-100 expression was more common in poorly differentiated PPGL and associated with metastases or relapse (P < 0.01). However, no significant difference in the Ki-67 index between the clinically malignant and benign group was observed. GAPP is thus helpful for evaluations of the biological behavior of PPGL.

Coping profiles predict long-term anxiety trajectory in breast cancer survivors.

This study examined the long-term trajectory patterns of anxiety in breast cancer survivors and identified its predicting variables, especially the type of coping profile. Eighty-one patients who completed all four questionnaires from the 10-year study were included in the analysis. Anxiety scores from the Hospital Anxiety and Depression Scale were used in latent class growth analysis to chart the anxiety trajectory of cancer survivors. Demographic variables, clinical variables, depression level, and coping profiles (adaptive versus maladaptive) were used as predictors. Our study identified a two-class model of long-term anxiety trajectory among breast cancer survivors, with a resistant group (85.2%) and a distress group (14.8%). Demographic and clinical variables were not associated with anxiety trajectory paths. On the other hand, maladaptive coping characterized by higher scores in helplessness/hopelessness, cognitive avoidance, and anxious preoccupation, and lower scores in fighting spirit and fatalism in the Mini-MAC was a significant predictor of distressed anxiety. Coping profiles identified using the Mini-MAC were predictive of long-term anxiety trajectory among breast cancer survivors in our survey. Early interventions on coping with cancer could reduce long-term anxiety problems.

Human astrocytes and astrocytoma respond differently to resveratrol.

A fundamental problem in oncology is that anticancer chemotherapeutics kill both cancer and healthy cells in the surrounding tissues. Resveratrol is a natural antioxidant with intriguing and opposing biological properties: it reduces viability of some cancer cells but not of non-transformed ones (in equimolar concentrations). Therefore, we examined resveratrol in human non-transformed primary astrocytes and astrocytoma. Resveratrol reduced reactive oxygen species in astrocytes, but not in astrocytoma. Such cell-type dependent response is particularly evident with analyses at the single cell level showing clear population difference in high and low glutathione levels. Due to resveratrol's poor aqueous solubility that limits its use in clinics, we incorporated it into stimulus-responsive micelles assembled from miktoarm polymers. This could be an attractive chemotherapeutic delivery strategy in nano-oncology. As a proof of principle, we show that these formulations containing resveratrol markedly decrease astrocytoma viability, particularly in combination with temozolomide, a first line chemotherapeutic for astrocytoma.

Cognitive vulnerabilities to anxiety symptoms among Chinese adolescents: A 3-year longitudinal study.

OBJECTIVE: This study established cognitive vulnerability of anxiety symptoms among high school students. METHOD: A total of 72 grade 9-11 students completed measures on levels of anxiety sensitivity (AS), selective attentional processing, and anxiety symptoms annually between 2016 and 2018. RESULTS: Latent class growth analysis (unconditional model) showed a four-class model: High (stable) (6.94%), low (stable) (11.11%), medium (decreasing) (61.11%), and medium (increasing) (20.83%). The conditioned model controlling for the physical-concerns dimension of AS and negative attentional bias demonstrated that a two-class model consisted of a low anxiety class (n = 59, 81.9%) and a high anxiety class (n = 13, 18.1%) provided the best fit for the data. Negative attentional bias is a significant factor related to the development of anxiety trajectories. CONCLUSION: Attentional bias modification to disengage from negative stimuli may serve as a potential target of intervention to reduce chronic anxiety among high school students.

Asymmetric Carbene-Catalyzed Oxidation of Functionalized Aldimines as 1,4-Dipoles.

The use of functionalized aldimines has been demonstrated as newly structural 1,4-dipole precursors under carbene catalysis. More importantly, enantiodivergent organocatalysis has been successfully developed using carbene catalysts with the same absolute configuration, leading to both (R)- and (S)- enantiomers of six-membered heterocycles with quaternary carbon centers. This strategy features a broad substrate scope, mild reaction conditions, and good enantiomeric ratio. DFT calculation results indicated that hydrogen bond C-H⋅⋅⋅F interactions between the catalyst and substrate are the key factors for controlling and even switching the enantioselectivity. These new 1,4-dipoles can also react with isatin and its imines under carbene catalysis, allowing for access to the spiro oxindoles with excellent enantiomeric ratios.

Dendrimers as Modulators of Brain Cells.

Nanostructured hyperbranched macromolecules have been extensively studied at the chemical, physical and morphological levels. The cellular structural and functional complexity of neural cells and their cross-talk have made it rather difficult to evaluate dendrimer effects in a mixed population of glial cells and neurons. Thus, we are at a relatively early stage of bench-to-bedside translation, and this is due mainly to the lack of data valuable for clinical investigations. It is only recently that techniques have become available that allow for analyses of biological processes inside the living cells, at the nanoscale, in real time. This review summarizes the essential properties of neural cells and dendrimers, and provides a cross-section of biological, pre-clinical and early clinical studies, where dendrimers were used as nanocarriers. It also highlights some examples of biological studies employing dendritic polyglycerol sulfates and their effects on glia and neurons. It is the aim of this review to encourage young scientists to advance mechanistic and technological approaches in dendrimer research so that these extremely versatile and attractive nanostructures gain even greater recognition in translational medicine.

Highly Stereo-Controlled Synthesis of Fatty Acid-Derived Cyclic Carbonates by Using Iron(II) Complex and Nucleophilic Halide.

A catalytic system comprising an iron(II) complex and a nucleophilic halide for the preparation of a series of stereo-bio-derived cyclic carbonates was developed. The protocol allows for the coupling of various cis-fatty acid-derived epoxides. For example, cis-epoxy substrates can be reacted with CO2 to selectively yield either the cis- or trans-cyclic carbonates by adjusting the nucleophilic halide. These reactions are highly stereoselective and have excellent yields even under mild reaction conditions. The stereochemical divergence in the fatty acid-derived products strongly depends on the leaving ability of the halide anion in the cocatalyst.

Progress of Large-Scale Synthesis and Electronic Device Application of Two-Dimensional Transition Metal Dichalcogenides.

The recent exploration of semiconducting two-dimensional (2D) transition metal dichalcogenides (TMDs) with atomic thickness has taken both the scientific and technological communities by storm. Extensively investigated TMD that are accessible by large-scale synthetic methods materials are remarkably stable, such as MoS2 and WSe2 . They allow superior gate control due to their 2D nature and favorable electronic transport properties, thus suggesting a bright future for digital and RF electronics. In this review, the latest developments in the controlled synthesis of large scale TMDs are firstly introduced by discussing various approaches. The major obstacles that must be overcome to achieve wafer-scale, uniform, and high-quality TMD films for practical electronic applications are included. Advances in the electronic transport studies of TMDs are presented, such as doping, contact engineering, and mobility improvement, which contribute to overall device performance. A perspective and a look at the future for this field is provided in closing.

MIDA as a simple and highly efficient ligand for palladium-catalyzed Hiyama cross-coupling of aryl halides.

N-Methyliminodiacetic acid (MIDA) as a simple, air stable and water-soluble ligand has been used in the palladium-catalyzed Hiyama cross-coupling reaction of trimethoxyphenylsilane with aryl halides. The yield of the corresponding Hiyama coupling products is high up to around 90% in water and isopropanol under an ambient atmosphere in the presence of KOH and NaF.

Coping styles and the developmental trajectories of anxiety symptoms in children during transition into early adolescence.

The study investigated how active and avoidant coping styles predicted the trajectory membership of anxiety symptoms during the transition into early adolescence. A total of 321 Chinese children aged 9 to 10 years were recruited from a primary school in mainland China. Self-reported trait anxiety, coping styles and anxiety symptoms were assessed at baseline. After 6 months, self-reported anxiety symptoms were measured at three follow-up assessments with an interval of 6 months. Latent class growth modelling revealed high (18.7%) and low (81.3%) trajectories of anxiety symptoms in children during the transition into early adolescence. After controlling for trait anxiety, depression and sex, high active coping style predicted the trajectory of high anxiety symptoms, which was not moderated by trait anxiety. Before controlling for these covariates, the relation between active coping style and anxiety symptoms was in the opposite direction. A high avoidant coping style showed a trend to predict the trajectory of high anxiety symptoms only for children with low trait anxiety. These findings add a developmental context to the relationships of active and avoidant coping styles to anxiety symptoms and suggest that trait anxiety may moderate these relationships.

Spectral intensity drift correction of Spark Mapping Analysis for large-size metal materials.

BACKGROUND: Spectral intensity drift is a frequent issue in analytical processes, especially in long time excitation scanning for large size metal materials, which can significantly adversely impact the accuracy and stability of analysis results. Spectral intensity drift correction is the process of preprocessing spectral data using mathematical algorithms in order to facilitate the subsequent qualitative and quantitative analysis of spectra, especially in combination with stoichiometric methods. Up to now, spectral intensity drift correction within prolonged excitation has not been reported yet. RESULTS: We propose an intensity drift correction method for element content of large-size samples using the Spark Mapping Analysis for Large Samples (SMALS) technique. By considering the row-by-row and column-by-column mapping modes of the SMALS, this includes curve fitting baseline correction for in-row and in-column correction, as well as total average value correction for inter-row and inter-column correction. The final measurement values are derived by coupling rows with columns. The careful implementation of correction steps can enhance baseline correction performance, effectively reducing measurement errors a drift errors. Application of this method to characterize the cross and longitudinal sections of an oversized steel billet indicates high agreement with composition distribution obtained by micro-beam X-ray fluorescence (µ-XRF). The corrected longitudinal and cross-sectional data also exhibit strong alignment. Comparison of statistical analysis results pre- and post-correction demonstrates significant improvements in the clarity of elements segregation pattern. SIGNIFICANCE: This intensity drift correction method not only enhances the spectral quality but also improves the accuracy and robustness of quantitative and qualitative spectral analysis. This study contributes to establishing a robust foundation for component characterization of large-size metal materials using the SMALS technique. The novel spectral intensity correction method shows theoretical significance and practical value for large-scale, long-duration excitation scanning analysis.

Development and Comprehensive Benchmark of a High-Quality AMBER-Consistent Small Molecule Force Field with Broad Chemical Space Coverage for Molecular Modeling and Free Energy Calculation.

Biomolecular simulations have become an essential tool in contemporary drug discovery, and molecular mechanics force fields (FFs) constitute its cornerstone. Developing a high quality and broad coverage general FF is a significant undertaking that requires substantial expert knowledge and computing resources, which is beyond the scope of general practitioners. Existing FFs originate from only a limited number of groups and organizations, and they either suffer from limited numbers of training sets, lower than desired quality because of oversimplified representations, or are costly for the molecular modeling community to access. To address these issues, in this work, we developed an AMBER-consistent small molecule FF with extensive chemical space coverage, and we provide Open Access parameters for the entire modeling community. To validate our FF, we carried out benchmarks of quantum mechanics (QM)/molecular mechanics conformer comparison and free energy perturbation calculations on several benchmark data sets. Our FF achieves a higher level of performance at reproducing QM energies and geometries than two popular open-source FFs, OpenFF2 and GAFF2. In relative binding free energy calculations for 31 protein-ligand data sets, comprising 1079 pairs of ligands, the new FF achieves an overall root-mean-square error of 1.19 kcal/mol for ΔΔG and 0.92 kcal/mol for ΔG on a subset of 463 ligands without bespoke fitting to the data sets. The results are on par with those of the leading commercial series of OPLS FFs.

Patterns of attentional biases in children and emotional symptoms during the COVID-19 pandemic: a two-wave longitudinal study.

BACKGROUND: It is unknown how the patterns of negative and positive attentional biases in children predict fear of COVID-19, anxiety symptoms, and depression symptoms during the COVID-19 pandemic. The study identified profiles of negative and positive attentional biases in children and examined their association with emotional symptoms during the COVID-19 pandemic. METHOD: 264 children (girls: 53.8% and boys: 46.2%) of 9-10 years born in Hong Kong or mainland China from a primary school in Shenzhen, People's Republic of China were involved in a two-wave longitudinal study. Children completed the COVID-19 Fear Scale, the Revised Child Anxiety and Depression Scale, and the Attention to Positive and Negative Information Scale to measure fear of COVID-19, anxiety and depression symptoms, and negative and positive attentional biases in classrooms. After six months, they completed the second assessment of fear of COVID-19, anxiety symptoms, and depression symptoms in classrooms. Latent profile analysis was conducted to reveal distinct profiles of attentional biases in children. A series of repeated MANOVA was performed to examine the association of profiles of attentional biases to fear of COVID-19, anxiety symptoms, and depression symptoms across 6 months. RESULTS: Three profiles of negative and positive attentional biases were revealed in children. Children with a "moderate positive and high negative attentional biases" profile had significantly higher fear of the COVID-19 pandemic, anxiety symptoms, and depression symptoms than children with a "high positive and moderate negative attentional biases" profile. Children with a "low positive and negative attentional biases" profile were not significantly different in fear of COVID-19, anxiety symptoms, and depression symptoms than those with the other two profiles. CONCLUSIONS: Patterns of negative and positive attentional biases were related to emotional symptoms during the COVID-19 pandemic. It might be important to consider children's overall patterns of negative and positive attentional biases to identify children at risk of higher emotional symptoms.

The Moderation Effect of Cognitive Tendencies on the Relationship Between Fear of COVID-19 and Post-Traumatic Stress Symptoms in Chinese Children.

COVID-19 pandemic is a public health emergency with potential traumatizing effects on children. However, not many studies have been devoted to investigating the association between fear of COVID-19 and post-traumatic stress symptoms (PTSS) in children during the pandemic. Also, if the association is modulated by cognitive tendencies to focus on negative and positive information in children is unknown. The study recruited 122 native Chinese children from a primary school in mainland China. Self-reported psychological inventories were used to assess the above variables. The prevalence rate of PTSS in Chinese children was approximately 15.1%. Girls had higher post-traumatic stress levels than boys. Fear of COVID-19 was associated with higher level of post-traumatic stress in children. Only cognitive tendency to focus on negative information was significantly associated with the level of post-traumatic stress in children. Moreover, cognitive tendency to focus on negative information was a significant moderator of the relationship between fear of COVID-19 and PTSS. Generalization of the results to adults should be cautious. It was concluded that stronger fear of COVID-19 was related to more PTSS in children. The association of fear of COVID-19 with PTSS was significant only for children with strong tendency to focus on negative information. Cognitive interventions for PTSS may need to be delivered to children who have both strong fear of COVID-19 and strong tendency to focus on negative information.

Mariculture structure adjustment to achieve China's carbon neutrality and mitigate climate change.

China is responsible for the biggest shellfish and macroalgae production in the world. In this study, comprehensive methods were used to assess the CO2 release and sequestration by maricultured shellfish and macroalgae in China. Through considering CaCO3 production and CO2 release coefficient (Φ, moles of CO2 released per mole of CaCO3 formed) in different waters, we find that cultured shellfish released 0.741 ± 0.008 Tg C yr-1 through calcification based on the data of 2016-2020. In addition to calcification, maricultured shellfish released 0.580 ± 0.004 Tg C yr-1 by respiration. Meanwhile, shellfish sequestered 0.145 ± 0.001 and 0.0387 ± 0.0004 Tg C yr-1 organic carbon in sediments and shells, respectively. Therefore, the net released CO2 by maricultured shellfish was 1.136 ± 0.011 Tg C yr-1, which is about four times higher than that maricultured macroalgae could sequester (0.280 ± 0.010 Tg C yr-1). To achieve carbon neutrality within the mariculture system, shellfish culture may need to be restricted and meanwhile the expansion of macroalgae cultivation should be carried out. The mean carbon sequestration rate of seven kinds of macroalgae was 174 ± 6 g m-2 yr-1 while some cultivated macroalgae had higher CO2 sequestration rates, e.g. 356 ± 24 g C m-2 yr-1 for Gracilariopsis lemaneiformis and 331 ± 17 g C m-2 yr-1 for Undaria pinnatifida. In scenario 0.5 (CCUS (Carbon Capture, Utilization and Storage) sequesters 0.5 Gt CO2 per year), using macroalgae culture cannot achieve China's carbon neutrality by 2060 but in scenarios 1.0 and 1.5 (CCUS sequesters 1.0 and 1.5 Gt CO2 per year, respectively) it is feasible to achieve carbon neutrality using some macroalgae species with high carbon sequestration rates. This study provides important insights into how to develop mariculture in the context of carbon-neutrality and climate change mitigation.