Expeditious assembly of fused dihydropyranones via N-heterocyclic carbene-catalyzed tandem Michael addition/lactonization.

A convergent and efficient NHC-catalyzed enantioselective tandem Michael addition/lactonization sequence of ynals with 1,2-dione as the dual-nucleophile is disclosed. This straightforward strategy expeditiously assembles the synthetically and pharmaceutically valuable optically active fused dihydropyranones in good to high yields (70-88%) and with excellent enantioselectivities (92-99% ee).

Recent advances in N-heterocyclic carbene catalyzed achiral synthesis.

N-Heterocyclic carbenes (NHCs) have emerged as powerful and elegant organocatalysts in a variety of newly developed and unprecedented enantioselective transformations due to their unique umpolung capacity. As a supplement to conventional enantioselective organocatalysis, NHC-induced non-asymmetric catalysis has gradually attracted much interest in recent years. Herein, this review aims to reveal the recent developments in NHC-promoted non-asymmetric umpolung transformations resulting in the expeditious construction of versatile achiral natural heterocycles, carbocycles and acylated products.

Impact of the Russia-Ukraine Conflict on International Staple Agrifood Trade Networks.

The Russia-Ukraine conflict is a growing concern worldwide and poses serious threats to regional and global food security. Using monthly trade data for maize, rice, and wheat from 2016/1 to 2023/12, this paper constructs three international crop trade networks and an aggregate international food trade network. We aim to examine the structural changes following the occurrence of the Russia-Ukraine conflict. We find significant shifts in the number of edges, average in-degree, density, and efficiency in the third quarter of 2022, particularly in the international wheat trade network. Additionally, we have shown that political reasons have caused more pronounced changes in the trade connections between the economies of the North Atlantic Treaty Organization and Russia than with Ukraine. This paper could provide insights into the negative impact of geopolitical conflicts on the global food system and encourage a series of effective strategies to mitigate the negative impact of the conflict on global food trade.

Temporal rich club phenomenon and its formation mechanisms.

The temporal rich club (TRC) phenomenon is widespread in real systems, forming a tight and continuous collection of the prominent nodes that control the system. However, there is still a lack of sufficient understanding of the mechanisms of TRC formation. Here we use the international N-nutrient trade network as an example of an in-depth identification, analysis, and modeling of its TRC phenomenon. The system exhibits a statistically significant TRC phenomenon, with eight economies forming the cornerstone club. Our analysis reveals that node degree is the most influential factor in TRC formation compared to other variables. The mathematical evolution models we constructed propose that the TRC in the N-nutrient trade network arises from the coexistence of degree-homophily and path-dependence mechanisms. By comprehending these mechanisms, we introduce a different perspective on TRC formation. Although our analysis is limited to the international trade system, the methodology can be extended to analyze the mechanisms underlying TRC emergence in other systems.

Distribution of the active components from Xianglian Pill in tissues of healthy and antibiotic-associated diarrhea model mice and the mechanism study.

Antibiotic-associated diarrhea (AAD) is a common side effect of antibiotic therapy, characterized by intestinal inflammation which reduces the quality of life of patients. Xianglian Pill (XLP) has long been used to treat abdominal pain, diarrhea, bacillary dysentery and enteritis. Studies found that XLP has curative effect on AAD; however, the chemical constituents and mechanism of XLP have not been fully elucidated because of the lack of in vitro and in vivo studies. In this study, ultra-high performance liquid chromatography mass spectrometry method (UPLC-Q-Exactive-Orbitrap-HRMS) was used to examine the components of the XLP. Then, the binding between active compounds and the key targets was studied using network pharmacology and molecular docking. A comparative tissue distribution study was established for the simultaneous determination of the 10 active components in healthy and AAD mouse models. Forty-six components were characterized from XLP. According to the network pharmacology degree value, a prediction was made that encompassed 42 components and 14 core targets, which were intricately involved in crucial biological pathways, such as the AGE-RAGE signaling, cellular senescence, and MAPK signaling. Tissue distribution analysis showed that the 10 components were widely distributed in the heart, liver, spleen, lungs, kidneys, small intestine, and large intestine of mice, with varying concentrations in healthy and AAD mice. Molecular docking analysis also indicated that the active compounds in the tissue distribution could bind tightly to key targets of network pharmacological studies. This study provides a reference for further investigations of the relationships between the chemical components and pharmacological activities of XLP.

Magnetic Field-Optimized Paramagnetic Nanoprobe for T2/T1 Switchable Histopathological-Level MRI.

Traditional magnetic resonance imaging (MRI) contrast agents (CAs) are a type of "always on" system that accelerates proton relaxation regardless of their enrichment region. This "always on" feature leads to a decrease in signal differences between lesions and normal tissues, hampering their applications in accurate and early diagnosis. Herein, we report a strategy to fabricate glutathione (GSH)-responsive one-dimensional (1-D) manganese oxide nanoparticles (MONPs) with improved T2 relaxivities and achieve effective T2/T1 switchable MRI imaging of tumors. Compared to traditional contrast agents with high saturation magnetization to enhance T2 relaxivities, 1-D MONPs with weak Ms effectively increase the inhomogeneity of the local magnetic field and exhibit obvious T2 contrast. The inhomogeneity of the local magnetic field of 1-D MONPs is highly dependent on their number of primary particles and surface roughness according to Landau-Lifshitz-Gilbert simulations and thus eventually determines their T2 relaxivities. Furthermore, the GSH responsiveness ensures 1-D MONPs with sensitive switching from the T2 to T1 mode in vitro and subcutaneous tumors to clearly delineate the boundary of glioma and metastasis margins, achieving precise histopathological-level MRI. This study provides a strategy to improve T2 relaxivity of magnetic nanoparticles and construct switchable MRI CAs, offering high tumor-to-normal tissue contrast signal for early and accurate diagnosis.

Soy biodiesel and petrodiesel emissions differ in size, chemical composition and stimulation of inflammatory responses in cells and animals.

Debate about the biological effects of biodiesel exhaust emissions exists due to variation in methods of exhaust generation and biological models used to assess responses. Because studies in cells do not necessarily reflect the integrated response of a whole animal, experiments were conducted in two human cell lines representing bronchial epithelial cells and macrophages and female mice using identical particle suspensions of raw exhaust generated by a Volkswagen light-duty diesel engine using petrodiesel (B0) and a biodiesel blend (B20: 20% soy biodiesel/80% B0 by volume). Tailpipe particle emissions measurement showed B0 generated two times more particle mass, larger ultrafine particle number distribution modes, and particles of more nonpolar organic composition than the B20 fuel. Biological assays (inflammatory mediators, oxidative stress biomarkers) demonstrated that particulate matter (PM) generated by combustion of the two fuels induced different responses in in vitro and in vivo models. Concentrations of inflammatory mediators (Interleukin-6, IL-6; Interferon-gamma-induced Protein 10, IP-10; Granulocyte-stimulating factor, G-CSF) in the medium of B20-treated cells and in bronchoalveolar lavage fluid of mice exposed to B20 were ∼20-30% higher than control or B0 PM, suggesting that addition of biodiesel to diesel fuels will reduce PM emissions but not necessarily adverse health outcomes.

Does environmental management system certification affect green innovation performance?-Based on a moderated mediating effects model.

What is the impact of environmental management system certification on green innovation performance, and is it a futile endeavor or a profitable one? Grounded in the principles of ecological civilization construction and green development, this study embarks on a comprehensive examination. Initially, it investigates the varying impacts of environmental management system certification on both traditional innovation performance and green innovation performance. Subsequently, it dissects the underlying mechanisms and moderating factors influencing the latter, including an exploration of intermediary effects. The empirical findings of this study are as follows: (i) Environmental management system certification emerges as a catalyst for innovation performance, with the primary impact observed in the realm of green innovation performance. (ii) Social responsibility disclosure is identified as a mediating factor in the relationship between environmental management system certification and green innovation performance. (iii) Larger enterprises, those equipped with robust equity incentives, and those operating in less competitive markets are more prone to benefit from the impact of environmental management system certification on social responsibility disclosure. This, in turn, amplifies the promotion of green innovation performance. However, the moderating effect of property rights on the mediating path remains statistically insignificant. (iv) Environmental management system certification exerts a more pronounced influence on green innovation performance in regions characterized by lower economic development. Moreover, it particularly stimulates exploratory green innovation performance, surpassing its impact on exploitative green innovation performance.

Effectiveness and safety of first-line immune checkpoint inhibitors for patients with extensive-stage small cell lung carcinoma: A systematic review and network meta-analysis.

Objective: In recent years, the introduction of immune checkpoint inhibitors (ICIs) has revolutionized the treatment of extensive-stage small cell lung carcinoma (ES-SCLC), but the optimal combination of ICI and standard chemotherapy strategy is yet to be established. The aim of this network meta-analysis (NMA) was to identify which first-line combination strategy is optimal for patients with ES-SCLC. Methods: PubMed, Embase, Cochrane Library, and the proceedings of international conferences, including American Society of Clinical Oncology and European Society for Medical Oncology meetings, were searched for randomized controlled trials (RCTs) published through October 31, 2022. The collected primary outcomes were overall survival (OS), progression-free survival (PFS), and grade 3-5 treatment-related adverse events (TRAEs). Results: Our NMA study included six phase 3 and three phase 2 RCTs including 4037 patients and 10 first-line regimens. Regarding effectiveness, the addition of programmed cell death 1 (PD-1) or programmed cell death ligand 1 (PD-L1) inhibitors to standard chemotherapy provided greater efficacy than chemotherapy alone. However, cytotoxic T lymphocyte-associated antigen-4 inhibitors were not associated with satisfactory prognoses. Serplulimab plus carboplatin-etoposide (vs. standard chemotherapy, hazard ratio [HR] = 0.63; 95% CI = 0.49-0.82) and nivolumab plus platinum-etoposide (HR = 0.65; 95% confidence interval [CI] = 0.46-0.91) displayed the greatest benefit regarding OS. In terms of PFS, serplulimab plus carboplatin-etoposide yielded the best benefit of all treatments (HR = 0.48; 95% CI = 0.39-0.6). The combination of ICIs and chemotherapy caused more toxicity in general, but durvalumab plus platinum-etoposide (odds ratio [OR] = 0.98; 95% CI = 0.68-1.4), atezolizumab plus carboplatin-etoposide (OR = 1.04; 95% CI = 0.68-1.6), and adebrelimab plus platinum-etoposide (OR = 1.02; 95% CI = 0.52-2) displayed similar safety as standard chemotherapy. Subgroup analysis by race illustrated that serplulimab plus carboplatin-etoposide was associated with the best OS in Asian patients. And in non-Asian patients, the combination of PD-1/PD-L1 inhibitors and chemotherapy (pembrolizumab plus platinum-etoposide, durvalumab plus platinum-etoposide, and durvalumab and tremelimumab plus platinum-etoposide) displayed superiority to standard chemotherapy. Conclusions: The results of our NMA study suggested that serplulimab plus carboplatin-etoposide and nivolumab plus platinum-etoposide are associated with the best OS as first-line treatments for patients with ES-SCLC. Serplulimab plus carboplatin-etoposide was associated with the best PFS. In Asian patients, serplulimab plus carboplatin-etoposide had the best OS. Systematic review registration: This study is registered with PROSPERO, number CRD42022345850.

Switchable ROS Scavenger/Generator for MRI-Guided Anti-Inflammation and Anti-Tumor Therapy with Enhanced Therapeutic Efficacy and Reduced Side Effects.

Photosensitizer in photodynamic therapy (PDT)  accumulates in both tumor and adjacent normal tissue due to low selective biodistribution, results in undesirable side effect with limited clinic application. Herein, an intelligent nanoplatform is reported that selectively acts as reactive oxygen species (ROS) scavenger in normal tissue but as ROS generator in tumor microenvironment (TME) to differentially control ROS level in tumor and surrounding normal tissue during PDT. By down-regulating the produced ROS with dampened cytokine wave in normal tissue after PDT, the nanoplatform reduces the inflammatory response of normal tissue in PDT, minimizing the side effect and tumor metastasis in PDT. Alternatively, the nanoplatform switches from ROS scavenger to generator through the glutathione (GSH) responsive degradation in TME, which effectively improves the PDT efficacy with reduced GSH level and amplified oxidative stress in tumor. Simultaneously, the released Mn ions provide real-time and in situ signal change of magnetic resonance imaging (MRI) to monitor the reversal process of catalysis activity and achieve accurate tumor diagnosis. This TME-responsive ROS scavenger/generator with activable MRI contrast may provide a new dimension for design of next-generation PDT agents with precise diagnosis, high therapeutic efficacy, and low side effect.

The health risk of acetochlor metabolite CMEPA is associated with lipid accumulation induced liver injury.

Liver injury may cause many diseases, such as non-alcoholic fatty liver disease (NAFLD). Acetochlor is one of the representative chloroacetamide herbicides, and its metabolite 2-chloro-N-(2-ethyl-6-methyl phenyl) acetamide (CMEPA) is the main form of exposure in the environment. It has been shown that acetochlor can cause mitochondrial damage of HepG2 cells and induce apoptosis by activating Bcl/Bax pathway (Wang et al., 2021). But there has been less research on CMEPA. we explored the possibility of CMEPA and liver injury through biological experiments. In vivo, CMEPA (0-16 mg/L) induced liver damage in zebrafish larvae, including increased lipid droplets, changes in liver morphology (>1.3-fold) and increased TC/TG content (>2.5-fold). In vitro, we selected L02 (human normal liver cells) as the model, and explored its molecular mechanism. We found that CMEPA (0-160 mg/L) induced apoptosis (similar to 40%), mitochondrial damage and oxidative stress in L02 cells. CMEPA induced intracellular lipid accumulation by inhibiting AMPK/ACC/CPT-1A signaling pathway and activating SREBP-1c/FAS signaling pathway. Our study provides evidence of a link between CMEPA and liver injury. This raises concerns regarding the health risks of pesticide metabolites to liver health.

Differential activation of the inflammasome in THP-1 cells exposed to chrysotile asbestos and Libby "six-mix" amphiboles and subsequent activation of BEAS-2B cells.

Inflammatory responses of THP-1 cells (macrophage cell line) exposed to chrysotile asbestos (Chry) and Libby six-mix (LIB) and the subsequent impact on bronchial epithelial cells were determined. Direct treatment of THP-1 cells with Chry caused cell death, activation of caspase-1 and release of IL-1ß, while the addition of caspase-1 inhibitor, Z-YVAD-FMK, reduced IL-1ß, suggesting that Chry activated the caspase-1 mediated Nod-like receptor protein 3 (NLRP3) inflammasome; by comparison, LIB had less effects on all of these parameters. Expression of antioxidant enzymes, protein oxidation and nitration, and lipid peroxides in THP-1 cells treated with the two particles suggest that LIB generated more reactive oxygen species (ROS) than the same dose of Chry. Differences in fiber length and surface area suggest a possible role for particulate size in the differential activation of the inflammasome. BEAS-2B cells, representing the bronchial epithelium, treated with supernatants of medium from Chry- or LIB-treated THP-1 cells (conditioned medium) activated the MAPK cascade, increased phosphorylation of ERK and Cot (MAP3K8), increased AP-1 binding activity and induced IL-6 release. To verify that IL-1ß from THP-1 cells was responsible for activation of BEAS-2B, conditioned medium with added IL-1Ra, an IL-1ß antagonist, was applied to BEAS-2B. Results show that IL-1Ra attenuated effects of conditioned medium, supporting a role of IL-1ß, as a secondary mediator, in the transduction of inflammatory signaling from the macrophage to epithelial cells. The effects of LIB-conditioned medium appeared to be less dependent on IL-1ß. In conclusion, Chry and LIB induce differential inflammatory responses in THP-1 cells that subsequently lead to differential effects in epithelial cells.

Pd-Catalyzed coupling of benzyl bromides with BMIDA-substituted N-tosylhydrazones: synthesis of trans-alkenyl MIDA boronates.

A palladium-catalyzed stereoselective synthesis of alkenyl boronates from N-methyliminodiacetyl boronate (BMIDA)-substituted N-tosylhydrazone and benzyl bromides is developed. A range of trans-alkenyl MIDA boronates as single stereoisomers were obtained in moderate yields with good functional group compatibility. The resultant boronate products may be transformed to other boron-containing compounds and may also be directly used in cross-coupling reactions.

Recent advances in engineering iron oxide nanoparticles for effective magnetic resonance imaging.

Iron oxide nanoparticle (IONP) with unique magnetic property and high biocompatibility have been widely used as magnetic resonance imaging (MRI) contrast agent (CA) for long time. However, a review which comprehensively summarizes the recent development of IONP as traditional T 2 CA and its new application for different modality of MRI, such as T 1 imaging, simultaneous T 2/T 1 or MRI/other imaging modality, and as environment responsive CA is rare. This review starts with an investigation of direction on the development of high-performance MRI CA in both T 2 and T 1 modal based on quantum mechanical outer sphere and Solomon-Bloembergen-Morgan (SBM) theory. Recent rational attempts to increase the MRI contrast of IONP by adjusting the key parameters, including magnetization, size, effective radius, inhomogeneity of surrounding generated magnetic field, crystal phase, coordination number of water, electronic relaxation time, and surface modification are summarized. Besides the strategies to improve r 2 or r 1 values, strategies to increase the in vivo contrast efficiency of IONP have been reviewed from three different aspects, those are introducing second imaging modality to increase the imaging accuracy, endowing IONP with environment response capacity to elevate the signal difference between lesion and normal tissue, and optimizing the interface structure to improve the accumulation amount of IONP in lesion. This detailed review provides a deep understanding of recent researches on the development of high-performance IONP based MRI CAs. It is hoped to trigger deep thinking for design of next generation MRI CAs for early and accurate diagnosis.

Engineering manganese ferrite shell on iron oxide nanoparticles for enhanced T1 magnetic resonance imaging.

Doping Mn (II) ions into iron oxide (IO) as manganese ferrite (MnIO) has been proved to be an effective strategy to improve T1 relaxivity of IO nanoparticle in recent years; however, the high T2 relaxivity of MnIO nanoparticle hampers its T1 contrast efficiency and remains a hurdle when developing contrast agent for early and accurate diagnosis. Herein, we engineered the interfacial structure of IO nanoparticle coated with manganese ferrite shell (IO@MnIO) with tunable thicknesses. The Mn-doped shell significantly improve the T1 contrast of IO nanoparticle, especially with the thickness of ∼0.8 nm. Compared to pristine IO nanoparticle, IO@MnIO nanoparticle with thickness of ∼0.8 nm exhibits nearly 2 times higher T1 relaxivity of 9.1 mM-1s-1 at 3 T magnetic field. Moreover, exclusive engineering the interfacial structure significantly lower the T2 enhancing effect caused by doped Mn (II) ions, which further limits the impairing of increased T2 relaxivity to T1 contrast imaging. IO@MnIO nanoparticles with different shell thicknesses reveal comparable T1 relaxation rates but obvious lower T2 relaxivities and r2/r1 ratios to MnIO nanoparticles with similar sizes. The desirable T1 contrast endows IO@MnIO nanoparticle to provide sufficient signal difference between normal and tumor tissue in vivo. This work provides a detailed instance of interfacial engineering to improve IO-based T1 contrast and a new guidance for designing effective high-performance T1 contrast agent for early cancer diagnosis.

Molecular changes during arsenic-induced cell transformation.

Arsenic and its derivatives are naturally occurring metalloid compounds widely distributed in the environment. Arsenics are known to cause cancers of the skin, liver, lung, kidney, and bladder. Although numerous carcinogenic pathways have been proposed, the exact molecular mechanisms remain to be delineated. To further characterize the role of oxidative stress in arsenite-induced cell transformation via the reactive oxygen species (ROS)-mediated Ras/Erk pathway, here we demonstrated arsenite-induced rat lung epithelial cell (LEC) transformation, epithelial-mesenchymal transition, stimulation of the extracellular signal-regulated kinase signaling pathway, and enhancement of cell proliferation. However, there was no evidence of activation of the phosphoinositide 3-kinase/protein kinase B pathway in arsenite-induced transformed LECs. Since ROS is involved in arsenite-induced LEC cell transformation, Redox-status regulatory proteins (Cu/Zn SOD and thioredoxin) and arsenite-induced LEC cell transformation were significantly inhibited by concurrent treatment with the antioxidants. Our experimental results clearly demonstrated that induction of p-ERK and cell proliferation by arsenite is mediated via oxidative stress, since antioxidants can inhibit arsenite-induced cell transformation.

Opening the Black Box: The Impacts of Environmental Regulations on Technological Innovation.

Environmental regulations (ERs) that can stimulate technological innovation (TI) are the key to enabling a win-win strategy that benefits both economic development and environmental protection. This study seeks to analyze the impacts of ERs on TI. Previous literature highlighted that the black box of TI can be decomposed into technology investment and technology transformation, but empirical studies on such a decomposition have largely been ignored. Moreover, a detailed discussion of the links between ERs and the decomposed components of TI has not been conducted in developing countries such as China. Our study attempts to address these research gaps by (i) decomposing TI using a novel data envelopment analysis (DEA) procedure and further analyzing the impacts of ERs on the decomposed components of TI and (ii) applying this novel methodology to Chinese context. Accordingly, this study is conducted in two stages. First, a novel application of the slack-based measure Network DEA model is developed to uncover the black box of TI using Chinese data in order to estimate the overall efficiency of technological innovation (TIE) and to decompose it into the efficiency of technology investment (TVE) and the efficiency of technology transformation (TTE). Second, a random effect Tobit model is applied to (i) investigate both the linear and nonlinear impacts of ERs on TIE in all sectors and (ii) examine whether the impacts of ERs on TVE and TTE in different subprocesses are heterogeneous or not. Our results have showed the benefits of decomposing TI: while technology transformation in China closely follows the trend of TI, the trend of technology investment is somewhat different. The estimation results further indicate that the impacts of ERs on TIE are nonlinear. Besides, ERs have heterogeneous impacts on the decomposed components of TI. The impacts of ERs on TVE are nonlinear, whereas the impacts of ERs on TTE are statistically insignificant.

Identification and characterization of an Nrf2-mediated ARE upstream of the rat glutamate cysteine ligase catalytic subunit gene (GCLC).

The antioxidant response element (ARE) is an essential component of upstream regulatory sequences present on genes for most phase II detoxification enzymes, including the glutamate cysteine ligase catalytic subunit (GCLC). NF-E2-related factor 2 (Nrf2) is a principal transcription factor that binds to the ARE and plays a key role in cellular responses to stress via the Keap1-Nrf2-ARE pathway. However, the ARE that mediates human GCLC gene expression has not been found in the rat. Thus, how the ARE-mediated Keap1-Nrf2-ARE pathway regulates glutathione homeostasis in the rat remains a puzzle. We have identified a putative ARE sequence approximately 4 kb upstream in the rat GCLC. We further defined the rat GCLC-ARE in the category with the most ARE characters, that is, this rat GCLC-ARE is a sequence-specific site that significantly enhances promoter activity in reporter genes. The rat GCLC-ARE is an Nrf2-mediated element to which binding has been demonstrated in nuclear extracts and induced by tert-butylhydroquinone. Given the central role that rat models play in toxicology and pathology, this first discovery of the rat GCLC-ARE enhancer similar to that found in the human gene has broad implications for the study of antioxidant defenses and their regulation in a number of different fields.

Effect of time and temperature on stability of progestagens, testosterone and cortisol in Asian elephant blood stored with and without anticoagulant.

The value of biological samples collected in the field is compromised if storage conditions result in analyte degradation, especially in warmer climates like Thailand. We evaluated the effects of time and temperature on immunoactive steroid hormone stability in Asian elephant (Elephas maximus) blood stored with and without an anti-coagulant before centrifugation. For each elephant (5 male, 5 female), whole blood was aliquoted (n = 2 ml each) into 13 red top (without anticoagulant) or purple top (with anticoagulant) tubes. One tube from each treatment was centrifuged immediately and the serum or plasma frozen at -20°C (Time 0, T0). The remaining 12 aliquots were divided into stored temperature groups: 4°C, room temperature (RT, ~22°C), and 37°C, and centrifuged after 6, 24, 48 and 62 h of storage. Serum and plasma concentrations of progestagens in females, testosterone in males and cortisol in both sexes were quantified by validated enzyme immunoassays. Steroid concentration differences from T0 were determined by a randomized complete block ANOVA and Dunnett's tests. The only evidence of hormone degradation was cortisol and testosterone concentrations in serum stored at 37°C. Testosterone concentrations declined by 34% at 48 h and 52% at 62 h, cortisol was decreased by 19% after 48 h and 27% after 62 h at 37°C, respectively. None of the other aliquots displayed significant changes over time at any temperature. In conclusion, steroids appear to be stable in blood for nearly 3 days at room or refrigeration temperatures before centrifugation; steroids in samples with ethylenediaminetetraacetic acid were particularly stable. However, warmer temperatures may negatively affect steroids stored without anti-coagulant, perhaps due to red blood cell metabolism. Thus, under field conditions with no access to cold or freezer temperatures, collection of plasma is a better choice for elephants up to at least 62 h before centrifugation.