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Design and application of a novel integrated electrochemical hydride generation cell for the determination of arsenic in seaweeds by atomic fluorescence spectrometry.
An integrated electrochemical hydride generation cell, mainly composed of three components (a gas liquid separator, a graphite tube cathode and a reticulate Pt wire anode), was laboratory constructed and employed for the detection of arsenic by coupling to atomic fluorescence spectrometry. This integrated cell was free of ion-exchange membrane and individual anolyte, with the virtues of low-cost, easy assembly and environmental-friendly. Using flow injection mode, the sample throughput could come to 120 h(-1) attributed to the small dimension of the cathode chamber. The operating conditions for the electrochemical hydride generation of arsenic were investigated in detail and the potential interferences from oxygen or various ions were also evaluated. Under the optimized conditions, no obvious oxygen quenching effects were observed. The limit of detection of As (III) for the sample blank solution was 0.2 ng mL(-1) (3sigma) and the relative standard deviation was 3.1% for nine consecutive measurements of 5 ng mL(-1) As (III) standard solution. The calibration curve was linear up to 100 ng mL(-1). The accuracy of the method was verified by the determination of arsenic in the reference materials GBW08517 (Laminaria Japonica Aresch) and GBW10023 (Porphyra crispata) and the developed method was successfully applied to determine trace amounts of arsenic in edible seaweeds.
Ultrasonic slurry sampling electrothermal atomic absorption spectrometry with a metal tube atomizer has been applied to the determination of lead in Bangladeshi fish samples. The slurry sampling conditions, such as slurry stabilizing agent, slurry concentration, pyrolysis temperature for the slurried fish samples, particle size and ultrasonic agitation time, were optimized for electrothermal atomic absorption spectrometry with the Mo tube atomizer. Thiourea was used as the chemical modifier for the interference of matrix elements. The detection limit was 53 fg (3S/N). The determined amount of lead in Bangladeshi fish samples was consistent with those measured in the dissolved acid-digested samples. The advantages of the proposed methods are easy calibration, simplicity, low cost and rapid analysis.
Design and application of a novel integrated electrochemical hydride generation cell for the determination of arsenic in seaweeds by atomic fluorescence spectrometry.
An integrated electrochemical hydride generation cell, mainly composed of three components (a gas liquid separator, a graphite tube cathode and a reticulate Pt wire anode), was laboratory constructed and employed for the detection of arsenic by coupling to atomic fluorescence spectrometry. This integrated cell was free of ion-exchange membrane and individual anolyte, with the virtues of low-cost, easy assembly and environmental-friendly. Using flow injection mode, the sample throughput could come to 120 h(-1) attributed to the small dimension of the cathode chamber. The operating conditions for the electrochemical hydride generation of arsenic were investigated in detail and the potential interferences from oxygen or various ions were also evaluated. Under the optimized conditions, no obvious oxygen quenching effects were observed. The limit of detection of As (III) for the sample blank solution was 0.2 ng mL(-1) (3sigma) and the relative standard deviation was 3.1% for nine consecutive measurements of 5 ng mL(-1) As (III) standard solution. The calibration curve was linear up to 100 ng mL(-1). The accuracy of the method was verified by the determination of arsenic in the reference materials GBW08517 (Laminaria Japonica Aresch) and GBW10023 (Porphyra crispata) and the developed method was successfully applied to determine trace amounts of arsenic in edible seaweeds.
A straightforward method for measuring two aquatic inorganic species of selenium, selenate, Se(VI), and selenite, Se(IV), was developed in this study. Selenium toxicity and bioaccumulation in food chain are strongly dependent on its speciation. Therefore, it is important to measure selenium species as part of any selenium risk assessment practice. In this method, total selenium was first measured using graphite furnace atomic absorption spectrometry, and then, chemical procedures in the literature were used to reduce selenite, Se(IV), to hydrogen selenide (H<sub>2</sub>Se). Total selenium of the same solution was measured again with the analytical instrument after stripping H<sub>2</sub>Se from the solution. The difference of total selenium measured gave Se(IV) concentration. The two main species in natural waters are Se(VI) and Se(IV). Therefore, it can be assumed that after removing Se(IV) from the solution, the remaining total selenium is Se(VI). The two inorganic selenium species of (IV) and (VI) in purified waters and synthetic irrigation waters both spiked with Se(VI) and Se(IV) were determined using this method. Recovery of spiked samples in diluted synthetic irrigation water was 97% for Se(VI) and 99% for Se(IV). Detection limits of the method were 0.32&#160;&#181;g L<sup>-1</sup> for Se(VI) and 0.11&#160;&#181;g L<sup>-1</sup> for Se(IV). The advantages of the method developed in this study are that it employs a straightforward simple chemical reaction combined with acidification and stripping, requires only one instrument (graphite furnace atomic absorption spectrometry), and does not require extensive sample pretreatment.
Design and application of a novel integrated electrochemical hydride generation cell for the determination of arsenic in seaweeds by atomic fluorescence spectrometry.
An integrated electrochemical hydride generation cell, mainly composed of three components (a gas liquid separator, a graphite tube cathode and a reticulate Pt wire anode), was laboratory constructed and employed for the detection of arsenic by coupling to atomic fluorescence spectrometry. This integrated cell was free of ion-exchange membrane and individual anolyte, with the virtues of low-cost, easy assembly and environmental-friendly. Using flow injection mode, the sample throughput could come to 120 h(-1) attributed to the small dimension of the cathode chamber. The operating conditions for the electrochemical hydride generation of arsenic were investigated in detail and the potential interferences from oxygen or various ions were also evaluated. Under the optimized conditions, no obvious oxygen quenching effects were observed. The limit of detection of As (III) for the sample blank solution was 0.2 ng mL(-1) (3sigma) and the relative standard deviation was 3.1% for nine consecutive measurements of 5 ng mL(-1) As (III) standard solution. The calibration curve was linear up to 100 ng mL(-1). The accuracy of the method was verified by the determination of arsenic in the reference materials GBW08517 (Laminaria Japonica Aresch) and GBW10023 (Porphyra crispata) and the developed method was successfully applied to determine trace amounts of arsenic in edible seaweeds.
A screen-printed silver strip with three-electrode configuration of Ag-working, Ag-counter and Ag/Ag(x)O reference electrodes was developed for simultaneous determination of chloride, bromide and iodide in aqueous solutions. It was fabricated simply by screen-printing silver ink onto a polypropylene (PP) base. The in-situ prepared Ag/Ag(x)O reference electrode can avoid the leaching interference in chloride detection while using a conventional Ag/AgCl reference electrode. A single drop of analyte (50 microl) is enough to determine iodide, bromide and chloride by measuring the well-separated oxidation peak currents of respective silver halides. The calibration graph was linear from 10 microM to 20 mM for iodide and bromide and 100 microM to 20 mM for chloride and the detection limit (S/N=3) was 3.05 microM, 2.95 microM and 18.83 microM for iodide, bromide and chloride, respectively. The strip is designed to be disposable and as such manual polishing is not necessary. The proposed sensor is not only simple to manufacture and easy to operate but also fast and precise with little detection volume. It is successfully applied to the determination of halide ions in real samples.
Design and application of a novel integrated electrochemical hydride generation cell for the determination of arsenic in seaweeds by atomic fluorescence spectrometry.
An integrated electrochemical hydride generation cell, mainly composed of three components (a gas liquid separator, a graphite tube cathode and a reticulate Pt wire anode), was laboratory constructed and employed for the detection of arsenic by coupling to atomic fluorescence spectrometry. This integrated cell was free of ion-exchange membrane and individual anolyte, with the virtues of low-cost, easy assembly and environmental-friendly. Using flow injection mode, the sample throughput could come to 120 h(-1) attributed to the small dimension of the cathode chamber. The operating conditions for the electrochemical hydride generation of arsenic were investigated in detail and the potential interferences from oxygen or various ions were also evaluated. Under the optimized conditions, no obvious oxygen quenching effects were observed. The limit of detection of As (III) for the sample blank solution was 0.2 ng mL(-1) (3sigma) and the relative standard deviation was 3.1% for nine consecutive measurements of 5 ng mL(-1) As (III) standard solution. The calibration curve was linear up to 100 ng mL(-1). The accuracy of the method was verified by the determination of arsenic in the reference materials GBW08517 (Laminaria Japonica Aresch) and GBW10023 (Porphyra crispata) and the developed method was successfully applied to determine trace amounts of arsenic in edible seaweeds.
Colloidal palladium was used as chemical modifier in the determination of blood thallium by graphite furnace atomic absorption spectrometry.
Packaging of Diisopropyl Fluorophosphatase (DFPase) in Bacterial Outer Membrane Vesicles Protects Its Activity at Extreme Temperature.
Enzymatic decontamination of organophosphate compounds offers a biofriendly pathway to the neutralization of highly dangerous compounds. Environmental dissemination of enzymes, however, is an ongoing problem considering the costly process of production and chemical modification for stability that can diminish catalytic activity. As a result, there is interest in the potential for enzymatic encapsulation in situ or into nascent bacterial membrane vesicles to improve catalytic stability across various environmental challenges associated with storage and field deployment. In this study, we have engineered bacterial outer membrane vesicles (OMVs) to encapsulate the diisopropyl fluorophosphatase (DFPase), an enzyme originally isolated from squid <i>Loligo vulgaris</i> and capable of hydrolyzing diisopropyl fluorophosphate (DFP) and other organophosphates compounds. Here we employed a recombinant lipopeptide anchor to direct recruitment of DFPase into OMVs, which were isolated from culture media and tested for catalytic activity against both diisopropyl fluorophosphate and paraoxon. Our encapsulation strategy prevented the loss of catalytic activity despite lyophilization, extended storage time (2 days), and extreme temperatures up to 80 &#176;C. These data underscore the appeal of DFPase as a biodecontaminant of organophosphates as well as the potential for OMV packaging in stabilized field deployment applications.
An increasing number of people are infected with antibiotic-resistant bacteria each year, sometimes with fatal consequences. In this manuscript, we report a novel urea-functionalized crown ether that can bind to the bacterial lipid phosphatidylethanolamine (PE), facilitate PE flip-flop and displays antibacterial activity against the Gram-positive bacterium Bacillus cereus with a minimum inhibitory concentration comparable to that of the known PE-targeting lantibiotic duramycin.
Packaging of Diisopropyl Fluorophosphatase (DFPase) in Bacterial Outer Membrane Vesicles Protects Its Activity at Extreme Temperature.
Enzymatic decontamination of organophosphate compounds offers a biofriendly pathway to the neutralization of highly dangerous compounds. Environmental dissemination of enzymes, however, is an ongoing problem considering the costly process of production and chemical modification for stability that can diminish catalytic activity. As a result, there is interest in the potential for enzymatic encapsulation in situ or into nascent bacterial membrane vesicles to improve catalytic stability across various environmental challenges associated with storage and field deployment. In this study, we have engineered bacterial outer membrane vesicles (OMVs) to encapsulate the diisopropyl fluorophosphatase (DFPase), an enzyme originally isolated from squid <i>Loligo vulgaris</i> and capable of hydrolyzing diisopropyl fluorophosphate (DFP) and other organophosphates compounds. Here we employed a recombinant lipopeptide anchor to direct recruitment of DFPase into OMVs, which were isolated from culture media and tested for catalytic activity against both diisopropyl fluorophosphate and paraoxon. Our encapsulation strategy prevented the loss of catalytic activity despite lyophilization, extended storage time (2 days), and extreme temperatures up to 80 &#176;C. These data underscore the appeal of DFPase as a biodecontaminant of organophosphates as well as the potential for OMV packaging in stabilized field deployment applications.
Membrane fusion is an important process for the survival of eukaryotes. The shape of lipids plays an important role in fusion by stabilizing nonlamellar fusion intermediates. Lipids with intrinsic positive curvature such as lysophosphatidylcholine and others inhibit hemifusion formation, whereas lipids having intrinsic negative curvature, e.g., phosphatidylethanolamine and cholesterol (CH), are known to promote hemifusion formation. In this work, we have measured the effect of dioleoylphosphatidylethanolamine (DOPE) and CH on the depth-dependent organization, dynamics, and fusion of dioleoylphosphatidylcholine membranes. Both DOPE and CH promote hemifusion formation despite their ability to order the interfacial and acyl chain region of the membrane and block water percolation at these regions. Generally, membrane ordering and inhibition of water percolation at the acyl chain region are detrimental to membrane fusion. This clearly emphasizes the importance of intrinsic negative curvature of lipids in membrane fusion. Interestingly, DOPE and CH show differential effects on the rate of hemifusion formation, which might be owing to their ability to induce order at the interfacial region and intrinsic negative curvature. Overall, our result is significant in understanding the role of lipidic shape in membrane fusion.
Packaging of Diisopropyl Fluorophosphatase (DFPase) in Bacterial Outer Membrane Vesicles Protects Its Activity at Extreme Temperature.
Enzymatic decontamination of organophosphate compounds offers a biofriendly pathway to the neutralization of highly dangerous compounds. Environmental dissemination of enzymes, however, is an ongoing problem considering the costly process of production and chemical modification for stability that can diminish catalytic activity. As a result, there is interest in the potential for enzymatic encapsulation in situ or into nascent bacterial membrane vesicles to improve catalytic stability across various environmental challenges associated with storage and field deployment. In this study, we have engineered bacterial outer membrane vesicles (OMVs) to encapsulate the diisopropyl fluorophosphatase (DFPase), an enzyme originally isolated from squid <i>Loligo vulgaris</i> and capable of hydrolyzing diisopropyl fluorophosphate (DFP) and other organophosphates compounds. Here we employed a recombinant lipopeptide anchor to direct recruitment of DFPase into OMVs, which were isolated from culture media and tested for catalytic activity against both diisopropyl fluorophosphate and paraoxon. Our encapsulation strategy prevented the loss of catalytic activity despite lyophilization, extended storage time (2 days), and extreme temperatures up to 80 &#176;C. These data underscore the appeal of DFPase as a biodecontaminant of organophosphates as well as the potential for OMV packaging in stabilized field deployment applications.
The hallmark of the gram-negative bacterial envelope is the presence of the outer membrane (OM). The OM is asymmetric, comprising lipopolysaccharides (LPS) in the outer leaflet and phospholipids (PLs) in the inner leaflet; this critical feature confers permeability barrier function against external insults, including antibiotics. To maintain OM lipid asymmetry, the OmpC-Mla system is believed to remove aberrantly localized PLs from the OM and transport them to the inner membrane (IM). Key to the system in driving lipid trafficking is the MlaFEDB ATP-binding cassette transporter complex in the IM, but mechanistic details, including transport directionality, remain enigmatic. Here, we develop a sensitive point-to-point in&#160;vitro lipid transfer assay that allows direct tracking of [<sup>14</sup>C]-labeled PLs between the periplasmic chaperone MlaC and MlaFEDB reconstituted into nanodiscs. We reveal that MlaC spontaneously transfers PLs to the IM transporter in an MlaD-dependent manner that can be further enhanced by coupled ATP hydrolysis. In addition, we show that MlaD is important for modulating productive coupling between ATP hydrolysis and such retrograde PL transfer. We further demonstrate that spontaneous PL transfer also occurs from MlaFEDB to MlaC, but such anterograde movement is instead abolished by ATP hydrolysis. Our work uncovers a model where PLs reversibly partition between two lipid-binding sites in MlaC and MlaFEDB, and ATP binding and/or hydrolysis shift this equilibrium to ultimately drive retrograde PL transport by the OmpC-Mla system. These mechanistic insights will inform future efforts toward discovering new antibiotics against gram-negative pathogens.
Packaging of Diisopropyl Fluorophosphatase (DFPase) in Bacterial Outer Membrane Vesicles Protects Its Activity at Extreme Temperature.
Enzymatic decontamination of organophosphate compounds offers a biofriendly pathway to the neutralization of highly dangerous compounds. Environmental dissemination of enzymes, however, is an ongoing problem considering the costly process of production and chemical modification for stability that can diminish catalytic activity. As a result, there is interest in the potential for enzymatic encapsulation in situ or into nascent bacterial membrane vesicles to improve catalytic stability across various environmental challenges associated with storage and field deployment. In this study, we have engineered bacterial outer membrane vesicles (OMVs) to encapsulate the diisopropyl fluorophosphatase (DFPase), an enzyme originally isolated from squid <i>Loligo vulgaris</i> and capable of hydrolyzing diisopropyl fluorophosphate (DFP) and other organophosphates compounds. Here we employed a recombinant lipopeptide anchor to direct recruitment of DFPase into OMVs, which were isolated from culture media and tested for catalytic activity against both diisopropyl fluorophosphate and paraoxon. Our encapsulation strategy prevented the loss of catalytic activity despite lyophilization, extended storage time (2 days), and extreme temperatures up to 80 &#176;C. These data underscore the appeal of DFPase as a biodecontaminant of organophosphates as well as the potential for OMV packaging in stabilized field deployment applications.
Membranes&#9472;cells' essential scaffolds&#9472;are valid molecular targets for substances with an antimicrobial effect. While certain substances, such as octenidine, have been developed to target membranes for antimicrobial purposes, the recently reported molecule, fabimycin (F2B)&#9472;a novel agent targeting drug-resistant Gram-negative bacteria&#9472;has not received adequate attention regarding its activity on membranes in the literature. The following study aims to investigate the effects of F2B on different bacterial membrane models, including simple planar bilayers and more complex bilayer systems that mimic the <i>Escherichia coli</i> shell equipped with double inner and outer bilayers. Our results show that F2B exhibited more pronounced interactions with bacterial membrane systems compared to the control PC system. Furthermore, we observed significant changes in local membrane property homeostasis in both the inner and outer membrane models, specifically in the case of lateral diffusion, membrane thickness, and membrane resilience (compressibility, tilt). Finally, our results showed that the effect of F2B differed in a complex system and a single membrane system. Our study provides new insights into the multifaceted activity of F2B, demonstrating its potential to disrupt bacterial membrane homeostasis, indicating that its activity extends the currently known mechanism of FabI enzyme inhibition. This disruption, coupled with the ability of F2B to penetrate the outer membrane layers, sheds new light on the behavior of this antimicrobial molecule. This highlights the importance of the interaction with the membrane, crucial in combating bacterial infections, particularly those caused by drug-resistant strains.
Packaging of Diisopropyl Fluorophosphatase (DFPase) in Bacterial Outer Membrane Vesicles Protects Its Activity at Extreme Temperature.
Enzymatic decontamination of organophosphate compounds offers a biofriendly pathway to the neutralization of highly dangerous compounds. Environmental dissemination of enzymes, however, is an ongoing problem considering the costly process of production and chemical modification for stability that can diminish catalytic activity. As a result, there is interest in the potential for enzymatic encapsulation in situ or into nascent bacterial membrane vesicles to improve catalytic stability across various environmental challenges associated with storage and field deployment. In this study, we have engineered bacterial outer membrane vesicles (OMVs) to encapsulate the diisopropyl fluorophosphatase (DFPase), an enzyme originally isolated from squid <i>Loligo vulgaris</i> and capable of hydrolyzing diisopropyl fluorophosphate (DFP) and other organophosphates compounds. Here we employed a recombinant lipopeptide anchor to direct recruitment of DFPase into OMVs, which were isolated from culture media and tested for catalytic activity against both diisopropyl fluorophosphate and paraoxon. Our encapsulation strategy prevented the loss of catalytic activity despite lyophilization, extended storage time (2 days), and extreme temperatures up to 80 &#176;C. These data underscore the appeal of DFPase as a biodecontaminant of organophosphates as well as the potential for OMV packaging in stabilized field deployment applications.
The distribution of phospholipids across the inner membrane (IM) of Gram-negative bacteria is unknown. We demonstrate that the IMs of <i>Escherichia coli</i> and <i>Yersinia pseudotuberculosis</i> are asymmetric, with a 75%/25% (cytoplasmic/periplasmic leaflet) distribution of phosphatidylethanolamine (PE) in rod-shaped cells and an opposite distribution in <i>E. coli</i> filamentous cells. In initially filamentous PE-lacking <i>E. coli</i> cells, nascent PE appears first in the periplasmic leaflet. As the total PE content increases from nearly zero to 75%, cells progressively adopt a rod shape and PE appears in the cytoplasmic leaflet of the IM. The redistribution of PE influences the distribution of the other lipids between the leaflets. This correlates with the tendency of PE and cardiolipin to regulate antagonistically lipid order of the bilayer. The results suggest that PE asymmetry is metabolically controlled to balance temporally the net rates of synthesis and translocation, satisfy envelope growth capacity, and adjust bilayer chemical and physical properties.
Mapping capacity to conduct health technology assessment in Central, Eastern and South-Eastern Europe.
To provide insights into the capacity to conduct health technology assessment (HTA) in Central, Eastern, and South-Eastern Europe (CESEE), taking account of technical, financial, networking, and human resources.
To establish a transborder virtual tumor board (VTB) fostering state-of-the-art management of cancer patients by exchanging knowledge and expertise among oncologists in Central and Southeastern Europe (CEE).
Mapping capacity to conduct health technology assessment in Central, Eastern and South-Eastern Europe.
To provide insights into the capacity to conduct health technology assessment (HTA) in Central, Eastern, and South-Eastern Europe (CESEE), taking account of technical, financial, networking, and human resources.
Recent developments in the geographical sciences and technologies, namely geographical epidemiology, geographical information systems, global positioning systems and spatial data analysis brings about a unique opportunity to investigate the role of "place" in human health in a scientific manner. The aim of the present communication is to open a discussion about the application of these advances to study the geography of public health problems within the Eastern Mediterranean Region of the World Health Organization.
Mapping capacity to conduct health technology assessment in Central, Eastern and South-Eastern Europe.
To provide insights into the capacity to conduct health technology assessment (HTA) in Central, Eastern, and South-Eastern Europe (CESEE), taking account of technical, financial, networking, and human resources.
The objectives of this study were to review current methodological guidelines for economic evaluations of all types of technologies in the 33 countries with organizations involved in the European Network for Health Technology Assessment (EUnetHTA), and to provide a general framework for economic evaluation at a European level.
Mapping capacity to conduct health technology assessment in Central, Eastern and South-Eastern Europe.
To provide insights into the capacity to conduct health technology assessment (HTA) in Central, Eastern, and South-Eastern Europe (CESEE), taking account of technical, financial, networking, and human resources.
E-health has been a recurrent topic in health reform, yet its implementation, ultimate role and feasibility are yet to be clearly defined. Organisations such as the Royal Flying Doctor Service South East Section (RFDS SE) are in a position to utilise technology to enhance the effectiveness of existing clinical services for remote communities. The study aim was to explore the readiness of the remote population of far-west New South Wales, Australia, and RFDS SE as a monopoly service provider to take up e-health innovations.
Mapping capacity to conduct health technology assessment in Central, Eastern and South-Eastern Europe.
To provide insights into the capacity to conduct health technology assessment (HTA) in Central, Eastern, and South-Eastern Europe (CESEE), taking account of technical, financial, networking, and human resources.
Community emergency medicine (CEM) aims to bring highly skilled, expert medical care to the patient outside of the traditional ED setting. Currently, there are several different CEM models in existence within the UK and Ireland which confer multiple benefits including provision of a senior clinical decision-maker early in the patient's journey, frontloading of time-critical interventions, easing pressure on busy EDs and reducing inpatient bed days. This is achieved through increased community-based management supplemented by utilisation of alternative care pathways. This study aimed to undertake a national comparison of CEM services currently in operation.
[Research progress on nonspecific immune microenvironment in breast cancer].
The immune microenvironment plays an important role in the occurrence and development of breast cancer. The infiltrating immune cells and the produced inflammatory cytokines in the tumor microenvironment regulate the growth, proliferation and metastasis of breast cancer. In this article, the roles and related mechanisms of nonspecific immune microenvironment in breast cancer are summarized, focusing on the natural killer cells, dendritic cells, myeloid derived suppressor cells, tumor associated macrophages, interleukins, chemokines, tumor necrosis factor-α, transforming growth factor-β and so on.
The tumor microenvironment significantly influences malignant behavior and progression. Many components are involved in the tumor microenvironment, including extracellular matrix, stromal cells, immune and inflammatory cells, as well as cytokines that promote tumor development with complex interactions through the exchange of molecular information. It is now known that tumor immune escape may be influenced by the tumor microenvironment. The aim of this work is to conduct a review of the tumor immune-microenvironment in gastric cancer.
[Research progress on nonspecific immune microenvironment in breast cancer].
The immune microenvironment plays an important role in the occurrence and development of breast cancer. The infiltrating immune cells and the produced inflammatory cytokines in the tumor microenvironment regulate the growth, proliferation and metastasis of breast cancer. In this article, the roles and related mechanisms of nonspecific immune microenvironment in breast cancer are summarized, focusing on the natural killer cells, dendritic cells, myeloid derived suppressor cells, tumor associated macrophages, interleukins, chemokines, tumor necrosis factor-α, transforming growth factor-β and so on.
Breast cancer is a complex disease with a highly immunosuppressive tumor microenvironment, and has limited clinical response to immune checkpoint blockade (ICB) therapy. T-helper 2 (Th2) cells, an important component of the tumor microenvironment (TME), play an essential role in regulation of tumor immunity. However, the deep relationship between Th2-mediated immunity and immune evasion in breast cancer remains enigmatic.
[Research progress on nonspecific immune microenvironment in breast cancer].
The immune microenvironment plays an important role in the occurrence and development of breast cancer. The infiltrating immune cells and the produced inflammatory cytokines in the tumor microenvironment regulate the growth, proliferation and metastasis of breast cancer. In this article, the roles and related mechanisms of nonspecific immune microenvironment in breast cancer are summarized, focusing on the natural killer cells, dendritic cells, myeloid derived suppressor cells, tumor associated macrophages, interleukins, chemokines, tumor necrosis factor-α, transforming growth factor-β and so on.
Tumour immune microenvironment (TIME) has long been a key direction of tumour research. Understanding the occurrence, metastasis and other processes of cervical cancer (CC) is of great significance in the diagnosis and prognosis of tumours.
[Research progress on nonspecific immune microenvironment in breast cancer].
The immune microenvironment plays an important role in the occurrence and development of breast cancer. The infiltrating immune cells and the produced inflammatory cytokines in the tumor microenvironment regulate the growth, proliferation and metastasis of breast cancer. In this article, the roles and related mechanisms of nonspecific immune microenvironment in breast cancer are summarized, focusing on the natural killer cells, dendritic cells, myeloid derived suppressor cells, tumor associated macrophages, interleukins, chemokines, tumor necrosis factor-α, transforming growth factor-β and so on.
Tumor microenvironment denotes the non-cancerous cells and components presented in the tumor, including molecules produced and released by them. The constant interactions between tumor cells and the tumor microenvironment play decisive roles in tumor initiation, progression, metastasis, and response to therapies. The tumor microenvironment as a therapeutic target in cancer has attracted great research and clinical interest. Here we summarize the current progress in targeting the tumor microenvironment in both drug development and clinical trials; highlight challenges in targeting the tumor microenvironment to achieve therapeutic efficacy; explore new technologies and approaches to better decipher the tumor microenvironment; and discuss strategies to intervene in the pro-tumor microenvironment and maximize therapeutic benefits.
[Research progress on nonspecific immune microenvironment in breast cancer].
The immune microenvironment plays an important role in the occurrence and development of breast cancer. The infiltrating immune cells and the produced inflammatory cytokines in the tumor microenvironment regulate the growth, proliferation and metastasis of breast cancer. In this article, the roles and related mechanisms of nonspecific immune microenvironment in breast cancer are summarized, focusing on the natural killer cells, dendritic cells, myeloid derived suppressor cells, tumor associated macrophages, interleukins, chemokines, tumor necrosis factor-α, transforming growth factor-β and so on.
Currently, tumor-infiltrating B lymphocytes have been recognized as a new hallmark of breast cancer (BC). The function seems to be controversial, either with positive, negative, or no significance in BC's prediction and prognosis. Moreover, B-cell infiltrates regulate tumor process through productions of antibodies and interleukin-10. The interactions with other lymphocytes and programmed death-1/PD-1 ligand axis are also documented. The regulatory mechanisms will eventually be incorporated into diagnostic and therapeutic algorithms, thus give guide to clinical treatment. In this review, we give new insights into clinical impacts and regulatory mechanisms of tumor-infiltrating B cells, which heralds a new era in immuno-oncology in BC treatment.
[Progress in digital PCR technology and application].
Digital PCR is an emerging analysis technology for absolute quantification after realtime-PCR. Through digital PCR, single DNA molecules are distributed into isolated reactions, and the product with fluorescence signal can be detected and analyzed after amplification. With the advantages of higher sensitivity and accuracy, digital PCR, independent of a standard curve, is developing rapidly and applied widely to the next generation sequencing and detection fields, such as gene mutation, copy number variation, microorganism, and genetically modified food. In this article, we reviewed the quantitative method and research progress of digital PCR technology in the main application fields.
During the last years the technology used for gene expression analysis has changed dramatically. The old mainstay, DNA microarray, has served its due course and will soon be replaced by next-generation sequencing (NGS), the Swiss army knife of modern high-throughput nucleic acid-based analysis. Therefore preparation technologies have to adapt to suit the emerging NGS technology platform. Moreover, interpretation of the results is still time consuming and employs the use of high-end computers usually not found in molecular biology laboratories. Alternatively, cloud computing might solve this problem. Nevertheless, these new challenges have to be embraced for gene expression analysis in general.
[Progress in digital PCR technology and application].
Digital PCR is an emerging analysis technology for absolute quantification after realtime-PCR. Through digital PCR, single DNA molecules are distributed into isolated reactions, and the product with fluorescence signal can be detected and analyzed after amplification. With the advantages of higher sensitivity and accuracy, digital PCR, independent of a standard curve, is developing rapidly and applied widely to the next generation sequencing and detection fields, such as gene mutation, copy number variation, microorganism, and genetically modified food. In this article, we reviewed the quantitative method and research progress of digital PCR technology in the main application fields.
To introduce the recent advances of the application of computer technology in tissue engineering.
[Progress in digital PCR technology and application].
Digital PCR is an emerging analysis technology for absolute quantification after realtime-PCR. Through digital PCR, single DNA molecules are distributed into isolated reactions, and the product with fluorescence signal can be detected and analyzed after amplification. With the advantages of higher sensitivity and accuracy, digital PCR, independent of a standard curve, is developing rapidly and applied widely to the next generation sequencing and detection fields, such as gene mutation, copy number variation, microorganism, and genetically modified food. In this article, we reviewed the quantitative method and research progress of digital PCR technology in the main application fields.
The introduction of new medical technologies such as sensors has accelerated the process of collecting patient data for relevant clinical decisions, which has led to the introduction of a new technology known as digital biomarkers.
[Progress in digital PCR technology and application].
Digital PCR is an emerging analysis technology for absolute quantification after realtime-PCR. Through digital PCR, single DNA molecules are distributed into isolated reactions, and the product with fluorescence signal can be detected and analyzed after amplification. With the advantages of higher sensitivity and accuracy, digital PCR, independent of a standard curve, is developing rapidly and applied widely to the next generation sequencing and detection fields, such as gene mutation, copy number variation, microorganism, and genetically modified food. In this article, we reviewed the quantitative method and research progress of digital PCR technology in the main application fields.
With the focus on technology for this issue of Molecular Cell, a group of scientists working in different areas of molecular biology provide their perspective on the most recent important technological advance in their field, where the field is lacking, and their wish list for future technology development.
[Progress in digital PCR technology and application].
Digital PCR is an emerging analysis technology for absolute quantification after realtime-PCR. Through digital PCR, single DNA molecules are distributed into isolated reactions, and the product with fluorescence signal can be detected and analyzed after amplification. With the advantages of higher sensitivity and accuracy, digital PCR, independent of a standard curve, is developing rapidly and applied widely to the next generation sequencing and detection fields, such as gene mutation, copy number variation, microorganism, and genetically modified food. In this article, we reviewed the quantitative method and research progress of digital PCR technology in the main application fields.
Over the past 20 years,clinical molecular diagnostic technology has made rapid development,and became the most promising field in clinical laboratory medicine.In particular,with the development of genomics,clinical molecular diagnostic methods will reveal the nature of clinical diseases in a deeper level,thus guiding the clinical diagnosis and treatments.Many molecular diagnostic projects have been routinely applied in clinical works.This paper reviews the advances on application of clinical diagnostic techniques in infectious disease,tumor and genetic disorders,including nucleic acid amplification,biochip,next-generation sequencing,and automation molecular system,and so on.
EQCM biosensors based on DNA aptamers and antibodies for rapid detection of prions.
Novel affinity biosensors for detecting cellular prions, PrP(C), based on DNA aptamers and antibodies immobilized onto the carbon nanotubes have been designed and compared in accordance with their binding ability and analytical performance. The biosensors allowed us to detect PrP(C) with the limits of detection of 20 to 50 pM.
The main advances in control and early diagnosis of cancer is greatly aided by low level detection of tumor markers in biological samples. Extensive efforts have been devoted to developing some ultrasensitive electrochemical biosensors for detection of cancer markers with high selectivity. These efforts include the development of the bioreceptors with high specificity and affinity, synthesis of novel signal amplifiers based on nanomaterials and the exploration of appropriate design strategies. Electrochemical measurement protocols are suitable for mass fabrication of miniaturized devices. They have a major role in the move towards rapid and simplified testing for point-of-care usage. This review discusses the remarkable advances of the last 6 years in the electrochemical affinity biosensors for determination of protein and glycoprotein tumor markers, with a particular focus on antibodies and aptamers as biorecognition probes.
EQCM biosensors based on DNA aptamers and antibodies for rapid detection of prions.
Novel affinity biosensors for detecting cellular prions, PrP(C), based on DNA aptamers and antibodies immobilized onto the carbon nanotubes have been designed and compared in accordance with their binding ability and analytical performance. The biosensors allowed us to detect PrP(C) with the limits of detection of 20 to 50 pM.
Herein, an isothermal padlock probe-based assay for the simple and portable detection of pathogens coupled with a glucose oxidase (GOx)-based electrochemical readout is reported. Infectious diseases remain a constant threat on a global scale, as in recurring pandemics. Rapid and portable diagnostics hold the promise to tackle the spreading of diseases and decentralising healthcare to point-of-care needs. Ebola, a hypervariable RNA virus causing fatalities of up to 90% for recent outbreaks in Africa, demands immediate attention for bedside diagnostics. The design of the demonstrated assay consists of a rolling circle amplification (RCA) technique, responsible for the generation of nucleic acid amplicons as RCA products (RCPs). The RCPs are generated on magnetic beads (MB) and subsequently, connected via streptavidin-biotin bonds to GOx. The enzymatic catalysis of glucose by the bound GOx allows for an indirect electrochemical measurement of the DNA target. The RCPs generated on the surface of the MB were confirmed by scanning electron microscopy, and among other experimental conditions such as the type of buffer, temperature, concentration of GOx, sampling and measurement time were evaluated for the optimum electrochemical detection. Accordingly, 125&#160;&#956;g&#160;mL<sup>-1</sup> of GOx with 5&#160;mM glucose using phosphate buffer saline (PBS), monitored for 1&#160;min were selected as the ideal conditions. Finally, we assessed the analytical performance of the biosensing strategy by using clinical samples of Ebola virus from patients. Overall, this work provides a proof-of-concept bioassay for simple and portable molecular diagnostics of emerging pathogens using electrochemical detection, especially in resource-limited settings.
EQCM biosensors based on DNA aptamers and antibodies for rapid detection of prions.
Novel affinity biosensors for detecting cellular prions, PrP(C), based on DNA aptamers and antibodies immobilized onto the carbon nanotubes have been designed and compared in accordance with their binding ability and analytical performance. The biosensors allowed us to detect PrP(C) with the limits of detection of 20 to 50 pM.
The recent surge of effort in nucleic-acid-based electrochemical (EC) sensors has been fruitful, yet there remains a need for more generalizable EC platforms for sensing multiple classes of clinically relevant targets. We recently reported a nucleic acid nanostructure for simple, economical, and more generalizable EC readout of a range of analytes, including small molecules, peptides, proteins, and antibodies. The nanostructure is built through on-electrode enzymatic ligation of three oligonucleotides for attachment, binding, and signaling. However, the generalizable detection of larger proteins remains a challenge. Here, we adapted the sensor to quantify larger proteins in a more generic manner through conjugating the protein's minimized antibody-binding epitope to the central DNA strand. This concept was verified using creatine kinase (CK-MM), a biomarker of muscle damage and several disorders for which rapid clinical sensing is important. DNA-epitope conjugates permitted a competitive immunoassay for the CK protein at the electrode via square-wave voltammetry (SWV). Sensing through a signal-off mechanism, the anti-CK antibody limit of detection (LOD) was 5 nM with a response time as low as 3 min. Antibody displacement by native protein analytes gave a signal-on response with the CK sensing range from the LOD of 14 nM up to 100 nM, overlapping with the normal (nonelevated) human clinical range (3-37 nM), and the sensor was validated in 98% human serum. While a need for improved DNA-epitope conjugate purification was identified, overall, this approach allows the quantification of a generic protein- or peptide-binding antibody and should facilitate future quantitative EC readouts of clinically relevant proteins that were previously inaccessible to EC techniques.
EQCM biosensors based on DNA aptamers and antibodies for rapid detection of prions.
Novel affinity biosensors for detecting cellular prions, PrP(C), based on DNA aptamers and antibodies immobilized onto the carbon nanotubes have been designed and compared in accordance with their binding ability and analytical performance. The biosensors allowed us to detect PrP(C) with the limits of detection of 20 to 50 pM.
DNA biosensors are attractive tools for genetic analysis as there is an increasing need for rapid and low-cost DNA analysis, primarily driven by applications in personalized pharmacogenomics, clinical diagnostics, rapid pathogen detection, food traceability and forensics. A rapid electrochemical genosensor detection methodology exploiting a combination of modified primers for solution-phase isothermal amplification, followed by rapid detection via hybridization on gold electrodes is reported. Modified reverse primers, exploiting a C18 spacer between the primer-binding site and an engineered single stranded tail, are used in a recombinase polymerase amplification reaction to produce an amplicon with a central duplex flanked by two single stranded tails. These tails are designed to be complementary to a gold electrode tethered capture oligo probe as well as a horseradish peroxidase labelled reporter oligo probe. The time required for hybridization of the isothermally generated amplicons with each of the immobilized and reporter probes was optimised to be 2 min, in both cases. The effect of amplification time and the limit of detection were evaluated using these hybridization times for both single stranded and double stranded DNA templates. The best detection limit of 70 fM for a ssDNA template was achieved using 45 min amplification, whilst for a dsDNA template, just 30 min amplification resulted in a slightly lower detection limit of 14 fM, whilst both 20 and 45 min amplification times were observed to provide detection limits of 71 and 72 fM, respectively, but 30 and 45 min amplification resulted in a much higher signal and sensitivity. The genosensor was applied to genomic DNA and real patient and control blood samples for detection of the coeliac disease associated DQB1*02 HLA allele, as a model system, demonstrating the possibility to carry out molecular diagnostics, combining amplification and detection in a rapid and facile manner.
EQCM biosensors based on DNA aptamers and antibodies for rapid detection of prions.
Novel affinity biosensors for detecting cellular prions, PrP(C), based on DNA aptamers and antibodies immobilized onto the carbon nanotubes have been designed and compared in accordance with their binding ability and analytical performance. The biosensors allowed us to detect PrP(C) with the limits of detection of 20 to 50 pM.
By combination of the aptamer biorecognition with the colorimetric signal transduction of a DNAzyme-functionalized nanoprobe, a new biosensing method was developed for the rapid and sensitive detection of chloramphenicol (CAP). The nanoprobe was prepared through the functionalization of gold nanoparticles with the complementary oligonucleotide against aptamer and high-content hemin/G-quadruplex DNAzyme. When one-step incubating the nanoprobe and CAP at a constructed aptamer-magnetic bead (MB) biosensing platform, due to the competitive biorecognition reaction, the nanoprobes related with CAP amounts were quantitative captured onto the MB surface. Based on the catalytic reaction of the peroxidase-mimicking DNAzyme, a colored substance was produced for the colorimetric signal transduction of the method. Due to the great signal amplification of the nanoprobe, a very low detection limit down to 0.13 pg/mL was obtained. Considering the excellent performance of the aptasensing method and satisfactory results for milk sample experiments, it indicates good reliability for practical applications.
Resolving the origins of secretory products and anthelmintic responses in a human parasitic nematode at single-cell resolution.
Nematode excretory-secretory (ES) products are essential for the establishment and maintenance of infections in mammals and are valued as therapeutic and diagnostic targets. While parasite effector proteins contribute to host immune evasion and anthelmintics have been shown to modulate secretory behaviors, little is known about the cellular origins of ES products or the tissue distributions of drug targets. We leveraged single-cell approaches in the human parasite <i>Brugia malayi</i> to generate an annotated cell expression atlas of microfilariae. We show that prominent antigens are transcriptionally derived from both secretory and non-secretory cell and tissue types, and anthelmintic targets display distinct expression patterns across neuronal, muscular, and other cell types. While the major classes of anthelmintics do not affect the viability of isolated cells at pharmacological concentrations, we observe cell-specific transcriptional shifts in response to ivermectin. Finally, we introduce a microfilariae cell culture model to enable future functional studies of parasitic nematode cells. We expect these methods to be readily adaptable to other parasitic nematode species and stages.
Host innate immunity plays a central role in detecting and eliminating microbial pathogenic infections in both vertebrate and invertebrate animals. Entomopathogenic or insect pathogenic nematodes are of particular importance for the control of insect pests and vectors of pathogens, while insect-borne nematodes cause serious diseases in humans. Recent work has begun to use the power of insect models to investigate host-nematode interactions and uncover host antiparasitic immune reactions. This review describes recent findings on innate immune evasion strategies of parasitic nematodes and host cellular and humoral responses to the infection. Such information can be used to model diseases caused by human parasitic nematodes and provide clues indicating directions for research into the interplay between vector insects and their invading tropical parasites.
Resolving the origins of secretory products and anthelmintic responses in a human parasitic nematode at single-cell resolution.
Nematode excretory-secretory (ES) products are essential for the establishment and maintenance of infections in mammals and are valued as therapeutic and diagnostic targets. While parasite effector proteins contribute to host immune evasion and anthelmintics have been shown to modulate secretory behaviors, little is known about the cellular origins of ES products or the tissue distributions of drug targets. We leveraged single-cell approaches in the human parasite <i>Brugia malayi</i> to generate an annotated cell expression atlas of microfilariae. We show that prominent antigens are transcriptionally derived from both secretory and non-secretory cell and tissue types, and anthelmintic targets display distinct expression patterns across neuronal, muscular, and other cell types. While the major classes of anthelmintics do not affect the viability of isolated cells at pharmacological concentrations, we observe cell-specific transcriptional shifts in response to ivermectin. Finally, we introduce a microfilariae cell culture model to enable future functional studies of parasitic nematode cells. We expect these methods to be readily adaptable to other parasitic nematode species and stages.
Important insights have recently been gained in our understanding of the intricate relationship in the intestinal milieu between the vertebrate host mucosal immune response, commensal bacteria, and helminths. Helminths are metazoan worms (macrobiota) and trigger immune responses that include potent regulatory components capable of controlling harmful inflammation, protecting barrier function and mitigating tissue damage. They can secrete a variety of products that directly affect immune regulatory function but they also have the capacity to influence the composition of microbiota, which can also then impact immune function. Conversely, changes in microbiota can affect susceptibility to helminth infection, indicating that crosstalk between these two disparate groups of endobiota can play an essential role in host intestinal immune function and homeostasis.
Resolving the origins of secretory products and anthelmintic responses in a human parasitic nematode at single-cell resolution.
Nematode excretory-secretory (ES) products are essential for the establishment and maintenance of infections in mammals and are valued as therapeutic and diagnostic targets. While parasite effector proteins contribute to host immune evasion and anthelmintics have been shown to modulate secretory behaviors, little is known about the cellular origins of ES products or the tissue distributions of drug targets. We leveraged single-cell approaches in the human parasite <i>Brugia malayi</i> to generate an annotated cell expression atlas of microfilariae. We show that prominent antigens are transcriptionally derived from both secretory and non-secretory cell and tissue types, and anthelmintic targets display distinct expression patterns across neuronal, muscular, and other cell types. While the major classes of anthelmintics do not affect the viability of isolated cells at pharmacological concentrations, we observe cell-specific transcriptional shifts in response to ivermectin. Finally, we introduce a microfilariae cell culture model to enable future functional studies of parasitic nematode cells. We expect these methods to be readily adaptable to other parasitic nematode species and stages.
Animals often suffer from multiple parasite attacks in natural conditions (i.e., polyparasitism). The community of these parasites, which simultaneously or sequentially infest given host species, has rarely been investigated as a parasitic pressure per se. From this perspective, and despite the impressive number of immunoecological or comparative studies, the impacts of polyparasitism on immune responses are far from being appreciated. Focusing on helminths across a wide range of mammalian species and using a phylogenetic comparative method, we show, for the first time, that an increase in the number of helminth parasite species is positively correlated with an increase in basal immune investment (estimated by the counts of white blood cells) across mammal species. After discussing inherent limits of this comparative approach, we put this result in the evolutionary perspective of multiple parasitic infestations.
Resolving the origins of secretory products and anthelmintic responses in a human parasitic nematode at single-cell resolution.
Nematode excretory-secretory (ES) products are essential for the establishment and maintenance of infections in mammals and are valued as therapeutic and diagnostic targets. While parasite effector proteins contribute to host immune evasion and anthelmintics have been shown to modulate secretory behaviors, little is known about the cellular origins of ES products or the tissue distributions of drug targets. We leveraged single-cell approaches in the human parasite <i>Brugia malayi</i> to generate an annotated cell expression atlas of microfilariae. We show that prominent antigens are transcriptionally derived from both secretory and non-secretory cell and tissue types, and anthelmintic targets display distinct expression patterns across neuronal, muscular, and other cell types. While the major classes of anthelmintics do not affect the viability of isolated cells at pharmacological concentrations, we observe cell-specific transcriptional shifts in response to ivermectin. Finally, we introduce a microfilariae cell culture model to enable future functional studies of parasitic nematode cells. We expect these methods to be readily adaptable to other parasitic nematode species and stages.
The development and integration of DNA-based methods in research and clinical microbiology laboratories have enabled standardised and comprehensive detection and differentiation of the microbes colonising our guts. For instance, the single-celled parasites Blastocystis and Dientamoeba appear to be much more common than previously thought, especially so in healthy individuals. While increasing evidence appears to suggest limited pathogenicity of these parasites, next-generation-sequencing-based studies have helped us to appreciate links between parasite colonisation and certain host phenotypical characteristics and gut microbial profiles. The fundamental question remains as to whether such parasites are merely indicators or active manipulators of gut microbiota structure and function. In this article, we collate existing evidence that these parasites are, at minimum, indicators of intestinal microbiota structure.
Resolving the origins of secretory products and anthelmintic responses in a human parasitic nematode at single-cell resolution.
Nematode excretory-secretory (ES) products are essential for the establishment and maintenance of infections in mammals and are valued as therapeutic and diagnostic targets. While parasite effector proteins contribute to host immune evasion and anthelmintics have been shown to modulate secretory behaviors, little is known about the cellular origins of ES products or the tissue distributions of drug targets. We leveraged single-cell approaches in the human parasite <i>Brugia malayi</i> to generate an annotated cell expression atlas of microfilariae. We show that prominent antigens are transcriptionally derived from both secretory and non-secretory cell and tissue types, and anthelmintic targets display distinct expression patterns across neuronal, muscular, and other cell types. While the major classes of anthelmintics do not affect the viability of isolated cells at pharmacological concentrations, we observe cell-specific transcriptional shifts in response to ivermectin. Finally, we introduce a microfilariae cell culture model to enable future functional studies of parasitic nematode cells. We expect these methods to be readily adaptable to other parasitic nematode species and stages.
Vertebrates and helminths have co-evolved for 500 million years, developing mutual adaptation mechanisms between parasites and hosts. Today, however, helminth diseases are among the most neglected communicable diseases. Epidemiological evidence shows that exposure to helminth parasites is inversely correlated with allergy incidence, and helminths induce immune hyporeactivity in both the innate and adaptive systems. The mechanisms include parasite-derived regulatory molecules, the study of which opens new avenues for the control of allergic and autoimmune diseases.
Grazing intensity differentially regulates ANPP response to precipitation in North American semiarid grasslands.
Grazing intensity elicits changes in the composition of plant functional groups in both shortgrass steppe (SGS) and northern mixed-grass prairie (NMP) in North America. How these grazing intensity-induced changes control aboveground net primary production (ANPP) responses to precipitation remains a central open question, especially in light of predicted climate changes. Here, we evaluated effects of four levels (none, light, moderate, and heavy) of long-term (&gt;30&#160;yr) grazing intensity in SGS and NMP on: (1) ANPP; (2) precipitation-use efficiency (PUE, ANPP : precipitation); and (3) precipitation marginal response (PMR; slope of a linear regression model between ANPP and precipitation). We advance prior work by examining: (1) the consequences of a range of grazing intensities (more grazed vs. ungrazed); and (2) how grazing-induced changes in ANPP and PUE are related both to shifts in functional group composition and physiological responses within each functional group. Spring (April-June) precipitation, the primary determinant of ANPP, was only 12% higher in NMP than in SGS, yet ANPP and PUE were 25% higher. Doubling grazing intensity in SGS and nearly doubling it in NMP reduced ANPP and PUE by only 24% and 33%, respectively. Increased grazing intensity reduced C<sub>3</sub> graminoid biomass and increased C<sub>4</sub> grass biomass in both grasslands. Functional group shifts affected PUE through biomass reductions, as PUE was positively associated with the relative abundance of C<sub>3</sub> species and negatively with C<sub>4</sub> species across both grasslands. At the community level, PMR was similar between grasslands and unaffected by grazing intensity. However, PMR of C<sub>3</sub> graminoids in SGS was eightfold higher in the ungrazed treatment than under any grazed level. In NMP, PMR of C<sub>3</sub> graminoids was only reduced under heavy grazing intensity. Knowing the ecological consequences of grazing intensity provides valuable information for mitigation and adaptation strategies in response to predicted climate change. For example, moderate grazing (the recommended rate) in SGS would sequester the same amount of aboveground carbon as light grazing because ANPP was nearly the same. In contrast, reductions in grazing intensity in NMP from moderate to light intensity would increase the amount of aboveground carbon sequestrated by 25% because of increased ANPP.
Nitrogen (N) loading and extreme drought strongly alter biomass production, species composition and carbon and water fluxes of temperate grasslands. Such changes at the community level are often attributed to species- and functional group-specific responses in phenology and/or physiology. In a multifactorial field experiment, we studied the responses of three abundant grassland species (forb Centaurea jacea, grasses Arrhenatherum elatius and Dactylis glomerata) to N loading and extreme drought, focusing on responses of carbon and water relations at the leaf level. We analysed (1) changes in bulk leaf N (uptake efficiency of additional N), (2) adaptation of plant water status (leaf water potential) and (3) impact on leaf carbon and water fluxes. We observed more efficient N utilization in the two grasses compared to C. jacea. Naturally occurring summer drought significantly impacted the plant water status of all species, while extreme drought treatment only further affected water status during and after summer drought. C. jacea was able to maintain much lower leaf water potentials compared to the grasses during drought. Despite these clear species-specific responses to N loading and drought, the species were able to maintain homeostasis of leaf carbon and water fluxes. Thus, strong declines in the (community) carbon sequestration observed at this site during the (natural) summer drought were not related to leaf physiological responses in assimilation, but were driven by phenological adaptions of the species community: the drought-sensitive grasses, even though exhibiting higher N uptake efficiency, responded with a shortened life cycle to severe summer drought.
Grazing intensity differentially regulates ANPP response to precipitation in North American semiarid grasslands.
Grazing intensity elicits changes in the composition of plant functional groups in both shortgrass steppe (SGS) and northern mixed-grass prairie (NMP) in North America. How these grazing intensity-induced changes control aboveground net primary production (ANPP) responses to precipitation remains a central open question, especially in light of predicted climate changes. Here, we evaluated effects of four levels (none, light, moderate, and heavy) of long-term (&gt;30&#160;yr) grazing intensity in SGS and NMP on: (1) ANPP; (2) precipitation-use efficiency (PUE, ANPP : precipitation); and (3) precipitation marginal response (PMR; slope of a linear regression model between ANPP and precipitation). We advance prior work by examining: (1) the consequences of a range of grazing intensities (more grazed vs. ungrazed); and (2) how grazing-induced changes in ANPP and PUE are related both to shifts in functional group composition and physiological responses within each functional group. Spring (April-June) precipitation, the primary determinant of ANPP, was only 12% higher in NMP than in SGS, yet ANPP and PUE were 25% higher. Doubling grazing intensity in SGS and nearly doubling it in NMP reduced ANPP and PUE by only 24% and 33%, respectively. Increased grazing intensity reduced C<sub>3</sub> graminoid biomass and increased C<sub>4</sub> grass biomass in both grasslands. Functional group shifts affected PUE through biomass reductions, as PUE was positively associated with the relative abundance of C<sub>3</sub> species and negatively with C<sub>4</sub> species across both grasslands. At the community level, PMR was similar between grasslands and unaffected by grazing intensity. However, PMR of C<sub>3</sub> graminoids in SGS was eightfold higher in the ungrazed treatment than under any grazed level. In NMP, PMR of C<sub>3</sub> graminoids was only reduced under heavy grazing intensity. Knowing the ecological consequences of grazing intensity provides valuable information for mitigation and adaptation strategies in response to predicted climate change. For example, moderate grazing (the recommended rate) in SGS would sequester the same amount of aboveground carbon as light grazing because ANPP was nearly the same. In contrast, reductions in grazing intensity in NMP from moderate to light intensity would increase the amount of aboveground carbon sequestrated by 25% because of increased ANPP.
Changes in livestock loads and eutrophication associated with human activities can modify the stability of grassland's aboveground net primary productivity (ANPP), by modifying the mean (&#956;) and/or standard deviation (&#963;) of ANPP. The changes in attributes of the plant community (i.e., species richness, species asynchrony, dominance) might in turn explain the ecosystem temporal (inter-annual) stability of grassland production. Here, we evaluated the interactive effects of changes in livestock loads and chronic nutrient addition on the temporal stability of ANPP (estimated as &#956;/&#963;) in temperate grasslands. We also assessed the role of different attributes of the plant community on ecosystem stability. We carried out a factorial experiment of domestic livestock exclusion and nutrient addition (10&#160;g.m<sup>-2</sup>.year<sup>-1</sup> of nitrogen, phosphorus, and potassium; n&#160;=&#160;6 blocks) during five consecutive years in a natural grassland devoted to cattle production (Flooding Pampa, Argentina). Domestic livestock exclusion reduced ANPP stability by 65%, regardless of nutrient load, mainly by the increase of ANPP standard deviation. This reduction in ANPP stability after livestock exclusion was associated mostly with higher plant species dominance and also with reductions in plant effective richness and in the asynchrony of grassland's species. Despite not finding direct negative effects of eutrophication on ANPP stability, chronic nutrient addition decreased effective species richness and asynchrony, which may translate into reductions in ANPP stability in the future. Our findings highlight that the presence of livestock maintains the temporal stability of ANPP mainly by lowering the dominance of the plant community. However, increases in nutrient loads in grasslands devoted to livestock production may threaten grassland's stability.