Point-of-care PCR testing of SARS-CoV-2 in the emergency department: Influence on workflow and efficiency.

PROBLEM: Regarding transmissible viral diseases such as those caused by SARS-CoV-2 virus, one of the key challenges is isolation management until final diagnosis. This study investigates the influence of SARS-CoV-2 point-of-care (POC) PCR on workflow and efficiency in an emergency department (ED) of a tertiary university hospital. METHOD: An analysis of 17,875 ED patients receiving either SARS-CoV-2 POC PCR (rapid PCR, 11,686 patients) or conventional laboratory SARS-CoV-2 PCR (conventional PCR, 6,189 patients) was performed. The pathways for both groups were mapped and compared, and process times from admission to diagnosis were measured. Effects on resource management within the ED were quantified. Direct costs due to isolation, loss of capacities, and revenues were calculated for inpatients. RESULTS: The mean time from admission to result was 1.62 h with rapid PCR and 16.08 h with conventional PCR (p < 0.01), reducing the isolation time by 14.46 h. In the first 2 h after testing, test results were available for > 75% of the rapid PCR group and none of the conventional PCR group. Ninety percent of the results were available within 3 h for the rapid PCR and within 21 h for the conventional PCR group. For the conventional PCR group, an increase in direct costs of €35.74 and lost revenues of €421.06 for each inpatient case was detected. CONCLUSION: Rapid PCR significantly reduces the time-to-results and time for isolation relative to conventional PCR. Although testing costs for rapid PCR are higher, it benefits workflow, reduces total costs, and frees up ward capacity.

Interacting Bioenergetic and Stoichiometric Controls on Microbial Growth.

Microorganisms function as open systems that exchange matter and energy with their surrounding environment. Even though mass (carbon and nutrients) and energy exchanges are tightly linked, there is a lack of integrated approaches that combine these fluxes and explore how they jointly impact microbial growth. Such links are essential to predicting how the growth rate of microorganisms varies, especially when the stoichiometry of carbon- (C) and nitrogen (N)-uptake is not balanced. Here, we present a theoretical framework to quantify the microbial growth rate for conditions of C-, N-, and energy-(co-) limitations. We use this framework to show how the C:N ratio and the degree of reduction of the organic matter (OM), which is also the electron donor, availability of electron acceptors (EAs), and the different sources of N together control the microbial growth rate under C, nutrient, and energy-limited conditions. We show that the growth rate peaks at intermediate values of the degree of reduction of OM under oxic and C-limited conditions, but not under N-limited conditions. Under oxic conditions and with N-poor OM, the growth rate is higher when the inorganic N (NInorg)-source is ammonium compared to nitrate due to the additional energetic cost involved in nitrate reduction. Under anoxic conditions, when nitrate is both EA and NInorg-source, the growth rates of denitrifiers and microbes performing the dissimilatory nitrate reduction to ammonia (DNRA) are determined by both OM degree of reduction and nitrate-availability. Consistent with the data, DNRA is predicted to foster growth under extreme nitrate-limitation and with a reduced OM, whereas denitrifiers are favored as nitrate becomes more available and in the presence of oxidized OM. Furthermore, the growth rate is reduced when catabolism is coupled to low energy yielding EAs (e.g., sulfate) because of the low carbon use efficiency (CUE). However, the low CUE also decreases the nutrient demand for growth, thereby reducing N-limitation. We conclude that bioenergetics provides a useful conceptual framework for explaining growth rates under different metabolisms and multiple resource-limitations.

Molecular Epidemiology, Clinical Course, and Implementation of Specific Hygiene Measures in Hospitalised Patients with Clostridioides difficile Infection in Brandenburg, Germany.

(1) Background: Clostridioides difficile infections (CDI) have increased worldwide, and the disease is one of the most common healthcare-associated infections (HAI). This study aimed to evaluate the molecular epidemiology of C. difficile, the clinical outcome, and the time of initiation of specific hygiene measures in patients with CDI in a large tertiary-care hospital in Brandenburg. (2) Methods: Faecal samples and data from hospitalised patients diagnosed with CDI were analysed from October 2016 to October 2017. The pathogens were isolated, identified as toxigenic C. difficile, and subsequently subtyped using PCR ribotyping and whole genome sequencing (WGS). Data regarding specific hygiene measures for handling CDI patients were collected. (3) Results: 92.1% of cases could be classified as healthcare-associated (HA)-CDI. The recurrence rate within 30 and 90 days after CDI diagnosis was 15.7% and 18.6%, and the mortality rate was 21.4% and 41.4%, respectively. The most frequent ribotypes (RT) were RT027 (31.3%), RT014 (18.2%), and RT005 (14.1%). Analysis of WGS data using cgMLST showed that all RT027 isolates were closely related; they were assigned to two subclusters. Single-room isolation or barrier measures were implemented in 95.7% patients. (4) Conclusions: These data show that RT027 is regionally predominant, thus highlighting the importance of specific hygiene measures to prevent and control CDI and the need to improve molecular surveillance of C. difficile at the local and national level.

[SARS-CoV-2 IgG seroprevalence in personnel of the extraclinical fight against the COVID-19 pandemic]. / SARS-CoV-2-IgG-Antikörperseroprävalenz bei Personal in der außerklinischen Bekämpfung der COVID-19-Pandemie.

BACKGROUND AND OBJECTIVES: The SARS-CoV­2 pandemic and the different manifestations of the coronavirus disease 2019 (COVID-19) are a major challenge for health systems worldwide. Medical personnel have a special role in containing the pandemic. The aim of the study was to investigate the SARS-CoV­2 IgG antibody prevalence in extraclinical personnel depending on their operational area in the fight against the COVID-19 pandemic. METHODS: On May 28 and 29, 2020, serum samples were taken from 732 of 1183 employees (61.9%) of the professional fire brigade and aid organizations in the city area and tested for SARS-CoV­2 IgG antibodies. The employees were divided into four categories according to their type of participation. category 1: decentralized PCR sampling teams, category 2: rescue service, category 3: fire protection, category 4: situation center. Some employees participated in more than one operational area. RESULTS: SARS-CoV­2 IgG antibodies were detected in 8 of 732 serum samples. This corresponds to a prevalence of 1.1%. A previous COVID-19 infection was known in 3 employees. In order to make a separate assessment of the other employees possible and to diagnose unknown infections, a corrected collective of 729 employees with 6 SARS-CoV­2 antibody detection was considered separately. The prevalence in the corrected collective is 0.82%. After subdividing the collective into areas of activity, the prevalence was low (1: 0.77%, 2: 0.9%, 3: 1.00%, 4: 1.58%). CONCLUSIONS: The seroprevalence of SARS-CoV­2 in the study collective is low at 1.1% and 0.82%, respectively. There is an increased seroprevalence in operational areas with a lower risk of virus exposure in comparison to operational areas with a higher risk.

A rapid test recognizing mucosal SARS-CoV-2-specific antibodies distinguishes prodromal from convalescent COVID-19.

The COVID-19 pandemic poses enormous challenges to global healthcare sectors. To prevent the overburden of medical systems, it is crucial to distinguish individuals approaching the most infectious early phase from those in the declining non-infectious phase. However, a large fraction of transmission events occur during pre- or asymptomatic phases. Especially in the absence of symptoms, it is difficult to distinguish prodromal from late phases of infection just by RT-PCR since both phases are characterized by low viral loads and corresponding high Ct values (>30). We evaluated a new rapid test detecting IgG antibodies recognizing SARS-CoV-2 nucleocapsid protein using two commercial antibody assays and an in-house neutralization test before determining suitability for testing clinical swab material. Our analyses revealed the combination of the well-known RT-PCR and the new rapid antibody test using one single clinical nasopharyngeal swab specimen as a fast, cost-effective, and reliable way to discriminate prodromal from subsiding phases of COVID-19.

SARS-CoV-2 Seroprevalence in Healthcare Workers in Germany: A Follow-Up Study.

SARS-CoV-2 is a worldwide challenge for the medical sector. Healthcare workers (HCW) are a cohort vulnerable to SARS-CoV-2 infection due to frequent and close contact with COVID-19 patients. However, they are also well trained and equipped with protective gear. The SARS-CoV-2 IgG antibody status was assessed at three different time points in 450 HCW of the University Hospital Essen in Germany. HCW were stratified according to contact frequencies with COVID-19 patients in (I) a high-risk group with daily contacts with known COVID-19 patients (n = 338), (II) an intermediate-risk group with daily contacts with non-COVID-19 patients (n = 78), and (III) a low-risk group without patient contacts (n = 34). The overall seroprevalence increased from 2.2% in March-May to 4.0% in June-July to 5.1% in October-December. The SARS-CoV-2 IgG detection rate was not significantly different between the high-risk group (1.8%; 3.8%; 5.5%), the intermediate-risk group (5.1%; 6.3%; 6.1%), and the low-risk group (0%, 0%, 0%). The overall SARS-CoV-2 seroprevalence remained low in HCW in western Germany one year after the outbreak of COVID-19 in Germany, and hygiene standards seemed to be effective in preventing patient-to-staff virus transmission.

A Novel In-Cell ELISA Assay Allows Rapid and Automated Quantification of SARS-CoV-2 to Analyze Neutralizing Antibodies and Antiviral Compounds.

The coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently the most pressing medical and socioeconomic challenge. Constituting important correlates of protection, the determination of virus-neutralizing antibodies (NAbs) is indispensable for convalescent plasma selection, vaccine candidate evaluation, and immunity certificates. In contrast to standard serological ELISAs, plaque reduction neutralization tests (PRNTs) are laborious, time-consuming, expensive, and restricted to specialized laboratories. To replace microscopic counting-based SARS-CoV-2 PRNTs by a novel assay exempt from genetically modified viruses, which are inapplicable in most diagnostics departments, we established a simple, rapid, and automated SARS-CoV-2 neutralization assay employing an in-cell ELISA (icELISA) approach. After optimization of various parameters such as virus-specific antibodies, cell lines, virus doses, and duration of infection, SARS-CoV-2-infected cells became amenable as direct antigen source for quantitative icELISA. Antiviral agents such as human sera containing NAbs or antiviral interferons dose dependently reduced the SARS-CoV-2-specific signal. Applying increased infectious doses, the icELISA-based neutralization test (icNT) was superior to PRNT in discriminating convalescent sera with high from those with intermediate neutralizing capacities. In addition, the icNT was found to be specific, discriminating between SARS-CoV-2-specific NAbs and those raised against other coronaviruses. Altogether, the SARS-CoV-2 icELISA test allows rapid (<48 h in total, read-out in seconds) and automated quantification of virus infection in cell culture to evaluate the efficacy of NAbs and antiviral drugs using reagents and equipment present in most routine diagnostics departments.

A framework for modelling soil structure dynamics induced by biological activity.

Soil degradation is a worsening global phenomenon driven by socio-economic pressures, poor land management practices and climate change. A deterioration of soil structure at timescales ranging from seconds to centuries is implicated in most forms of soil degradation including the depletion of nutrients and organic matter, erosion and compaction. New soil-crop models that could account for soil structure dynamics at decadal to centennial timescales would provide insights into the relative importance of the various underlying physical (e.g. tillage, traffic compaction, swell/shrink and freeze/thaw) and biological (e.g. plant root growth, soil microbial and faunal activity) mechanisms, their impacts on soil hydrological processes and plant growth, as well as the relevant timescales of soil degradation and recovery. However, the development of such a model remains a challenge due to the enormous complexity of the interactions in the soil-plant system. In this paper, we focus on the impacts of biological processes on soil structure dynamics, especially the growth of plant roots and the activity of soil fauna and microorganisms. We first define what we mean by soil structure and then review current understanding of how these biological agents impact soil structure. We then develop a new framework for modelling soil structure dynamics, which is designed to be compatible with soil-crop models that operate at the soil profile scale and for long temporal scales (i.e. decades, centuries). We illustrate the modelling concept with a case study on the role of root growth and earthworm bioturbation in restoring the structure of a severely compacted soil.

SARS-CoV-2-specific antibody detection in healthcare workers in Germany with direct contact to COVID-19 patients.

BACKGROUND: The novel coronavirus SARS-CoV-2 is associated with a severe respiratory manifestation, COVID-19, and presents a challenge for healthcare systems worldwide. Healthcare workers are a vulnerable cohort for SARS-CoV-2 infection due to frequent and close contact to patients with COVID-19. STUDY DESIGN: Serum samples from 316 healthcare workers of the University Hospital Essen, Germany were tested for SARS-CoV-2-IgG antibodies. A questionnaire was used to collect demographic and clinical data. Healthcare workers were grouped depending on the frequency of contact to COVID-19 patients in high-risk-group (n = 244) with daily contact to known or suspected SARS-CoV-2 positive patients, intermediated-risk-group (n = 37) with daily contact to patients without known or suspected SARS-CoV-2 infection at admission and low-risk-group (n = 35) without patient contact. RESULTS: In 5 of 316 (1.6 %) healthcare workers SARS-CoV-2-IgG antibodies could be detected. The seroprevalence was higher in the intermediate-risk-group vs. high-risk-group (2/37 (5.4 %) vs. 3/244 (1.2 %), p = 0.13). Four of the five subject were tested negative for SARS-CoV-2 via PCR. One (20 %) subject was not tested via PCR since he was asymptomatic. CONCLUSION: The overall seroprevalence of SARS-CoV-2 in healthcare workers of a tertiary hospital in Germany is low (1.6 %). The data indicate that the local hygiene standard might be effective.

Energy use efficiency of root growth - a theoretical bioenergetics framework.

Metabolic efficiency of root growth is a crucial physiological parameter, contributing to the amount of photosynthate that plants need to invest into soil exploration. Common measurements of metabolic efficiency usually rely on CO2 respiration measurements with the underlying assumption that all metabolic processes are taking place under aerobic conditions. In this conceptual paper, we introduce energy use efficiency based on the quantification of heat dissipation and energy fluxes as an alternative metric to quantify the metabolic efficiency of root growth. In a theoretical framework, we adopted recently published heat dissipation data from wheat seedlings and show that energy use efficiency decreases in response to (i) soil hypoxia and (ii) increased soil penetration resistance. In contrast to traditional CO2 respiration measurements, heat dissipation measurements account for both aerobic as well as anaerobic respiration in growing roots. Hence, we advocate that the quantification of heat dissipation provides a more complete picture of the metabolic efficiency of root growth than CO2 respiration measurements alone. We therefore propose that energy use efficiency should be included in future studies assessing the metabolic efficiency of root growth.

Cortical Cell Diameter Is Key To Energy Costs of Root Growth in Wheat.

Root growth requires substantial amounts of energy and thus carbohydrates. The energy costs of root growth are particularly high in both dry and compacted soil, due to high soil penetration resistance. Consequently, more carbon must be allocated from aboveground plant tissue to roots, which limits crop productivity. In this study, we tested the utility of root cortical cell diameter as a potential selection target to reduce the energy costs of root growth. Isothermal calorimetry was adopted for in situ quantification of the energy costs of root growth of 16 wheat (Triticum aestivum) genotypes under three levels of penetration resistance. We show that cortical cell diameter is a pivotal and heritable trait, which is strongly related to the energy costs of root growth. Genotypic diversity was found for cortical cell diameter and the energy costs of root growth. A large root cortical cell diameter correlated with reduced energy costs of root growth, particularly under high soil penetration resistance. Moreover, significant correlations were found between the ability to radially enlarge cortical cells upon greater penetration resistance (i.e. phenotypic plasticity) and the responsiveness in the energy costs of root growth. A higher degree of phenotypic plasticity in cortical cell diameter was associated with reduced energy costs of root growth as soil penetration resistance increased. We therefore suggest that genotypic diversity and phenotypic plasticity in cortical cell diameter should be harnessed to adapt crops to dry and compacted soils.

Isothermal microcalorimetry for thermal viable count of microorganisms in pure cultures and stabilized formulations.

BACKGROUND: Quantification of viable microorganisms is an important step in microbiological research as well as in microbial product formulation to develop biological control products or probiotics. Often, the efficiency of the resulting product is dependent on the microbial cell density and their viability, which may decrease over time. Commonly, the number of viable cells is determined by serial dilution and plating techniques or flow cytometry. In 2017, we developed a mathematical model for isothermal microcalorimetry (IMC) data analysis and showed that the new method allows for a more rapid quantification of viable fresh and freeze-dried anaerobic Lactobacillus reuteri cells than traditional viable count methods. RESULTS: This study developed the new method further by applying it to well-known aerophilic plant-beneficial microbial species (Pseudomonas brassicacearum, Bacillus amyloliquefaciens subsp. plantarum and Clonostachys rosea) used in biological control products. We utilized IMC to quantify viable cells in microbial pure cultures as well as when coated onto wheat seeds. The results from this study confirmed that thermal viable count methods are more rapid and sensitive than traditional viable count techniques. Most interestingly, a thermal viable count method was able to quantify microbes coated on seeds despite the presence of the natural microbiota of the seeds. Our results also showed that, in contrast to plating techniques for which clustered cells skew the results, IMC does not require single cells for accurate viable counts. CONCLUSIONS: Thermal viable count methods are novel methods for the rapid quantification of divergent bacterial and fungal species and enhance the speed, sensitivity, and accuracy of routine viable counts of pure cultures and controlled microbiomes such as plant seed coatings.

Does less surgical trauma result in better outcome in management of iatrogenic tracheobronchial laceration?

BACKGROUND: Iatrogenic tracheobronchial injury is a rare, but severe complication of endotracheal intubation. Risk factors are emergency intubation, percutaneous dilatational tracheostomy and intubation with double lumen tube. Regarding these procedures, underlying patients often suffer from severe comorbidities. The aim of this study was to evaluate the results of a standardized treatment algorithm in a referral center with focus on the surgical approach. METHODS: Sixty-four patients with iatrogenic tracheal lesion were treated in our department by standardized management adopted to clinical findings between 2003 and 2019. Patients with superficial laceration were treated conservatively. In the case of transmural injury of the tracheal wall and necessity of mechanical ventilation, patients underwent surgery. We decided on a cervical surgical approach for lesions limited to the trachea. In case of involvement of a main bronchus we performed thoracotomy. Data were evaluated retrospectively. RESULTS: In 19 patients the tracheal lesion occurred in elective intubation and in 17 patients during emergency intubation. In 23 cases a tracheal tear occurred during percutaneous dilatational tracheostomy and in three patients at replacement of a tracheostomy tube. Two patients received laceration during bronchoscopy. Twenty-nine patients underwent surgery with cervical approach and 14 underwent thoracotomy. There was no difference in the mortality of these groups. Treatment of tracheal tear was successful in 62 individuals. Nine patients died of multi organ dysfunction syndrome (MODS), two of them during surgery. CONCLUSIONS: Iatrogenic tracheal laceration is a life-threatening complication and the mortality after tracheal injury is high, even in a specialized thoracic unit. Conservative management in patients with superficial tracheal lesion is a feasible procedure. In case of complete laceration of tracheal wall, surgical therapy is recommendable, whereby several approaches of surgical management seem to be equivalent.

Role of BK polyomavirus (BKV) and Torque teno virus (TTV) in liver transplant recipients with renal impairment.

PURPOSE: Renal impairment is a common complication after liver transplantation (LT). While BK polyomavirus (BKV) has been linked to renal failure in kidney transplant recipients, Torque teno virus (TTV) is a surrogate marker for immunosuppression that does not have a clear association with any human disease. The impact of BKV and TTV on renal impairment after LT is unknown. METHODOLOGY: In this retrospective study, urine and serum samples from 136 liver transplant recipients were screened for BKV and TTV by quantitative PCR. In addition, serum was screened for BKV-specific antibodies and the VP1 typing region was sequenced for BKV genotyping. All parameters were correlated with clinical data.Results/Key findings. BK viruria was detected up to 21 years after transplantation in 16.9 % of cases. BK viraemia was detected in 8.7 % of patients with BK viruria up to 4 years after LT. BKV-specific antibodies were detected in 93.6 % of all LT recipients and correlated with BKV viral load in urine. There was no correlation between renal impairment and the detection of BK DNA in urine (OR 0.983). TTV DNA was detected in 84.6 % of serum samples and in 66.6 % of urine samples. The TTV viral load in serum correlated with the BKV viral load but had no impact on renal impairment. CONCLUSION: Our data indicate that the detection of BKV and TTV is not a risk factor for renal impairment after LT. A correlation of TTV and BKV viral load seems to be an indicator for the immune status of the host.

HBV reactivation in allogeneic stem cell transplant recipients: Risk factors, outcome, and role of hepatitis B virus mutations.

Hepatitis B virus (HBV) reactivation (HBVr) in recipients of allogeneic hematopoetic stem cells (aHSCs) appears heterogeneously with respect to its frequency, manifestation, and outcome. The aim of this study was to present data from a large German cohort of recipients of aHSC transplantation (aHSCT), focusing on the incidence of HBVr in antibody to hepatitis B core antigen (anti-HBc)-positive aHSCT recipients, its clinical outcome, and the role of mutations in HBV. Between 2005 and 2015, 1,871 patients received aHSCT at University Hospital Essen. A follow-up of at least 6 months after transplant was available in 55 patients who were anti-HBc-positive; clinical and virologic data were analyzed. The HBV genome was sequenced with next generation technology from serum samples of 8 patients with HBVr. Thirteen out of 55 (23.6%) patients developed HBVr at a median of 26 months after aHSCT. After initiation of antiviral treatment, complete HBV DNA suppression was achieved in 7/10 (70%) patients 1 to 40 months after HBVr. Nine of 13 patients had increased alanine aminotransferase; 3 patients had compromised coagulation and model for end-stage liver disease scores of 18-27, and 1 of these patients died due to liver failure 5 weeks after HBVr. As a risk factor for HBVr, we identified anti-HBc signal to cut-off ration (S/CO) ≥7.5 before transplantation. Complete HBV DNA suppression was achieved in 7/10 patients; therapy-relevant mutations were found in 1 patient. In 4/8 patients, immune escape mutations were detected either as majority or minority variants. Conclusion: HBVr is common in anti-HBc-positive aHRCT recipients and can lead to severe hepatitis with compromised coagulation. The level of anti-HBc S/CO before transplantation is a risk factor for HBVr. Complete virologic response under adequate antiviral treatment could not be achieved in all patients. (Hepatology Communications 2017;1:1014-1023).

Isothermal microcalorimetry provides new insight into terrestrial carbon cycling.

Energy is continuously transformed in environmental systems through the metabolic activities of living organisms, but little is known about the relationship between the two. In this study, we tested the hypothesis that microbial energetics are controlled by microbial community composition in terrestrial ecosystems. We determined the functional diversity profiles of the soil biota (i.e., multiple substrate-induced respiration and microbial energetics) in soils from an arable ecosystem with contrasting long-term management regimes (54 years). These two functional profiling methods were then related to the soils' microbial community composition. Using isothermal microcalorimetry, we show that direct measures of energetics provide a functional link between energy flows and the composition of below-ground microbial communities at a high taxonomic level (Mantel R = 0.4602, P = 0.006). In contrast, this link was not apparent when carbon dioxide (CO2) was used as an aggregate measure of microbial metabolism (Mantel R = 0.2291, P = 0.11). Our work advocates that the microbial energetics approach provides complementary information to soil respiration for investigating the involvement of microbial communities in below-ground carbon dynamics. Empirical data of our proposed microbial energetics approach can feed into carbon-climate based ecosystem feedback modeling with the suggested conceptual ecological model as a base.

Long-term fertilization of a boreal Norway spruce forest increases the temperature sensitivity of soil organic carbon mineralization.

Boreal ecosystems store one-third of global soil organic carbon (SOC) and are particularly sensitive to climate warming and higher nutrient inputs. Thus, a better description of how forest managements such as nutrient fertilization impact soil carbon (C) and its temperature sensitivity is needed to better predict feedbacks between C cycling and climate. The temperature sensitivity of in situ soil C respiration was investigated in a boreal forest, which has received long-term nutrient fertilization (22 years), and compared with the temperature sensitivity of C mineralization measured in the laboratory. We found that the fertilization treatment increased both the response of soil in situ CO2 effluxes to a warming treatment and the temperature sensitivity of C mineralization measured in the laboratory (Q10). These results suggested that soil C may be more sensitive to an increase in temperature in long-term fertilized in comparison with nutrient poor boreal ecosystems. Furthermore, the fertilization treatment modified the SOC content and the microbial community composition, but we found no direct relationship between either SOC or microbial changes and the temperature sensitivity of C mineralization. However, the relation between the soil C:N ratio and the fungal/bacterial ratio was changed in the combined warmed and fertilized treatment compared with the other treatments, which suggest that strong interaction mechanisms may occur between nutrient input and warming in boreal soils. Further research is needed to unravel into more details in how far soil organic matter and microbial community composition changes are responsible for the change in the temperature sensitivity of soil C under increasing mineral N inputs. Such research would help to take into account the effect of fertilization managements on soil C storage in C cycling numerical models.

Application of nanoscale secondary ion mass spectrometry to plant cell research.

Imaging resource flow in soil-plant systems remains central to understanding plant development and interactions with the environment. Typically, subcellular resolution is required to fully elucidate the compartmentation, behavior, and mode of action of organic compounds and mineral elements within plants. For many situations this has been limited by the poor spatial resolution of imaging techniques and the inability to undertake studies in situ. Here we demonstrate the potential of Nanoscale Secondary Ion Mass Spectrometry (NanoSIMS), which is capable of the quantitative high-resolution spatial imaging of stable isotopes (e.g. (12) C, (13) C, (14) N, (15) N, (16) O, (18) O, (31) P, (34) S) within intact plant-microbial-soil systems. We present examples showing how the approach can be used to investigate competition for (15) N-labeled nitrogen compounds between plant roots and soil microorganisms living in the rhizosphere and the spatial imaging of (31) P in roots. We conclude that NanoSIMS has great potential to elucidate the flow of isotopically-labeled compounds in complex media (e.g. soil) and opens up countless new opportunities for studying plant responses to abiotic stress (e.g. (18) O3, elevated (13) CO2), signal exchange, nutrient flow and plant-microbial interactions.

In situ mapping of nutrient uptake in the rhizosphere using nanoscale secondary ion mass spectrometry.

Plant roots and microorganisms interact and compete for nutrients within the rhizosphere, which is considered one of the most biologically complex systems on Earth. Unraveling the nitrogen (N) cycle is key to understanding and managing nutrient flows in terrestrial ecosystems, yet to date it has proved impossible to analyze and image N transfer in situ within such a complex system at a scale relevant to soil-microbe-plant interactions. Linking the physical heterogeneity of soil to biological processes marks a current frontier in plant and soil sciences. Here we present a new and widely applicable approach that allows imaging of the spatial and temporal dynamics of the stable isotope (15)N assimilated within the rhizosphere. This approach allows visualization and measurement of nutrient resource capture between competing plant cells and microorganisms. For confirmation we show the correlative use of nanoscale secondary ion mass spectrometry, and transmission electron microscopy, to image differential partitioning of (15)NH(4)(+) between plant roots and native soil microbial communities at the submicron scale. It is shown that (15)N compounds can be detected and imaged in situ in individual microorganisms in the soil matrix and intracellularly within the root. Nanoscale secondary ion mass spectrometry has potential to allow the study of assimilatory processes at the submicron level in a wide range of applications involving plants, microorganisms, and animals.