Bacteria face trade-offs in the decomposition of complex biopolymers.

Although depolymerization of complex carbohydrates is a growth-limiting bottleneck for microbial decomposers, we still lack understanding about how the production of different types of extracellular enzymes affect individual microbes and in turn the performance of whole decomposer communities. In this work we use a theoretical model to evaluate the potential trade-offs faced by microorganisms in biopolymer decomposition which arise due to the varied biochemistry of different depolymerizing enzyme classes. We specifically consider two broad classes of depolymerizing extracellular enzymes, which are widespread across microbial taxa: exo-enzymes that cleave small units from the ends of polymer chains and endo-enzymes that act at random positions generating degradation products of varied sizes. Our results demonstrate a fundamental trade-off in the production of these enzymes, which is independent of system's complexity and which appears solely from the intrinsically different temporal depolymerization dynamics. As a consequence, specialists that produce either exo- or only endo-enzymes limit their growth to high or low substrate conditions, respectively. Conversely, generalists that produce both enzymes in an optimal ratio expand their niche and benefit from the synergy between the two enzymes. Finally, our results show that, in spatially-explicit environments, consortia composed of endo- and exo-specialists can only exist under oligotrophic conditions. In summary, our analysis demonstrates that the (evolutionary or ecological) selection of a depolymerization pathway will affect microbial fitness under low- or high substrate conditions, with impacts on the ecological dynamics of microbial communities. It provides a possible explanation why many polysaccharide degraders in nature show the genetic potential to produce both of these enzyme classes.

Distinct microbial communities are linked to organic matter properties in millimetre-sized soil aggregates.

Soils provide essential ecosystem services and represent the most diverse habitat on Earth. It has been suggested that the presence of various physico-chemically heterogeneous microhabitats supports the enormous diversity of microbial communities in soil. However, little is known about the relationship between microbial communities and their immediate environment at the micro- to millimetre scale. In this study, we examined whether bacteria, archaea, and fungi organize into distinct communities in individual 2-mm-sized soil aggregates and compared them to communities of homogenized bulk soil samples. Furthermore, we investigated their relationship to their local environment by concomitantly determining microbial community structure and physico-chemical properties from the same individual aggregates. Aggregate communities displayed exceptionally high beta-diversity, with 3-4 aggregates collectively capturing more diversity than their homogenized parent soil core. Up to 20%-30% of ASVs (particularly rare ones) were unique to individual aggregates selected within a few centimetres. Aggregates and bulk soil samples showed partly different dominant phyla, indicating that taxa that are potentially driving biogeochemical processes at the small scale may not be recognized when analysing larger soil volumes. Microbial community composition and richness of individual aggregates were closely related to aggregate-specific carbon and nitrogen content, carbon stable-isotope composition, and soil moisture, indicating that aggregates provide a stable environment for sufficient time to allow co-development of communities and their environment. We conclude that the soil microbiome is a metacommunity of variable subcommunities. Our study highlights the necessity to study small, spatially coherent soil samples to better understand controls of community structure and community-mediated processes in soils.

Acute and Long-Term Toxicity after Planned Intraoperative Boost and Whole Breast Irradiation in High-Risk Patients with Breast Cancer-Results from the Targeted Intraoperative Radiotherapy Boost Quality Registry (TARGIT BQR).

In the context of breast cancer treatment optimization, this study prospectively examines the feasibility and outcomes of utilizing intraoperative radiotherapy (IORT) as a boost in combination with standard external beam radiotherapy (EBRT) for high-risk patients. Different guidelines recommend such a tumor bed boost in addition to whole breast irradiation with EBRT for patients with risk factors for local breast cancer recurrence. The TARGIT BQR (NCT01440010) is a prospective, multicenter registry study aimed at ensuring the quality of clinical outcomes. It provides, for the first time, data from a large cohort with a detailed assessment of acute and long-term toxicity following an IORT boost using low-energy X-rays. Inclusion criteria encompassed tumors up to 3.5 cm in size and preoperative indications for a boost. The IORT boost, administered immediately after tumor resection, delivered a single dose of 20 Gy. EBRT and systemic therapy adhered to local tumor board recommendations. Follow-up for toxicity assessment (LENT SOMA criteria: fibrosis, teleangiectasia, retraction, pain, breast edema, lymphedema, hyperpigmentation, ulceration) took place before surgery, 6 weeks to 90 days after EBRT, 6 months after IORT, and then annually using standardized case report forms (CRFs). Between 2011 and 2020, 1133 patients from 10 centers were preoperatively enrolled. The planned IORT boost was conducted in 90%, and EBRT in 97% of cases. Median follow-up was 32 months (range 1-120, 20.4% dropped out), with a median age of 61 years (range 30-90). No acute grade 3 or 4 toxicities were observed. Acute side effects included erythema grade 1 or 2 in 4.4%, palpable seroma in 9.1%, punctured seroma in 0.3%, and wound healing disorders in 2.1%. Overall, chronic teleangiectasia of any grade occurred in 16.2%, fibrosis grade ≥ 2 in 14.3%, pain grade ≥ 2 in 3.4%, and hyperpigmentation in 1.1%. In conclusion, a tumor bed boost through IORT using low-energy X-rays is a swift and feasible method that demonstrates low rates in terms of acute or long-term toxicity profiles in combination with whole breast irradiation.

Tuning Charge-Transfer States by Interface Electric Fields.

Intermolecular charge-transfer (CT) states are extended excitons with a charge separation on the nanometer scale. Through absorption and emission processes, they couple to the ground state. This property is employed both in light-emitting and light-absorbing devices. Their conception often relies on donor-acceptor (D-A) interfaces, so-called type-II heterojunctions, which usually generate significant electric fields. Several recent studies claim that these fields alter the energetic configuration of the CT states at the interface, an idea holding prospects like multicolor emission from a single emissive interface or shifting the absorption characteristics of a photodetector. Here, we test this hypothesis and contribute to the discussion by presenting a new model system. Through the fabrication of planar organic p-(i-)n junctions, we generate an ensemble of oriented CT states that allows the systematic assessment of electric field impacts. By increasing the thickness of the intrinsic layer at the D-A interface from 0 to 20 nm and by applying external voltages up to 6 V, we realize two different scenarios that controllably tune the intrinsic and extrinsic electric interface fields. By this, we obtain significant shifts of the CT-state peak emission of about 0.5 eV (170 nm from red to green color) from the same D-A material combination. This effect can be explained in a classical electrostatic picture, as the interface electric field alters the potential energy of the electric CT-state dipole. This study illustrates that CT-state energies can be tuned significantly if their electric dipoles are aligned to the interface electric field.

The NLRP3 inflammasome is essential for IL-18 production in a murine model of macrophage activation syndrome.

Hyperinflammatory disease is associated with an aberrant immune response resulting in cytokine storm. One such instance of hyperinflammatory disease is known as macrophage activation syndrome (MAS). The pathology of MAS can be characterised by significantly elevated serum levels of interleukin-18 (IL-18) and interferon gamma (IFNγ). Given the role for IL-18 in MAS, we sought to establish the role of inflammasomes in the disease process. Using a murine model of CpG-oligonucleotide-induced MAS, we discovered that the expression of the NLRP3 inflammasome was increased and correlated with IL-18 production. Inhibition of the NLRP3 inflammasome or the downstream caspase-1 prevented MAS-mediated upregulation of IL-18 in the plasma but, interestingly, did not alleviate key features of hyperinflammatory disease including hyperferritinaemia and splenomegaly. Furthermore blockade of IL-1 receptor with its antagonist IL-1Ra did not prevent the development of CpG-induced MAS, despite being clinically effective in the treatment of MAS. These data demonstrate that, during the development of MAS, the NLRP3 inflammasome was essential for the elevation in plasma IL-18 - a key cytokine in clinical cases of MAS - but was not a driving factor in the pathogenesis of CpG-induced MAS.

The NLRP3 inflammasome is essential for IL-18 production in a murine model of macrophage activation syndrome.

Hyperinflammatory disease is associated with an aberrant immune response resulting in cytokine storm. One such instance of hyperinflammatory disease is known as macrophage activation syndrome (MAS). The pathology of MAS can be characterised by significantly elevated serum levels of interleukin (IL)-18 and interferon (IFN)-γ. Given the role for IL-18 in MAS, we sought to establish the role of inflammasomes in the disease process. Using a murine model of CpG-DNA induced MAS, we discovered that the expression of the NLRP3 inflammasome was increased and correlated with IL-18 production. Inhibition of the NLRP3 inflammasome, or downstream caspase-1, prevented MAS-mediated upregulation of plasma IL-18 but interestingly did not alleviate key features of hyperinflammatory disease including hyperferritinaemia and splenomegaly. Furthermore IL-1 receptor blockade with IL-1Ra did not prevent the development of CpG-induced MAS, despite being clinically effective in the treatment of MAS. These data demonstrate that in the development of MAS, the NLRP3 inflammasome was essential for the elevation in plasma IL-18, a key cytokine in clinical cases of MAS, but was not a driving factor in the pathogenesis of CpG-induced MAS.

Soil warming increases the number of growing bacterial taxa but not their growth rates.

Soil microorganisms control the fate of soil organic carbon. Warming may accelerate their activities putting large carbon stocks at risk of decomposition. Existing knowledge about microbial responses to warming is based on community-level measurements, leaving the underlying mechanisms unexplored and hindering predictions. In a long-term soil warming experiment in a Subarctic grassland, we investigated how active populations of bacteria and archaea responded to elevated soil temperatures (+6°C) and the influence of plant roots, by measuring taxon-specific growth rates using quantitative stable isotope probing and 18O water vapor equilibration. Contrary to prior assumptions, increased community growth was associated with a greater number of active bacterial taxa rather than generally faster-growing populations. We also found that root presence enhanced bacterial growth at ambient temperatures but not at elevated temperatures, indicating a shift in plant-microbe interactions. Our results, thus, reveal a mechanism of how soil bacteria respond to warming that cannot be inferred from community-level measurements.

Surgical Management and System Therapy of the Most Giant Known Malignant Metastatic Breast Phyllodes Tumor: A Case Report and Review of the Literature.

INTRODUCTION: Phyllodes tumors belong to uncommon fibroepithelial breast tumors with a range of biological behaviors. Phyllodes tumors are responsible for less than 1 percent of all neoplasms of the breast. CASE PRESENTATION: A 66-year-old woman presented to our Breastcancer Unit in March 2021 because of a huge mass of her left breast with bleeding out of a tumor necrosis. Five years ago in 2016, a benign phyllodes tumor was diagnosed externally. When we started the treatment, the tumor had a weight of 18.6 kg. CONCLUSION: We describe the surgical management and the systemic treatment of metastatic disease.

Identification of inulin-responsive bacteria in the gut microbiota via multi-modal activity-based sorting.

Prebiotics are defined as non-digestible dietary components that promote the growth of beneficial gut microorganisms. In many cases, however, this capability is not systematically evaluated. Here, we develop a methodology for determining prebiotic-responsive bacteria using the popular dietary supplement inulin. We first identify microbes with a capacity to bind inulin using mesoporous silica nanoparticles functionalized with inulin. 16S rRNA gene amplicon sequencing of sorted cells revealed that the ability to bind inulin was widespread in the microbiota. We further evaluate which taxa are metabolically stimulated by inulin and find that diverse taxa from the phyla Firmicutes and Actinobacteria respond to inulin, and several isolates of these taxa can degrade inulin. Incubation with another prebiotic, xylooligosaccharides (XOS), in contrast, shows a more robust bifidogenic effect. Interestingly, the Coriobacteriia Eggerthella lenta and Gordonibacter urolithinfaciens are indirectly stimulated by the inulin degradation process, expanding our knowledge of inulin-responsive bacteria.

Do Barrier Films Impact Long-Term Skin Toxicity following Whole-Breast Irradiation? Objective Follow-Up of Two Randomised Trials.

Purpose: Hydrofilm, a polyurethane-based barrier film, can be used to prevent acute radiation dermatitis (RD) in adjuvant whole-breast irradiation (WBI) for breast cancer. This cost-effective prophylactic measure is currently being recommended to a growing number of patients, yet long-term safety data and its impact on late radiation-induced skin toxicity such as pigmentation changes and fibrosis have not been investigated. Methods: We objectively evaluated patients who were previously enrolled in either of two intrapatient-randomised (lateral versus medial breast halve) controlled trials on the use of Hydrofilm for RD prevention (DRKS00029665; registered on 19 July 2022). Results: Sixty-two patients (47.7% of the initial combined sample size) provided consent for this post-hoc examination, with a median follow-up time (range) of 58 (37-73) months. Following WBI, there was a significant increase in yellow skin tones of the entire breast when compared to baseline measurements before WBI (p < 0.001) and a significant increase of cutis, subcutis, and oedema thickness (p < 0.001, p < 0.001, and p = 0.004, respectively). At follow-up, there were no significant differences in either pigmentation changes or skin fibrosis between the Hydrofilm and standard of care breast halves. Conclusion: These data suggest that Hydrofilm can be safely used in the context of acute RD prevention, without affecting late side effects, supporting its widespread use.

Microbial growth under drought is confined to distinct taxa and modified by potential future climate conditions.

Climate change increases the frequency and intensity of drought events, affecting soil functions including carbon sequestration and nutrient cycling, which are driven by growing microorganisms. Yet we know little about microbial responses to drought due to methodological limitations. Here, we estimate microbial growth rates in montane grassland soils exposed to ambient conditions, drought, and potential future climate conditions (i.e., soils exposed to 6 years of elevated temperatures and elevated CO2 levels). For this purpose, we combined 18O-water vapor equilibration with quantitative stable isotope probing (termed 'vapor-qSIP') to measure taxon-specific microbial growth in dry soils. In our experiments, drought caused >90% of bacterial and archaeal taxa to stop dividing and reduced the growth rates of persisting ones. Under drought, growing taxa accounted for only 4% of the total community as compared to 35% in the controls. Drought-tolerant communities were dominated by specialized members of the Actinobacteriota, particularly the genus Streptomyces. Six years of pre-exposure to future climate conditions (3 °C warming and + 300 ppm atmospheric CO2) alleviated drought effects on microbial growth, through more drought-tolerant taxa across major phyla, accounting for 9% of the total community. Our results provide insights into the response of active microbes to drought today and in a future climate, and highlight the importance of studying drought in combination with future climate conditions to capture interactive effects and improve predictions of future soil-climate feedbacks.

Barrier films for the prevention of acute radiation dermatitis in breast cancer: A systematic review and meta-analysis of randomised controlled trials.

PURPOSE: Radiation dermatitis (RD) is the most common side effect of adjuvant whole-breast or chest wall irradiation, majorly impacting quality of life in numerous patients. The use of barrier films (polyurethane dressings such as Hydrofilm® and Mepitel® film remaining on the skin for the duration of the radiation treatment) has been investigated as a prophylactic measure in several prospective trials. Here, we critically appraise the available evidence behind preventive barrier film application in the context of breast cancer treatment. METHODS: International literature was reviewed and high-quality randomised controlled trials (RCTs) were included in this meta-analysis. RESULTS: The results of 5 RCTs (663 patients; >90% Caucasian) were analysed. Overall, barrier films lead to improved clinician- and patient-reported outcomes: fewer grade ≥2 RD (11% vs. 42%; OR = 0.16; p < 0.001) and moist desquamation (2% vs. 16%; OR = 0.12; p = 0.006), as well as less patient-reported pain (standardised mean difference [SMD] -0.51; p < 0.001), itching (SMD -0.52; p = 0.001), burning (SMD -0.41; p = 0.011), and limitations in daily activities (SMD -0.20; p = 0.007). Furthermore, barrier films have a high acceptance rate among patients, as well as a favourable cost-benefit ratio. Possible side effects due to its application are mild and mostly self-limiting. Overall, there was a lack of information on the radiation treatment techniques used. CONCLUSION: The evidence presented in this meta-analysis suggests that barrier films are an excellent tool in the prevention of RD among Caucasian patients receiving whole-breast or chest wall irradiation. Its use should therefore be considered routinely in these patients.

Objective, Clinician- and Patient-Reported Evaluation of Late Toxicity Following Adjuvant Radiation for Early Breast Cancer: Long-Term Follow-Up Results of a Randomised Series.

Background and Purpose: This study aimed to differentially assess the frequency and severity of late radiation-induced toxicity following adjuvant whole-breast irradiation for early breast cancer with conventional fractionation (CF) and moderate hypofractionation (mHF). Materials and Methods: Patients recruited in a previous randomised controlled trial comparing acute toxicity between CF and mHF without disease recurrence were included in a post hoc analysis. Spectrophotometric and ultrasonographic examinations were performed for an objective evaluation and subsequent comparison of long-term skin toxicity. Furthermore, patient- and clinician-reported outcomes were recorded. Results: Sixty-four patients with a median age of 58 (37-81) years were included. The median follow-up was 57 (37-73) months. A total of 55% underwent CF and 45% mHF. A total of 52% received a sequential boost to the tumour bed. A significant decrease in mean L* (p = 0.011) and an increase in a* (p = 0.040) and b* values (p < 0.001) were observed, indicating hyperpigmentation. In comparison with the non-irradiated breast, there was a significant increase in both cutis (+14%; p < 0.001) and subcutis (+17%; p = 0.011) thickness, significantly more pronounced in CF patients (p = 0.049). In CF patients only, a sequential boost significantly increased the local cutis thickness and oedema compared to non-boost regions in the same breast (p = 0.001 and p < 0.001, respectively). Conclusions: mHF objectively resulted in reduced long-term skin toxicity compared to CF. A sequential boost increased the local fibrosis rate in CF, but not in mHF. This might explain the subjectively reported better cosmetic outcomes in patients receiving mHF and reinforces the rationale for favouring mHF as the standard of care.

Decision regret in breast cancer patients after adjuvant radiotherapy.

PURPOSE: Breast cancer patients often engage in shared decision-making to select an individualized treatment regimen from multiple options. However, dissatisfaction with treatment outcomes can lead to decision regret. We evaluated decision regret and physical and psychological well-being among breast cancer patients who underwent adjuvant radiotherapy and explored their associations with patient, tumor, treatment, and symptom characteristics. METHODS: This cross-sectional study involved retrospectively obtaining clinical data and data collected through interviews carried out as part of regular long-term medical aftercare. Decision regret regarding the radiotherapy was assessed using the Ottawa Decision Regret Scale, physical and psychological well-being were assessed using the PROMIS Global Health-10 questionnaire, and patients were asked about their treatment outcomes and symptoms. The questionnaire was administered 14 months to 4 years after completion of radiotherapy. RESULTS: Of the 172 included breast cancer patients, only 13.9% expressed high decision regret, with most patients expressing little or no decision regret. More decision regret was associated with volumetric modulated arc therapy, chest wall irradiation, use of docetaxel as a chemotherapy agent, lymphangiosis carcinomatosa, new heart disease after radiotherapy, and lower psychological well-being. CONCLUSION: Although most patients reported little or no decision regret, we identified several patient, treatment, and symptom characteristics associated with more decision regret. Our findings suggest that psychological well-being influences patients' satisfaction with therapy decisions, implying that practitioners should pay special attention to maintaining psychological well-being during shared decision-making and ensuring that psychological assessment and treatment is provided after cancer therapy to deal with long-term effects of radiotherapy.

Impact of patient nationality on the severity of early side effects after radiotherapy.

BACKGROUND: Major demographical changes in Germany commenced in the 1960s. Ongoing humanitarian crises in the Ukraine with subsequent immigration will have also long-range effects on national provision of cancer treatment. Ensuring the best possible outcomes for each cancer patient undergoing radiotherapy requires the prediction and prevention of unfavorable side effects. Given that recent research has primarily focused on clinical outcome indicators solely, less is known regarding sociodemographic predictors of therapeutic outcomes, such as patient nationality. Here, we investigated whether the severity of early side effects after radiotherapy are associated with patient nationality and other sociodemographic and clinical characteristics. METHODS: Out of 9187 patients treated at a German university medical center between 2017 and 2021, 178 German and 178 non-German patients were selected for matched-pair analysis based on diagnostic and demographic criteria. For all 356 patients, data on side effects from follow-up care after radiotherapy were collected. RESULTS: Non-German patients were more likely to have severe side effects than German patients. Side effect severity was also associated with tumor entity, concomitant therapy, body mass index, and age. CONCLUSION: Foreign cancer patients are at higher risk of experiencing severe side effects of radiotherapy, suggesting a need to develop and implement targeted preventive measures for these patients. Further research investigating factors predicting the occurrence of radiotherapy side effects, including other sociodemographic characteristics, is needed to better personalize therapy regimens for cancer.

Both abundant and rare fungi colonizing Fagus sylvatica ectomycorrhizal root-tips shape associated bacterial communities.

Ectomycorrhizal fungi live in close association with their host plants and form complex interactions with bacterial/archaeal communities in soil. We investigated whether abundant or rare ectomycorrhizal fungi on root-tips of young beech trees (Fagus sylvatica) shape bacterial/archaeal communities. We sequenced 16S rRNA genes and fungal internal transcribed spacer regions of individual root-tips and used ecological networks to detect the tendency of certain assemblies of fungal and bacterial/archaeal taxa to inhabit the same root-tip (i.e. modularity). Individual ectomycorrhizal root-tips hosted distinct fungal communities associated with unique bacterial/archaeal communities. The structure of the fungal-bacterial/archaeal association was determined by both, dominant and rare fungi. Integrating our data in a conceptual framework suggests that the effect of rare fungi on the bacterial/archaeal communities of ectomycorrhizal root-tips contributes to assemblages of bacteria/archaea on root-tips. This highlights the potential impact of complex fine-scale interactions between root-tip associated fungi and other soil microorganisms for the ectomycorrhizal symbiosis.

Resemblance of the Recurrence Patterns in Primary Systemic, Primary Surgery and Secondary Oncoplastic Surgery.

PURPOSE: Surgical interventions tend to have an effect on the generation of recurrences in tumor patients due to the anesthesia involved as well as tissue damage and subsequent inflammation. This can also be found in patients with breast cancer. METHODS: In this multicenter study, we investigated data of 632 patients with breast cancer and the subsequent diagnosis of a recurrence. The patient data were acquired from 1 January 2006 to 31 December 2019 in eight different centers in Germany. The data sets were separated into those with primary surgery, primary systemic therapy with subsequent surgery, and reconstructive surgery. Three different starting points for observation were defined: the date of diagnosis, the date of first surgery, and the date of reconstructive surgery, if applicable. The observational period was divided into steps of six months and maxima of recurrences were compared. Furthermore, the variance was calculated using the difference of the distribution in percent. RESULTS: The descriptive analysis showed no resemblance between the groups. The variance of the difference of the recurrence rates analysis using the surgical date as the starting point showed similarities in the age subgroup. CONCLUSION: Our clinical analysis shows different metastatic behavior in different analysis and treatment regimes. These findings justify further investigations on a larger database. These results may possibly identify an improved follow-up setting depending on tumor stage, biology, treatment, and patient factors (i.e., age, …).

Non-Invasive Physical Plasma for Preventing Radiation Dermatitis in Breast Cancer: A First-In-Human Feasibility Study.

Radiation dermatitis (RD) is the most common acute side effect of breast irradiation. More than a century following the therapeutic utilisation of X-rays, potent preventative and therapeutic options are still lacking. Non-invasive physical plasma (NIPP) is an emerging approach towards treatment of various dermatological disorders. In this study, we sought to determine the safety and feasibility of a NIPP device on RD. Thirty patients undergoing hypofractionated whole-breast irradiation were included. Parallel to radiation treatment, the irradiated breast was treated with NIPP with different application regimens. RD was assessed during and after NIPP/radiation, using clinician- and patient-reported outcomes. Additionally, safety and feasibility features were recorded. None of the patients was prescribed topical corticosteroids and none considered the treatment to be unpleasant. RD was less frequent and milder in comparison with standard skin care. Neither NIPP-related adverse events nor side effects were reported. This proven safety and feasibility profile of a topical NIPP device in the prevention and treatment of RD will be used as the framework for a larger intrapatient-randomised double-blind placebo-controlled trial, using objective and patient-reported outcome measures as an endpoint.

From diversity to complexity: Microbial networks in soils.

Network analysis has been used for many years in ecological research to analyze organismal associations, for example in food webs, plant-plant or plant-animal interactions. Although network analysis is widely applied in microbial ecology, only recently has it entered the realms of soil microbial ecology, shown by a rapid rise in studies applying co-occurrence analysis to soil microbial communities. While this application offers great potential for deeper insights into the ecological structure of soil microbial ecosystems, it also brings new challenges related to the specific characteristics of soil datasets and the type of ecological questions that can be addressed. In this Perspectives Paper we assess the challenges of applying network analysis to soil microbial ecology due to the small-scale heterogeneity of the soil environment and the nature of soil microbial datasets. We review the different approaches of network construction that are commonly applied to soil microbial datasets and discuss their features and limitations. Using a test dataset of microbial communities from two depths of a forest soil, we demonstrate how different experimental designs and network constructing algorithms affect the structure of the resulting networks, and how this in turn may influence ecological conclusions. We will also reveal how assumptions of the construction method, methods of preparing the dataset, and definitions of thresholds affect the network structure. Finally, we discuss the particular questions in soil microbial ecology that can be approached by analyzing and interpreting specific network properties. Targeting these network properties in a meaningful way will allow applying this technique not in merely descriptive, but in hypothesis-driven research. Analysing microbial networks in soils opens a window to a better understanding of the complexity of microbial communities. However, this approach is unfortunately often used to draw conclusions which are far beyond the scientific evidence it can provide, which has damaged its reputation for soil microbial analysis. In this Perspectives Paper, we would like to sharpen the view for the real potential of microbial co-occurrence analysis in soils, and at the same time raise awareness regarding its limitations and the many ways how it can be misused or misinterpreted.

Contrasting drivers of belowground nitrogen cycling in a montane grassland exposed to a multifactorial global change experiment with elevated CO2 , warming, and drought.

Depolymerization of high-molecular weight organic nitrogen (N) represents the major bottleneck of soil N cycling and yet is poorly understood compared to the subsequent inorganic N processes. Given the importance of organic N cycling and the rise of global change, we investigated the responses of soil protein depolymerization and microbial amino acid consumption to increased temperature, elevated atmospheric CO2 , and drought. The study was conducted in a global change facility in a managed montane grassland in Austria, where elevated CO2 (eCO2 ) and elevated temperature (eT) were stimulated for 4 years, and were combined with a drought event. Gross protein depolymerization and microbial amino acid consumption rates (alongside with gross organic N mineralization and nitrification) were measured using 15 N isotope pool dilution techniques. Whereas eCO2  showed no individual effect, eT had distinct effects which were modulated by season, with a negative effect of eT on soil organic N process rates in spring, neutral effects in summer, and positive effects in fall. We attribute this to a combination of changes in substrate availability and seasonal temperature changes. Drought led to a doubling of organic N process rates, which returned to rates found under ambient conditions within 3 months after rewetting. Notably, we observed a shift in the control of soil protein depolymerization, from plant substrate controls under continuous environmental change drivers (eT and eCO2 ) to controls via microbial turnover and soil organic N availability under the pulse disturbance (drought). To the best of our knowledge, this is the first study which analyzed the individual versus combined effects of multiple global change factors and of seasonality on soil organic N processes and thereby strongly contributes to our understanding of terrestrial N cycling in a future world.