Forest floor vegetation response to nitrogen deposition in Europe.

Chronic nitrogen (N) deposition is a threat to biodiversity that results from the eutrophication of ecosystems. We studied long-term monitoring data from 28 forest sites with a total of 1,335 permanent forest floor vegetation plots from northern Fennoscandia to southern Italy to analyse temporal trends in vascular plant species cover and diversity. We found that the cover of plant species which prefer nutrient-poor soils (oligotrophic species) decreased the more the measured N deposition exceeded the empirical critical load (CL) for eutrophication effects (P = 0.002). Although species preferring nutrient-rich sites (eutrophic species) did not experience a significantly increase in cover (P = 0.440), in comparison to oligotrophic species they had a marginally higher proportion among new occurring species (P = 0.091). The observed gradual replacement of oligotrophic species by eutrophic species as a response to N deposition seems to be a general pattern, as it was consistent on the European scale. Contrary to species cover changes, neither the decrease in species richness nor of homogeneity correlated with nitrogen CL exceedance (ExCLemp N). We assume that the lack of diversity changes resulted from the restricted time period of our observations. Although existing habitat-specific empirical CL still hold some uncertainty, we exemplify that they are useful indicators for the sensitivity of forest floor vegetation to N deposition.

Quantification of the effect of environmental changes on the brownification of Lake Kukkia in southern Finland.

The browning of surface waters due to the increased terrestrial loading of dissolved organic carbon is observed across the northern hemisphere. Brownification is often explained by changes in large-scale anthropogenic pressures (including acidification, and climate and land-use changes). We quantified the effect of environmental changes on the brownification of an important lake for birds, Kukkia in southern Finland. We studied the past trends of organic carbon loading from catchments based on observations taken since the 1990s. We created hindcasting scenarios for deposition, climate and land-use change in order to simulate their quantitative effect on brownification by using process-based models. Changes in forest cuttings were shown to be the primary reason for the brownification. According to the simulations, a decrease in deposition has resulted in a slightly lower leaching of total organic carbon (TOC). In addition, runoff and TOC leaching from terrestrial areas to the lake was smaller than it would have been without the observed increasing trend in temperature by 2 °C in 25 years.

Role of land cover in Finland's greenhouse gas emissions.

We present regionally aggregated emissions of greenhouse gases (GHG) from five land cover categories in Finland: artificial surfaces, arable land, forest, waterbodies, and wetlands. Carbon (C) sequestration to managed forests and unmanaged wetlands was also assessed. Models FRES and ALas were applied for emissions (CO2, CH4, N2O) from artificial surfaces and agriculture, and PREBAS for forest growth and C balance. Empirical emission coefficients were used to estimate emissions from drained forested peatland (CH4, N2O), cropland (CO2), waterbodies (CH4, CO2), peat production sites and undrained mires (CH4, CO2, N2O). We calculated gross emissions of 147.2 ± 6.8 TgCO2eq yr-1 for 18 administrative units covering mainland Finland, using data representative of the period 2017-2025. Emissions from energy production, industrial processes, road traffic and other sources in artificial surfaces amounted to 45.7 ± 2.0 TgCO2eq yr-1. The loss of C in forest harvesting was the largest emission source in the LULUCF sector, in total 59.8 ± 3.3 TgCO2eq yr-1. Emissions from domestic livestock production, field cultivation and organic soils added up to 12.2 ± 3.5 TgCO2eq yr-1 from arable land. Rivers and lakes (13.4 ± 1.9 TgCO2eq yr-1) as well as undrained mires and peat production sites (14.7 ± 1.8 TgCO2eq yr-1) increased the total GHG fluxes. The C sequestration from the atmosphere was 93.2 ± 13.7 TgCO2eq yr-1. with the main sink in forest on mineral soil (79.9 ± 12.2 TgCO2eq yr-1). All sinks compensated 63% of total emissions and thus the net emissions were 53.9 ± 15.3 TgCO2eq yr-1, or a net GHG flux per capita of 9.8 MgCO2eq yr-1.

Modelling the regional potential for reaching carbon neutrality in Finland: Sustainable forestry, energy use and biodiversity protection.

The EU aims at reaching carbon neutrality by 2050 and Finland by 2035. We integrated results of three spatially distributed model systems (FRES, PREBAS, Zonation) to evaluate the potential to reach this goal at both national and regional scale in Finland, by simultaneously considering protection targets of the EU biodiversity (BD) strategy. Modelling of both anthropogenic emissions and forestry measures were carried out, and forested areas important for BD protection were identified based on spatial prioritization. We used scenarios until 2050 based on mitigation measures of the national climate and energy strategy, forestry policies and predicted climate change, and evaluated how implementation of these scenarios would affect greenhouse gas fluxes, carbon storages, and the possibility to reach the carbon neutrality target. Potential new forested areas for BD protection according to the EU 10% protection target provided a significant carbon storage (426-452 TgC) and sequestration potential (- 12 to - 17.5 TgCO2eq a-1) by 2050, indicating complementarity of emission mitigation and conservation measures. The results of the study can be utilized for integrating climate and BD policies, accounting of ecosystem services for climate regulation, and delimitation of areas for conservation.

Sources and sinks of greenhouse gases in the landscape: Approach for spatially explicit estimates.

Climate change mitigation is a global response that requires actions at the local level. Quantifying local sources and sinks of greenhouse gases (GHG) facilitate evaluating mitigation options. We present an approach to collate spatially explicit estimated fluxes of GHGs (carbon dioxide, methane and nitrous oxide) for main land use sectors in the landscape, to aggregate, and to calculate the net emissions of an entire region. Our procedure was developed and tested in a large river basin in Finland, providing information from intensively studied eLTER research sites. To evaluate the full GHG balance, fluxes from natural ecosystems (lakes, rivers, and undrained mires) were included together with fluxes from anthropogenic activities, agriculture and forestry. We quantified the fluxes based on calculations with an anthropogenic emissions model (FRES) and a forest growth and carbon balance model (PREBAS), as well as on emission coefficients from the literature regarding emissions from lakes, rivers, undrained mires, peat extraction sites and cropland. Spatial data sources included CORINE land use data, soil map, lake and river shorelines, national forest inventory data, and statistical data on anthropogenic activities. Emission uncertainties were evaluated with Monte Carlo simulations. Artificial surfaces were the most emission intensive land-cover class. Lakes and rivers were about as emission intensive as arable land. Forests were the dominant land cover in the region (66%), and the C sink of the forests decreased the total emissions of the region by 72%. The region's net emissions amounted to 4.37 ± 1.43 Tg CO2-eq yr-1, corresponding to a net emission intensity 0.16 Gg CO2-eq km-2 yr-1, and estimated per capita net emissions of 5.6 Mg CO2-eq yr-1. Our landscape approach opens opportunities to examine the sensitivities of important GHG fluxes to changes in land use and climate, management actions, and mitigation of anthropogenic emissions.

Assessing critical load exceedances and ecosystem impacts of anthropogenic nitrogen and sulphur deposition at unmanaged forested catchments in Europe.

Anthropogenic emissions of nitrogen (N) and sulphur (S) compounds and their long-range transport have caused widespread negative impacts on different ecosystems. Critical loads (CLs) are deposition thresholds used to describe the sensitivity of ecosystems to atmospheric deposition. The CL methodology has been a key science-based tool for assessing the environmental consequences of air pollution. We computed CLs for eutrophication and acidification using a European long-term dataset of intensively studied forested ecosystem sites (n = 17) in northern and central Europe. The sites belong to the ICP IM and eLTER networks. The link between the site-specific calculations and time-series of CL exceedances and measured site data was evaluated using long-term measurements (1990-2017) for bulk deposition, throughfall and runoff water chemistry. Novel techniques for presenting exceedances of CLs and their temporal development were also developed. Concentrations and fluxes of sulphate, total inorganic nitrogen (TIN) and acidity in deposition substantially decreased at the sites. Decreases in S deposition resulted in statistically significant decreased concentrations and fluxes of sulphate in runoff and decreasing trends of TIN in runoff were more common than increasing trends. The temporal developments of the exceedance of the CLs indicated the more effective reductions of S deposition compared to N at the sites. There was a relation between calculated exceedance of the CLs and measured runoff water concentrations and fluxes, and most sites with higher CL exceedances showed larger decreases in both TIN and H+ concentrations and fluxes. Sites with higher cumulative exceedance of eutrophication CLs (averaged over 3 and 30 years) generally showed higher TIN concentrations in runoff. The results provided evidence on the link between CL exceedances and empirical impacts, increasing confidence in the methodology used for the European-scale CL calculations. The results also confirm that emission abatement actions are having their intended effects on CL exceedances and ecosystem impacts.

Competitive protein adsorption of albumin and immunoglobulin G from human serum onto polymer surfaces.

Competitive protein adsorption from human serum onto unmodified polyethylene terephthalate (PET) surfaces and plasma-polymerized PET surfaces, using the monomer diethylene glycol vinyl ether (DEGVE), has been investigated using radioactive labeling. Albumin and immunoglobulin G (IgG) labeled with two different iodine isotopes have been added to human serum solutions of different concentrations, and adsorption has been performed using adsorption times from approximately 5 s to 24 h. DEGVE surfaces showed indications of being nonfouling regarding albumin and IgG adsorption during competitive protein adsorption from diluted human serum solutions with relatively low protein concentrations, but the nonfouling character was weakened when less diluted human serum solutions with higher protein concentrations were used. The observed adsorption trend is independent of adsorption time, indicating that the protein concentration has a stronger influence on observed adsorption characteristics of the material than the adsorption time has.

Identifying multiple stressors that influence eutrophication in a Finnish agricultural river.

In Finland, a recent ecological classification of surface waters showed that the rivers and coastal waters need attention to improve their ecological state. We combined eco-hydrological and empirical models to study chlorophyll-a concentration as an indicator of eutrophication in a small agricultural river. We used a modified story-and-simulation method to build three storylines for possible changes in future land use due to climate change and political change. The main objective in the first storyline is to stimulate economic activity but also to promote the sustainable and efficient use of resources. The second storyline is based on the high awareness but poor regulation of environmental protection, and the third is to survive as individual countries instead of being part of a unified Europe. We assumed trade of agricultural products to increase to countries outside Europe. We found that chlorophyll-a concentration in the river depended on total phosphorus concentration. In addition, there was a positive synergistic interaction between total phosphorus and water temperature. In future storylines, chlorophyll-a concentration increased due to land use and climate change. Climate change mainly had an indirect influence via increasing nutrient losses from intensified agriculture. We found that well-designed agri-environmental measures had the potential to decrease nutrient loading from fields, as long as the predicted increase in temperature remained under 2 °C. However, we were not able to achieve the nutrient reduction stated in current water protection targets. In addition, the ecological status of the river deteriorated. The influence of temperature on chlorophyll-a growth indicates that novel measures for shading rivers to decrease water temperature may be needed in the future.

Ecosystem Services Related to Carbon Cycling - Modeling Present and Future Impacts in Boreal Forests.

Forests regulate climate, as carbon, water and nutrient fluxes are modified by physiological processes of vegetation and soil. Forests also provide renewable raw material, food, and recreational possibilities. Rapid climate warming projected for the boreal zone may change the provision of these ecosystem services. We demonstrate model based estimates of present and future ecosystem services related to carbon cycling of boreal forests. The services were derived from biophysical variables calculated by two dynamic models. Future changes in the biophysical variables were driven by climate change scenarios obtained as results of a sample of global climate models downscaled for Finland, assuming three future pathways of radiative forcing. We introduce continuous monitoring on phenology to be used in model parametrization through a webcam network with automated image processing features. In our analysis, climate change impacts on key boreal forest ecosystem services are both beneficial and detrimental. Our results indicate an increase in annual forest growth of about 60% and an increase in annual carbon sink of roughly 40% from the reference period (1981-2010) to the end of the century. The vegetation active period was projected to start about 3 weeks earlier and end ten days later by the end of the century compared to currently. We found a risk for increasing drought, and a decrease in the number of soil frost days. Our results show a considerable uncertainty in future provision of boreal forest ecosystem services.

Modelling study of soil C, N and pH response to air pollution and climate change using European LTER site observations.

Current climate warming is expected to continue in coming decades, whereas high N deposition may stabilize, in contrast to the clear decrease in S deposition. These pressures have distinctive regional patterns and their resulting impact on soil conditions is modified by local site characteristics. We have applied the VSD+ soil dynamic model to study impacts of deposition and climate change on soil properties, using MetHyd and GrowUp as pre-processors to provide input to VSD+. The single-layer soil model VSD+ accounts for processes of organic C and N turnover, as well as charge and mass balances of elements, cation exchange and base cation weathering. We calibrated VSD+ at 26 ecosystem study sites throughout Europe using observed conditions, and simulated key soil properties: soil solution pH (pH), soil base saturation (BS) and soil organic carbon and nitrogen ratio (C:N) under projected deposition of N and S, and climate warming until 2100. The sites are forested, located in the Mediterranean, forested alpine, Atlantic, continental and boreal regions. They represent the long-term ecological research (LTER) Europe network, including sites of the ICP Forests and ICP Integrated Monitoring (IM) programmes under the UNECE Convention on Long-range Transboundary Air Pollution (LRTAP), providing high quality long-term data on ecosystem response. Simulated future soil conditions improved under projected decrease in deposition and current climate conditions: higher pH, BS and C:N at 21, 16 and 12 of the sites, respectively. When climate change was included in the scenario analysis, the variability of the results increased. Climate warming resulted in higher simulated pH in most cases, and higher BS and C:N in roughly half of the cases. Especially the increase in C:N was more marked with climate warming. The study illustrates the value of LTER sites for applying models to predict soil responses to multiple environmental changes.

Boreal forests can have a remarkable role in reducing greenhouse gas emissions locally: Land use-related and anthropogenic greenhouse gas emissions and sinks at the municipal level.

Ecosystem services have become an important concept in policy-making. Carbon (C) sequestration into ecosystems is a significant ecosystem service, whereas C losses can be considered as an ecosystem disservice. Municipalities are in a position to make decisions that affect local emissions and therefore are important when considering greenhouse gas (GHG) mitigation. Integrated estimations of fluxes at a regional level help local authorities to develop land use policies for minimising GHG emissions and maximising C sinks. In this study, the Finnish national GHG accounting system is modified and applied at the municipal level by combining emissions and sinks from agricultural land, forest areas, water bodies and mires (land use-related GHG emissions) with emissions from activities such as energy production and traffic (anthropogenic GHG emissions) into the LUONNIKAS calculation tool. The study area consists of 14 municipalities within the Vanajavesi catchment area located in Southern Finland. In these municipalities, croplands, peat extraction sites, water bodies and undrained mires are emission sources, whereas forests are large carbon sinks that turn the land use-related GHG budget negative, resulting in C sequestration into the ecosystem. The annual land use-related sink in the study area was 78tCO2eqkm(-2) and 2.8tCO2eq per capita. Annual anthropogenic GHG emissions from the area amounted to 250tCO2eqkm(-2) and 9.2tCO2eq per capita. Since forests are a significant carbon sink and the efficiency of this sink is heavily affected by forest management practices, forest management policy is a key contributing factor for mitigating municipal GHG emissions.

Nebulin mutations in autosomal recessive nemaline myopathy: an update.

We report mutational analysis of the last 42 exons of the nebulin gene (NEB) in 77 patients with various forms of nemaline myopathy. In addition to the previously described six mutations in five families, we identified 12 novel recessive mutations in 13 families. Affected individuals were homozygous for the mutations in five families and compound heterozygous in two, while in the remaining cases only one heterozygous mutation was identified. The majority of the mutations were frameshifts due to small deletions or insertions; also common were point mutations causing premature stop codons or abnormal splicing, while missense mutations appeared rare. There were no obvious mutational hotspots, although four unrelated patients showed mutations in the differentially expressed exon 177d, and another three showed mutations in exon 184. Most of the mutations are predicted to result in truncated or internally deleted proteins. Mutations in the differentially expressed exons are expected to reduce the nebulin isoform diversity necessary for normal muscle development.

Altered response to A(H1N1)pnd09 vaccination in pregnant women: a single blinded randomized controlled trial.

BACKGROUND: Pregnant women were suspected to be at particular risk when H1N1pnd09 influenza became pandemic in 2009. Our primary objective was to compare the immune responses conferred by MF59®-adjuvanted vaccine (Focetria®) in H1N1pnd09-naïve pregnant and non-pregnant women. The secondary aims were to compare influences of dose and adjuvant on the immune response. METHODS: The study was nested in the Copenhagen Prospective Studies on Asthma in Childhood (COPSAC2010) pregnancy cohort in 2009-2010 and conducted as a single-blinded block-randomised [1∶1∶1] controlled clinical trial in pregnant women after gestational week 20: (1) 7.5 µg H1N1pnd09 antigen with MF59-adjuvant (Pa7.5 µg); (2) 3.75 µg antigen half MF59-adjuvanted (Pa3.75 µg); (3) 15 µg antigen unadjuvanted (P15 µg); and in non-pregnant women receiving (4) 7.5 µg antigen full adjuvanted (NPa7.5 µg). Blood samples were collected at baseline, 3 weeks, 3 and 10 months after vaccination, adverse events were recorded prospectively. RESULTS: 58 pregnant women were allocated to Pa7.5 µg and 149 non-pregnant women were recruited to NPa7.5 µg. The sero-conversion rate was significantly increased in non-pregnant (NPa7.5 µg) compared with pregnant (Pa7.5 µg) women (OR = 2.48 [1.03-5.95], p = 0.04) and geometric mean titers trended towards being higher, but this difference was not statistically significant (ratio 1.27 [0.85-1.93], p = 0.23). The significant titer increase rate showed no difference between pregnant (Pa7.5 µg) and non-pregnant (NPa7.5 µg) groups (OR = 0.49 [0.13-1.85], p = 0.29). CONCLUSION: Our study suggests the immune response to the 7.5 µg MF59-adjuvanted Focetria® H1N1pnd09 vaccine in pregnant women may be diminished compared with non-pregnant women. TRIAL REGISTRATION: ClinicalTrials.gov NCT01012557.

Increased adsorption of histidine-tagged proteins onto tissue culture polystyrene.

In this study we compare histidine-tagged and native proteins with regards to adsorption properties. We observe significantly increased adsorption of proteins with an incorporated polyhistidine amino acid motif (HIS-tag) onto tissue culture polystyrene (TCPS) compared to similar proteins without a HIS-tag. The effect is not observed on polystyrene (PS). Adsorption experiments have been performed at physiological pH (7.4) and the effect was only observed for the investigated proteins that have pI values below or around 7.4. Competitive adsorption experiments with imidazole and ethylenediaminetetraacetic acid (EDTA), as well as adsorption performed at different pH and ionic strength indicates that the high adsorption is caused by electrostatic interaction between negatively charged carboxylate groups on the TCPS surface and positively charged histidine residues in the proteins. Pre-adsorption of bovine serum albumin (BSA) does not decrease the adsorption of HIS-tagged proteins onto TCPS. Our findings identify a potential problem in using HIS-tagged signalling molecule in assays with cells cultured on TCPS, since the concentration of the molecule in solution might be affected and this could critically influence the assay outcome.

Fibrinogen adsorption on blocked surface of albumin.

We have investigated the adsorption of albumin and fibrinogen onto PET (polyethylene terephthalate) and glass surfaces and how pre-adsorption of albumin onto these surfaces can affect the adsorption of later added fibrinogen. For materials and devices being exposed to blood, adsorption of fibrinogen is often a non-wanted event, since fibrinogen is part of the clotting cascade and unspecific adsorption of fibrinogen can have an influence on the activation of platelets. Albumin is often used as blocking agent for avoiding unspecific protein adsorption onto surfaces in devices designed to handle biological samples, including protein solutions. It is based on the assumption that proteins adsorbs as a monolayer on surfaces and that proteins do not adsorb on top of each other. By labelling albumin and fibrinogen with two different radioactive iodine isotopes that emit gamma radiation with different energies, the adsorption of both albumin and fibrinogen has been monitored simultaneously on the same sample. Information about topography and coverage of adsorbed protein layers has been obtained using AFM (Atomic Force Microscopy) analysis in liquid. Our studies show that albumin adsorbs in a multilayer fashion on PET and that fibrinogen adsorbs on top of albumin when albumin is pre-adsorbed on the surfaces.

Competitive protein adsorption--multilayer adsorption and surface induced protein aggregation.

In this study, competitive adsorption of albumin and IgG (immunoglobulin G) from human serum solutions and protein mixtures onto polymer surfaces is studied by means of radioactive labeling. By using two different radiolabels (125I and 131I), albumin and IgG adsorption to polymer surfaces is monitored simultaneously and the influence from the presence of other human serum proteins on albumin and IgG adsorption, as well as their mutual influence during adsorption processes, is investigated. Exploring protein adsorption by combining analysis of competitive adsorption from complex solutions of high concentration with investigation of single protein adsorption and interdependent adsorption between two specific proteins enables us to map protein adsorption sequences during competitive protein adsorption. Our study shows that proteins can adsorb in a multilayer fashion onto the polymer surfaces and that the outcome of IgG adsorption is much more sensitive to surface characteristics than the outcome of albumin adsorption. Using high concentrations of protein solution and hydrophobic polymer surfaces during adsorption can induce IgG aggregation, which is observed as extremely high IgG adsorptions. Besides using a more hydrophilic substrate, surface-induced IgG aggregation can be inhibited by changing the adsorption sequence of albumin and IgG.

Enhancing the formation and shear resistance of nitrifying biofilms on membranes by surface modification.

Polypropylene (PP) membranes and polyethylene (PE) surfaces were modified to enhance formation and shear resistance of nitrifying biofilms for wastewater treatment applications. A combination of plasma polymerization and wet chemistry was employed to ultimately introduce poly(ethyleneglycol) (PEG) chains with two different functional groups (-PEG-NH(2) and -PEG-CH(3)). Biofilm growth experiments using a mixed nitrifying bacterial culture revealed that the specific combination of PEG chains with amino groups resulted in most biofilm formation on both PP and PE samples. Detachment experiments showed similar trends: biofilms on -PEG-NH(2) modified surfaces were much stronger compared to the other modifications and the unmodified reference surfaces. Electrostatic interactions between the protonated amino group and negatively charged bacteria as well as PEG chain density which can affect the surface structure might be possible explanations of the superiority of the -PEG-NH(2) modification. The success of the-PEG-NH(2) modification was independent of the original surface and might, therefore, be used in wastewater treatment bioreactors to improve reactor performance by making biofilm formation more stable and predictable.

Competitive protein adsorption to polymer surfaces from human serum.

Surface modification by "soft" plasma polymerisation to obtain a hydrophilic and non-fouling polymer surface has been validated using radioactive labelling. Adsorption to unmodified and modified polymer surfaces, from both single protein and human serum solutions, has been investigated. By using different radioisotopes, albumin and Immunoglobulin G (IgG) adsorption has been monitored simultaneously during competitive adsorption processes, which to our knowledge has not been reported in the literature before. Results show that albumin and IgG adsorption is dependent on adsorption time and on the presence and concentration of other proteins in bulk solutions during adsorption. Generally, lower albumin and IgG adsorption was observed on the modified and more hydrophilic polymer surfaces, but otherwise the modified and unmodified polymer surfaces showed the same adsorption characteristics.

Protein aggregation and degradation during iodine labeling and its consequences for protein adsorption to biomaterials.

Protein adsorption on modified and unmodified polymer surfaces investigated through radiolabeling experiments showed a tendency for higher than expected albumin and immunoglobulin G (IgG) adsorption. Possible enhanced protein aggregation and degradation caused by the iodine labeling method used were analyzed through chromatography and spectroscopy techniques. Results show that the iodine labeling method using chloramine-T (CAT) as an oxidizing agent can cause both enhanced aggregation and fragmentation of proteins. Albumin shows an enhanced tendency to aggregate after iodine labeling using the CAT method, and higher amounts of fragmentation are observed for CAT-labeled IgG molecules relative to unlabeled IgG molecules as well as to IgG molecules labeled using the Iodo-Gen method. These results show that the widely applied method of radioisotope labeling for quantitative assessment of protein adsorption should be used with caution and preferably should be validated by a label-free methodology for each combination of radiolabel and protein. The results obtained in this study can be used to optimize investigation of protein adsorption on surfaces of materials for biomedical devices.

Optimal control of gas exchange.

A major difficulty in evaluating the optimization theory of leaf gas exchange under conditions of water deficit has been that of obtaining suitable experimental data. Mathematical solutions to three formulations of optimal stomatal control are presented which can be tested experimentally. First, it is assumed that the movement of stomata and changes in environmental factors are slow compared to changes in the internal CO(2) concentration. The optimization problem is solved under this assumption, and the procedures for testing the solution experimentally are described. Second, instantaneous stomatal response is postulated and the solution suggests that very rapid oscillations provide optimal CO(2) uptake. Third, variable stomatal dynamics are postulated and the mathematical solution shown to be similar to that of the second case. The second and third cases can also be tested empirically.