Effects of a hydrological protection zone on the restoration of a raised bog: a case study from Northeast-Germany 1997–2008

We investigated the changes of water level and vegetation in a restored cut-over raised bog in response to a hydrological protection zone established around the bog. The restoration began 1997 and techniques involved ditch blocking within and around the bog to stimulate a return to conditions of intact bog ecosystems. In order to monitor the rehabilitation of the raised bog, water levels and vegetation have been recorded since before restoration measures began. The monitoring is ongoing, but an assessment of 15 year’s data (1994–2008) is presented. A hydrological protection zone with continuous high water levels could be established around the raised bog which minimizes the runoff of precipitation. Shortly after the first measures, the water levels increased significantly at all dipwells. Parallel to the increasing water levels a vascular plant species assemblage and a diverse Sphagnum community developed. In particular Sphagnum fimbriatum, S. palustre, S. recurvum and S. squarrosum spread efficiently. The cover of trees decreased significantly because of high water levels and ongoing acidification by Sphagnum spp. The high water levels have stimulated the re-vegetation and the hydrology self-regulation of the acrotelm. The successful regeneration of the acrotelm particularly became apparent in years with below-average precipitations (e.g. 2008), when the water levels in the central parts of the raised bog did not fall back to the low level reached in previous years, which had also remarkably water deficits (e.g. 2003).     

Potential hotspots identified by social LCA–Part 2: Reflections on a study of a complex product

Purpose

We present experiences and reflections from social life cycle assessment (S-LCA) case study, the aim of which was to identify social hotspots, test and evaluate the methodology and propose improvements. This paper discusses the usability and applicability of the methodology used based on our experiences from the study. The main issues considered are whether the gathering of data and other information is feasible and straightforward to perform, whether the method provides added value and relevant results and how these can be presented.

Method

We have conducted a generic hotspot assessment on a laptop computer according to the Guidelines for Social Life Cycle Assessment of Products (Benoît and Mazijn 2009). The experiences presented were gathered throughout the case study. The supply chain of the laptop was simplified, and we focused on a limited number of materials. The impacts were assessed in relation to the area of protection on human well-being and to affected stakeholders. Social impacts from the actual use of the product were not included. Methodological sheets were used for guidance on inventory indicators and data sources for data collection. Country-specific data were collected and entered into a spreadsheet. The process has been guided by regular meetings in a reference group, composed of representatives of all stakeholder groups.

Results and discussion

The data collection process was impaired by a lack of data and low data quality. In order to relate the data collected to the product assessed, each country's share of the activity performed in each phase was determined, and the activity percentage was calculated. In order to consider and relate all the phases in the product system, we used an estimated activity variable due to the lack of data. We developed a new approach to impact assessment. By determining the combination of the most extensive activity, as well as the most negative in the range of possible values for involved countries, we identified the hotspots. The results were not further aggregated in order to promote transparency.

Conclusions

We found the S-LCA methodology to be feasible and useful. By handling all relevant issues within one study using a systems perspective on the product life cycle, knowledge can be gained. However, there are still some major challenges. The definition of relevant indicators, data availability, impact pathways, activity variables, results presentation and possible aggregation, the handling of stakeholder context and the restricted assessment of the use phase were identified as major issues to deal with in further studies. Communication, and hence use of the results, is a crucial issue to enable the outcome of a study to result in actions that actually improve human well-being.     

Alfy-dependent elimination of aggregated proteins by macroautophagy: Can there be too much of a good thing?

Degradation of different cargo by macroautophagy is emerging as a highly selective process which relies upon specific autophagy receptors and adapter molecules that link the cargo with the autophagic molecular machinery. We have recently reported that the large phsophatidylinositol-3-phosphate (PtdIns(3)P)-binding protein Alfy (Autophagy-linked FYVE protein) is required for selective degradation of aggregated proteins. Although depletion of Alfy inhibits Atg5-dependent aggregate degradation, overexpression of Alfy results in Atg5-dependent aggregate clearance and neuroprotection. Alfy-mediated degradation requires the ability of Alfy to directly interact with Atg5. This ability to interact with the core autophagic machinery may cause Alfy to diminish the responsiveness to nonselective autophagic degradation as measured by long-lived protein degradation. Thus, increasing Alfy-mediated protein degradation may be beneficial in some organs, but may be detrimental in others.Key words: autophagy, protein aggregates, neurodegeneration, Alfy, aggregation, degradation     

Reduction of β-thiopyruvic acid by lactate dehydrogenase: a kinetic study

Summary In this paper a steady-state kinetic study on the system lactate dehydrogenase--thiopyruvate--thiolactate is presented and the possible mechanistic and physiological implications are discussed.At pH 7.4 the equilibrium between -thiopyruvate and -thiolactate, in the presence of NADH and lactate dehydrogenase is largerly shifted towards the formation of -thiolactate as in the case of pyruvate and lactate. This can be relevant in connection with the mixed disulfide between cysteine and -thiolactate that is observed to be present in the mammalian body fluids.The catalytic mechanism is of the bi-bi compulsory order type, and rapid equilibrium conditions for the binding of the first substrate (NADH) are shown to apply. A complex inhibition pattern of inhibitions by both substrates, however, prevents simple suggestions about the nature of the dead-end species involved.Abbreviations TPA -thiopyruvic acid - TLA -thiolactic acid - E enzyme This work is gratefully dedicated to Prof. Alessandro Rossi Fanelli on the occasion of his 75th birthday.     

Characterization of a novel lytic bacteriophage from an industrial <Emphasis Type="Italic">Escherichia coli</Emphasis> fermentation process and elimination of virulence using a heterologous CRISPR–Cas9 system

Bacterial–bacteriophage interactions are a well-studied and ecologically-important aspect of microbiology. Many commercial fermentation processes are susceptible to bacteriophage infections due to the use of high-density, clonal cell populations. Lytic infections of bacterial cells in these fermentations are especially problematic due to their negative impacts on product quality, asset utilization, and fouling of downstream equipment. Here, we report the isolation and characterization of a novel lytic bacteriophage, referred to as bacteriophage DTL that is capable of rapid lytic infections of an Escherichia coli K12 strain used for commercial production of 1,3-propanediol (PDO). The bacteriophage genome was sequenced and annotated, which identified 67 potential open-reading frames (ORF). The tail fiber ORF, the largest in the genome, was most closely related to bacteriophage RTP, a T1-like bacteriophage reported from a commercial E. coli fermentation process in Germany. To eliminate virulence, both a fully functional Streptococcus thermophilus CRISPR3 plasmid and a customized S. thermophilus CRISPR3 plasmid with disabled spacer acquisition elements and seven spacers targeting the bacteriophage DTL genome were constructed. Both plasmids were separately integrated into a PDO production strain, which was subsequently infected with bacteriophage DTL. The native S. thermophilus CRISPR3 operon was shown to decrease phage susceptibility by approximately 96%, while the customized CRISPR3 operon provided complete resistance to bacteriophage DTL. The results indicate that the heterologous bacteriophage-resistance system described herein is useful in eliminating lytic infections of bacteriophage DTL, which was prevalent in environment surrounding the manufacturing facility.     

Inhibition of the thrombin–fibrinogen reaction by a macromolecular factor from rat liver

1. The supernatant obtained by centrifugation of a rat liver homogenate at 100000g for 1h contained a heat-labile macromolecular inhibitor of the thrombin-fibrinogen reaction. 2. The inhibitor was purified to electrophoretic homogeneity by repeated preparative polyacrylamide disc electrophoresis. Inhibition was observed with purified inhibitor equivalent to about 1mug of protein/ml. 3. The inhibitor had a pI of 3.50-3.75, a molecular weight (from sodium dodecyl sulphate-polyacrylamide-gel electrophoresis) of 72000+/-3000 and was inactivated by p-hydroxymercuribenzoate or 5,5'-dithiobis-(2-nitrobenzoic acid). 4. Kinetic studies revealed a non-competitive inhibition, with the inhibitor probably acting on the thrombin-fibrinogen complex.     

Challenges in deep basin sequence stratigraphy: a case study from the Early–Middle Cretaceous of SW Zagros

Thick Neocomian–middle Albian successions include the most significant hydrocarbon source rocks in the Zagros Basin. These successions, named the Garau Formation, are composed of about 700 m of limestone, shaly (argillaceous) limestone, and bedded cherts with high organic matter (OM) content. Considering several lines of evidence, including the presence of partially dissolved planktonic foraminifer shells, thin-bedded radiolarian cherts, and the absence of index palynomorphs and shallow-marine fauna and facies, the depositional environment is interpreted as a deep basin (probably up to 1,000 m deep). Due to its bathymetry, facies changes are subtle, which in turn makes it challenging to reconstruct the sequence stratigraphic framework. Identification of five microfacies related to the deep basin along with palynological factors (AOM ratio, AOM%, phytoclast ratio, and phytoclast %) and their variations in the stratigraphic column led to identification of the relative sea-level changes during this time span. Consequently, various sequence surfaces and ten third-order sequences are recognized. These sequences are clearly correlatable with the shallow-water sequences of the Arabian Plate in their numbers and ages. Using Rock-Eval pyrolysis, organic content of the Garau facies is determined. TOC data show an elevated OM content (1.648 %) with a marked increase during the Early Aptian, which could be ascribed to the OAE1a.     

Comparative study of metal uptake by Eichhornia crassipes growing in ponds from mining and nonmining areas—a field study

The study deals with metal (Cu, Mn, Pb, Cd) concentrations in sediment, water, and corresponding leaf samples of Eichhornia crassipes obtained from ponds in nonmining (P1) and mining (P2, P3, P4) regions. In spite of significant high metal concentrations in sediments from mining regions rather than from nonmining regions, the unelevated SQG-I (sediment quality guideline index) values proved low levels of toxicity. Irrespective of the wide range of metal concentration in sediments, the levels in water had been nearly consistent in all the ponds. Concentration of metals in leaves decreased with an increase in concentration in the substrate. Mn, Cu, and Cd accumulated within the range of MAC (maximum allowable concentration) for plants, whereas Pb accumulated above the limit. BAF_sl (bioaccumulation factor with respect to sediment) values for Mn (0.20–0.27) were highest, followed by Cu (0.13–0.20) and Pb (0.03–0.20), whereas BAF_wl (bioaccumulation factor with respect to water) was highest for Cu (428–3205), followed by Mn (285–1100), Pb (242–506), and Cd (7–130). This study concludes that E. crassipes plays a very important role in removing the metals from the pond ecosystem, whereas leaves of this plant can be used effectively for biomonitoring surveys. E. crassipes can be used for phytoremediation of polluted wetlands through proper management strategies.     

Rule-based simulation of temperate bacteriophage infection: Restriction–modification as a limiter to infection in bacterial populations

An individual-based model (IbM) for bacterial adaptation and evolution, COSMIC-Rules, has been employed to simulate interactions of virtual temperate bacteriophages (phages) and their bacterial hosts. Outcomes of infection mimic those of a phage such as lambda, which can enter either the lytic or lysogenic cycle, depending on the nutritional status of the host. Infection of different hosts possessing differing restriction and modification systems is also simulated. Phages restricted upon infection of one restricting host can be adapted (by host-controlled modification of the phage genome) and subsequently propagate with full efficiency on this host. However, such ability is lost if the progeny phages are passaged through a new host with a different restriction and modification system before attempted re-infection of the original restrictive host. The simulations show that adaptation and re-adaptation to a particular host-controlled restriction and modification system result in lower efficiency and delayed lysis of bacterial cells compared with infection of non-restricting host bacteria.     

Encapsulation in a sol–gel matrix of lipase from Aspergillus niger obtained by bioconversion of a novel agricultural residue

Lipase from Aspergillus niger was obtained from the solid-state fermentation of a novel agroindustrial residue, pumpkin seed flour. The partially purified enzyme was encapsulated in a sol–gel matrix, resulting in an immobilization yield of 71.4 %. The optimum pH levels of the free and encapsulated enzymes were 4.0 and 3.0, respectively. The encapsulated enzyme showed greater thermal stability at temperatures of 45 and 60 °C than the free enzyme. The positive influence of the encapsulation process was observed on the thermal stability of the enzyme, since a longer half-life t _1/2 and lower deactivation constant were obtained with the encapsulated lipase when compared with the free lipase. Kinetic parameters were found to follow the Michaelis–Menten equation. The K _m values indicated that the encapsulation process reduced enzyme–substrate affinity and the V _max was about 31.3 % lower than that obtained with the free lipase. The operational stability was investigated, showing 50 % relative activity up to six cycles of reuse at pH 3.0 at 37 °C. Nevertheless, the production of lipase from agroindustrial residue associated with an efficient immobilization method, which promotes good catalytic properties of the enzyme, makes the process economically viable for future industrial applications.     

Misconceptions in biology: a meta-synthesis study of research, 2000–2014

Abstract

Teachers need to be aware of biology misconceptions in their classrooms and how to address them. In response, researchers and science educators have suggested and examined effective practices to prevent and ameliorate misconceptions. An extensive review of the literature gives researchers and educators insights into trends, practices, and gaps in the misconceptions research and helps decide which issues to address and why. The current study shares how researchers in Turkey conduct a content analysis of published misconception research in Turkey by using a form. The analysis resulted in a meta-synthesis (thematic content analysis) that inventoried and compared the purposes, research methods, data collection instruments, and findings of the selected publications. Biology educators in other regions of the world can inform their practice by using this instrument and research methods to learn about trends and patterns in misconception research. Researchers will gain insights into effective methods that have been used to examine misconceptions and will be able to identify biology misconceptions that have been under-investigated and need further analysis.     

Production of ��-poly-l-lysine using a novel two-stage pH control strategy by Streptomyces sp. M-Z18 from glycerol

The production of ε-poly-l-lysine (ε-PL) by Streptomyces sp. M-Z18 from glycerol was investigated in a 5-L jar-fermenter. Batch fermentations by Streptomyces sp. M-Z18 at various pH values ranging from 3.5 to 4.5 were studied. Based on the analysis of the time course of specific cell growth rate and specific ε-PL formation rate, a novel two-stage pH control strategy was developed to improve ε-PL production by shifting the culture pH from 3.5 to 3.8 after 36 h of cultivation. By applying the strategy, the maximal ε-PL concentration and productivity had a significant improvement and reached 9.13 g L⁻¹ and 4.76 g L⁻¹ day⁻¹, respectively, compared with those in one-stage pH control process where the pH value is controlled at 3.5 (7.83 g L⁻¹ and 3.13 g L⁻¹ day⁻¹). Fed-batch fermentation with two-stage pH control strategy was also applied to produce ε-PL; final ε-PL concentration of 30.11 g L⁻¹ was obtained, being 3.3-fold greater than that of batch fermentation. To our knowledge, it is the first report on production of ε-PL from glycerol in fermenter scale and achievement of high ε-PL production with two-stage pH control strategy.     

Adsorption of HCN on reduced graphene oxides: a first–principles study

Journal of Molecular Modeling - Over the past few years, time-dependent ultrafast fluorescence spectroscopy method has been applied to the study of protein dynamics. However, observations from...     

Reconstruction of the contamination of the Techa River in 1949–1951 as a result of releases from the “MAYAK” Production Association

More accurate reconstruction of the radioactive contamination of the Techa River system in 1949–1951 has been made on the basis of refined data on the amounts and the rate of discharge of radionuclides into the Techa River from the Mayak Production Association; this has led to the development of a modified Techa River model that describes the transport of radionuclides through the up-river ponds and along the Techa River and deposition of radionuclides in the river-bottom sediments and flooded areas. The refined Techa River source-term data define more precisely the time-dependent rates of release and radionuclide composition of the releases that occurred during 1949–1951. The Techa River model takes into account the time-dependent characteristics of the releases and considers (a) the transport of radionuclides adsorbed on solid particles originally contained in the discharges or originating in the up-river ponds as a result of stirring up of contaminated bottom sediments and (b) the transport of radionuclides in soluble form. The output of the Techa River model provides concentrations of all source-term radionuclides in the river water, bottom sediments, and floodplain soils at different distances from the site of radioactive releases for the period of major contamination in 1950–1951. The outputs of the model show good agreement with historical measurements of water and sediment contamination. In addition, the river-model output for ⁹⁰Sr concentration in the river water is harmonized with retrospective estimates derived from the measurements of ⁹⁰Sr in the residents of the Techa Riverside villages. Modeled contamination of the floodplain soils by ¹³⁷Cs is shown to be in agreement with the values reconstructed from late measurements of this radionuclide. Reconstructed estimates of the Techa River contamination are being used for the quantification of internal and external doses received by residents of the Techa Riverside communities.     

Hair cortisol: a parameter of chronic stress? Insights from a radiometabolism study in guinea pigs

Measurement of hair cortisol has become popular in the evaluation of chronic stress in various species. However, a sound validation is still missing. Therefore, deposition of radioactivity in hair and excretion into feces and urine after repeated injection of (3)H-cortisol was studied in guinea pigs (n?=?8). Each animal was given intraperitoneally 243.6?kBq (3)H-cortisol/day on 3 successive days. After the first injection, all voided excreta were collected for 3?days. After the second injection, hair was shaved off the animals' back and newly grown hair was obtained on day 7. Following methanol extraction, radiolabeled and unlabeled glucocorticoid metabolites (GCM) in fecal and hair samples were characterized by high-performance liquid chromatography (HPLC) and enzyme immunoassays (EIA). In feces, maximum radioactivity was reached 8?h (median) post each injection, whereas maxima in urine were detected in the first samples (median 2.5?h). Metabolites excreted into feces (13.3?%?±?3.7) or urine (86.7?%) returned nearly to background levels. HPLC of fecal extracts showed minor variation between individuals and sexes. In hair, small amounts of radioactivity were present. However, two EIAs detected large amounts of unlabeled GCM, including high levels at the position of the cortisol standard; radioactivity was absent in this fraction, demonstrating that (3)H-cortisol was metabolized. Furthermore, large amounts of immunoreactivity coinciding with a radioactive peak at the elution position of cortisone were found. These results show for the first time that only small amounts of systemically administered radioactive glucocorticoids are deposited in hair of guinea pigs, while measurement of large amounts of unlabeled GCM strongly suggests local production of glucocorticoids in hair follicles.     

Potential hotspots identified by social LCA—part 1: a case study of a laptop computer

Purpose

A generic hotspot assessment of social impacts from a product was conducted, using a laptop computer as a case. The aims of the case study were to identify social hotspots of the laptop and to test and evaluate the methodology.

Methods

The case study was based on the social LCA methodology described in the Guidelines for social LCA and included the product system from ‘cradle to grave’ as well as the impacts on all relevant stakeholders. We focused on a simplified list of materials and used mainly country-specific data.

Results and discussion

A new method for impact assessment of hotspots was developed. The total activity in each phase was distributed among countries. The countries were divided into groups related to the extent of activity in the product system, as well as to their performance on a subcategory. High values in both groups were highlighted and hotspots were identified. The results revealed some hotspots, some hot countries and some hot issues, all indicating a risk of negative social impacts in the product system of a laptop. It also identified workers and the local community as the stakeholders most at risk of negative social impacts. Among the hotspots identified, the following subcategories were of importance: safe and healthy living conditions, social benefit/social security, access to material resources, involvement in areas with armed conflicts, community engagement (lack of), corruption, and access to immaterial resources.

Conclusions

The study showed it is possible to conduct a social LCA on a generic complex product using the Guidelines, even though data collection was impaired by lack of data and low data quality. It identified methodological issues that need further attention, for example the indicator impact pathways. Still, it is clear that new insights can be gained by social LCA, where the life cycle perspective and the systematic approach help users identify potentially important aspects that could otherwise have been neglected.     

Structure of the UreD–UreF–UreG–UreE complex in Helicobacter pylori: a model study

The molecular details of the protein complex formed by UreD, UreF, UreG, and UreE, accessory proteins for urease activation in the carcinogenic bacterium Helicobacter pylori, have been elucidated using computational modeling. The calculated structure of the complex supports the hypothesis of UreF acting as a GTPase activation protein that facilitates GTP hydrolysis by UreG during urease maturation, and provides a rationale for the design of new drugs against infections by ureolytic bacterial pathogens.     

Risk of febrile seizures after first dose of measles–mumps–rubella–varicella vaccine: a population-based cohort study

Background:

The combination measles–mumps–rubella–varicella (MMRV) vaccine currently used in Canada (Priorix-Tetra) may increase the risk of febrile seizures relative to the separate vaccines (MMR and varicella) previously administered. We determined the risk of febrile seizure after the first dose of MMRV, as well as any additional risk for children at high risk for seizures because of pre-existing medical conditions.

Methods:

In this retrospective, population-based cohort study, we compared the risk of seizures after the first dose of MMRV with the risk after same-day administration of separate MMR and varicella vaccines (MMR+V) in children 12 to 23 months of age in the province of Alberta. We deterministically linked vaccination data to health service utilization data for seizures. We used Poisson regression, with adjustment for age and calendar year, to determine the risk for the full cohort and for high-risk children.

Results:

The risk of seizures 7 to 10 days after vaccination was twice as high with MMRV as with MMR+V (relative risk [RR] 1.99, 95% confidence interval [CI] 1.30–3.05). The excess absolute risk of seizures was 3.52 seizures per 10 000 doses of MMRV relative to MMR+V. In high-risk children, the risk was not differentially higher for MMRV (RR 1.30, 95% CI 0.60–2.79).

Interpretation:

Despite an increased risk of febrile seizures following MMRV (compared with MMR+V), the absolute level of risk was small. Policy-makers need to balance these findings with the potential benefits of administering the combination vaccine or determine whether the choice of vaccine rests with clinicians and/or parents.Combination measles–mumps–rubella–varicella (MMRV) vaccines were developed as an alternative to separate MMR and varicella (chickenpox) vaccines. Two such vaccines, with different formulations, are available in North America. ProQuad (Merck) is used in the United States, whereas Priorix-Tetra (GlaxoSmithKline) is the formulation currently used in Canada. Priorix-Tetra is also used in Australia, Italy and Germany, and it has market authorization in most member states of the European Union.¹ Postlicensure vaccine safety studies of ProQuad have identified an increased risk of febrile seizures in children 12–23 months old after the first dose² but not after the subsequent preschool dose.³Priorix-Tetra was approved for use in Canada in July 2007.⁴ Currently, 9 of 13 Canadian provinces and territories administer this vaccine as part of their routine childhood immunization schedule. The province of Alberta started administering the vaccine in September 2010, to replace the first dose of MMR and varicella vaccines due at 12 months of age.⁵Prelicensure clinical trials of Priorix-Tetra indicated that it had a similar safety profile to co-administration of the separate vaccines, except for a higher incidence of fever.^4,6 Determination of the less common risk of febrile seizures requires postlicensure monitoring in a large population and is an identified research priority of the Canadian National Advisory Committee on Immunization.⁴ Only 1 previous study, conducted in Germany, has assessed febrile seizure risk after Priorix-Tetra,⁷ considering only cases treated in hospital. It is also a priority to determine if the risk is amplified among children with pre-existing medical conditions that may predispose them to seizures.⁸Our objective was to determine the risk of febrile seizures after the first dose of the combination MMRV vaccine (Priorix-Tetra) administered to children in Alberta, relative to same-day administration of separate MMR and varicella (MMR+V) vaccines. A secondary objective was to determine if children considered to be at “high risk” for seizures were at increased risk for febrile seizures following administration of the combination vaccine.     

Nosocomial candidemia at a general hospital: The change of epidemiological and clinical characteristics. A comparative study of 2 cohorts (1993–1998 versus 2002–2005)

BackgroundNosocomial candidemia (NC) is associated with high mortality, increased hospital stay and greater economical cost.AimsTo evaluate epidemiological and clinical aspects of 2 different cohorts of non-paediatric patients with NC.MethodsA retrospective observational and comparative study of patients with NC. Patients were identified by review of results of blood cultures from the hospital microbiology laboratory. We analysed epidemiological, clinical, microbiological and laboratory data and changes in the 2 cohorts: 1993–1998 (P1) and from 2002 to 2005 (P2).ResultsEighty patients were studied during P1 and 107 during P2; incidence was 9/10,000 in P1 and 15.8/10,000 admitted patients in P2 (p<0.05). Mean age was 52 years in P1 and 61 years in P2 (p<0.05); 66% and 49% NC were due to Candida albicans in P1 and P2, respectively (p<0.05); diabetes was present in 12% in P1 and in 25% in P2 (p<0.05). All of the patients had previously received at least one course of broad-spectrum antibiotics. A statistically significant difference (p<0.05) in predisposing conditions was identified in central intravenous line rate (100% in P1 and 91% in P2) and previous surgery (43% in P1 and 78% in P2). Acute severity of illness at onset and complications were more frequent in P2 (p<0.05). Mortality rate was similar in P1 and P2 (51% and 49.5%, respectively).ConclusionsFrequency of NC has increased and non-albicans Candida is now more frequent than C. albicans. Although acute severity of illness at onset and complications are now more frequent, mortality remains the same.