No evidence of an association between size and levels of four per- and polyfluorinated substances (PFAS) in perch (Perca fluviatilis).

It was assessed how the size of perch (Perca fluviatilis) is related to levels of four per- and polyfluorinated substances (PFAS) in its muscle tissue. These were PFOS, PFNA, PFOA, and PFHxS, for which the sum, denoted as ΣPFAS4, has a tolerable intake derived by the European Food Safety Authority. The results indicate that, in contrast to, e.g., mercury levels, ΣPFAS4 levels in perch muscle do not increase with increasing weight of the fish, which implies that consuming larger perch does not increase the risk of exceeding the TWI of ΣPFAS4, in relation to consuming smaller perch. Therefore, for risk assessment, analyzing samples of smaller perch is sufficient, demanding less effort to catch. The credibility of the results was strengthened by applying the same statistical model to mercury levels in the same samples. As expected, larger fish had generally higher levels than small fish for mercury.

Cellulose nanofibers (CNFs) in the recycling of nickel and cadmium battery metals using electrodeposition.

Cellulose nanofibers (CNFs) were employed in the aqueous electrodeposition of nickel and cadmium for battery metal recycling. The electrowinning of mixed Ni-Cd metal ion recycling solutions demonstrated that cadmium with a purity of over 99% could be selectively extracted while leaving the nickel in the solution. Two types of CNFs were evaluated: negatively charged CNFs (a-CNF) obtained through acid hydrolysis (-75 µeq. g-1) and positively charged CNFs (q-CNF) functionalized with quaternary ammonium groups (+85 µeq. g-1). The inclusion of CNFs in the Ni-Cd electrolytes induced growth of cm-sized dendrites in conditions where dendrites were otherwise not observed, or increased the degree of dendritic growth when it was already present to a lesser extent. The augmented dendritic growth correlated with an increase in deposition yields of up to 30%. Additionally, it facilitated the formation of easily detachable dendritic structures, enabling more efficient processing on a large scale and enhancing the recovery of the toxic cadmium metal. Regardless of the charged nature of the CNFs, both negatively and positively charged CNFs led to a significant formation of protruding cadmium dendrites. When deposited separately, dendritic growth and increased deposition yields remained consistent for the cadmium metal. However, dendrites were not observed during the deposition of nickel; instead, uniformly deposited layers were formed, albeit at lower yields (20%), when positively charged CNFs were present. This paper explores the potential of utilizing cellulose and its derivatives as the world's largest biomass resource to enhance battery metal recycling processes.

Using Fish as a Sentinel in Risk Management of Contaminated Sediments.

Sediments polluted by historical emissions from anthropogenic point sources are common in industrialized parts of the world and pose a potential threat to the function of aquatic ecosystems. Gradient studies using fish as a bioindicator are an option to assess the ecological impact of locally polluted areas. This study investigates the remaining effects of historical emissions on sediments outside ten Swedish pulp and paper mills using perch (Perca fluviatilis). The aim has been to obtain a general picture of the impact area of local deposits of cellulose fiber-rich sediments containing elevated levels of trace metals, e.g., Hg, and organochlorines, e.g., dioxins. In addition to analyzing contaminant levels in muscle and liver tissue, morphological measures in the fish that constitute biomarkers for health and reproductivity were measured. Another aim was to augment existing historical data sets to observe possible signs of environmental recovery. Overall, the results indicate only a minor elevation in contaminant levels and a minor impact on the fish health status in the polluted areas, which in several cases is an improvement from historical conditions. However, exceptions exist. Differences in the ecosystems' responses to pollution loads are primarily explained by abiotic factors such as water turnover rate, bottom dynamic conditions, and water chemistry. Weaknesses in the sampling methodology and processing of data were identified. After minor modifications, the applied survey strategy has the potential to be a management tool for decision-makers working on the remediation of contaminated areas.

Using a dynamic mass balance model to predict fate and transport of PCBs in a polluted boreal lake in Sweden.

In 2013, a screening survey including fish (European perch, Perca fluviatilis) from 20 locations in the Stockholm region of Sweden indicated exceptionally high levels of PCBs (>450 ng ΣPCB7/g ww) in Lake Oxundasjön. An extensive sampling program was launched to define the magnitude and area of impact of PCBs. Moreover, a dynamic mass balance model approach was applied to identify and quantify key transport processes and predict the long-term turnover of PCBs given various remediation scenarios. Based on the dating of sediment profiles, primary emissions of PCBs to Lake Oxundasjön have likely occurred from the end of the 1940s until 1980, reaching the lake via one of its tributaries. Presently, the main source of PCBs is diffusion from the lake sediments. From the lake outlet, >400 g ΣPCB7/yr are transported to Lake Mälaren (the third largest lake in Sweden), supplying drinking water for parts of the Stockholm area. Remediation actions are necessary to reduce the PCB levels in fish below today's marketing limits and environmental quality standards. With natural recovery, our results indicate that the PCB levels in non-migratory fish from Lake Oxundasjön will be elevated for decades to come. The mass of PCBs stored in the lake sediments was estimated, and to our knowledge, Lake Oxundasjön is the most heavily PCB contaminated lake in Sweden. The system constitutes a unique opportunity to test and develop a mathematical mass balance model for PCBs, with substantial data acquired from different aquatic matrices. The model presented in the paper is applicable for risk assessments of PCBs, and the results contribute to the general understanding of the transport and turnover dynamics of PCBs in aquatic ecosystems.

Molecular Details of a Coupled Binding and Folding Reaction between the Amyloid Precursor Protein and a Folded Domain.

Intrinsically disordered regions in proteins often function as binding motifs in protein-protein interactions. The mechanistic aspects and molecular details of such coupled binding and folding reactions, which involve formation of multiple noncovalent bonds, have been broadly studied theoretically, but experimental data are scarce. Here, using a combination of protein semisynthesis to incorporate phosphorylated amino acids, backbone amide-to-ester modifications, side chain substitutions, and binding kinetics, we examined the interaction between the intrinsically disordered motif of amyloid precursor protein (APP) and the phosphotyrosine binding (PTB) domain of Mint2. We show that the interaction is regulated by a self-inhibitory segment of the PTB domain previously termed ARM. The helical ARM linker decreases the association rate constant 30-fold through a fast pre-equilibrium between an open and a closed state. Extensive side chain substitutions combined with kinetic experiments demonstrate that the rate-limiting transition state for the binding reaction is governed by native and non-native hydrophobic interactions and hydrogen bonds. Hydrophobic interactions were found to be particularly important during crossing of the transition state barrier. Furthermore, linear free energy relationships show that the overall coupled binding and folding reaction involves cooperative formation of interactions with roughly 30% native contacts formed at the transition state. Our data support an emerging picture of coupled binding and folding reactions following overall chemical principles similar to those of folding of globular protein domains but with greater malleability of ground and transition states.

Factor XIII activity at onset of labour and association with postpartum haemorrhage: an exploratory post-hoc study.

BACKGROUND: Platelets, fibrinogen and factor XIII (FXIII) are required to form a stable clot in case of haemorrhage. The aims of this study were to evaluate a possible association between FXIII activity at the onset of labour and postpartum haemorrhage (PPH), and to ascertain whether FXIII activity at labour onset differs from after delivery. METHODS: FXIII activity in 239 women with PPH (blood loss >1 L) and in 76 women without PPH was compared, as was activity before and after delivery in a third group of 80 women. RESULTS: FXIII activity at onset of labour was significantly lower in the PPH group compared with the control group (mean ±â€¯SD 0.98 ±â€¯0.20 vs 1.05 ±â€¯0.17 kIU/L; P=0.0006). The difference was significantly greater in subgroups having vaginal delivery with no oxytocin stimulation or uterine exploration (absolute difference 0.131; 95% CI 0.055 to 0.206), compared with a subgroup experiencing any complication (0.04; 95% CI -0.023 to 0.104; interaction P-value 0.098). There was a weak but statistically significant inverse correlation between FXIII and estimated blood loss (r=-0.25; P=0.030) in the control group but not the PPH group. There was no significant difference between FXIII activity at onset of labour and after delivery (mean ±â€¯SD 1.03 ±â€¯0.17 vs 1.04 ±â€¯0.19 kIU/L; P=0.093). CONCLUSIONS: At the onset of labour women with a subsequent PPH had significantly lower mean FXIII activity than that of women without PPH. This difference was small and within normal limits. FXIII activity did not change during normal delivery. The importance of FXIII during PPH requires study.

Binding Kinetics of the Intrinsically Disordered p53 Family Transactivation Domains and MDM2.

Because of their prominent roles in cell-cycle regulation and cancer, the interaction between MDM2 and the intrinsically disordered transactivation domain (TAD) of p53 is exceptionally well-studied. However, although there are numerous computational studies on the interaction mechanism, there is a paucity of experimental data regarding the kinetics and mechanism. We have used stopped flow fluorescence to investigate the binding reaction between MDM2 and TAD from p53 as well as from its paralogs p63 and p73, and in particular, focused on the salt dependence of the interaction. The observed kinetics are consistent with a two-state mechanism within the time frame of the stopped flow methodology; thus, any conformational changes including the previously identified MDM2 lid dynamics must occur on a time scale <5 ms at 10 °C. The association rate constants are similar for the three TADs, and differences in the dissociation rate constants determine the various affinities with MDM2. In contrast to previous studies, we found a relatively small ionic-strength dependence for all three interactions, highlighting the large variation in the role of electrostatics among binding reactions of intrinsically disordered proteins (IDPs). The basal association rate constants in the absence of electrostatic interactions were relatively high (≥2 × 106 M-1 s-1 at 10 °C), suggesting that a large number of initial contacts may lead to a productive complex. Our findings support an emerging picture of "conformational funneling" occurring in the initial stages of interactions involving IDPs and that these early binding events can rely on hydrophobic as well as charge-charge interactions.

Antarctic seals: Molecular biomarkers as indicators for pollutant exposure, health effects and diet.

Weddell (Leptonychotes weddellii), Ross (Ommatophoca rossii) and crabeater seals (Lobodon carcinophaga) are phocid seals with a circumpolar distribution around Antarctica. As long-lived and large top predators, they bioaccumulate contaminants and are considered as sentinels of ecosystem health. Antarctic seals are increasingly exposed to climate change, pollution, shipping and fisheries. To reveal and understand possible anthropogenic impacts on their immune and health status, this study investigates sensitive biomarkers of the xenobiotic metabolism and immune system in relation to mercury (Hg) burden. Gene-transcription studies using minimally-invasive blood samples are useful to monitor physiological processes in wildlife that can be related to different stressors. Blood samples of 72 wild-caught seals (Weddell n=33; Ross n=12; crabeater n=27) in the Amundsen and Ross Seas in 2008-2011 were investigated. Copy numbers per µl mRNA transcription of xenobiotic biomarkers (aryl hydrocarbon receptor (AHR)), aryl hydrocarbon receptor nuclear translocator (ARNT) and peroxisome proliferator-activated receptor (PPARα) and immune relevant cell mediators (cytokines interleukin-2 (IL-2), interleukin-10 (IL-10) and heat-shock-protein 70 (HSP70)) were measured using reference genes Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide (YWHAZ) and ribosomal protein L4 (RPL4) by real time RT-qPCR. Hg concentration was analysed in fur. Hg concentration increased with body weight and standard length in all species. Crabeater seals showed a lower Hg concentration than Ross and Weddell seals. Species-specific differences in gene-transcription were found between all species with highest levels of AHR, ARNT and PPARα in crabeater seals. Ross seals showed highest IL-10 and HSP70 transcription, while HSP70 was exceptionally low in crabeater seals. Between Hg and HSP70 a clear negative relationship was found in all species. The species-specific, age and sex-dependent gene-transcription probably reflect dietary habits, pollutant exposure and immune status.

Post-operative pain relief using local infiltration analgesia during open abdominal hysterectomy: a randomized, double-blind study.

BACKGROUND: Post-operative pain is common and often severe after open abdominal hysterectomy, and analgesic consumption high. This study assessed the efficacy of local infiltration analgesia (LIA) injected systematically into different tissues during surgery compared with saline on post-operative pain and analgesia. METHODS: Fifty-nine patients were randomized to Group LIA (n = 29) consisting of 156 ml of a mixture of 0.2% ropivacaine + 30 mg ketorolac + 0.5 mg (5 ml) adrenaline, where the drugs were injected systematically in the operating site, around the proximal vagina, the ligaments, in the fascia and subcutaneously, or to saline and intravenous ketorolac, Group C (Control, n = 28), in a double-blind study. Post-operative pain, analgesic consumption, side-effects, and home discharge were analysed. RESULTS: Median dose of rescue morphine given 0-24 h after surgery was significantly lower in group LIA (18 mg, IQR 5-25 mg) compared with group C (27 mg, IQR 15-43 mg, P = 0.028). Median time to first analgesic injection was significantly longer in group LIA (40 min, IQR 20-60 min) compared with group C (20 min, IQR 12-30 min, P = 0.009). NRS score was lower in the group LIA compared with group C in the direct post-operative period (0-2 h). No differences were found in post-operative side-effects or home discharge between the groups. DISCUSSION: Systematically injected local infiltration analgesia for pain management was superior to saline in the primary endpoint, resulting in significantly lower rescue morphine requirements during 0-24 h, longer time to first analgesic request and lower early post-operative pain intensity.

Improved affinity at the cost of decreased specificity: a recurring theme in PDZ-peptide interactions.

The E6 protein from human papillomavirus (HPV) plays an important role during productive infection and is a potential drug target. We have previously designed a high affinity bivalent protein binder for the E6 protein, a fusion between a helix from the E6 associated protein and PDZØ9, an engineered variant (L391F/K392M) of the second PDZ domain from synapse associated protein 97 (SAP97 PDZ2). How the substitutions improve the affinity of SAP97 PDZ2 for HPV E6 is not clear and it is not known to what extent they affect the specificity for cellular targets. Here, we explore the specificity of wild type SAP97 PDZ2 and PDZØ9 through proteomic peptide phage display. In addition, we employ a double mutant cycle of SAP97 PDZ2 in which the binding kinetics for nine identified potential cellular peptide ligands are measured and compared with those for the C-terminal E6 peptide. The results demonstrate that PDZØ9 has an increased affinity for all peptides, but at the cost of specificity. Furthermore, there is a peptide dependent coupling free energy between the side chains at positions 391 and 392. This corroborates our previous allosteric model for PDZ domains, involving sampling of intramolecular energetic pathways.

Ligand binding to the PDZ domains of postsynaptic density protein 95.

Cellular scaffolding and signalling is generally governed by multidomain proteins, where each domain has a particular function. Postsynaptic density protein 95 (PSD-95) is involved in synapse formation and is a typical example of such a multidomain protein. Protein-protein interactions of PSD-95 are well studied and include the following three protein ligands: (i)N-methyl-d-aspartate-type ionotropic glutamate receptor subunit GluN2B, (ii) neuronal nitric oxide synthase and (iii) cysteine-rich protein (CRIPT), all of which bind to one or more of the three PDZ domains in PSD-95. While interactions for individual PDZ domains of PSD-95 have been well studied, less is known about the influence of neighbouring domains on the function of the respective individual domain. We therefore performed a systematic study on the ligand-binding kinetics of PSD-95 using constructs of different size for PSD-95 and its ligands. Regarding the canonical peptide-binding pocket and relatively short peptides (up to 15-mer), the PDZ domains in PSD-95 by and large work as individual binding modules. However, in agreement with previous studies, residues outside of the canonical binding pocket modulate the affinity of the ligands. In particular, the dissociation of the 101 amino acid CRIPT from PSD-95 is slowed down at least 10-fold for full-length PSD-95 when compared with the individual PDZ3 domain.

Design of a PDZbody, a bivalent binder of the E6 protein from human papillomavirus.

Chronic infection by high risk human papillomavirus (HPV) strains may lead to cancer. Expression of the two viral oncoproteins E6 and E7 is largely responsible for immortalization of infected cells. The HPV E6 is a small (approximately 150 residues) two domain protein that interacts with a number of cellular proteins including the ubiquitin ligase E6-associated protein (E6AP) and several PDZ-domain containing proteins. Our aim was to design a high-affinity binder for HPV E6 by linking two of its cellular targets. First, we improved the affinity of the second PDZ domain from SAP97 for the C-terminus of HPV E6 from the high-risk strain HPV18 using phage display. Second, we added a helix from E6AP to the N-terminus of the optimized PDZ variant, creating a chimeric bivalent binder, denoted PDZbody. Full-length HPV E6 proteins are difficult to express and purify. Nevertheless, we could measure the affinity of the PDZbody for E6 from another high-risk strain, HPV16 (Kd = 65 nM). Finally, the PDZbody was used to co-immunoprecipitate E6 protein from HPV18-immortalized HeLa cells, confirming the interaction between PDZbody and HPV18 E6 in a cellular context.

Fibrinogen plasma concentration before delivery is not associated with postpartum haemorrhage: a prospective observational study.

BACKGROUND: Low plasma fibrinogen concentration has been linked to postpartum haemorrhage. The primary aim of this study was to assess whether fibrinogen concentration at admission before labour is associated with severe postpartum haemorrhage. Secondary aims were to describe fibrinogen concentration before and after labour and to identify predictors for severe postpartum haemorrhage. METHODS: 1951 healthy women were included in a prospective observational study. Fibrinogen concentration was determined at admission to the labour ward and in a subgroup of women (n=80) also after the placenta was delivered. Bleeding volume postpartum was estimated by weighing surgical sponges and pads and by measuring collected blood. Predictors for severe postpartum haemorrhage (>1000 ml) were identified with bivariate and multivariate regression analyses. RESULTS: Mean fibrinogen concentration was 5.3 (SD 0.8) g litre(-) (1). Median estimated blood loss was 450 (range 70-4400) ml and 250 (12.8%) women bled >1000 ml. Fibrinogen concentration was not correlated with postpartum haemorrhage in the entire cohort (r(s)=0.003, P=0.90) or in any subgroup. Fibrinogen concentration was not associated with bleeding >1000 ml (odds ratio 1.01 (CI 95% 0.85-1.19), P=0.93) and did not differ significantly before and after delivery. Oxytocin stimulation, instrumental delivery, Caesarean section and exploration of uterus were identified as independent predictors of haemorrhage >1000 ml. CONCLUSIONS: Fibrinogen plasma concentration at admission before labour does not predict severe postpartum haemorrhage in a general obstetric population. Fibrinogen concentration does not decrease significantly during normal labour. Excessive postpartum bleeding is mainly as a result of obstetric complications.

Major obstetric haemorrhage: monitoring with thromboelastography, laboratory analyses or both?

BACKGROUND: Haemorrhage is a common cause of morbidity and mortality in the obstetric population. The aim of this study was to compare the use of thromboelastography and laboratory analyses to evaluate haemostasis during major obstetric haemorrhage. A secondary aim was to evaluate correlations between the results of thromboelastography, laboratory analyses and estimated blood loss. METHODS: Forty-five women with major obstetric haemorrhage and 49 women with blood loss <600 mL were included. The following thromboelastography analyses were performed: time to start of clotting (TEG-R), time to 20 mm of clot firmness (TEG-K), rate of clot growth (TEG-Angle), maximum amplitude of clot (TEG-MA) and lysis after 30 min (TEG-LY30). In addition, platelet count, activated partial thromboplastin time, prothrombin time, fibrinogen, antithrombin and D-dimer were measured. RESULTS: Thromboelastography variables reflecting clot stability and fibrinolysis were decreased in women with massive obstetric haemorrhage compared to women with normal bleeding, while clot initiation was accelerated. Laboratory analyses also showed impaired haemostasis with the most pronounced differences in platelet count, fibrinogen concentration and antithrombin activity. The strongest correlations existed between fibrinogen and TEG-MA and between estimated blood loss and TEG-MA, fibrinogen and antithrombin, respectively. CONCLUSIONS: Impaired haemostasis, demonstrated by thromboelastography and laboratory analyses, was found after an estimated blood loss of 2000 mL. Thromboelastography provides faster results than standard laboratory testing which is advantageous in the setting of on-going obstetric haemorrhage. However, laboratory analyses found greater differences in coagulation variables, which correlated better with estimated blood loss.

High-throughput sequencing in veterinary infection biology and diagnostics.

Sequencing methods have improved rapidly since the first versions of the Sanger techniques, facilitating the development of very powerful tools for detecting and identifying various pathogens, such as viruses, bacteria and other microbes. The ongoing development of high-throughput sequencing (HTS; also known as next-generation sequencing) technologies has resulted in a dramatic reduction in DNA sequencing costs, making the technology more accessible to the average laboratory. In this White Paper of the World Organisation for Animal Health (OIE) Collaborating Centre for the Biotechnology-based Diagnosis of Infectious Diseases in Veterinary Medicine (Uppsala, Sweden), several approaches and examples of HTS are summarised, and their diagnostic applicability is briefly discussed. Selected future aspects of HTS are outlined, including the need for bioinformatic resources, with a focus on improving the diagnosis and control of infectious diseases in veterinary medicine.

The transition state of coupled folding and binding for a flexible ß-finger.

Flexible and fully disordered protein regions that fold upon binding mediate numerous protein-protein interactions. However, little is known about their mechanism of interaction. One such coupled folding and binding occurs when a flexible region of neuronal nitric oxide synthase adopts a ß-finger structure upon binding to its protein ligand, a PDZ [PSD-95 (postsynaptic density protein-95)/Discs large/ZO-1] domain from PSD-95. We have analyzed this binding reaction by protein engineering combined with kinetic experiments. Mutational destabilization of the ß-finger changed mainly the dissociation rate constant of the proteins and, to a lesser extent, the association rate constant. Thus, mutation affected late events in the coupled folding and binding reaction. Our results therefore suggest that the native binding interactions of the ß-finger are not present in the rate-limiting transition state for binding but form on the downhill side in a cooperative manner. However, by mutation, we could destabilize the ß-finger further and change the rate-limiting step such that an initial conformational change becomes rate limiting. This switch in rate-limiting step shows that multistep binding mechanisms are likely to be found among flexible and intrinsically disordered regions of proteins.

Side-chain interactions form late and cooperatively in the binding reaction between disordered peptides and PDZ domains.

Intrinsically disordered proteins are very common and mediate numerous protein-protein and protein-DNA interactions. While it is clear that these interactions are instrumental for the life of the mammalian cell, there is a paucity of data regarding their molecular binding mechanisms. Here we have used short peptides as a model system for intrinsically disordered proteins. Linear free energy relationships based on rate and equilibrium constants for the binding of these peptides to ordered target proteins, PDZ domains, demonstrate that native side-chain interactions form mainly after the rate-limiting barrier for binding and in a cooperative fashion. This finding suggests that these disordered peptides first form a weak encounter complex with non-native interactions. The data do not support the recent notion that the affinities of intrinsically disordered proteins toward their targets are generally governed by their association rate constants. Instead, we observed the opposite for peptide-PDZ interactions, namely, that changes in K(d) correlate with changes in k(off).

Structure-related distribution of PCDD/Fs, PCBs and HCB in a river-sea system.

Water concentrations of PCDD/Fs, HCB, and non-ortho, mono-ortho, and non-dioxin-like PCBs were measured four times during 1 year in a coastal area of the Baltic Sea, to investigate background levels and distribution behaviour. Sampling sites included two rivers, an estuary, and the sea. Particulate and apparently dissolved concentrations were determined using active sampling (filters+PUFs), while freely dissolved concentrations were determined using passive sampling (POM-samplers). The distribution between particulate+colloidal and freely dissolved phases, in the form of TOC-normalized distribution ratios (K(TOC)), was found to be near or at equilibrium. The observed K(TOC) were not significantly different between sampling sites or seasons. For PCDD/Fs, the concentrations were significantly correlated to suspended particulate matter (SPM), while no correlation to organic carbon (TOC) was observed. In the estuary and the sea, PCB concentrations were correlated to TOC. The sorption of various congeners to SPM and TOC appeared to be related to both hydrophobicity and 3D-structure. The PCDD/F concentration in the sea decreased to one third in May, likely connected to the increased vertical flux of particles during the spring bloom.

Indications of recovery from hypoxia in the inner Stockholm archipelago.

Improved benthic conditions compared to the 1990s were found during benthic investigations, including sediment and benthic macrofauna in the inner Stockholm archipelago during 2008. In the 1990s, these areas were dominated by black and laminated surface sediments and very sparse fauna. A clear relationship was found when comparing sediment status with the benthic macrofauna. Reduced surface sediment and impoverished macroinvertebrate community was only found at one sampling station representing an enclosed part of the inner archipelago, whereas the other seven stations, with depths ranging from 20 to 50 m, had oxidized surface sediments and considerable biomasses of benthic macrofauna (6-65 g m(-2)) dominated by the invading polychaete Marenzelleria neglecta. An extrapolation of the results shows that, within the investigated area, the coverage of reduced surface sediments had decreased from approximately 17% in the late 1990s to 4% in 2008.