The genera Cryptoglena and Monomorphina also formed a well-supported monophyletic clade. Euglena and the recently erected genus Euglenaria emerged as sister groups. However, Euglena proxima branched off at the base of the Euglenaceae. The Phacaceae clade was also a monophyletic group with high support values and subdivided into three clades, the Discoplastis, Phacus, and Lepocinclis

clades. The genus Discoplastis branched first, and then Phacus and Lepocinclis emerged as sister groups. These genera shared a common characteristic, numerous small discoid chloroplasts without pyrenoids. These results clearly separated the Phacaceae clade from the Euglenaceae clade. Therefore, we propose to limit the family Euglenaceae to the members of the Euglena clade and erect a new family, the Phacaceae, to house the genera Phacus, Lepocinclis, and Discoplastis. “
“Little selleck chemicals is known about the UV photobiology of psychrophilic dinoflagellates, particularly Dabrafenib in freshwater

systems. We addressed the life strategies of Borghiella dodgei Moestrup, Gert. Hansen et Daugbjerg to cope with ambient levels of ultraviolet radiation (UVR) under cold conditions. Several physiological parameters related to growth, metabolism, and UVR protection were determined for 4 d in UVR-exposed and control cells by applying stable isotope analysis, spectrophotometry, and liquid chromatography–mass spectrometry (LC/MS). In UVR-exposed cells, assimilation of 15N and 13C and content of chl a and carotenoids, specifically diatoxanthin with respect to dinoxanthin and diadinoxanthin, were increased; furthermore, catalase activity showed a cyclic pattern with a strong increase after UVR exposure but a rapid return to preexposure levels. Both in UVR-exposed and control cells, no lipid peroxidation of galactolipids was observed. However, in UVR-exposed cells, content of galactolipids was higher

and linked to an increase in monogalactosyldiacylglycerols (MGDGs). We concluded that Borghiella’s adaptation to UVR depended on a general metabolic enhancement and efficient scavenging of oxygen radicals to mitigate and counteract damage. While Borghiella seemed to be well adapted to ambient UVR, the interactive effects of higher temperature check details and UVR on psychrophilic species in front of climate change merit further investigation. “
“The green macroalga Ulva L. is well known as having an alternation of isomorphic biphases: gametophyte and sporophyte. However, an examination of the temporal alternation in phase dominance has not been carried out. By inducing reproduction of thallus samples in the laboratory, this study reports the temporal changes in the two phases and sex ratios in a natural population of Ulva pertusa Kjellm. in the Seto Inland Sea, Japan, over a period of >3 years. The results showed that a temporal alternation in phase dominance occurred after 11–20 months; seasonal changes in phase dominance were not observed in the Ulva population.

A product created by a process that incorporates two viral reduction steps should not automatically be considered better than one that only has one specific viral inactivation step. If only one step is used, this step should preferably inactivate viruses AG-014699 order with and without lipid envelopes. FVIII concentrates are the treatment of choice for hemophilia A. All plasma-derived products currently in the market are listed in the WFH Registry of Clotting Factor Concentrates [3]. Consult the product insert for specific details. Vials of factor concentrates are available in dosages ranging from approximately 250–3000 units each. In the absence of an inhibitor, each unit of FVIII per kilogram of body weight infused

intravenously will raise the plasma FVIII level approximately 2 IU dL −1 . (Level 4) [ [11] ] The half-life of FVIII is approximately 8–12 h. The patient’s factor level should be measured 15 min after the infusion to verify the calculated dose. (Level 4) [ [11] ] The dose is calculated by multiplying the patient’s weight in kilograms by the factor level in IU dL−1 desired, multiplied by 0.5. Example:

50 kg × 40 (IU dL−1 level desired) × 0.5 = 1,000 units of FVIII. Refer to Tables 7-1 and 7-2 for suggested factor level and duration of replacement required based on type of hemorrhage. FVIII should be infused by slow IV injection at a rate not to exceed 3 mL per min in adults Lapatinib purchase and 100 units per min in young children, or as specified in the product information leaflet. (Level 5) [ [12] ] Subsequent doses should ideally be based on the half-life of FVIII and on the recovery in an individual patient for a particular product. It is best to use the entire vial of FVIII once reconstituted, although many products have been shown to have extended selleck chemical stability after reconstitution. Continuous infusion avoids peaks and troughs and is considered by some to be advantageous and more convenient. However, patients must be monitored frequently for pump failure. (Level 3) [ [13, 14] ] Continuous

infusion may lead to a reduction in the total quantity of clotting factor concentrates used and can be more cost-effective in patients with severe hemophilia [15]. However, this cost-effectiveness comparison can depend on the doses used for continuous and intermittent bolus infusions [16]. Dose for continuous infusion is adjusted based on frequent factor assays and calculation of clearance. As FVIII concentrates of very high purity are stable in IV solutions for at least 24–48 h at room temperature with less than 10% loss of potency, continuous infusion for a similar number of hours is possible. FIX concentrates are the treatment of choice for hemophilia B. All plasma-derived products currently in the market are listed in the WFH Registry of Clotting Factor Concentrates [3]. Consult the product information guide for specific details.

The frequency of adverse events was similar in both the placebo and N7-GP groups and no neutralizing antibodies against N7-GP were detected [34]. N9-GP, a recombinant FIX molecule, obtained by site-directed glycoPEGylation

where a 40-kDa PEG molecule is attached to the activation peptide of FIX underwent a first human dose trial in patients with haemophilia B. The toxicology programme did not identify any PEG-related safety findings [35].The clinical study investigated the safety and pharmacokinetic properties of a single IV dose of N9-GP in 16 patients. None of the patients developed inhibitors. Treatment emergent adverse events were reported in six (37.5%) patients. Ten of these events in five patients were rated as moderate or mild and three were rated as probably or possibly related to N9-GP. These three events were fatigue (two events in one patient) http://www.selleckchem.com/products/LY294002.html and myalgia. One buy Staurosporine serious adverse event was reported in one patient and probably related to N9-GP.

The event was a hypersensitivity reaction, which occurred during administration of N9-GP in a 25-year-old male patient who had no history of inhibitors, nor any history of allergic reactions to his previous pdFIX (plasma-derived FIX) product. The patient fully recovered within 8 h after onset of the hypersensitivity event with supportive care and antihistamines. No antibodies (Abs) were detected on analyses of the patient’s pre and postdose blood samples for FIX inhibitors, N9-GP binding Abs, IgE against N9-GP, IgE against rFIX, IgE against CHO cells,

IgE against hamster epithelium, Ig against host cell proteins and Ig against murine IgG. After the event, the patient learn more continued with his previous pdFIX product without any complications [35]. Recently, a summary of toxicology and preclinical results were reported for BAX 855, a full-length rFVIII. Assessment of toxicity was based on mortality, clinical observations, ophthalmic examination, clinical pathology, assessment on male fertility in rats, organ weights and pathology evaluations. In addition to safety endpoints, toxico-kinetics and the formation of antiproduct antibodies were assessed. No PEG-related effects were observed [36]. One issue that may have been overlooked by the community is that PEG is also present in some plasma-derived FVIII products, which have been used in patients for a long time [37]. (BAY 94–9027; Bayer HealthCare), a site-specific PEGylated B-domain deleted rFVIII (60 kDa-PEG-BDD-rFVIII) with a branched 60 kDa PEG, is currently being investigated in a pivotal clinical trial prior to registration. In vivo studies using BAY 94–9027 or PEG-60 alone were conducted at Bayer laboratories under Good Laboratory Practice, they followed applicable laws and regulations and internal standards for husbandry and ethical treatment of animals. An initial single high dose acute toxicity study in male Sprague Dawley rats with doses up to 210 mg kg−1 PEG-60 was conducted.

Alpha granule disorders.  Gray platelet syndrome is associated with macrothrombocytopenia, absence

of platelet granules visible using light microscopy, and variably impaired aggregation responses to thrombin and collagen [17]. Proteins synthesized AG 14699 in megakaryocytes and destined for α-granules are not appropriately stored, resulting in empty granules and the release of coagulation and growth factors into the marrow, increasing the risk of myelofibrosis. The molecular defect is unknown, although the trafficking defect may involve SNARE proteins that mediate vesicle membrane fusion. In Quebec platelet disorder, α-granule proteins are abnormally degraded because of aberrant expression and storage of the fibrinolytic enzyme urokinase plasminogen activator (uPA). The genetic abnormality has recently been identified as a tandem duplication in the uPA gene PLAU [18]. The unique feature of this disorder is delayed-onset bleeding that responds to antifibrinolytic drug therapy. Several rare defects are the result of abnormalities Staurosporine cost in the platelet contractile cytoskeleton. Cytoskeletal components support the plasma membrane and maintain the shape of resting platelets. Reorganization of the cytoskeleton following platelet activation results in the extension of filipodia and platelet spreading. The cytoskeleton also

plays an essential role in proplatelet formation by megakaryocytes. Wiskott–Aldrich Syndrome: defects in actin assembly.  X-linked Wiskott–Aldrich syndrome (WAS) is characterized by thrombocytopenia, small platelets, eczema, immunodeficiency and an increased risk of lymphoid malignancy. WAS is caused by mutations in the WAS gene leading to defects or absence of the WAS protein (WASp).

Mutations resulting in absent or truncated protein give rise to the classic WAS phenotype; missense mutations with residual protein are associated with a milder phenotype, X-linked thrombocytopenia. WASp regulates the assembly of actin monomers into filaments and thus, cytoskeletal organization selleck chemicals and motility of cells. WASp has a role in the regulation of actin polymerization and the structure and dynamics of actin filament networks. WASp defects lead to abnormalities of cytoskeletal organization, which affect proplatelet formation by megakaryocytes, granule content, and cell spreading [19]. MYH9 disorders: defects in myosin heavy chain.  The MYH9-related disorders (May-Hegglin anomaly, Fechtner, Epstein and Sebastian syndromes) typically present with macrothrombocytopenia and mild-to-moderate bleeding symptoms [20]. These disorders, although identified as separate entities, all result from mutations in the MYH9 gene that encodes non-muscle myosin-heavy chain-IIA. Multiple different mutations have been detected in the MYH9 gene but most affect dimerization of the protein and its assembly into filaments.

Intriguingly, lingering CK19 expression indicated a persistent ductal phenotype. Thus, the Lgr5+ cells are truly bipotential in this cell population, although bias toward induction of a default biliary phenotype was GSK3 inhibitor observed (Fig. 1).

It would have been more convincing if a direct comparison of stemness and differentiation of Lgr5+ cells to Sox9+/Lgr5- or CK19+/Lgr5- cells could be made in the organoid cultures, as it would underscore the heterogeneity of biliary epithelial cells in terms of their stem cell characteristics. Finally, Huch et al. transplanted organoids derived from single Lgr5+ cells cultured in hepatocyte differentiation media for 9 days, into the fumarylacetoacetate hydrolase (Fah−/−) mutant mice. Fah+ nodules representing transplanted cell-derived colonies were found within the liver in only 5 of the 15 mice. The repopulation

ranged anywhere between 0.1 to 1% of total hepatic parenchyma and led to only a partial rescue of the enzymatic defect in Fah−/− animals. This was drastically lower than engraftment and rescue of Fah−/− animals by transplantation of freshly isolated hepatocytes. However, selleckchem the engrafted Lgr5+ derived hepatocytes increased recipient animal survival significantly and did not lead to any oncogenic events. Similarly, it was interesting to note that the in vivo hepatic milieu led to sufficient differentiation of organoids to hepatocytes, since no CK19 expression was detected in engrafted Lgr5-derived cells after transplantation. The current in vitro organoid culture system is an important tool to understand the biology of liver stem cells. It should be emphasized that this model represents the bipotentiality of a single cell and can now allow interrogation of the biology of stemness, differentiation, learn more and maturation. Furthermore, assuming that the engraftment pitfalls can be adequately addressed and the differentiation protocols optimized, these adult organ-derived cells

may provide an important candidate for tissue engineering and regenerative therapies. The appearance of Lgr5+ stem cells in the liver following injury is intriguing since this marker has shown to be expressed in stem cells of the gut, hair follicles, and other tissues.[8] Based on the presented injury models, Lgr5+ cells may represent a dynamic stem cell compartment for hepatic repair as well.[9] Several possible origins for these cells are outlined in Fig. 2, and there may be alternate scenarios that are not fully understood at this time. Whatever the source, the relative contribution of Lgr5+ progenitors to either cell compartment appears to be context-specific, depending on the mode and severity of hepatic injury. In addition, the exact mechanism by which Lgr5 may be regulating stemness remains a mystery.

They were randomly assigned into two groups. Eleven patients were treated Ruxolitinib by two injections of BT at four weeks interval.

In another group, EO was injected 3 times with two weeks intervals. All patients were followed by achalasia symptom score (ASS) and timed barium esophagram (TBE). We defined good response as decrease in ASS (≤4), and reduction of height and volume of barium in TBE > 80% of baseline at 1.5 months after last injection. Relapse was defined as increase in ASS (>4) after initial good response. Results: Mean age of patients was 63.14 ± 13.2 years (Min: 26, Max: 81). All patients in EO group and ten patients in BT group revealed good initial response Five patients in EO and four in BT groups, who had relapsed, were treated by re-injection. AG 14699 The mean duration of follow up was 27.38 months Finally, in BT group, six patients had good response (ASS ≤ 4) and five had poor response, but in EO group good responses were nine and only one patient had a poor response.(P value: 0.049) Conclusion: This study revealed that both BT and EO are effective

in the treatment of IA, but in long term follow up more patients in EO group remained in remission. Moreover, the cost of BT is approximately 20 times more than EO. We suggest the use of EO in selected patients with IA. Key Word(s): 1. Achalasia; 2. Botulinum Toxin; 3. Ethanolamine oleate; Presenting Author: FANDONG MENG Additional Authors: WENYAN LI, QIAOZHI ZHOU, YONGDONG WU, MING JI, SHUTIAN ZHANG Corresponding Author: FANDONG MENG Affiliations: Capital Medical University, Beijing Friendship Hospital Objective: Achalasia is divided into 3 subtypes selleck chemical using the Chicago classification for high-resolution manometry (HRM). Aim of this study was to apply the achalasia subtypes classification to a retrospective cohort of patients referred for esophageal manometry and to compare clinical and manometric characteristics between the 3 subtypes. Methods: Patients referred for esophageal manometry and diagnosed with achalasia on HRM

were retrospectively identified in Beijing friendship hospital. Only untreated patients before HRM were included in the retrospective study. Symptoms (dysphagia, chest pain, regurgitation) were collected at the time of HRM. Three achalasia subtypes were determined based on the Chicago classification. Clinical characteristics and manometric parameters were compared. Results: From January 2012 to March 2013 achalasia was diagnosed in 33 patients, 25 were untreated achalasia. 20% of patients were classified as type I, 60% as type II and 20% as type III. 100% of patients complained of dysphagia, 27% of type II patients presented chest pain. 53% of type I patients presented regurgitations compared to 20% of type II and 48% of type III (p = 0.36). Dilatation of esophagus was shown in 60% of type I patents, 73% for type II and 20% for type III.

Disclosures: The following people have nothing to disclose: Susan L. Zickmund, Michael K. Chapko, Barbara H. Hanusa, Pexidartinib cost Ada O. Youk, Galen E. Switzer, Mary Ann Sevick, David S.

Obrosky, Nichole K. Bayliss, Carolyn L. Zook, Robert A. Arnold Introduction: HCV-infected patients from endemic regions of the world immigrate to Canada and are subsequently referred to viral hepatitis clinics for management. Cultural differences and language barriers are potential obstacles to receiving HCV treatment. We set out to describe the racial / ethnic diversity of a HCV-infected population receiving care at a tertiary care, hospital-based viral hepatitis clinic and to identify differences in investigations, HCV therapy access and HCV therapy outcomes between Canadian-born and foreign-born patients. Methods: The Ottawa Hospital Viral Hepatitis Program Clinical Database (SPSS 17.0) was utilized to identify HCV-infected patients followed between June 2000 and May 2013. Information on immigration history, country of origin and race is contained within this database. Information on HCV work-up,

treatment and outcome CB-839 mw [i.e. Sustained Virological Response (SVR)] was compared by these parameters (Chi square, Student’s t test). Results: 3229 HCV-infected patients were assessed (68% male; 80% white, 9% black, 7% Asian, 4% Aboriginal). 24% were born outside of Canada (Sub-Saharan Africa-18%, South East Asia-11%). this website A median 16 years (Quartiles: 5,28) passed between immigration and assessment. The mean age at the time of first evaluation was 50.1 for Canadian-born and 40.1 years for immigrant referrals (p<0.001). The median biopsy stage (2) and grade (2) did not differ by group. Access to liver biopsy and HCV antiviral therapy initiation did not differ by race.

SVR was 38% in blacks compared to 43% for other races. Conclusion: Access to care and treatment was similar irrespective of immigrant status suggesting that a multidisci-plinary approach to HCV care with a commitment to culturally-sensitive care, including providing services in the patient’s language of choice, can overcome barriers to care. Disclosures: Curtis Cooper – Advisory Committees or Review Panels: Vertex, MERCK, Roche; Grant/Research Support: MERCK, Roche; Speaking and Teaching: Roche, MERCK Kimberly Corace – Advisory Committees or Review Panels: Gilead; Grant/ Research Support: Pfizer; Speaking and Teaching: Vertex, Janssen, Reckitt-Benck-iser Gary Garber – Advisory Committees or Review Panels: Gilead; Grant/Research Support: Pfizer; Speaking and Teaching: Reckett The following people have nothing to disclose: Crystal D.

7A), indicating that the SIRPα-CD47 interaction may not involve tumor immunosurveillance against Hepa1-6 cells in syngeneic mice. In contrast, knockdown of SIRPα on Mψ promoted Hepa1-6 cell proliferation even without cell-cell direct interaction, suggesting that the content released by Mψ may have an important role in tumor progression (Supporting Fig. 7B-E). In summary, our results suggest that there is a fine-tuned collaborative action between SIRPα expression on Mψ and tumor

progression. Mψ with SIRPα-KD have the powerful potential to migrate and survive in tumor sites. Soluble factors derived from tumors trigger transient activation of newly recruited Mψ and reduce SIRPα expression, thereby inducing these Ruxolitinib nmr cells to produce a large amount of cytokines, in turn leading to the down-expression of SIRPα on Mψ and ultimately create an inflammatory environment supporting tumor progression. Our findings provide new insight into the importance of SIRPα in tumor progression, selleck chemicals llc which may be helpful for new antitumor drug design. We thank Dr. Bin Gao (Laboratory of Liver Diseases, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, MD) and Dr. G.S. Feng (School of Medicine, Section of Molecular Biology,

Division of Biological Sciences, University of California, San Diego) for helpful discussion and suggestions. Additional Supporting Information may be found in the online version of this article. “
“The pathogenesis of non-alcoholic fatty liver disease (NAFLD) is now focusing on its organ cross-talk with not only adipose

tissue but also systemic skeletal muscle. Cross-sectional and longitudinal studies were conducted to determine the role of intramuscular adipose tissue content (IMAC) measured by computed tomography on the severity of NAFLD/non-alcoholic steatohepatitis (NASH). Two hundred eight Japanese patients with NAFLD/NASH diagnosed find more by liver biopsy were enrolled into a cross-sectional study. Twenty-one patients were enrolled in a longitudinal study and received a programmed diet and exercise intervention, in some cases the combination of pharmacotherapy. We measured IMAC in the multifidus muscle and biochemical parameters, and conducted liver histology to assess NAFLD/NASH status. Histopathological stage in terms of simple steatosis and Brunt’s classification was significantly correlated with IMAC (P < 0.01). Multivariate logistic regression analysis indicated that risk factors associated with the severity of NASH were IMAC and aging (IMAC: odds ratio = 2.444, P < 0.05; Age: odds ratio = 2.355, P < 0.05). The interventions improved histopathological changes in 11 patients with NASH as well as IMAC. These results suggest that skeletal muscle fat accumulation may have been linked to the pathogenesis and severity of NASH.

Our culture and field observations indicate that there is a previously unrecognized stage in the life cycle of P. antarctica G. Karst. This stage comprises nonmotile cells that Cetuximab range in size from ∼4.2 to 9.8 μm in diameter and form aggregates in which interstitial spaces between cells are small or absent. The aggregates (hereafter called attached aggregates, AAs) adhere to available surfaces. In field samples, small AAs, surrounded by a colony skin, adopt an epiphytic lifestyle and adhere in most cases to setae or spines of diatoms. These AAs, either directly or via other life stages, produce the colonial life stage. Culture studies indicate that bloom-forming, colonial stages release flagellates (microzoospores)

that fuse and form AAs, which can proliferate on the bottom of culture vessels and can eventually reform free-floating colonies. We propose that these AAs are a new stage in the

life cycle of P. antarctica, which we believe to be the zygote, thus documenting sexual reproduction in this species for the first time. “
“Chroococcidiopsis Geitler (Geitler 1933) is a genus of cyanobacteria containing desiccation and radiation resistant strains. Members of the genus live in habitats ranging from hot and cold deserts to fresh and saltwater environments. Morphology and cell division pattern have historically been used to define the genus. To better understand the evolution and ability of the Chroococcidiopsis genus to survive Cabozantinib mw in diverse environments we investigated how salt tolerance varies among 15 strains previously isolated from different locations, and if salt tolerant strains are monophyletic to those isolated from freshwater and land environments. Four markers were sequenced from these 15 strains, the 16S rRNA, rbcL, desC1, and gltX

genes. Phylogenetic trees were generated which identified a distinct clade of salt-tolerant strains. This study demonstrates that the genus is selleck kinase inhibitor polyphyletic based on saltwater and freshwater phenotypes. To understand the resistance to salt in more details, the strains were grown on a range of sea salt concentrations which demonstrated that the freshwater strains were salt-intolerant whilst the saltwater strains required salt for growth. This study shows an increased resolution of the phylogeny of Chroococcidiopsis and provides further evidence that the genus is polyphyletic and should be reclassified to improve clarity in the literature. “
“As the process of ocean acidification alters seawater carbon chemistry, physiological processes such as skeletal accretion are expected to become more difficult for calcifying organisms. The crustose coralline red algae (Corallinales, Rhodophyta) form an important guild of calcifying primary producers in the temperate Northeast Pacific. The morphology of important ecological traits, namely, skeletal density and thallus thickness near the growing edge, was evaluated in Pseudolithophyllum muricatum (Foslie) Steneck & R.T.

These results may have been, in part, reflective of the imbalance in BMI between the groups in that study, as well as differences in environmental and dietary factors between the two study cohorts. In addition, the lack of classification of patients in each group based

on liver histology may have also affected the results of the study by Zhu et al.29 Lastly, differences in age may have also played a role, as discussed below. There are various theories to support an inverse correlation between Bacteroidetes and steatohepatitis. First, Bacteroidetes carry 45% of the lean metabolic potential in a study comparing the microbiome of lean and obese Ipatasertib chemical structure adults.21 A lower percentage of Bacteroidetes could have affected energy balance by facilitating metabolic dominance of other bacteria

that are more efficient in extracting energy from the diet. Jumpertz et al.11 showed that a 20% increase in fecal Bacteroidetes is associated with a 150 kcal decrease in energy harvest from the diet. A second theory is that an initial hit causes the cell death Gefitinib chemical structure of Bacteroides leading to LPS release from their cell wall and subsequent endotoxemia.19 The latter leads to the development of NASH.19, 27 It is not clear what would cause the death of these microorganisms. Changes in diet could play a role, as shown by studies in obese subjects, whose baseline lower fecal Bacteroidetes increase when placed on a hypocaloric diet or after bariatric selleck kinase inhibitor surgery.38, 39 There is literature supporting an increase in intestinal permeability of subjects with IR, such as in obesity40 and diabetes.41 Recently, Zhu et al.29 reported higher serum ethanol levels in children

with NASH, which was thought to be bacterially derived and, hence, potentially also contributing to increased intestinal permeability. In addition, there is scientific evidence linking endotoxemia with states of glucose intolerance, such as NAFLD.41, 42, 43 Animals and humans exposed to low levels of endotoxin develop IR.19, 22 Exploratory analysis from our study also suggested a potential link between the intestinal microbiota and IR by showing a trend toward a negative association between Bacteroidetes and IR when controlling for BMI. This requires further studies. We did not find lower bifidobacteria counts or higher Firmicutes-to-Bacteroidetes ratio in NASH compared to SS and HC. This is in contrast to some of the previously published literature in the field of obesity9, 14, 44 and the recent study by Zhu et al. on children with NASH.29 The inability to show differences in these bacteria may have been due to the sample size; however, the size of our cohort was similar to that of other cross-sectional studies on IM in obesity9, 11, 12, 37 and NAFLD.29 Our results on Firmicutes-to-Bacteroidetes ratio are in line with other smaller projects, which also failed to replicate the findings of Ley et al.