High accuracy of predicting hybrid performance of Fusarium head blight resistance by mid-parent values in wheat

Key message

Mid-parent values of Fusarium head blight (FHB) resistance tested across several locations are a good predictor of hybrid performance caused by a preponderance of additive gene action in wheat.

Abstract

Hybrid breeding is intensively discussed as one solution to boost yield and yield stability including an enhanced biotic stress resistance. Our objectives were to investigate (1) the heterosis for Fusarium head blight (FHB) resistance, (2) the importance of general (GCA) vs. specific combining ability (SCA) for FHB resistance, and (3) the possibility to predict the FHB resistance of the hybrids by the parental means. We re-analyzed phenotypic data of a large population comprising 1604 hybrids and their 120 female and 15 male parental lines evaluated in inoculation trials across seven environments. Mid-parent heterosis of FHB severity averaged ?9%, with a range from ?36 to +35%. Mean better parent heterosis was 2% and 78 of the hybrids significantly (P < 0.05) outperformed the best commercial check variety included in our study. FHB resistance was not correlated with grain yield in healthy status for lines (r = 0.01) and hybrids (r = 0.09, P < 0.01). While a preponderance of GCA variance (P < 0.01) was found, SCA variance was not significantly different from zero. Accuracy to predict hybrid performance of FHB severity based on mid-parent values and on GCA effects was high (r = 0.70 and 0.86, respectively; P < 0.01). Similarly, line per se performance and GCA effects were significantly correlated (r = 0.77; P < 0.01). The substantial level of mid-parent heterosis in the desired direction of decreased susceptibility and the negligible better parent heterosis suggest that hybrids are an attractive alternative variety type to improve FHB resistance.

     

Characterization of <Emphasis Type="Italic">Gladiolus</Emphasis> Germplasm Using Morphological,Physiological, and Molecular Markers

Estimation of variability and genetic relationships among breeding materials is one of the important strategies in crop improvement programs. Morphological (plant height, spike length, a number of florets/spike), physiological (chlorophyll content, chlorophyll fluorescence, and rapid light curve parameters) and Directed amplification of minisatellite DNA (DAMD) markers were used to investigate the relationships among 50 Gladiolus cultivars. Cluster analysis based on morphological data, physiological characteristics, molecular markers, and cumulative data discriminated all cultivars into seven, five, seven, and six clusters in the unweighted pair-group method using arithmetic mean (UPGMA) dendrogram, respectively. The results of the principal coordinate analysis (PCoA) also supported UPGMA clustering. Variations among the Gladiolus cultivars at phenotypic level could be due to the changes in physiology, environmental conditions, and genetic variability. DAMD analysis using 10 primers produced 120 polymorphic bands with 80% polymorphism showing polymorphic information content (PIC = 0.28), Marker index (MI = 3.37), Nei’s gene diversity (h = 0.267), and Shannon’s information index (I = 0.407). Plant height showed a positive significant correlation with Spike length and Number of florets/spike (r = 0.729, p < 0.001 and r = 0.448, p = 0.001 respectively). Whereas, Spike length showed positive significant correlation with Number of florets/spike (r = 0.688, p < 0.001) and Chlorophyll content showed positive significant correlation with Electron transport rate (r = 0.863, p < 0.001). Based on significant morphological variations, high physiological performance, high genetic variability, and genetic distances between cultivars, we have been able to identify diverse cultivars of Gladiolus that could be the potential source as breeding material for further genetic improvement in this ornamental crop.     

Milk and Dairy Products Intake Is Associated with Low Levels of Lead (Pb) in Workers highly Exposed to the Metal

Lead (Pb) is a toxic metal, frequently associated with occupational exposure, due to its widespread use in industry and several studies have shown high Pb levels in workers occupationally exposed to the metal. The aim of this study was to evaluate the influence of milk and dairy products (MDP) on Pb levels in blood (B-Pb), plasma (P-Pb), and urine (U-Pb), in workers from automotive battery industries in Brazil. The study included 237 male workers; information concerning diet and lifestyle were gathered through a questionnaire, and B-Pb, P-Pb, and U-Pb were determined by ICP-MS. Mean B-Pb, P-Pb, and U-Pb were 21 ± 12, 0.62 ± 0.73 μg/dL, and 39 ± 47 μg/g creatinine, respectively. Forty three percent of participants declared consuming ≤3 portions/week of MDP (classified as low-MDP intake), while 57% of individuals had >3portions/week of MDP (high-MDP intake). B-Pb and P-Pb were correlated with working time (r _s  = 0.21; r _s  = 0.20; p < 0.010). Multivariable linear regressions showed a significant influence of MDP intake on B-Pb (β = ?0.10; p = 0.012) and P-Pb (β = ?0.16; p < 0.010), while no significance was seen on U-Pb. Our results suggest that MDP consumption may modulate Pb levels in individuals highly exposed to the metal; these findings may be due to the Pb-Ca interactions, since the adverse effects of Pb are partially based on its interference with Ca metabolism and proper Ca supplementation may help to reduce the adverse health effects induced by Pb exposure.     

Feasibility and relevance of compound strain imaging in non-stenotic arteries: comparison between individuals with cardiovascular diseases and healthy controls

Background

Compound strain imaging is a novel method to noninvasively evaluate arterial wall deformation which has recently shown to enable differentiation between fibrous and (fibro-)atheromatous plaques in patients with severe stenosis. We tested the hypothesis that compound strain imaging is feasible in non-stenotic arteries and provides incremental discriminative power to traditional measures of vascular health (i.e., distensibility coefficient (DC), central pulse wave velocity [cPWV], and intima-media thickness [IMT]) for differentiating between participants with and without a history of cardiovascular diseases (CVD).

Methods

Seventy two participants (60 ± 7 years) with non-stenotic arteries (IMT < 1.1 mm) were categorized in healthy participants (CON, n = 36) and CVD patients (n = 36) based on CVD history. Participants underwent standardised ultrasound-based assessment (DC, cPWV, and IMT) and compound strain imaging (radial [RS] and circumferential [CS] strain) in left common carotid artery. Area under receiver operating characteristics (AROC)-curve was used to determine the discriminatory power between CVD and CON of the various measures.

Results

CON had a significantly (P < 0.05) smaller carotid IMT (0.68 [0.58 to 0.76] mm) than CVD patients (0.76 [0.68 to 0.80] mm). DC, cPWV, RS, and CS did not significantly differ between groups (P > 0.05). A higher CS or RS was associated with a higher DC (CS: r = ?0.32;p < 0.05 and RS: r = 0.24;p < 0.05) and lower cPWV (CS: r = 0.24;p < 0.05 and RS: r = ?0.25;p < 0.05). IMT could identify CVD (AROC: 0.66, 95%-CI: 0.53 to 0.79), whilst the other measurements, alone or in combination, did not significantly increase the discriminatory power compared to IMT.

Conclusions

In non-stenotic arteries, compound strain imaging is feasible, but does not seem to provide incremental discriminative power to traditional measures of vascular health for differentiation between individuals with and without a history of CVD.

     

First-Rotation Performance of Five Short-Rotation Willow Cultivars on Different Soil Types and Along a Large Climate Gradient

The establishment of short-rotation willow coppice plantations for bioenergy production is proposed as a land management practice to reduce the use of fossil fuels. However, there is little information on the performance of different willow (Salix spp.) cultivars on various types of soils and over climate gradients. This study aimed to determine which set of soil, climatic conditions, and cultivars are conducive to greater growth in eastern Canada. The performance of five willow cultivars was followed over three growing seasons on eight sites representing a large hydroclimatic gradient. Both geographic location and cultivar had a significant effect on annual yields. Annual yields were on average greater in the southern part of the climatic gradient. Across all cultivars, annual yields were positively correlated to average annual temperature (r = 0.23), total annual precipitation (r = 0.77), average growing season temperature (r = 0.21), average growing season precipitation (r = 0.47), and degree days (r = 0.18), as well as soil pH (r = 0.37) and soil extractible P (r = 0.38), and negatively correlated to soil clay content (r = 0.33). Cultivars of the SX group (i.e., Salix miyabeana SX61, SX64, and SX67) showed greater yield than did cultivars of the SV group (i.e., Salix × dasyclados SV1 and Salix viminalis SV5027). These results indicate that at the landscape level, climate variables, especially climate-related variables, largely explain the yield of the selected willow cultivars. Nonetheless, soil pH, extractable P, and clay content likely play an important role in plantation yield.     

Genetic population structure and low genetic diversity in the over-exploited sea cucumber <Emphasis Type="Italic">Holothuria edulis</Emphasis> Lesson, 1830 (Echinodermata: Holothuroidea) in Okinawa Island

Understanding genetic connectivity is fundamental for ecosystem-based management of marine resources. Here we investigate the metapopulation structure of the edible sea cucumber Holothuria edulis Lesson, 1830 across Okinawa Island, Japan. This species is of economic and ecological importance and is distributed from the Red Sea to Hawai‘i. We examined sequence variation in fragments of mitochondrial cytochrome oxidase subunit I (COI) and 16S ribosomal RNA (16S), and nuclear histone (H3) at six locations across Okinawa Island. We found higher haplotype diversity for mtDNA (COI: Hd = 0.69 and 16S: Hd = 0.67) and higher heterozygosity of nDNA (H3: H _E = 0.39) in populations from the west coast of Okinawa compared to individuals from populations on the east coast (COI: Hd = 0.40; 16S: Hd = 0.21; H3: H _E = 0.14). Overall population structure was significant (AMOVA results for COI: Φ _ST = 0.49, P < 0.0001; 16S: Φ _ST = 0.34, P < 0.0001; H3: Φ _ST = 0.12, P < 0.0001). One population in the east, Uruma, showed elevated pairwise Φ _ST values in comparisons with all other sites and a marked reduction of genetic diversity (COI: Hd = 0.25 and 16S: Hd = 0.24), possibly as a consequence of a shift to a more dominant asexual reproduction mode. Recent reports have indicated that coastal development in this area influences many marine organisms, and ecosystem degradation in this location could cause the observed decrease of genetic diversity and isolation of H. edulis in Uruma. Our study should provide valuable data to help with the urgently needed management of sea cucumber populations in Okinawa, and indicates particular attention needs to be paid to vulnerable locations.     

Association between the blood concentrations of ammonia and carnitine/amino acid of schizophrenic patients treated with valproic acid

Background

Administration of valproic acid (VPA) is complicated with approximately 0.9% of patients developing hyperammonemia, but the pathogenesis of this adverse effect remains to be clarified. The aim of the present study was to search for mechanisms associated with VPA-induced hyperammonemia in the light of changes in serum amino acids concentrations associated with the urea cycle of schizophrenic patients.

Method

Blood samples (10 mL) were obtained from 37 schizophrenic patients receiving VPA for the prevention of violent behaviors in the morning after overnight fast. Blood concentrations of ammonia, VPA, free carnitine, acyl-carnitine, and 40 amino acids including glutamate and citrulline were measured for each patient. Univariate and multivariate regression analyses were performed to identify amino acids or concomitantly administered drugs that were associated with variability in the blood concentrations of ammonia.

Result

The blood ammonia level was positively correlated with the serum glutamate concentration (r = 0.44, p < 0.01) but negatively correlated with glutamine (r = ?0.41, p = 0.01), citrulline (r = ?0.42, p = 0.01), and glycine concentrations (r = ?0.54, p < 0.01). It was also revealed that the concomitant administration of the mood stabilizers (p = 0.04) risperidone (p = 0.03) and blonanserin (p < 0.01) was positively associated with the elevation of the blood ammonia level.

Conclusion

We hypothisized that VPA would elevate the blood ammonia level of schizophrenic patients. The observed changes in serum amino acids are compatible with urea cycle dysfunction, possibly due to reduced carbamoyl-phosphate synthase 1 (CPS1) activity. We conclude that VPA should be prudently prescribed to schizophrenic patients, particularly those receiving mood stabilizers or certain antipsychotics.

     

Serum histones as biomarkers of the severity of heatstroke in dogs

Heatstroke is associated with systemic inflammatory response syndrome, leading to multiple organ dysfunction and death. Currently, there is no specific treatment decreasing hyperthermia-induced inflammatory/hemostatic derangements. Emerging studies indicate that histones leaking from damaged cells into the extracellular space are toxic, pro-inflammatory, and pro-thrombotic. We therefore hypothesize that serum histones (sHs) are elevated during heatstroke and are associated with the severity of the disease. Sixteen dogs with heatstroke and seven healthy controls were included in the study. Median serum histones (sHs) upon admission in dogs with heatstroke were significantly higher (P = 0.043) compared to that in seven controls (13.2 vs. 7.3 ng/mL, respectively). sHs level was significantly higher among non-survivors and among dogs with severe hemostatic derangement (P = 0.049, median 21.4 ng/mL vs. median 8.16 ng/mL and P = 0.038, 19.0 vs. 7.0 ng/mL, respectively). There were significant positive correlation between sHs and urea (r = 0.8, P = 0.02); total CO2 (r = 0.661, P = 0.05); CK (r = 0.678, P = 0.04); and prothrombin time (PT) 12 h post presentation (r = 0.888, P = 0.04). The significant positive correlation between sHs and other heatstroke severity biomarkers, and significant increase among severely affected dogs, implies its role in inflammation/oxidation/coagulation during heatstroke. sHs, unlike other prognostic and severity biomarkers in heatstroke, can be pharmacologically manipulated, offering a potential therapeutic target.     

<Emphasis Type="Italic">A MCP</Emphasis>-<Emphasis Type="Italic">1</Emphasis> promoter polymorphism at G-2518A is associated with spontaneous preterm birth

Monocyte chemoattractant protein-1 (MCP-1) is an important chemokine involved in the pathogenesis of spontaneous preterm birth (SPTB). We examined whether the MCP-1 G-2518A polymorphism is associated with the risk of SPTB in a Chinese population. The MCP-1 G-2518A polymorphism was genotyped in 569 preterm singleton neonates and in 673 term neonates using polymerase chain reaction–restriction fragment length polymorphism analysis. The distribution of the MCP-1 G-2518A genotype and the allele frequencies between the SPTB patients and the controls were not significantly different in the overall sample. However, we found that the AA genotype was associated with significantly increased susceptibility to very SPTB (<32 weeks) [odds ratio (OR) 2.07; 95 % confidence interval (CI), 1.27–3.36; P = 0.005) and extremely SPTB (<28 weeks) (OR 2.74; 95 % CI, 1.10–6.72; P = 0.014) compared with ?2518G-positive genotypes (GG + GA genotypes). When extremely preterm neonates and very preterm neonates were combined, the AA genotype was also significantly associated with increased susceptibility to SPTB (OR 2.23; 95 % CI, 1.40–3.54; P < 0.001). The MCP-1 G-2518A polymorphism was not associated with increased susceptibility to SPTB in patients with premature rupture of the membranes (PROM) or in those without PROM. Our findings suggest that the MCP-1 G-2518A polymorphism may plays a role in mediating the susceptibility to SPTB in the Chinese population. Knowledge of genetic factors contributing to the pathogenesis of SPTB may have implications for screening and treatment of this disorder.     

Sweet Sorghum Juice and Bagasse as Feedstocks for the Production of Optically Pure Lactic Acid by Native and Engineered <Emphasis Type="Italic">Bacillus coagulans</Emphasis> Strains

Sweet sorghum is a bioenergy crop that produces large amounts of soluble sugars in its stems (3–7 Mg ha^?1) and generates significant amounts of bagasse (15–20 Mg ha^?1) as a lignocellulosic feedstock. These sugars can be fermented not only to biofuels but also to bio-based chemicals. The market potential of the latter may be higher given the current prices of petroleum and natural gas. The yield and rate of production of optically pure d-(?)- and l-(+)-lactic acid as precursors for the biodegradable plastic polylactide was optimized for two thermotolerant Bacillus coagulans strains. Strain 36D1 fermented the sugars in unsterilized sweet sorghum juice at 50 °C to l-(+)-lactic acid (～150 g L^?1; productivity, 7.2 g L^?1 h^?1). B. coagulans strain QZ19-2 was used to ferment sorghum juice to d-(?)-lactic acid (～125 g L^?1; productivity, 5 g L^?1 h^?1). Carbohydrates in the sorghum bagasse were also fermented after pretreatment with 0.5 % phosphoric acid at 190 °C for 5 min. Simultaneous saccharification and co-fermentation of all the sugars (SScF) by B. coagulans resulted in a conversion of 80 % of available carbohydrates to optically pure lactic acid depending on the B. coagulans strain used as the microbial biocatalyst. Liquefaction of pretreated bagasse with cellulases before SScF (L + SScF) increased the productivity of lactic acid. These results show that B. coagulans is an effective biocatalyst for fermentation of all the sugars present in sweet sorghum juice and bagasse to optically pure lactic acid at high titer and productivity as feedstock for bio-based plastics.     

Effects of VEGF levels on anti-VEGF therapy for patients with idiopathic choroidal neovascularization

The objective of this study is to investigate the levels of vascular endothelial growth factor (VEGF) and other cytokines in aqueous humor of patients with idiopathic choroidal neovascularization (CNV) and their effects together with central retinal thickness (CRT) on the response to intravitreal injection of anti-VEGF antibody ranibizumab. This clinical study recruited 32 eyes from 32 patients with CNV under or besides fovea. VEGF, interleukin (IL)-6, IL-8, and monocyte chemoattractant protein (MCP)-1 levels were detected in aqueous humor (0.1 ml) sampled during intravitreal injection. Aqueous humor controls were from nine cataract patients without any systemic disorders. The VEGF levels in aqueous humor were negatively related (r = ?0.373, p = 0.035) to CRT, which was positively related (r = 0.743, p < 0.001) to the number of injections. The VEGF levels before treatment and during the third injection in four patients with three or more injections were 13.42 ± 8.50 and 5.75 ± 3.68 (p = 0.055), respectively. The average best corrected visual acuity (BCVA) before and 12 months after treatment were 57.03 ± 16.15 and 75.16 ± 11.78 (p < 0.001), and the average CRT before and 12 months after treatment were 352.09 ± 84.15 and 251.13 ± 63.96 (p < 0.001), respectively. The visual improvement was negatively related (r = ?0.815, p < 0.001) to the visual baseline, and the vision 12 months after treatment was positively related (r = 0.581, p < 0.001) to that before treatment. No severe ocular or systemic complication appeared during treatment and follow-ups for all the patients. Intravitreal injection of anti-VEGF antibody ranibizumab is safe and effective for the treatment of idiopathic CNV through decreasing CRT. The patients with larger CRT baseline need more injections of ranibizumab.     

Regional chronologies of <Emphasis Type="Italic">Cedrela</Emphasis><Emphasis Type="Italic">fissilis</Emphasis> and <Emphasis Type="Italic">Cedrela angustifolia</Emphasis> in three forest types and their relation to climate

Key message

Although tree-ring chronologies of Cedrela fissilis and Cedrela angustifolia showed a common climatic signal, local conditions influence growth, suggesting that forest guidelines should be appropriate to the species and context.

Abstract

Cedrela species are highly valued because of the quality of their timber. Understanding the behaviour of each different Cedrela species and their ecology is of importance to ensuring that forest harvesting and management do not endanger the survival of natural populations. These species grow in a wide range of environmental gradients and different types of forests in Bolivia. This study used dendrochronological methods to analyse growth–precipitation relationships of two Cedrela species coming from three locations with different environmental conditions: dry Chiquitano (Concepción), Chiquitano transitional Amazonian (Guarayos), and Bolivian-Tucuman montane forests (Postrervalle). The rainy season in all locations runs from October to April and the dry season runs from May to September. Twelve Cedrela fissilis specimens were sampled from dry Chiquitano, 11 Cedrela fissilis specimens from Chiquitano transitional Amazonian, and 30 Cedrela angustifolia specimens from Bolivian-Tucuman montane forests. The samples were crossdated and exhibited a common signal between trees from three sites, despite tree rings from the Chiquitano transitional Amazonian forest being narrower and displaying blurred bands of parenchyma in the boundaries. Significant inter-series correlation was found for the C. fissilis species series from dry Chiquitano with r = 0.261 (p < 0.01) and Chiquitano transitional Amazonian forests with r = 0.284 (p < 0.01), and for Cedrela angustifolia from Bolivian-Tucuman montane forests with r = 0.374 (p < 0.01). Mean annual growth was 2.07, 1.92, and 2.82 mm year^?1 at the three sites, respectively. Cedrela species from dry Chiquitano and Bolivian-Tucuman montane forests were sensitive to precipitation from October to April of the current growth year (wettest season) and to low temperatures from May to July of the current growth year (driest season). Samples from Chiquitano transitional Amazonian were more sensitive to precipitation during late rainy season (March, April, and May of the current growth year) and high temperatures during the rainy months (November–December). Growth differences between sites and species in response to climate variations and local conditions should be taken into account and handled with different forest management guidelines.

     

Association study of protamine 2 (<Emphasis Type="Italic">PRM2</Emphasis>) gene polymorphism with male infertility in Chinese Han population

Protamine 2 (PRM2), an essential nuclear protein expressed in sperm, is known to be involved in the spermatogenesis. Although PRM2 defects have been reported to be involved in male infertility, studies for the relationship between male infertility and PRM2 polymorphisms are inconclusive. With the purpose to determine the association of PRM2 variant with male infertility in Chinese Han population, one single nucleotide polymorphism locus G398C in PRM2 which might play a role in semen quality was selected and the variant frequency was analyzed in 144 idiopathic infertile men (case group) and 111 proven-fertile men (control group) in the study. Three genotypes were discovered in the studied population and statistical analysis showed that the frequencies of GG and GC genotypes of PRM2 G398C were significantly different between the fertile and infertile men (P < 0.05) and GC genotype was associated with increased risk of male infertility (OR = 1.795, 95 % CI 1.070–3.013, P = 0.026). Further, the C allele distribution was significantly elevated in infertile group (OR = 1.484, 95 % CI 1.001–2.200, P = 0.049). Moreover, we discovered that sperm motility, progressive motility, sperm DNA integrity as well as nuclear maturity rate of GG genotype presented the highest values and were dramatically different with that of CC genotype (P < 0.05). Our results gave the first evidence that PRM2 G398C polymorphism was associated with the pathogenesis of male infertility and its genetic variation was in relation to semen quality in Chinese Han population.     

Repeated systemic administration of adipose tissue-derived mesenchymal stem cells prevents tracheal obliteration in a murine model of <Emphasis Type="Italic">bronchiolitis obliterans</Emphasis>

Objective

To investigate the effect of adipose tissue-derived mesenchymal stem cell (ASC) administered either systemically or locally in a murine model of bronchiolitis obliterans.

Results

When compared to controls, systemic treatment with 10⁶ ASCs on D0 and a second dose on D7 significantly prevented tracheal obliteration 28 days after heterotopic tracheal transplantation (median of 94 vs. 16%; P < 0.01). A single dose tended towards less stenosis than controls, but did not reach statistical significance (28 vs. 94%; P = 0.054). On the contrary, repeated local injection was incapable of preventing tracheal obliteration when compared to a single injection or controls (37 vs. 71 vs. 87%). Two intravenous doses also tended to be better than two local injections (16 vs. 37%; P = 0.058), and were better than a single local dose (16 vs. 71%; P < 0.01).

Conclusion

A second dose of ASC, given systemically after 7 days, reduces luminal obliteration in a heterotopic tracheal transplantation model in mice, suggesting that ASC can be used to prevent obliterative bronchiolitis after lung transplantation.

     

Serum Trace Elements and Electrolytes Are Associated with Fasting Plasma Glucose and HbA<Subscript>1c</Subscript> in Postmenopausal Women with Type 2 Diabetes Mellitus

The primary aim of the research was to assess the level of trace elements and electrolytes in serum of postmenopausal diabetic women. Sixty-four postmenopausal women with type 2 diabetes mellitus (DM2) and 64 age- and body mass index-matched controls were examined. Serum trace elements were assessed using inductively coupled plasma dynamic reaction cell mass spectrometry (ICP-DRC-MS). Fasting plasma glucose (FPG) and glycated hemoglobin (HbA_1c) levels were evaluated using Randox kits. The obtained data demonstrate that DM2 patients were characterized by 42 and 34 % higher FPG and HbA_1c levels, respectively (p < 0.001). The level of Cu and Se in diabetic postmenopausal women was increased by 10 and 15 % in comparison to the respective control values (p = 0.002 and <0.001). Serum Mn, Zn, and Ni concentrations were lower than the control ones by 32 % (p = 0.003), 8 % (p = 0.003), and 23 % (p = 0.046), respectively. FPG and HbA_1c levels directly correlated with serum Se (p < 0.001) and Cu (p = 0.014 and p = 0.028) concentrations and inversely related to Zn (p < 0.001) and Tl (p = 0.023 and p = 0.029) levels. Multiple regression analysis demonstrated a significant association between serum Zn and Se and FPG and HbA_1c levels. It is proposed that Zn and Se play an important role in DM2 pathogenesis. Further studies are required to assess the intimate mechanisms of the observed differences.