Reproductive strategy response of the fungi Sarocladium and the evaluation for remediation under stress of heavy metal Cd(II).

Cadmium (Cd) is documented as one of the most lethal metals and poses a major threat to all life forms in the environment due to its toxic effects. Bioremediation of hazardous metals has received considerable and growing interest over the years. The functional fungi with tolerance to the heavy metal Cd were screened from the mining soil samples. Two fungi isolates from coal mine soil were characterized as Sarocladium sp. M2 and Sarocladium sp. M6 based on morphological and partial ITS sequencing analysis. M2 and M6 exhibited high levels of resistance to cadmium, and they were investigated for their micro-morphology and application in heavy metal removal with different concentration Cd(II) (0, 50, 100, 150 and 200 mg/L). The colony morphology of M2 and M6 gradually become very similar to that of bacteria with the increase of cadmium concentration (150-200 mg/L). Micro-morphological studies showed that Cd(II) exposure caused the disappearance of conidial heads and the occurrence of hyphae breakage (100-200 mg/L Cd(II), which is consistent to the colony morphology results. The surface/volume ratio of the spores decreased with the presence of Cd(II). The removal potential of fungi for cadmium was quantified by atomic absorption spectrometry. M2 and M6 showed great potential as bioremediators for highly Cd(II)-contaminated environment. The highest Cd(II) biosorption capacity was 5.13 ± 0.21 mg/g for M2 and 6.04 ± 0.21 mg/g for M6. The highest heavy metal sorption by M2 removed 57.11% ± 4.45% Cd(II) while that of M6 removed 48.35% ± 1.44% Cd(II) in 200 mg/L initial concentration Cd(II). To the best of our knowledge, this is the first report that cadmium induced the change of reproduction mode of the Sarocladium, from conidia to arthrospores, which made the colony morphological modifications, from the fungi colony morphology to the bacteria colony morphology. The arthrospore-modified (hyphae breakage) seemed to accumulate greater amounts of heavy metals than filamentous hyphae formation.

Comment on the calculation of equilibrium constant and thermodynamic parameters by the distribution coefficient in Environmental Research 203 (2022) 111814.

This comment discussed the calculation of equilibrium constant and thermodynamic parameters in the paper of Kayalvizhi et al. (2022). This paper is of high academic value, however, there are some calculation and quotation errrors. The equilibrium constant K evolved from the distribution coefficient has a dimension of L/g, which is not the standard equilibrium constant, therefore, it cannot be used to calculate the thermodynamic parameters. This comment firstly analyzed the calculation errors made by Kayalvizhi et al. (2022), then established the relationship between distribution coefficient and standard equilibirum constant, and recalculated the thermodynamic parameters by using the correct distribution coefficient equation. As a research paper, authors not only provide the originality and novelty, but also give its correct results. This comment can bring to the attention of readers, authors, editors and reviewers about the thermodynamic calculation. At the same time, it is helpful to avoid the misuse and propagation of the incorrect equation in the area of adsorption thermodynamics.

Comment on the thermodynamic calculation using the non-standard equilibrium constant re Jemutai-Kimosop et al. (2020) and Conde-Cid et al. (2019).

This comment discussed the calculation of thermodynamic parameters about the paper of Jemutai-Kimosop et al. (2020) and Conde-Cid et al. (2019). Although these articles are valuable, in these cases, the equilibrium constant (K), which is used for the calculation of thermodynamic parameter is incorrect. The root reason for the errors in both articles is that the non-standard equilibrium constant was used to calculate the thermodynamic parameter, which is contrary to the principle of thermodynamics. This comment provided a correct method for the calculation of the standard equilibrium constant by using the distribution coefficient and Langmuir equation. This note can avoid the misuse and propagation of the incorrect equation in the area of adsorption thermodynamics.

The prevalence of human T-cell leukemia virus in blood donors in China.

BACKGROUND: China has not yet incorporated routine human T-lymphotropic virus (HTLV)-1/2 blood donor screening, even though HTLV has been reported in the southeastern coastal region. This study was conducted to investigate the prevalence of HTLV in five major regions across of China. METHODS: From January 2016 to December 2017, blood samples were collected in 20 blood centers located in different regions of China. These samples were screened for HTLV-1/2 antibodies using enzyme-linked immunosorbent assay (ELISA). If the test samples were reactive, the samples were confirmed with a western blot (WB) assay. If the results of WB were indeterminate, the donor was interviewed after a minimum lapse of 8 weeks. All follow-up samples from donors were tested for anti-HTLV-1/2 with ELISA and WB. RESULTS: There were 875,453 donor samples tested for anti-HTLV-1/2 by ELISA. In all, 365 samples tested negative, 22 samples tested positive by WB, and 14 samples with HTLV status undetermined due to being lost to follow-up. The prevalences were 11.09, 5.96, 3.16, 2.88 and 0.98 per 100,000 in Xiamen, Changsha, Beijing, Shenzhen, and Nanjing blood center, respectively. The prevalences were 0 per 100,000 for all 15 other blood centers. There was significant differences in the prevalence of HTLV in different regions of China (p = 0.0011). CONCLUSION: In China, HTLV-1 confirmed positive donors are mainly from southeastern coastal areas. It may be necessary to conduct HTLV screening in these areas to reduce the risk of transfusion-transmitted HTLV.

Inhibition of Suicidal Erythrocyte Death by Indirubin-3'-Monoxime.

BACKGROUND/AIMS: Qing Dai is a prized traditional Chinese medicine whose major component, indirubin, and its derivative, indirubin-3'-monoxime (IDM), have inhibitory effects on the growth of many human tumor cells and pronounced anti-leukemic activities. However, the effects of IDM on mature human erythrocytes are unclear. This study aimed to evaluate the potential impact of IDM on erythrocytes and the mechanisms underlying that impact. METHODS: Utilizing flow cytometry and confocal laser scanning microscopy, phosphatidylserine exposure at the cell surface was estimated by annexin V-fluorescein isothiocyanate (FITC). The relative cell size, expressed in arbitrary units, was evaluated by forward scatter in a flow cytometer. Fluo-3 fluorescence was used to bewrite changes in cytosolic Ca2+ activity, reactive oxygen species (ROS) formation was assessed by 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescence, and ceramide abundance was evaluated by FITC-conjugated specific antibodies. RESULTS: The 24-h exposure of human erythrocytes to IDM (12 µM) significantly decreased the percentage of annexin V-binding erythrocytes and the intracellular calcium concentration ([Ca2+]i). IDM (3-12 µM) did not significantly modify the ceramide level or DCFH-DA fluorescence. Energy depletion (removal of glucose for 24 hours) significantly increased annexin V binding and Fluo-3 fluorescence and diminished forward scatter, and these effects were significantly mitigated by IDM (12 µM). Moreover, the Ca2+ ionophore ionomycin (1 µM, 60 min) and oxidative stress (30 min exposure to 0.05 mM tert-butyl hydroperoxide, t-BHP) similarly triggered eryptosis, which was also significantly suppressed by IDM. CONCLUSIONS: IDM is a novel inhibitor of suicidal erythrocyte death following ionomycin treatment, t-BHP treatment and energy depletion. Thus, IDM may counteract anemia and impairment of microcirculation, at least in part, by inhibition of Ca2+ entry into erythrocytes.

Eryptosis as an Underlying Mechanism in Systemic Lupus Erythematosus-Related Anemia.

BACKGROUND: The progression of systemic lupus erythematosus (SLE) leads to anemia in patients, adversely affecting prognosis. The diverse causes of anemia may include excessive eryptosis or premature suicidal erythrocyte death characterized by cell shrinkage and phosphatidylserine (PS) exposure on the cell surface. The present study explored if SLE enhances eryptosis and the underlying mechanisms. MATERIALS AND METHODS: Eryptosis was assessed using flow cytometry in healthy volunteers (n = 20) and anemic patients hospitalized for SLE (n = 22), for parameters including PS exposure, cell volume, cytosolic calcium ion (Ca2+) levels and reactive oxygen species (ROS) and ceramide abundance. These indicators were measured in erythrocytes of experimental subjects and erythrocytes treated with plasma from healthy volunteers or SLE patients. RESULTS: The hemoglobin and hematocrit levels were significantly lower in anemic SLE patients than in healthy volunteers (***p<0.001, p<0.001, respectively). The percentage of PS-exposing erythrocytes was significantly higher in SLE patients than in healthy volunteers (p<0.001), accompanied by an increase in cytosolic Ca2+ levels, oxidative stress. The measurements of PS and Ca2+ levels were significantly higher in the erythrocytes of healthy volunteers following incubation in plasma of SLE patients than in plasma of healthy volunteers for 24h (***p<0.001, *p<0.05 respectively). CONCLUSION: Eryptosis is enhanced in SLE and may contribute to anemia. The probable underlying mechanisms may be an excessive formation of ROS in erythrocytes. Also, some plasma components may trigger eryptosis by increasing the cytosolic Ca2+ concentration.

Discussion on the thermodynamic calculation and adsorption spontaneity re Ofudje et al. (2023).

Accurate calculations and precise results are very important for the dissemination of scientific knowledge, whereas the errors of calculation will diminish the academic value of the paper. This discussion focuses on the calculation of thermodynamics and the determination of the spontaneity of adsorption processes in the paper of Ofudje et al. (2023). Ofudje et al. found that the apatite synthesized by chemical method (CHAp) has excellent adsorption properties for cadmium ions, which is an important contribution to the remediation of cadmium pollution. However, the calculation results of standard Gibbs free energy change (ΔGo), standard enthalpy change (ΔHo) and standard entropy change (ΔSo) of the adsorption of Cd2+ onto CHAp surface need to be corrected due to an incorrect calculation. Firstly, the partition coefficient (KD) with a dimension cannot be used for thermodynamic calculation. Secondly, the adsorbent mass (m) described by Ofudje et al. in different Sections is inconsistent, leading to incorrect results of Ko and ΔGo. When the appropriate value of the adsorbent mass is selected and the partition coefficient is converted to the standard adsorption equilibrium constant Ko, the calculated ΔGo is less than zero, which means that the adsorption is spontaneous. This discussion provides the correct calculation method of standard adsorption equilibrium constants and thermodynamic parameters, which can improve the reader's judgment and understanding of adsorption spontaneity.

Biotransformation of Bioactive Metabolites in Cassiae Semen by Endogenous Enzymes and Probiotics.

The present study evaluated the potential of endogenous enzymes and probiotics in transforming bioactive metabolites to reduce the purgative effect and improve the functional activity of Cassiae Semen and verified and revealed the biotransformation effect of endogenous enzymes. Although probiotics, especially Lactobacillus rhamnosus, exerted the transformation effect, the endogenous enzymes proved to be more effective in transforming the components of Cassiae Semen. After biotransformation by endogenous enzymes for 12 h, the levels of six anthraquinones in Cassiae Semen increased by at least 2.98-fold, and free anthraquinones, total phenolics, and antioxidant activity also showed significant improvement, accompanied by an 82.2% reduction in combined anthraquinones responsible for the purgative effect of Cassiae Semen. Further metabolomic analysis revealed that the biotransformation effect of endogenous enzymes on the bioactive metabolites of Cassiae Semen was complex and diverse, and the biotransformation of quinones and flavonoids was particularly prominent and occurred by three primary mechanisms, hydrolyzation, methylation, and dimerization, might under the action of glycosyl hydrolases, SAM-dependent methyltransferases, and CYP450s. Accordingly, biotransformation by endogenous enzymes emerges as a mild, economical, food safety risk-free, and effective strategy to modify Cassiae Semen into an excellent functional food.

Effects of Pb(II) and Zn(II) Contamination on Adsorption, Desorption and Degradation of Cry1Ac Toxin Identical to Bt Transgenic Poplar in Black Soil.

Bt transgenic white poplar has been commercially planted in China since 2002, and it showed obvious insect resistance in the field. However, the ecological risk of planting Bt transgenic poplar in a field contaminated with heavy metals has received little attention. The effects of Pb(II) and Zn(II) contamination on the adsorption, desorption and degradation of Bt toxin identical to Bt transgenic poplar in black soil were studied. The results showed that the adsorption of Bt toxin was enhanced and the desorption of Bt toxin was inhibited in black soil by Pb(II) and Zn(II) at concentrations between 0 and 1 mmol/L, and the effect of Pb(II) on Bt toxin was greater than that of Zn(II). In the presence of heavy metal ions, the Cry1Ac toxin molecules are oriented with domain I toward soil particles through the metal ion bridge. The promoting mechanism of Bt toxin adsorption by heavy metal ions in black soil is mainly attributed to cation-controlled electrostatic attraction (CCEA), which is different from patch-controlled electrostatic attraction (PCEA). With the increase in soil concentration from 1 to 4 mg/mL, the adsorption amount of Bt toxin showed a downward trend, and both Pb(II) and Zn(II) had the maximal promotion effect when the soil concentration was 2 mg/mL. The promoting effect of Zn(II) on the adsorption of Bt toxin increased with the increased temperature (5-45 °C), but the promoting effect of Pb(II) was maximal at 25 °C. Both Pb(II) and Zn(II) affected the degradation characteristics of Bt toxin in black soil. For the lead-contaminated black soil, the residual amount of Bt toxin increased in the early stage but decreased in the later stage compared to the control soil. For the zinc-contaminated black soil, the residual amount of Bt toxin decreased compared to the control soil except between the second and tenth days. In this study, it was observed that Bt toxin was degraded rapidly in the early stage, followed by a large amount of released Bt toxin and slow degradation in the middle and late stages.

Evaluation of nutritional value, bioactivity and mineral content of quinoa bran in China and its potential use in the food industry.

Quinoa bran is a by-product during quinoa processing, which is not well used due to its high content of antinutritional factors. The nutritional, antinutritional, antioxidative and mineral content were analyzed in quinoa bran from five producing areas (Hebei, Shanxi, Qinghai, Inner Mongolia and Gansu Province) in China. The results showed that the mean values of protein, starch, fat, fiber, reducing sugar, ash, moisture and energy in quinoa bran were 9.35%, 47.37%, 8.26%, 10.74%, 3.68%, 6.25%, 9.29% and 360.2 kcal/100 g, respectively. Although the protein content in quinoa bran is lower than that in quinoa grain, it is comparable to that in other grains (rice, corn, millet and sorghum) and brans (wheat, oat and rice), so it has the commercial potential to be processed into animal feed or other edible food. The contents of antioxidant flavonoids (460.9 mg/100g) and polyphenols (477.8 mg/100 g) in quinoa bran were higher than those in quinoa grain, suggesting that quinoa bran had better antioxidant capacity. The contents of saponins, tannins and phytic acid in quinoa bran were 18.65, 0.30 and 0.73%, respectively. The content of saponins was nearly one times higher than that in quinoa grain, the contents of tannins and phytic acid, however, were lower than those in quinoa grain. Therefore, the removal of saponins is the key to eliminate the antinutritional properties of quinoa bran. The contents of macroelements (sodium, potassium, calcium, magnesium, phosphorus) and microelements (iron, manganese, copper, zinc, cobalt, molybdenum, selenium, barium) in quinoa bran were generally higher than those in quinoa grain, which was consistent with the results of ash determination. In summary, quinoa bran was found to be a rich source of nutritional and bioactive components and minerals. If the antinutritional problem can be overcome, quinoa bran has great potential for application in the food industry.

Prediction of the potentially suitable areas of Leonurus japonicus in China based on future climate change using the optimized MaxEnt model.

Leonurus japonicus Houtt. is a traditional Chinese medicinal plant with high medicinal and edible value. Wild L. japonicus resources have reduced dramatically in recent years. This study predicted the response of distribution range of L. japonicus to climate change in China, which provided scientific basis for the conservation and utilization. In this study, 489 occurrence points of L. japonicus were selected based on GIS technology and spThin package. The default parameters of MaxEnt model were adjusted by using ENMeva1 package of R environment, and the optimized MaxEnt model was used to analyze the distribution of L. japonicus. When the feature combination in the model parameters is hing and the regularization multiplier is 1.5, the MaxEnt model has a higher degree of optimization. With the AUC of 0.830, our model showed a good predictive performance. The results showed that L. japonicus were widely distributed in the current period. The maximum temperature of warmest month, the min temperature of coldest month, the precipitation of wettest month, the precipitation of driest month, and altitude were the main environmental factors affecting the distribution of L. japonicus. Under the three climate change scenarios, the suitable distribution area of L. japonicus will range shift to high latitudes, indicating that the distribution of L. japonicus has a strong response to climate change. The regional change rate is the lowest under the SSP126-2090s scenario and the highest under the SSP585-2090s scenario.

Prediction of potential distribution areas and priority protected areas of Agastache rugosa based on Maxent model and Marxan model.

Agastache rugosa (Fisch. & C. A. Mey.) Kuntze has been widely studied because of its high medicinal and edible value. Establishing the priority protected area of wild A. rugosa can provide scientific basis for the protection of germplasm resources. In this study, we predicted the potential suitability distribution area of A. rugosa under the current and future climate scenarios with the MaxEnt model, and the dominant climate factors affecting the distribution of A. rugosa were analyzed. Based on the above results, we predicted the priority protected areas of A. rugosa with the Marxan model. The results showed that A. rugosa is mainly distributed in the eastern and central regions of China at present. In future, the suitable area of A. rugosa will increase, otherwise a few areas will shrink back and migrate to the high latitude areas as a whole. Hydrothermal conditions are the main environmental factors affecting the distribution of A. rugosa. The priority protected areas of A. rugosa are mainly distributed in Chongqing, eastern Sichuan, southern Guizhou, western Hunan and Hubei and southwestern Shaanxi, which are basically consistent with the highly suitable areas predicted by Maxent model. The results of this study are of great significance for the protection and rational utilization of species of Agastache.

Correction to the thermodynamic calculation using the Langmuir isotherm model by Saeed et al. (2022).

The thermodynamic parameters are usually used to analyze the spontaneity, thermal and random change of the adsorption process, therefore, it is important to obtain these parameters accurately. Recently, Saeed et al. (2022) published a high academic paper to reveal the adsorption properties and mechanism of dyes onto the chitosan composite of the iron metal-organic framework (CS/MOF-235). However, the thermodynamic parameters of ΔG and ΔS need to be corrected because their results affected the analyzed conclusions. In the present study, the thermodynamic parameters were recalculated by using the correct method through the Langmuir isotherm model. The recalculated thermodynamic parameters indicated that the change in free energy (ΔG) for the removal of methylene blue (MB) and methyl orange (MO) onto CS/MOF-235 is negative but not positive, meaning that the adsorption of dyes (MB and MO) is spontaneous but not non-spontaneous. The change in entropy (ΔS) is positive but not negative, implying that the randomness increases but not decreases during the adsorption process. The enthalpy (ΔH) maintained negative value and the entropy (ΔS) obtained positive value indicate that both of them are the driving forces of the adsorption.

Discussion on the thermodynamic calculation by distribution constant in Journal of Contaminant Hydrology 243 (2021) 103906.

This comment discussed the calculation of thermodynamic parameters about the paper published in Journal of Contaminant Hydrology 243 (2021) 103906. Despite the paper's novelty and innovation, the distribution constant (Kd) with a dimension of L/mg is not the standard equilibrium constant (Ko), therefore, it cannot be used to calculate the thermodynamic parameters. By analyzing the incorrect results and providing the reasonable method for calculating the thermodynamic parameters，this comment is helpful to understand the calculation principle of thermodynamic parameters by the distribution constant and avoid the misuse of incorrect formulas.

Comments on the calculation of the standard equilibrium constant using the Langmuir model in Journal of Hazardous Materials 422 (2022) 126863.

The calculation of equilibrium constant from the Langmuir model is widely used in the literature. However, the dimensional problem of the equilibrium constant has often been ignored. For example, a recent publication on the strong properties of the new adsorbent for tetracycline. Nevertheless, this used an improper calculation of the standard equilibrium constant, confusing solute and solution. This creates dimension for K0 obtained from Eq. (13). Unfortunately, K0 should be dimensionless. In this comment, the origin and background of this kind of error is analyzed and the method for correcting the error is presented. Specifically, we establish a clear relationship between the Langmuir constant (KL) and the standard equilibrium constant (K0). We hope this comment clarifies the essence of calculating the standard equilibrium constant by using the Langmuir model, so others avoid the propagation of this kind of error.

[Advances in effects of insecticidal crystal proteins released from transgenic Bt crops on soil ecology].

With the large scale cultivation of transgenic crops expressing Bacillus thuringiensis (Bt) insecticidal crystal proteins in the world, the problem of environmental safety caused by these Bt crops has received extensive attention. These insecticidal crystal proteins can be released into the soil continuously in the growing period of Bt plants. If their accumulation of the insecticidal crystal proteins exceeds consumption by insect larvae and degradation by the environmental factors, these insecticidal crystal proteins could constitute a hazard to non-target insects and soil microbiota. There are three main ways to release insecticidal crystal proteins into soil for Bt plants: root exudates, pollen falling, and crop reside returning. The Bt insecticidal crystal proteins released into soil can be adsorbed rapidly by active soil particles and the absorption equilibrium attained within 1-3 h. The adsorption protects Bt insecticidal crystal proteins against soil microbial degradation or enzyme degradation, which leads to remarkable prolong of the persistence of insecticidal activity. The change of soil microorganism species is an important index for evaluating the effect of Bt plants on soil ecology. The research showed that these insecticidal crystal proteins released by the Bt plant root exudates or Bt organism had no toxicity to the soil earthworms, nematodes, protozoa, bacteria and fungi; however, it could reduce the mycelium length of the arbuscular mycorrhizal fungi (AMF) and restrain AMF to form invasion unit. The influencing degree of Bt protein on soil enzyme activity varied with the releasing modes or growth period of Bt crops. Bt Cry1Ab protein can be taken up from soil by parts of following crops; however, different results were obtained with different commercial kits. To better understand the soil ecological evaluation about the insecticidal crystal proteins released from transgenic Bt crops, this review provides a comprehensive overview about the release, adsorption and residue of Bt insecticidal crystal proteins in soil, as well as their effects on soil protozoa, soil microorganism, soil enzyme activity and following crops.

Dynamical behavior of a hepatitis B virus transmission model with vaccination.

Hepatitis B virus (HBV) infection is a globally health problem. In 2005, the WHO Western Pacific Regional Office set a goal of reducing chronic HBV infection rate to less than 2% among children five years of age by 2012, as an interim milestone towards the final goal of less than 1%. Many countries made some plans (such as free HBV vaccination program for all neonates in China now) to control the transmission HBV. We develop a model to explore the impact of vaccination and other controlling measures of HBV infection. The model has simple dynamical behavior which has a globally asymptotically stable disease-free equilibrium when the basic reproduction number R(0)< or =1, and a globally asymptotically stable endemic equilibrium when R(0)>1. Numerical simulation results show that the vaccination is a very effective measure to control the infection and they also give some useful comments on controlling the transmission of HBV.

Correction to the calculation of Polanyi potential from Dubinnin-Rudushkevich equation.

The Dubinin-Radushkevitch (D-R) adsorption isotherm model has been widely used to measure adsorption, and it can be used to value the mean free adsorption energy (E). In order to do this, the first step is to calculate the Polanyi adsorption potential (Ɛ). However, we find that the computational formula of Polanyi potential from the D-R equation has a dimensinal problem. Specifically, the units of the equilibrium concentration of solute (Ce) used in the Polanyi potential equation are in chaos. Accurate calculation is the basic characteristic of scientific papers. This comment highlighted the possible flaws related to the calculation of Polanyi potential and analyzed the evolution and application of Polanyi potential equation, and then provided the modified form of Polanyi potential equation. This note offers deeper insights into the Polanyi potential equation, and can help avoid misuse and propagation of the Polanyi potential equation in adsorption research.

Functionally Graded Scaffolds with Programmable Pore Size Distribution Based on Triply Periodic Minimal Surface Fabricated by Selective Laser Melting.

Functional graded materials are gaining increasing attention in tissue engineering (TE) due to their superior mechanical properties and high biocompatibility. Triply periodic minimal surface (TPMS) has the capability to produce smooth surfaces and interconnectivity, which are very essential for bone scaffolds. To further enhance the versatility of TPMS, a parametric design method for functionally graded scaffold (FGS) with programmable pore size distribution is proposed in this study. Combining the relative density and unit cell size, the effect of design parameters on the pore size was also considered to effectively govern the distribution of pores in generating FGS. We made use of Gyroid to generate different types of FGS, which were then fabricated using selective laser melting (SLM), followed by investigation and comparison of their structural characteristics and mechanical properties. Their morphological features could be effectively controlled, indicating that TPMS was an effective way to achieve functional gradients which had bone-mimicking architectures. In terms of mechanical performance, the proposed FGS could achieve similar mechanical response under compression tests compared to the reference FGS with the same range of density gradient. The proposed method with control over pore size allows for effectively generating porous scaffolds with tailored properties which are potentially adopted in various fields.