However, the administration of dapagliflozin resulted in only 30–50% inhibition of reabsorption. This study was aimed at investigating the mechanism underlying the discrepancy between the expected and observed levels of glucose reabsorption. To this end, systems pharmacology models were developed to analyze the time profile of dapagliflozin, canagliflozin, ipragliflozin, empagliflozin, and tofogliflozin in the plasma and urine; their filtration and active secretion from the blood to the renal proximal tubules; reverse reabsorption; urinary excretion; and their inhibitory effect on SGLT2. The model shows that concentration levels of tofogliflozin, ipragliflozin, and empagliflozin are higher than levels of other inhibitors following administration of marketed SGLT2 inhibitors at labeled doses and non-marketed SGLT2 inhibitors at maximal doses (approved for phase 2/3 studies). All the compounds exhibited almost 100% inhibition of SGLT2. Based on the results of our model, two explanations for the observed low efficacy of SGLT2 inhibitors were supported: (1) the site of action of SGLT2 inhibitors is not in the lumen of the kidney's proximal tubules, but elsewhere ; and (2) there are other transporters that could facilitate glucose reabsorption under the conditions of SGLT2 inhibition .The Renal sodium-dependent glucose co-transporter 2 (SGLT2) is one of the most promising targets for the treatment of type 2 diabetes. Two SGLT2 inhibitors, dapagliflozin, and canagliflozin, have already been approved for use in USA and Europe; several additional compounds are also being developed for this purpose. Based on the  Type 2 diabetes mellitus (T2DM) is a metabolic disorder that is characterized by hyperglycemia resulting from insulin resistance and relative lack of insulin. Current therapies for T2DM primarily address endocrine pathogenesis of insulin resistance and b-cell dysfunction. Consequently, many patients receive multiple glucose-lowering therapies and eventually require exogenous insulin administration. However, in clinical practice patients often fail to meet the targets for glycemic control  is low.The site of action of the SGLT2 inhibitors is in the proximal tubule cells of the kidney, but not in the lumen.Other transporters facilitate glucose reabsorption under conditions of SGLT2 inhibition.Currently, two SGLT2 inhibitors are marketed  and several more are in development . Based on inhibitory concentration 50% (IC50) values measured To explore these hypotheses further, it is possible to adopt a systems pharmacology modeling (SPM) approach.Many mathematical models that describe development and treatment of T2DM appear in the literature, and Ajmera et al.  present The aim of this study was to construct a model that describes the active secretion of SGLT2 inhibitors from plasma into the lumen of the proximal tubules, reverse reabsorption, and urinary excretion. Using an SPM approach, our objective was to test the hypotheses used to explain the discrepancy between expected and observed levels of glucose reabsorption following administrations of SGLT2 inhibitors (see above). We also aimed to compare the efficacies of different SGLT2 inhibitors by simulating their concentration level in the lumen of the kidney's proximal tubules and estimating the level of inhibition produced during treatment in humans.A family of semi-mechanistic PK/PD models was developed to describe administration, degradation, transport, glomerular filtration, active secretion, reverse reabsorption, and urinary excretion of 5 SGLT2 inhibitors . The assumptions used for model development are presented in Table plasma, Vlumen, GFRV, and Qurine) that have equal values in each model. The physiological parameter values were taken  from the literature, as were those for 2 PK parameters (F and fup) and 6 PD parameters (IC50 for SGLT1–6). The remaining 7 or 5 parameters were fitted against PK data on the dynamics of compounds in plasma and urine. The 95% confidence intervals were calculated for fitted parameters. Parameter values, 95% confidence intervals, and the source of data for their identification are presented in Supplementary Table The strategy of model verification is presented in the Supplementary Materials. The dapagliflozin, ipragliflozin, and tofogliflozin models include 19 parameters: 4 physiological, 9 drug specific PK, and 6 drug specific PD. In the right hand side of canagliflozin or empagliflozin models, 17 parameters are included: 4 physiological, 7 drug specific PK, and 6 drug specific PD. The parameter values are specific to each SGLT2 inhibitor model, with the exception of 4 physiological parameters . In the models that described dapagliflozin, ipragliflozin, and tofogliflozin, distribution volume in peripheral compartments was fitted against PK data. Compartment volumes and the source of identification are presented in Supplementary Table Model simulations are presented as curves  with shadows (95% confidence bands) or as bars  with error bars (95% confidence bands). The description of simulating the 95% confidence bands is presented in the Supplementary Materials.All data available in the literature was used for model verification and validation Table . Models To analyze and compare the concentration of drugs in the lumen of the kidney's proximal tubules–the predicted site of inhibition–we simulated time profiles of the drugs resulting from oral administration at various dosages. Figure In order to investigate which of five compounds is most likely to accumulate at the potential site of action, we also simulated concentration levels of SGLT2 inhibitors in the lumen of the kidney's proximal tubules following a standardized dosage of the compounds equal to 20 mg. In this scenario, the model predicted that concentrations of empagliflozin and tofogliflozin were higher than the levels of dapagliflozin, canagliflozin, and ipragliflozin   predicted by the model is higher than glucose reabsorption inhibition level measured in clinical trials, and almost equal to 100%. Thus, if the lumen of the kidney's proximal tubules is indeed the site of action of SGLT2 inhibitors, and SGLT2 is the main transporter that facilitates reabsorption of glucose, then there is a contradiction between the simulations produced by the model and the clinical data. This contradiction leads us to support two hypotheses proposed by Liu et al. (The model shows that administration of labeled doses of marketed inhibitors (dapagliflozin and canagliflozin) and of maximal doses approved for phase 2/3 studies of tofogliflozin, ipragliflozin, and empagliflozin leads to almost complete inhibition of SGLT2 u et al. . Firstlyin vitro IC50 values. The effect of SGLT2 inhibitors on other transporters is also rather strong because of its high concentration in kidney's lumen. Canagliflozin is the strongest SGLT1 and SGLT6 inhibitor, and this is caused by canagliflozin's low IC50 . The most effective SGLT3 inhibitors are ipragliflozin and tofogliflozin because of their high in vitro potencies. All compounds have similar effects on SGLT4, except dapagliflozin. Dapagliflozin has the weakest influence on SGLT4 because of its low concentration in the kidney's lumen. Ipragliflozin and empagliflozin are the strongest SGLT5 inhibitors because of their low IC50 of for SGLT5, and the high concentration of ipragliflozin in the lumen.The model was applied to compare the efficacy of different SGLT2 inhibitors in respect of inhibiting other transporters in the SGLT family that are expressed in kidneys. Simulating the administration of equal doses of compounds (20 mg), labeled doses of marketed inhibitors, and maximal doses of other inhibitors approved for phase 2/3 studies showed that levels of SGLT2 inhibition are similar for all compounds and almost equal to 100%. This is caused by high concentrations of SGLT2 inhibitors in the lumen of the kidneys and low In conclusions, we have developed a systems pharmacology model for SGLT2 inhibitors that enables the estimation of its concentration in the lumen of the kidney's proximal tubules  and the prediction of SGLT2 inhibition levels during treatment in humans. We have shown that the concentration of SGLT2 inhibitors in the lumen of the proximal tubules is high, and that the level of SGLT2 inhibition during treatment in humans is almost 100%. Based on the results of our model, two explanations for the observed low efficacy of SGLT2 inhibitors were supported: (1) the site of action of SGLT2 inhibitors is not in the lumen of the kidney's proximal tubules, but elsewhere ; and (2) there are other transporters that could facilitate glucose reabsorption under the conditions of SGLT2 inhibition . It was found that all SGLT2 inhibitors have similar efficacy.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


AbstractAnastrephafraterculus, is a complex of cryptic species, but few studies have been performed on the morphology of its immature stages. An analysis of mandible shape and linear morphometric variability was applied to third-instar larvae of five morphotypes of the Anastrephafraterculus complex: Mexican, Andean, Ecuadorian, Peruvian and Brazilian-1. Outline geometric morphometry was used to study the mouth hook shape and linear morphometry analysis was performed using 24 linear measurements of the body, cephalopharyngeal skeleton, mouth hook and hypopharyngeal sclerite. Different morphotypes were grouped accurately using canonical discriminant analyses of both the geometric and linear morphometry. The shape of the mandible differed among the morphotypes, and the anterior spiracle length, number of tubules of the anterior spiracle, length and height of the mouth hook and length of the cephalopharyngeal skeleton were the most significant variables in the linear morphometric analysis. Third-instar larvae provide useful characters for studies of cryptic species in the Anastrephafraterculus complex.The occurrence of cryptic species among economically important fruit flies strongly affects the development of management tactics for these pests. Tools for studying cryptic species not only facilitate evolutionary and systematic studies, but they also provide support for fruit fly management and quarantine activities. Previous studies have shown that the South American fruit fly, PageBreak Tephritidae family are among the most important pests for agriculture because of their direct effects on fruit production and the quarantine restrictions imposed to prevent the transfer of foreign species from one region to another (sterile insect technique (SIT), the use of pheromones, the determination of pest-free or low-prevalence areas and quarantine measures or risk analysis , is a species of great economic importance within the genus and is subject to quarantine restrictions. It is widely distributed in America and is associated with a large number of host fruits . Through the use of geometric morphometry of the shape of the mouth hook and linear morphometry of larvae, we tested several variables and determined their usefulness in the differentiation of these morphotypes.The aim of this study was to perform a comparative analysis of third instar larvae of representatives of five morphotypes of the Biological material. The taxonomic identity of all larvae used in this study was fully known from associated reared adults and the diagnoses developed by PageBreakfrom natural populations. The samples from Colombia and Brazil came from colonies reared for a few generations on host fruit in laboratory conditions. The sample from Peru came from a laboratory colony maintained on an artificial diet since 2002 at the laboratories of the International Atomic Energy Agency, in Seibersdorf, Austria. The sample of the Brazilian-1 morphotype was collected in the field and identified by one of the co-authors (DS) in the same location at which previous genetic, cytogenetic and morphometric studies were conducted with adults of this morphotype . The mouth hook at 400× magnification resulted in a 3D figure with blurred edges; therefore, multiple shots (between six and 10) were taken at different focal planes and later assembled with the software Helicon Focus 6.0.18  (is (EFA) , for whiis (EFA)  were useLinear morphometry. Samples were compared with a discriminant function analysis (DFA) applied over either linear measures between two points or the ratio between them. Measurements suggested by Steck and Wharton (1989), The mouth hook morphology was observed carefully. Its shows a medial nub in the ventral curve, where the cuticle and muscles attaches, with a front and a rear notches next to it that extend to the top; a posterior apodema, like a neck, is also found. The anterior part of the dorsal apodema could be found where the slope turns greater Figure .All measurements were done on the digitized images of the structures. After variables were defined, measurements were performed three times by a single observer , but no differences in outcomes were found. Twenty-four variables were used, 15 of which corresponded to linear measurements, and nine to the ratios between various pairs Figure .: body lengthBL; : body width at the sixth abdominal tergiteBW; : cephalopharyngeal skeleton length, from the anterior apex of the mandible to the end of the ventral cornua, at lower end of the dorsal cornuaCSL; : hypopharyngeal sclerite length, from mouth hook joint to the rear distal pointHSL; and PageBreakPageBreaksclerite at the anterior base of the hypopharyngeal bridge, perpendicular to the upper edge: height of the hypopharyngeal HSH. The measurements of the mouth hook were : length from the apex to the ventral apodemeM1, : length from the apex to the dorsal most tip of neckM2, : length from the apex to the anterior base of the dorsal apodemeM3, : height from the apex of the ventral apodeme to the anterior base of the dorsal apodemeM4, : depth of ventral concavity from line M1 to tip of nubM5, : thickness of mouthhook at posterior base of nub by the posterior notchM6, : distance between the posterior base of nub and dorsal most tip of neckM7, and : width of the ventral apodeme at the base of the neck, in a line parallel to M1M8. : width of the left anterior spiracle between the apices of the most extreme tubulesASL, : number of tubules of the anterior spiraclesAST, : BL/BWX1, : M1/M4X2, : M2/M4X3, : M1/M5X4, : M2/M5X5, : M3/M4X6, : CSL/HSLX7, : CSL/M3X8, and : CSL/M1X9.Data analysis. The shape of the mouth hook was studied with an outline analysis in a two-dimensional plane, for which an EFA  on the standardized coefficients. Based on the first principal components obtained, a DFA was performed, and afterwards, each individual was reclassified through a Jackknife procedure.h an EFA  was usedDFA was performed on the complete dataset, with the forward stepwise method. A canonical analysis was done to determine the canonical variables and their significance through a Chi-squared test. All analyses were performed using Statistica 12 ; 10 of the variables resulted in statistically significant differences for the segregation of the morphotypes =12.224, p<0.0001) (Table body length (BL) was greater in the Peruvian, Ecuadorian and Mexican morphotypes, and body width (BW) was greater in the Andean morphotype; the ratio between these two measurements (X1) was lower in the Andean population. The HSL was, on average, longer in the Andean and Ecuadorian morphotypes. The mouth hook length between the apex and the ventral apodeme (M1) was shorter for the Peruvian morphotype; the mean width in the middle part of the mouth hook (M6) was greater for individuals of the Ecuadorian morphotype and smallest for the Peruvian morphotypes. The average basal width of the ventral apodeme (M8) was narrower in specimens of the Brazilian-1 and Peruvian morphotypes. The anterior spiracle PageBreakwas longer in specimens of the Andean morphotype, and the number of spiracle tubules was smaller in the Brazilian-1 and Peruvian morphotypes. The X9 ratio between the CSL and the length of the mouth hook from the apex to the ventral apodeme (M1) was greater for Peruvian individuals and smaller for Mexican individuals  and the number of tubules of the anterior spiracle (AST), followed in importance by the hypopharyngeal sclerite length (HSL), body width (BW), and dimensions of the mouth hook M3  and M8 . The most important variables for CV-2 were the ASL, BL/BW (X1) and mouth hook length/width (X2), followed by the BL and HSL (Table The canonical analysis resulted in four canonical roots, and the Chi-squared test showed statistical significance for all the roots. CV-1 had 51.6% of the discrimination power, CV-2 had 24.2%, CV-3 had 19.3% and CV-4 had 4.9%. In the first root, variables with major contribution to the separation of the groups were the PageBreakPageBreakPageBreakThe prediction model indicated that 96% of the individuals were correctly placed in their respective morphotypes; all of the Andean and Ecuadorian specimens were properly classified, and only two individuals from Brazilian-1, one from Mexico and one from Peru were incorrectly classified Table .The results obtained from our study of the mouth hook shape of the third-instar larvae established that variation exists in the shape of this structure that usefully separates the five morphotypes. Moreover, it was possible to confirm the presence of variability in the dorsal and ventral apodeme areas. Geometric morphometry is a sensitive tool to study the presence of cryptic species , 2014, aAnastrepha. The results obtained from our study suggest that the methods proposed here could be used for developing an identification system of this type that extends to larvae.Geometric morphometry has been used for differentiation of fruit flies , in all PageBreakof laboratory rearing (The results of the linear morphometry were also highly satisfactory, reaching a 96% accuracy of the predicted classification for the studied individuals. According to what was previously reported regarding size variation of insects through generations  rearing , it is pAnastrepha based on the study of third-instar larvae. The accuracy of the key produced was high for species differentiation, despite the high variability in some of the species. Samples of the nominal species Anastrephafraterculus from various parts of Latin America were included in that study, and the authors found that it was one of the species with highest variability in the characters analyzed. Currently it is well known that, in reality, the authors studied different cryptic species, hence the difficulty in identifying Anastrephafraterculus species. However, these authors could establish differences between species through normalization of variables by transformation and construction of linear discriminant functions. Our study included new variables to differentiate the cryptic species of Anastrephafraterculus, which could complement the study of Still these techniques have some difficulties. The most common errors made in this analysis result from poor mounting of structures, digital imaging and determination of landmarks . The priSpecimens studied here derive from different sources, either from wild samples reared on natural hosts or from lab strains reared on artificial diets, however we do not know the effects of this on the measured structures, and further studies are needed. Some authors have suggested that developmental conditions affect the size of insects . JaramilIn many cases, the use of morphological characters of immature stages of insects for phylogenetic studies has helped to improve the understanding of relationships among groups (see revision in PageBreakAnastrephafraterculus complex. The results obtained from this work with third-instar larvae should be expanded to include additional populations to strengthen the dataset and advance our tools to study cryptic species of economically important fruit flies.Outline geometric morphometry and linear morphometry proved to be useful tools for the study of cryptic species of the


Breast cancer (BC) is an important public health problem worldwide. In Brazil, breast cancer is the most frequently diagnosed tumor and the leading cause of cancer death in women. Hereditary cancer represents approximately 5 to 10 % of BC cases. Even outside the hereditary cancer context, the presence of polymorphisms acting as genetic modifiers may contribute to a better or worse prognosis. Not much is known about the hereditary BC epidemiology in Brazil or about the influence of polymorphisms on hereditary predisposition.BRCA1/2 genes; Group 2: women with variants of uncertain significance in BRCA1/2 and Group 3: women with no mutations in BRCA1/2.This study examined the role of five different polymorphisms in four groups of women with BC: Group 1: women with a germline mutation in the FGFR2 gene); rs3803662 (TNRC9); rs889312 (MAP3K1); rs3817198 (LSP1 gene); and rs13281615 (8q24). The analyses were performed using PCR amplification and bi-directional sequencing.The women included in groups 1, 2 and 3 were patients from the Department of Oncogenetics of the Barretos Cancer Hospital who had undergone genetic testing because of a clinical suspicion of hereditary predisposition syndrome. The constitutive DNA was analyzed for the presence of polymorphisms at rs2981582 .No differences were identified in the frequency of the polymorphisms that were analyzed among the three groups. However, some associations were identified, such as the occurrence of bilateral breast cancer and homozygosity for the G allele in rs13281615 as well as the correlation between the SNPs rs2981582 and rs13281615 and the number of cancer cases in the family. Regarding the G allele of rs13281615, we observed that the proportion of individuals who were homozygous for this allele increased with the number of generations affected by cancer, regardless of the group where the patients were included. Concerning the rs2981582 we could observe that individuals from group 1 and homozygous CC had fewer cancer  cases. Regarding the hormone receptors, we observed an increased frequency in C homozygotes (rs3803662) among estrogen receptor-negative individuals from groups 1 and 3. For rs2981582 (FGFR2), we observed an increased frequency of the T allele in women who were positive for the estrogen and progesterone receptors regardless of the The results presented here provide interesting data on the modifying effect of polymorphisms on a family history of cancer; this may be a variable to consider in the analysis of tumor diversity, and of the family history observed in families with hereditary breast cancer .The online version of this article (doi:10.1186/s13053-015-0042-1) contains supplementary material, which is available to authorized users. BRCA1 and BRCA2 are among the genes associated with hereditary breast cancer [BRCA1 gene show a lifetime cumulative risk (LCR) between 44 and 68 % of developing breast cancer until 70 years of age. Moreover, the LCR for ovarian cancer in these patients is also significantly higher and may reach 60 % by 70 years of age [BRCA2 gene, when altered, is responsible for approximately 30 to 40 % of all cases of hereditary breast cancer. The LCR for breast cancer in women harboring germline mutations in this gene is similar to the risk of carriers of germline mutations in BRCA1 (44 to 68 % until 70 years of age), whereas the risk of ovarian cancer ranges from 11 to 40 % [BRCA1/2 differ in terms of age at diagnosis, the number of family members affected, and tumor prognosis [In Brazil, breast neoplasms are the main cause of death by cancer among women . It is ct cancer . Women hs of age . The BRC to 40 % –8. Familrognosis . Currentrognosis –12.FGFR2, which encodes a tyrosine kinase receptor that acts in the development of the mammary glands; 2) rs3803662 (C > T), localized in a region that includes TNRC9; 3) rs889312 (A > C), localized in a region that includes MAP3K1 in addition to two putative genes, MGC33648 and MIER3; 4) rs3817198 (T > C) in gene LSP1; and 5) rs13281615 (A > G), which resides in a region that does not include any known gene (8q24) but where variants associated with an increased risk of prostate and colorectal cancer have been identified. The authors correlated the presence of the above mentioned polymorphisms with histopathological characteristics such as positivity or negativity for the estrogen and progesterone hormone receptors, stage of tumor development, nodes, size, histology and stage at diagnosis. Among the five SNPs mentioned above, three  were significantly associated with an increased risk of breast cancer in estrogen receptor-positive individuals. Women who were TT homozygous in SNP rs3803662 and who had estrogen-negative tumors exhibited a risk of developing breast cancer that was 1.28-fold (95 % CI = 1.13–1.45) higher than women who were homozygous for the wild-type allele (present in 53 % of the controls). Besides, Hunter and colleagues [In 2007, a large study was conducted by Easton and collaborators  involvinlleagues  identifiStudies involving rs3817198 have found that the least frequent allele (C) leads to an increase of approximately 10 % in the risk of breast cancer in Caucasian women; however, the same allele has a protective effect among women of African ascent , 16., and rs13281615 in women with breast cancer, with or without mutations in the BRCA1 and BRCA2 genes, and to correlate the frequency of the different polymorphisms with the family history of cancer in general and, more specifically with the history of breast and ovarian cancer and with the histopathological features of the tumors.Considering the factors described above and the possible modifying effect conferred by polymorphisms, the objective of this study was to evaluate the frequency of the polymorphisms rs2981582, rs3803662, rs889312, rs3817198BRCA1 and BRCA2 genes and, based on the results, were allocated to three different groups: a) Group 1: 51 women with a personal and family history of breast cancer with a pathogenic germline mutation in the BRCA1 and/or BRCA2 genes; b) Group 2: 53 women with a personal and family history of breast cancer with the presence of a variant of unknown clinical significance (VUS) identified in the BRCA1 and/or BRCA2 genes; c) Group 3: 100 women with a personal and family history of breast cancer without a pathogenic mutation and/or mutation of unknown clinical significance identified in the BRCA1 and/or BRCA2 genes.This project was approved by the Research Ethics Committee of the Barretos Cancer Hospital. All the participants in the study were treated at this institution and signed an informed consent form. The women from the oncogenetics department underwent genetic testing for mutations in the For the polymorphism analysis, DNA was extracted using the QIAamp Blood DNA Mini-Kit (Qiagen) according to the manufacturer's instructions. Genotyping was performed through PCR amplification using the Hot Start Taq enzyme (Qiagen). Following amplification, the products were purified with the ExoSap enzyme (USB products) and sequenced bidirectionally using the BigDye Terminator v3.1 Kit . Electrophoresis was run in the automated sequencer model 3500 .BRCA1 and BRCA2 genes and the subsequent separation of the participants into the three study groups, a multiplex PCR amplification of all coding exons of the BRCA1  and BRCA2  genes and their respective flanking intronic regions was performed, followed by bidirectional sequencing using two platforms (ABI 3500 XL sequencer) and a new generation sequencer . In addition, large rearrangements were investigated using the multiplex ligation-dependent probe amplification (MLPA) technique.For the analysis of mutations in the The program SPSS v.21.0 for Windows  was used for the statistical analysis. The categorical variables were described using absolute frequencies and relative percentage frequencies. The correlations were obtained using the Chi-square and Fisher’s exact tests. The level of significance adopted in all the tests was 5 %.BRCA1 and BRCA2 of all coding regions was performed for the 204 patients. Fifty-one had a pathogenic mutation identified (30 with BRCA1 mutations and 21 with a germline pathogenic mutation on BRCA2 gene), 53 had a VUS identified on BRCA1 and/or BRCA2 genes and 100 women were tested negative for both genes. All 204 women had a personal history of breast cancer (mean age at diagnosis = 38.4 years). In addition all participants had a positive family history of cancer, suggestive of Hereditary Breast and Ovarian Cancer Predisposition Syndrome . The main histological tumor types were invasive ductal carcinomas (91.8 %) and lobular carcinomas (3.9 %). In addition the great majority of the breast tumors (of the probands) were grade II, T1/T2, N0 and M0. Details about the family history of cancer and mutated gene can be found in Additional file Sequencing of FGFR2 , rs3803662 in gene TNRC9 , rs889312 in gene MAP3K1 , rs3817198 in gene LSP1  and rs13281615 at 8q24 .The following polymorphisms were considered in this study: rs2981582 in gene Table i) there was a higher frequency of TT homozygotes for rs3803662 in the groups comprising women without mutations in the BRCA1/BRCA2 genes; ii) regarding rs889312, an increase was observed in the frequency of heterozygous individuals among those without mutations or VUS in BRCA1/2; and iii) the Group3 (WT) had a relatively increased frequency of CC homozygotes for the SNP rs3817198 compared to groups 1 and 2.Although there were no marked differences in the frequency of the polymorphisms studied among the three groups, some findings are noteworthy: The analysis of the family history of cancer according to the different polymorphisms studied is detailed in Table When comparing the genotypes with the family history of cancer, some potential associations were identified, including the occurrence of bilateral breast cancer and homozygosity for the G allele in rs13281615 and “C” in rs889312 in which 85 % of women with bilateral breast cancer had at least one C allele . Likewise, 85 % of the women with bilateral breast cancer had at least one T allele in rs2981582. Furthermore, regarding allele G in rs13281615, we observed that the proportion of individuals who were homozygous for this allele increased with the number of generations affected by cancer. Moreover, we observed that in the case of rs3803662 and rs2981582, heterozygous TC individuals presented a higher number of cancer cases  and a higher number of breast cancer cases.BRCA1 and BRCA2 gene mutation status are found in Additional file Detailed data on the frequency of the different genotypes versus the family history of cancer with the women subgrouped according to their BRCA1 to those with mutations in BRCA2. Given the limited sample size of patients with a deleterious mutation identified in these genes, the group stratification per mutated gene limited the statistical analyses that were conducted. However, some trends could be observed, for example, all of the women with mutations in the BRCA2 gene with bilateral breast cancer were homozygous for the G allele in rs132281615, and among those with mutations in BRCA1 and with bilateral breast cancer, 86 % had at least one G allele. Moreover, in regard to rs3817198, all of the women with mutations in BRCA1 and with ovarian cancer had at least one T allele (with 88.9 % being TT homozygotes); however, the same pattern was not observed for the women with mutations in BRCA2 and a personal history of ovarian cancer (p = 0.006). To reach a conclusion on the association of these polymorphisms with the BRCA1/2 mutational status and with a family history of cancer, more patients with these characteristics must be genotyped.In addition, the frequency of the different genotypes was compared to the family history by separately considering the women with a mutation in The data correlating the genotypes of the five polymorphisms that were analyzed with the status of the hormone receptors  are shown in Table When the different polymorphisms were analyzed with the hormone receptors, we observed an increased frequency of heterozygotes for rs3803662 in individuals with estrogen receptor (ER)-positive tumors, in individuals with progesterone receptor (PR)- positive tumors and in individuals who were negative for the HER2 receptor. Regarding rs2981582, individuals with at least one T allele more frequently exhibited positivity for the three receptors . We also observed an increased frequency of the WT allele among ER-negative individuals. For the SNPs rs13281615, rs889312 and rs3817198, no association was found between their frequencies and the hormone receptors considered.FGFR2, we observed an association between group 2 (with VUS in the BRCA1/2 genes) with the ER in which the presence of TT homozygotes was not observed, and TC heterozygosity was present in 73.7 % of the ER- negative cases , with 50 % of individuals being AG heterozygotes vs. 25 % and 28.6 % of those from groups one and two respectively , in which 50.0 % of the ER-positive cases were heterozygous (TC) .An analysis of genotypes versus hormone receptors was conducted by separately considering the individuals with The association between the genotypes in the five SNPs considered and the age at diagnosis was evaluated. Although no significant association was identified, these data are shown in Additional file Genetic polymorphisms are being widely evaluated as modifiers of the genetic risk of disease by several research groups. A study conducted by Garcia-Closas and collaborators  evaluateBRCA1 and BRCA2 genes, women with VUS identified in one of those genes, and women WT for both genes. The genotype data from the five polymorphisms were then correlated with the family history of cancer and the histopathological features of the tumors, always considering a possible influence of the mutational status of BRCA1/2. A similar work was performed by Antoniou and collaborators in 2008,[FGFR2 (rs2981582), TNRC9 (rs3803662), and MAP3K1 (rs889312) SNPs in a sample of 10,358 mutation carriers from 23 studies.In this study, we compared the genotype distribution of the five polymorphisms that were previously reported in the literature to be associated with the risk of breast cancer , 17, 18  in 2008, where thTNRC9, there was an increased frequency of CC homozygotes among ER-negative individuals ; this increase in frequency among individuals positive for the hormone receptors (ER and PR) did not depend on the group to which the women belonged. The same association with the rs2981582 and positivity for estrogen and progesterone receptors was described by Garcia-Closas and collaborators in an analysis of the association among breast cancer risk, FGFR2 rs2981582 and clinical and pathological characteristics and they found that the effect of rs2981582 was stronger in ER/PR-positive patients [FGFR2 variants with high levels of endogenous sex hormones (FGFR2 is upregulated in ERα-positive breast tumors) [When we compared the genotypes of the polymorphisms analyzed with the pathological characteristics of the tumors, we noted that, in the case of the SNP rs3803662 in gene borators  in 2007)patients . Additio tumors) , especiaCASP8, TNRC9 and ESR1 genes. The authors also reported an association between survival and eight SNPs in six genes  and another SNP in a gene-free region in 8q24. For the SNPs in genes MMP7, TOX3 and MAP3K1, the survival effects did not depend on the known main clinical prognosis factors. The effect of the SNP in ESR1 on survival was more significant when only ER-positive tumors were evaluated. When the cases were stratified according to tumor features, it was observed that the SNPs in FGFR2 and TOX3 were associated with the disease. Finally, the study showed that several SNPs were associated with survival. In some cases, this association may occur because of an effect on the tumor characteristics that has an impact on prognosis; in other cases, the effect appears to be independent of these prognostic factors [There are several studies evaluating the effects of polymorphisms on the risk of cancer and their association with tumor features. According to the study published by Tapper and collaborators  in 2008, factors .TNRC9, FGFR2, MAP3K1, H19 and LSP1. The most strongly associated SNP is localized in gene FGFR2, which was overexpressed in 5-10 % of the cases of breast cancer. The SNP in gene TNRC9 showed a stronger association with breast cancer and appeared to be correlated with the presence of bone metastases and positivity for the estrogen receptor. The SNP rs889312 in gene MAP3K1 showed sensitivity only in individuals harboring mutations in BRCA2, and it was not associated with an increased risk in carriers of BRCA1 mutations. Several SNPs in LSP1 and H19 were most likely associated with the risk of developing cancer. Therefore, the study concluded that the identification of risk-modifying polymorphisms may lead to a better understanding of the biological mechanism of breast cancer, thus improving the prevention, detection and early treatment of the disease.Results published by Fanale and collaborators  in 2012 The results of the analyses of the presence and frequency of the five polymorphisms considered here were compared to the family history of cancer. Several associations were identified in that comparison, either with the number of cancer cases in the family (associated with the SNPs rs2981582 and rs13281615), with the presence of bilateral breast cancer (SNP rs13281615) or with the number of generations affected by cancer (SNP rs13281615). Moreover, regarding allele G of rs13281615, we observed that the proportion of individuals who were homozygous for this allele increased as the number of generations affected by cancer increased. Many of the findings described here corroborate data in the literature, such as the association found between SNP rs13281615 and a positive family history of cancer, which was a correlation previously reported by Gorodnova and collaborators in 2010 . BesidesFGFR2 and the average number of first- and second-degree relatives with breast and/or ovarian cancers (P = 0.05). The authors also found an association with the age at diagnosis, in which individuals who were heterozygous or homozygous for the least frequent allele for rs3803662 had a higher probability of having the cancer diagnosed at an age younger than 60 years.The effect of polymorphisms on the family history of cancer was also reported by Huijts and collaborators , who shoAn association with age at diagnosis was not observed in this study for any of the five polymorphisms considered. However, we should reinforce that these findings should be regarded with caution, given the relatively small size of our study, which is the main limitation of our study. For that reason the obtained data and the possible associations identified with the tumor pathological features and with the family history of cancer must be validated using a larger sample size. Nonetheless, if these associations are confirmed, we will have additional data to understand the epidemiology of hereditary and familial cancer in our population. In addition, the results contributed to the continued research efforts in the search for common variants for breast cancer that could help to explain the high variability observed among family history of families with hereditary susceptibility to breast cancer.BRCA1/BRCA2 has been identified, the influence of polymorphisms acting as genetic modifiers of cancer was observed, especially for the SNP rs13281615 whose presence the G allele was associated with the occurrence of bilateral breast cancer, as well as with an increased number of cancer cases in the family and with an increasing number of generations affected by cancer and, this association was, in general, independent of the mutational status of the BRCA1 or BRCA2 genes.Thus, although no significant association between the presence of the 5 analyzed polymorphisms and the occurrence of pathogenic mutation in Some potential associations were found between the five polymorphisms analyzed in this study previously reported in the literature as being associated and an increase in the risk of breast cancer, with an emphasis placed on the effect of these polymorphisms on the family history of cancer and on hormone receptor positivity/negativity. These findings must be interpreted with caution because of the limited sample size in this study. The data must be validated with a larger number of cases and, if confirmed, the clinical relevance of the associations identified and their applicability in medical practice must be considered carefully. However, the results presented here provide interesting information on the modifying effect of polymorphisms on the family history of cancer and may be a variable to consider in the analysis of tumor diversity, and of the family history observed in families with hereditary breast cancer .


The derivation of a conventional error model for the miniature gyroscope-based measurement while drilling (MGWD) system is based on the assumption that the errors of attitude are small enough so that the direction cosine matrix (DCM) can be approximated or simplified by the errors of small-angle attitude. However, the simplification of the DCM would introduce errors to the navigation solutions of the MGWD system if the initial alignment cannot provide precise attitude, especially for the low-cost microelectromechanical system (MEMS) sensors operated in harsh multilateral horizontal downhole drilling environments. This paper proposes a novel nonlinear error model (NNEM) by the introduction of the error of DCM, and the NNEM can reduce the propagated errors under large-angle attitude error conditions. The zero velocity and zero position are the reference points and the innovations in the states estimation of particle filter (PF) and Kalman filter (KF). The experimental results illustrate that the performance of PF is better than KF and the PF with NNEM can effectively restrain the errors of system states, especially for the azimuth, velocity, and height in the quasi-stationary condition. Horizontal drilling (HD), especially reentry multilateral horizontal well drilling (RMHWD), receives considerable attention because it provides great economic value in the oil and gas industry. The RMHWD technology was originally proposed in an industry consortium of operators and service companies in 1997 . RMHWD iAlternatively, gyroscope-based MWD (GWD) tools can continuously provide the attitude, velocity, and position of the drill bit regardless of the strong magnetic interferences. GWD tools with pure strapdown inertial navigation algorithm were proposed in ,10 and tetc. The ZUPT alignment can limit the position errors up to 40 m during a 90-min experiment by using a Litton LTN90-100 inertial measurement unit (IMU) [The stochastic errors of inertial sensors can be modeled by the first-order Gaussian–Markov (GM) model or the high-order autoregressive (AR) model . The Levit (IMU) . The velit (IMU) . An IDA it (IMU) ,23; the it (IMU) ,25. HoweThis paper proposes a NNEM for the MGWD system under large-angle attitude error conditions. The error matrix of DCM is introduced to represent the attitude errors. Because of the nonlinearity of the system state space model (SSM), the PF is selected as the state estimation approach. The body frame is the right, forward, and up direction of the MGWD system and the navigation frame is the north, east, and down direction in this paper.In The error model of the strapdown inertial navigation system (SINS), based on the discrete time differential equation and the error model, plays a fundamental role in the estimation of system states. Conventional linear error models (LEM) of SINS, such as the Phi-angle model, the Psi-angle model ,27, and The derivation of a linear model by the perturbation method alone can be implemented in light of the assumption that the attitude errors are small enough. The initial alignment of the MGWD system cannot provide an accurate attitude because of the low performance of the MEMS sensors and the sophisticated downhole drilling environments. Therefore, this paper proposes an accurate NNEM of the MGWD system under large-angle attitude error conditions.i.e.,:The key variable for the derivation of the SINS error model is the rotation vector, which is the eigenvector of the DCM associated with eigenvalue 1. The eigenvector of the DCM can be expressed as follows ,31:(1)RN=The output errors of accelerometers and gyroscopes (essed as :(15)δωiThrough differentiating the Equations (11) and (12), itten as :(17)δωiThe conventional velocity update equation can be written as follows :(20)V˙nposed in ).The velocity error model can be obtained by perturbing Equation (20), and the DCM can be approximated by the following expression:However, the above equation may introduce errors if large attitude errors exist, so the error matrix of DCM The velocity update equation with error interferences can be written as:The velocity errors The only unknown parameter in Equation (25) is essed as :(26)ΔRbThe velocity error equation is obtained by inserting Equation (26) into Equation (25). There is no relationship between the model position error inear as :(27)δP˙The nonlinear error model of the MGWD system can be achieved through Equations (7), (25) and (27). The observation equation is the linear ZUPT equation, which is expressed as follows:In this section, we briefly review the theory of the recursive Bayesian estimation. The system is generally modeled as an SSM, which includes a system equation and an observation equation in the discrete time domain; the SSM can be written as follows:A more general system model (Markov model) and observation model corresponding to the Equations (31) and (32) can be given by the following expressions:The definition of SSM in Equations (33) and (34) are based on the prior function and likelihood function. Both models are equivalent, but the general model is more convenient for the theoretical derivation.k−1, i.e., the conditional pdf i.e., the conditional pdf The assumptions of recursive Bayesian estimation are that (1) the state vector (1)Prediction: evaluating the prior pdf equation :(35)p(xgiven by :(36)p(x(2)Updating: the posterior pdf  follows :(37)p(xequation :(38)p(z(3)State estimation: once the posterior is obtained, the estimation of state given by :(39)x^kTo sum up, the evaluation of the Bayesian estimation is given by the following steps.Because the function  systems ,47. The  systems ,49. The The SIS algorithm steps from the literature ,51. The The first task to implement the SIS is the selection of the importance function Generally, the importance function is selected such that :(43)π(xNote that the Equation (41) can be factored as following equation according to the Bayesian rule :(44)p and (44) into Equation (42), yielding:As indicated in Equations (45) and (46), the posterior pdf depends on the likelihood function, the priori pdf, and the importance function. Since the likelihood function and the priori pdf can be obtained in terms of the SSM, the choice of importance function is essential for PF. The optimal function, the likelihood function, and the priori function are the components of the importance function .The optimal function ction in :(47)p(xSubstituting Equation (47) into Equation (45) yields:The drawbacks of using optimal importance function are: (1) it is difficult to draw samples from the optimal function As indicated in Equation (49), l in xki . In thisThen, the weight in Equation (45) can be updated by the following equation:However, the likelihood function as the importance function can only be used in the high signal-to-noise ratio (SNR) case. In this paper, the priori function is selected as the importance function because of the tradeoff between computational complexity and the feasibility of implementation. The importance function is written by:Then, the weights can be updated by:The above equation shows that the weights only depend on the previous weights and the likelihood function, which are available from the knowledge of the observation equation and the pdf of the observation noise. In this application, we assume that the measurement noise of the MGWD system is additive, Gaussian white noise, so the likelihood function can be evaluated in a simple way :(54)PNefThe particles should be resampled if etc. The resampling strategy in PF is sampling with replacement, that is, the particles drawn from the box will be replaced after the decimation. In this paper, the multinomial resampling approach is selected to alleviate particles’ degeneracy. The multinomial resampling can be considered as a series of Bernoulli trials. There are several ways to implement the resampling algorithm such as systematic resampling , multinoThen, the The problem of resampling is that only a few particles are drawn, which is also referred to as samples impoverishment. Therefore, the particles lack diversities because the selection may include many repeated particles. In order to address the samples impoverishment problem, the random noise is added to the particles after resampling. The posterior particles At this point, the particles after the roughing step can be propagated for the update of the weights and the posterior pdf in the next time cycle. The first two moments of estimation derived from the particles can be approximated by the following equations:The objectives of the designed experiments are to demonstrate that: (1) the performance of the PF with NEM or NNEM is better than KF with LEM under small-angle attitude error condition (Experiment 1); (2) PF with NNEM can provide more accurate states estimation for the MGWD system with large-angle attitude error (Experiment 2). The configuration of the MGWD device and the details of the experimental data collection are briefly described in the first part of The MGWD device consists of the azimuth MEMS sensor, the pitch MEMS sensor, and the microcontroller. The MEMS sensor  is a combined gyroscope and accelerometer component and the sensor is characterized by low cost, small diameter, and high performance. The microcontroller  is a high-speed System-on-Chip (SoC) combined with ARM cortex-M3 processor. The types of data communication between the MEMS sensor and microcontroller is the serial peripheral interfaces (SPI) interface with 115,200 bit/s data transmission rate. The data collection software evaluates the navigation solutions with angular rate and specific force received from MGWD device through the universal serial bus (USB) with 20 Hz sample rate.In the experiment, we collect original data from the static MGWD device for the period of 10 min to demonstrate the performance of the PF and the accuracy of the proposed NNEM. The aim of Experiment 1 is to confirm that the performance of the PF with NEM is better than the performance of KF with LEM under small-angle attitude error conditions. The assumption of KF is that all the states are Gaussian and the SSM is linear. However, the complicated drilling environments introduce such strong interference to the MGWD device that the linear SSM and Gaussian states are impossible.Initial covariance matrix of system noise: R = diag;Initial covariance matrix of observation noise: Q = diag;Initial position: latitude 126.6879Initial velocity: 0.The first 50 s are utilized for initial alignment; the results of the initial alignment for roll, pitch, and azimuth are −0.0221081, a2, b1, b2, c1, c2 show the particles of the state errors together with the corresponding weights drawn from the priori pdf 1, a2 show that both the latitude and the longitude estimated by KF and PF converge to 45.77761, b2 the north velocity, east velocity, and down velocity estimated by PF are close to zero  while the errors of north velocity, east velocity, and down velocity estimated by KF are up to 0.3702 m/s, 0.2839 m/s, and 2.2953 m/s respectively. Therefore, the PF with NEM or NNEM gives better performance than KF with LEM does for the velocity estimation under small-angle attitude error.In 1, c2 the roll, pitch, and azimuth of MGWD system estimated by PF with NEM or NNEM largely converge to −0.022125In The objective of Experiment 2 is the validation of the performance of NNEM with large-angle attitude error. The experimental data used in this section are the same as those in Experiment 1 and the initial parameter settings are also the same as in experiment 1 apart from the initial attitude. The assumption of this experiment is that the initial alignment fails to provide an accurate initial attitude. We set the initial roll, pitch, and azimuth as 01, a2, b1, b2, c1, c2 show the estimation of roll, pitch, and azimuth of the MGWD system and the corresponding estimation errors. The roll, pitch, and azimuth converge to 1, b1, and c1. 2, b2, c2 show that the error of roll and error of pitch estimated by PF with both NNEM and NEM are less than 1, a2, b1, b2, c1, c2 illustrate the results of the north velocity, east velocity, and down velocity and the corresponding estimation errors of the MGWD system. The velocity estimated by PF, regardless of NEM or NNEM, apparently converges to zero velocity while the velocity error estimated by PF with NNEM is approximately 10 times smaller than KF with LEM. Therefore, the velocity errors estimated by PF with NNEM can be reduced effectively.1, a2, b1, b2, c1, c2 describe the estimation of latitude, longitude, and height of the MGWD system and the corresponding estimation errors. The latitude and longitude estimated by KF with LEM, PF with NEM, and PF with NNEM are almost close to the reference values This paper proposes a NNEM for MGWD system under large-angle attitude error conditions. The error of DCM as the intermediate variable is introduced for the derivation of NNEM, which can effectively eliminate the approximation of errors existing in the derivation of conventional NEM. The evaluation of the state density shows that the system states are partially Gaussian, so the KF is suboptimal in this application. PF can improve the estimation performance of nonlinear problems by approximating the posterior pdf with the particles and weights. The PF with NEM gives a better performance than KF with LEM for the estimation of height, velocity, pitch, and azimuth in Experiment 1. The NNEM of the MGWD system provides an accurate estimation of position, velocity, and attitude compared with PF with NEM and KF with LEM in Experiment 2.


In this article, we have proposed a ratio chain-type exponential estimator for finite population mean of the study variable under double sampling scheme using auxiliary variables. The large sample properties of the suggested strategy are derived up to first order, of approximation, and its competence conditions are carried out under which the suggested estimator is performed better than the other existing estimators discussed in the literature. An empirical study shows that the suggested strategy is more efficient than the other relevant competing estimators under two phase sampling scheme. To increase the precision of estimators for population mean of the study variable under double sampling design, a lot of works have been done in the field of sample survey and when the study variable is strongly connected with the auxiliary variables the precision of the estimators can be more and more. Using the knowledge of the auxiliary variables several authors have proposed different estimation technique for finite population mean of the study variable, Sukhatme , have deN of different units y and x be the study and the auxiliary variable with corresponding values i-th unit U.Let us consider a finite population of size x and y.Let y and x be the study and the auxiliary variable with corresponding values i-th unit Let Also let y on x based on a sample of size n. Also Let The usual unbiased estimator to estimatethe population mean of the study variable isThe variance of the estimator The usual ratio and regression estimators in two phase sampling and their mean square error are, given as followsThe mean squared error and variance are given belowChand , proposeThe mean square error of the suggested estimator is, given asKiregyera , suggestThe mean square errors of the suggested estimators, up to first order of approximation are, given as followsSearls , proposeKhare and Rehman , have prThe mean square errors of the suggested estimators, are, given byAlso Singh et al. , recommeThe minimum mean square error of the suggested estimator is given as followsN units. To estimate the population mean y taking values x and z taking values ith unit y and x as compared to the correlation between y and z By  and 2),,2), \doc(ii)By  and 5),,5), \doc(iii)By  and 6),,6), \doc(iv)By  and 8),,8), \doc(v)By  and 11)11), \doc(vi)By  and 12)12), \doc(vii)By  and 17)17), \doc(viii)By  and 18)18), \doc(ix)By  and 20)20), \docTo verify the theoretical conditions under the efficiency comparison numerically, we have taken a real data set from the literature. The description and the necessary data statistics of the data is, given below.j = 1, 2, 3, 4, 5, 6, 7, 8 and m.For percent relative efficiency we use the following formula.y square feet of living space as an auxiliary variable x and annual taxes ($) as an additional variable z have been taken. The numerical values of the parameters of the population are given as.The data from the population of 100 records of resale of homes from February 15 to April 30, 1993 from the files maintained by the Albuquerque Board of realtors on selling price ($) as a study variable On the basis of mean square errors and the percent relative efficiencies of the estimators as shown in Tables


The pigs were sacrificed after one, three, or six weeks of the treatment (12 pigs per each time-point). Histological investigations revealed an increase in the mucosal thickness and the crypt depth as well as a decrease in the ratio of the villus height to the crypt depth in groups D and M after six weeks of exposure to the mycotoxins. The number of goblet cells in the villus epithelium was elevated in groups Z and M after one week and in group D after three weeks. The administration of ZEN increased the lymphocyte number in the villus epithelium after 1 week and the plasma cell quantity in the lamina propria after one, three, and six weeks of the experiment. DON treatment resulted in an increase in the lymphocyte number in the villus epithelium and the lamina propria after six weeks, and in the plasma cell quantity in the lamina propria after one, three, and six weeks of exposure. In group M, lymphocyte counts in the epithelium and the lamina propria increased significantly after six weeks. Neither mycotoxin induced significant adverse changes in the ultrastructure of the mucosal epithelium and the lamina propria or in the intestinal barrier permeability. Our results indicate that immune cells are the principal target of low doses of ZEN and DON.Immature gilts were administered  Fusarium mycotoxins that have been extensively studied because of their wide distribution in nature and their ability to cause pathological changes in animals and humans. Pigs are a good model for the investigation of various aspects of the toxic effects of ZEN and DON as they are highly sensitive to these compounds and the porcine digestive tract is similar to the human gastrointestinal system [Zearalenone (ZEN) and deoxynivalenol (DON) are l system ,2,3,4,5.l system ,7, metabl system ,9,10, anl system , is extel system ,12,13.Several mechanisms have been proposed for biological effects of DON ,17,18,19ZEN and its metabolites have estrogenic activity and compete with endogenous hormones for the binding sites of the estrogen receptors ,21. MoreIn many cases, a diet contains DON and ZEN simultaneously , howeverFusarium mycotoxins are absorbed in the proximal fragment of the small intestine [Fusarium mycotoxins are also partially metabolized in the jejunum [Most ntestine ,28,29,30 jejunum . Studies jejunum  and the  jejunum . The res jejunum ,11,16. A jejunum ,13,16;  and the group receiving DON + ZEN (M) compared to the control group (C) and the group receiving ZEN (Z) after six weeks of treatment A. No sigFusarium mycotoxins significantly affected the villus morphology [Fusarium mycotoxins, including DON [It is noteworthy that the ZEN and DON doses we used had no significant effect on the qualitative and quantitative characteristics of the villi. Morphological changes in the villi are generally considered as a robust indicator of the intestinal response to dietary modifications, the harmful effects of anti-nutritional factors and toxins in feed, and the digestive pathology ,38,39. Prphology ,11,19,31rphology . Similarrphology ,41. A derphology . In contrphology . Moreoverphology . In otheding DON ,45. Inteding DON .Fusarium mycotoxins [A significant decrease in the ratio of the villus height to the crypt depth found in our study after six weeks of DON administration alone or together with ZEN indicates changes in the function of the intestinal crypt-villus axis. A drop in this parameter of intestinal status is usually considered as a sign of insufficient cell proliferation in the growth zone in crypts or excessive exfoliation of cells in the villi ,39,47. Icotoxins ,38,39,47cotoxins  that is cotoxins . The chacotoxins .The occurrence of the quantitative changes in mucosa after six weeks of DON and DON + ZEN administration, observed in our study, may be attributed to the progressive accumulation of mycotoxins in the intestinal wall to a concentration that affects the mucosa .A significant increase in the number of goblet cells was noted in the epithelium lining jejunal villi in groups Z and M after one week of mycotoxin administration. The number of goblet cells in the villus epithelium was also significantly increased in group D at week three A. No siget al. [et al. [Other authors’ findings indicate that mycotoxins influence goblet cells in the intestinal epithelium, but the results obtained vary considerably between studies and are inconclusive ,40,42,43This study shows that changes in the relative number of goblet cells are transient and the time of their occurrence depends on the toxin administered. The increase in the percentage of goblet cells induced by ZEN was short-lived, and it was noted only in the initial period of the exposure. The changes caused by DON occurred after three weeks of administration and faded thereafter. Our data partially explain the discrepancies reported in the literature, indicating the duration of the administration of the mycotoxin as a source of the differences. An increase in the number of goblet cells is indicative of intensified mucus secretion and is most likely a protective mechanism against mycotoxins.The results of quantitative analyses revealed a significant increase in the relative number of lymphocytes in the epithelium covering villi of the jejunum in group Z after one week and in groups D and M after six weeks of exposure compared to group C A. In theThe mechanisms of ZEN action on intestinal immune functions are largely unknown. One possible mechanism might be a direct effect of ZEN on lymphocytes and macrophages because estrogen receptors are present in these cells ,49. In pSalmonella Typhimurium was potentiated when DON (1 µg/mL) was co-exposed with the bacteria [in vitro, DON (0.5–1 µg/mL) makes the intestinal epithelium more permeable to Salmonella Typhimurium [et al. [The mechanism of the DON-induced increase in the number of lymphocytes in the epithelium and the lamina propria is most likely related to the direct activation of cytokine synthesis by this mycotoxin ,17,51,52bacteria ,13 shoulhimurium . Zielonk [et al.  revealed [et al.  and Mare [et al. , such a  [et al. .Fusarium mycotoxins have an antagonistic effect on plasma cells when administered in combination. An increase in the number of plasma cells is usually closely correlated with infiltration of the lamina propria by B cells from the mesenteric lymph nodes. However, such a simple relationship was not found in our study; therefore, it is possible that mycotoxins affect the process of transformation of B cells into plasma cells. Literature data partially explain the action of ZEN on plasma cells. It has been demonstrated that long-term treatment with low doses of ZEN  decreases the expression of CD 21 on B cells and increases the percentage of the B1 cell subpopulation [Quantitative analysis revealed that, regardless of the duration of mycotoxin administration, the number of plasma cells in the lamina propria was significantly higher in groups Z and D than in groups C and M C. Our repulation . ZEN at pulation . Consequpulation ,56. On tMorphological changes in cells and tissues induced by toxins of natural origin and synthetic toxins start at the level of biological membranes and organelles, where they can be observed within minutes and hours after exposure. Then, depending on the toxin dose and the duration of its action, fine subcellular changes may lead to more pronounced cell damages, which are visible in histological analyses and later in anatomopathological examination. The morphological effects of low toxin doses might be restricted to fine structural changes. Therefore, we decided to perform ultrastructural studies of the samples taken after one, three, and six weeks after toxin administration.In the control pigs, the epithelium covering villi and forming crypts showed a typical organization in samples taken after one, three, and six weeks of the experiment. The absorptive cells were the major epithelial component and showed the polarity of cellular organization A. These The lamina propria had a classical ultrastructure with abundant immunocompetent and migratory cells in all of the control pigs. The crypts of Lieberkühn were simply tubular with a typical cellular composition.Treatment with ZEN, DON and DON + ZEN did not cause prominent pathological alternations in the epithelial cells forming villi and crypts; however, some differences in the ultrastructure between the experimental and control pigs were noted .In this study, we paid special attention to the microvilli of the absorptive cells because on one hand, they are exposed to the highest levels of toxin and on the other hand, they play a crucial role in nutrient adsorption. In the samples taken from the control and mycotoxin-treated pigs, well-developed, parallel microvilli covered the entire apical surface of the enterocytes A. In theThe next facet of our special interest included the cell junctions between epithelial cells because previous studies reported that DON can affect the intestinal barrier ,17. In aThe administered mycotoxins also did not change the internal organization of the epithelial cells and the morphology of organelles, including the mitochondria, the smooth and granular endoplasmic reticulum, and the Golgi apparatus .A characteristic feature of the epithelium covering the villi was the presence of numerous lymphocytes in samples taken from pigs that received ZEN for one week as well as ZEN, DON or DON + ZEN for three and six weeks .Some degenerating cells and apoptotic cells were found in the epithelium in all animals ; howeverThe lamina propria of the jejunum of pigs treated with mycotoxins was formed by a loose connective tissue with numerous cells: fibrocytes and fibroblasts, lymphocytes, plasma cells, mast cells, eosinophils, and macrophages . MyocyteFusarium mycotoxins on the intestine have been infrequently published [in vitro studies, incubation of jejunal explants with 10 μM DON resulted in an increase in intercellular spaces, a decrease in the size and number of microvilli, and a loss of junction complexes [In contrast to the histological data, the results of ultrastructural studies dealing with the effect of ublished ,42. Theromplexes .In order to check the effect of mycotoxins on permeability of the epithelial barrier, the samples were fixed using the lanthanum technique, which is the gold standard in the studies of tight junctions at an ultrastructural level ,58,59. IThe treatment of immature gilts with low doses of ZEN and DON induced changes in the jejunum morphology, which were dependent on the type of mycotoxin, the manner of administration (separately or in combination), and the duration of exposure. The effects of ZEN were observed during the initial period of experiment, and they were partially reduced in the successive weeks of exposure. In general, ZEN had a prominent effect on the local immune system, leading to an increase in the number of lymphocytes in the villus epithelium and plasma cells in the lamina propria. It also induced a transient increase in the percentage of goblet cells in the villus epithelium. ZEN did not affect the mucosal architecture. Moreover, an electron microscopy study failed to show an adverse effect of ZEN on cells located in the epithelium and the lamina propria, except for a sporadically observed formation of drop-like protrusions on the apical surface of the enterocytes. DON also affected the jejunum morphology, but unlike ZEN, its effects were mainly manifested after prolonged administration. The six-week-long treatment with DON led to an increase in the mucosal thickness and the depth of the intestinal crypts. Similarly to ZEN, the changes induced by DON mostly concerned the local immune system and included an increase in the number of lymphocytes in the villus epithelium and lymphocytes and plasma cells in the lamina propria. Electron microcopy demonstrated that the dose of DON used in this study did not cause any changes in the ultrastructure of the mucosal epithelium and the lamina propria nor disturb the intestinal barrier. The results of the joint administration of both mycotoxins indicated complex interactions between them.et al. [Based on the animal weight and the average feed intake, it could be estimated that the dose of DON used in this study corresponds to a toxin feed concentration of approximately 250 µg per kg, which is much lower than the limit (0.9 mg/kg of feed) recommended as acceptable for complementary and complete feedstuffs for pigs by the European Commission . In viewet al.  publisheThe study was performed on 36 clinically healthy mixed breed gilts (White Polish Big × Polish White Earhanging), with an initial body weight of 25 ± 2 kg, purchased from a farm on which they received feed without detectable amounts of ZEN, α-ZEL or DON. Tests for the presence of mycotoxins in the feed were performed as described previously . Serologn = 9 in each group) and a control group . The animals in group Z received ZEN at a dose of 40 μg/kg BW per day, the animals in group D received DON at a dose of 12 μg/kg BW per day, and the animals in group M received a mixture of ZEN and DON (40 μg ZEN/kg BW + 12 μg DON/kg BW per day). The mycotoxins were synthesized and standardized at the Department of Chemistry, Faculty of Wood Technology, Poznan University of Life Sciences, Poland. The mycotoxins were administered orally during the morning feeding in water-soluble capsules containing oat bran as a vehicle. The gilts were weighed each week to establish the amount of DON and ZEN given to each animal. The animals in group C received capsules without mycotoxins.The animals were divided into three experimental groups  and exsanguination after one, three, and six weeks after the beginning of the experiment. The tissue samples were taken from the middle part of the jejunum within 3 min after cardiac arrest.All procedures were carried out in compliance with Polish legal regulations determining the terms and methods for performing experiments on animals and the European Community Directive for the ethical use of experimental animals. The protocol was approved by the Local Ethical Council in Olsztyn (opinion No. 88/N of 16 December 2009).Tissue samples  were fixed in 4% paraformaldehyde in phosphate buffer for 48 h and embedded in paraffin. The 4-μm-thick sections were stained using the hematoxylin and eosin method (HE), the periodic acid Schiff method (PAS), and the methyl green-pyronine method (MGP) using an automated multistainer ST 5020 . The slides were labelled so that those who performed the microscopic analysis did not know the type and duration of the treatment. The specimens were analyzed and photographed in an Axioimager light microscope equipped with an AxioCam MRc5 camera .2 of the lamina propria. Goblet cells were counted in PAS-stained sections and plasma cells in MPG stained sections. All other measurements were performed in HE-stained sections. The linear measurements were repeated 20 times per animal, the percentages of lymphocytes and goblet cells were determined by counting their number per 50 epithelial cells in 10 randomly selected areas, and the density of lymphocytes and plasma cells in the lamina propria by counting the cells in 10 randomly selected areas of 6000 to 12,000 µm2 each. The measurements were performed using Pannoramic Viewer 1.15 software  and AxioVision 4.8 software (Carl Zeiss).For morphometrical evaluation, the sections were scanned in a Mirax Desk scanner (Carl Zeiss). The following parameters were determined: the thickness of mucosa, the length of villi, the crypt depth, the ratio of villus height to crypt depth, the percentage of goblet cells in epithelium covering villi and forming intestinal glands, the number of lymphocytes per 50 epithelial cells in the villus epithelium, the number of lymphocytes and plasma cells per 10,000 μmJejunal mucosal samples were immersion-fixed in a mixture of 1% paraformaldehyde and 2.5% glutaraldehyde in 0.2 M phosphate buffer (pH 7.4) for 2 h at 4 °C, washed and post-fixed in 2% osmium tetroxide for 2 h. After dehydration, the samples were embedded in Epon 812. Semi-thin sections were cut from each block of tissue, stained with 1% toluidine blue, and examined under a light microscope in order to choose the sites for preparation of ultrathin sections. Ultrathin sections, contrasted with uranyl acetate and lead citrate, were examined with a Tecnai 12 Spirit G2 BioTwin transmission electron microscope  equipped with two digital cameras: Veleta  and Eage 4 k (FEI).3)·6H2O, in cacodylate buffer (pH 7.2). Next, the samples were rinsed for 30 min in the cacodylate buffer containing 1% lanthanum nitrate and postfixed for 2 h in a freshly prepared mixture of 1% osmium tetroxide and 1% of lanthanum nitrate in cacodylate buffer. Then, the tissues were dehydrated and embedded in Epon 812. Unstained, ultrathin sections were examined with a Tecnai 12 Spirit G2 BioTwin transmission electron microscope (FEI).The mucosal specimens were fixed for 2 h in a freshly prepared mixture of 2.5% glutaraldehyde and 1% lanthanum nitrate, La.The data were analyzed using two-way ANOVA with Duncan’s test as a Fusarium mycotoxins along with other exogenous factors such as bacterial antigens play an important role in the control of homeostasis of the intestinal local immune system should also be considered.The obtained data show that low doses of ZEN and DON have no or a very weak effect on the epithelial cells covering the jejunal mucosa. However, these toxins significantly influence the intestinal immune system and may contribute to the development of subclinical inflammation and allergy. The possibility that very low doses of


To obtain detailed information about the effect of the solvent temperatures on protein dynamics, multiple long molecular dynamics (MD) simulations of serine protease proteinase K with the solute and solvent coupled to different temperatures (either 300 or 180 K) have been performed. Comparative analyses demonstrate that the internal flexibility and mobility of proteinase K are strongly dependent on the solvent temperatures but weakly on the protein temperatures. The constructed free energy landscapes (FELs) at the high solvent temperatures exhibit a more rugged surface, broader spanning range, and higher minimum free energy level than do those at the low solvent temperatures. Comparison between the dynamic hydrogen bond (HB) numbers reveals that the high solvent temperatures intensify the competitive HB interactions between water molecules and protein surface atoms, and this in turn exacerbates the competitive HB interactions between protein internal atoms, thus enhancing the conformational flexibility and facilitating the collective motions of the protein. A refined FEL model was proposed to explain the role of the solvent mobility in facilitating the cascade amplification of microscopic motions of atoms and atomic groups into the global collective motions of the protein. Proteins are important bio-macromolecules because they perform a vast variety of functions and involve virtually every process within cells. However, proteins are not static entities but undergo complex internal motions, which are essential for their functions ,2. ThereIn a protein–solvent system or under physiological conditions, protein molecules are immerged in an aqueous solvent and thus the dynamic behaviors of the water molecules and the protein–solvent interactions are fundamental to the structural, dynamic, and functional properties of the proteins ,4,5,6,7,i.e., the hydrophobic collapse that results in the molten globule intermediate, the solvent entropy maximization contributes substantially to the lowering of the system free energy [i.e., an organization that the low-tier wells reside within the high-tier wells, and the lower tier the wells are, the lower are barrier heights between these wells [The FEL, which is defined as a function describing the conformational states/substates of a protein with the corresponding Gibbs free energy of the protein–solvent system, is a seminal concept in characterizing and explaining many aspects of the protein dynamics and functions ,21,22,23e energy ,28,29. Ie energy ,30. The e energy ,32,33. Tse wells ,5,34,35.se wells . Becausese wells ,38, it ise wells ,40.et al. [The mobility of the atoms, and further, of the atomic groups and the molecules, is temperature dependent because the temperature is a fundamental determinant of the atomic thermal energy. A generally observed phenomenon is that the protein conformational flexibility increases/decreases with the elevated/decreased temperatures of the protein–solvent system. The fact that solvent fluctuations affect protein dynamics at various hierarchical levels raises a question of whether the temperature of the solvent, or that of the protein itself, is a primary factor in determining the internal motions of the protein. The second law of thermodynamics determines that the thermal energy always distributes as evenly as possible over the entire protein–solvent system, thus making it difficult to distinguish experimentally the effect of the solvent temperatures from that of the protein temperatures on protein dynamics . The metet al.  have shoTritirachium album limber with a broad-spectrum degradation capability to degrade proteins [In order to resolve the above issues, in the present work the proteinase K  in eproteins  and, in proteins ,50,51,52proteins ,54 were The time evolution of backbone RMSD relative to the starting structure was computed for each replica of the four simulation systems. The results  show thaThe cosine contents for the first three eigenvectors derived from ED analyses of the replicas and of the joined trajectories were calculated to assess the conformational sampling. The results  show thaWhen the solvent temperatures increase from 180 to 300 K while the protein temperatures remain unchanged, e.g., at 300 K (or 180 K), the NNC and number of residues in SSE decrease by 1.5% (0.9%) and 6.6% (3.3%), respectively, and the RMSD and SASA increase by 82.3% (45.2%) and 2.7% (1.8%), respectively; when the solvent temperatures remain at 300 K (or 180 K) while the protein temperatures increase from 180 to 300 K, the NNC and number of residues in SSE decrease by 1.2% (0.6%) and 5.4% (2.1%), respectively, and the RMSD and SASA increase by 37.8% (9.7%) and 1.9% (1.0%), respectively. These results reveal that, although the changes in protein temperatures have effects on structural properties of proteinase K, their effects are weaker and quantitatively smaller than those of the solvent temperature changes.The Rg of proteinase K is 16.6 and 16.7 Å when the solvent is at 180 and 300 K, respectively, independent of the protein temperature. In addition, for all of the structural properties, their standard deviations follow the order P180/S180 < P300/S180 < P180/S300 < P300/S300, implying that proteinase K experienced larger structural variations and hence, a stronger internal mobility, during simulations at the high solvent temperatures than at the low solvent temperatures.α root mean square fluctuation (RMSF) was computed from the joined trajectories to evaluate and compare the structural flexibility of proteinase K under the four different temperature combinations  the lack of structural packing environment for the surface-exposed atoms; and (ii) the direct interactions/contacts between the protein surface atoms and the water molecules. Nevertheless, the ordinal increase in RMSF values of the protein core from the low to the high solvent temperatures indicates that the mobility of the solvent can also determine the amplitude of atomic fluctuations in the protein core. The small increases in RMSF values from P180/S180 to P300/S180 reveal that elevating the temperature of the protein does make a contribution, to some extent, to its own mobility. Moreover, the degrees of these RMSF increases are relatively consistent over the entire protein structure (from the core to the surface), which may reflect a pure thermal relaxation of the protein arising from the elevated protein temperature. When compared to P300/S180, under the P180/S300 combination the protein shows not only significantly higher surface and near-surface RMSF values but also slightly higher core RMSF value. This result reveals that the high surface mobility caused by the high solvent temperature is not only transmitted to the protein core, but also improves the mobility of protein core.α atoms using the joined trajectories of the four simulation systems.In order to investigate the effect of the solvent temperatures on the collective motions  of proteinase K, ED analyses were performed on protein C2 for the combinations of P300/S300, P180/S300, P300/S180, and P180/S180, respectively, indicating that proteinase K experiences significantly larger atomic fluctuations when the solvent temperatures are at 300 K than when at 180 K, in agreement with the above structural flexibility analysis. When the temperature combination goes from P300/S180 to P300/S300 and from P180/S180 to P180/S300, the TMSF values increase by 3.64- and 1.83-fold, respectively; the TMSF values increase by 1.23- and 0.36-fold when the temperature combination goes from P180/S300 to P300/S300 and from P180/S180 to P300/S180, respectively. These results reveal that elevating the temperature of the solvent makes a substantial contribution to increasing protein fluctuations. Although elevating protein temperature also contributes to protein fluctuations, such a contribution is quantitatively smaller, especially when the solvent temperature is at 180 K.The total mean square fluctuation (TMSF) values obtained by diagonalization of the covariance matrices are 2.97, 1.33, 0.64, and 0.47 nmi.e., P300/S180 and P180/S180), the first few eigenvectors have eigenvalues only slightly higher than those of eigenvectors with higher index, implying that the insignificant anharmonic atomic motions predominate along the first few eigenvectors, and as thus, the collective motions of the protein are greatly suppressed.Combined ED analyses were performed to investigate the differences in ED properties. There are six possible pairwise combinations among the four joined trajectories; for purpose of clarity, we choose only the pair of P300/S180–P180/S300 to demonstrate the effect of the solvent temperatures on ED properties. Of particular note is that for each of the first few combined eigenvectors, the right half of the projection (corresponding to P180/S300) exhibits a larger amplitude of the fluctuations than the left half of the projection (corresponding to P300/S180) A. This rIn order to examine the influence of the solvent temperatures on the extent of conformational space explored by proteinase K, the four joined trajectories were projected onto a two-dimensional conformational subspace spanned by the first two eigenvectors . As showAs described above, elevating solvent temperature facilitates not only greatly the protein surface atomic fluctuations but also to a certain extent the fluctuations of atoms in the protein core B. In ordAs shown in HBs are the most important class of non-covalent interactions/forces that can contribute not only to the stability ,57 but avice versa  make a greater contribution than do the main chain–main chain HBs to the increase in the dynamic HB number . This isi.e., at temperatures above 200 K, the protein dynamic behavior is dominated by the large-scale collective motions involving the entire protein structure, while at temperatures below 200 K, the simple harmonic atomic vibrations predominate, thus resulting in the cease of the protein activity. Because the dried proteins do not show any dynamical transition [“Glass transition”  of proteins, like that of glass-forming liquids ,61, is aansition ,63,64 anansition ,65, the ansition , our simi.e., they are not “melted” by the high temperature of the protein), the high temperature of the protein can improve, to a certain extent, their diffusivity due to the physical contacts between the protein and the hydrated water molecules. Taken together, it can be concluded that the protein temperatures have only a very limited effect on the diffusivity (mobility) of the solvent.As a necessary complement to this work, we have also evaluated the influence of protein temperatures on the solvent mobility by calculating the mean square displacement (MSD) of the solvent in the P300/S180 and P180/S300 trajectories. The results  show thaα RMSF profiles  the surface mobility can transmit to the core periphery and further to the core; and (ii) the mobility and sampling degrees of the non-surface-exposed regions are proportional to those of the protein surface. In light of the above findings, we can propose the following rough scenario: the high mobility of the water molecules, which arises from the high solvent temperatures, can lead to more frequent collisions/interactions with protein surface when compared to water molecules at the low temperatures, and therefore results in larger atomic fluctuations and structural displacements of the protein surface, especially of the solvent-exposed loop regions that lack the structural constraints. Such a high mobility can further transmit via specific mechanic mechanism to the protein core periphery and finally to the protein core, thus causing the global collective motions of the protein.The number of dynamic HBs can be considered as a representative of competitive interactions within the protein and between the protein and the solvent because of the universal distribution of HBs in the protein–solvent system. Comparison between protein–solvent HB numbers reveals that the high solvent temperatures result in more intense competitive interactions between the water molecules and the protein surface atoms than do the low solvent temperatures. In addition, elevating solvent temperature also facilitates greatly the competitive interactions between atoms within the protein. In a previous theoretical study, Agarwal has investigated the transfer of energy from the solvent to the protein by adding kinetic energy to solvent molecules in the hydration shell, and the results show that within a short time, the solvent kinetic energy is transferred to the protein residues even more than 8 Å away from the protein surface, resulting in increased mobility of the protein . Taken ti.e., the ns-timescale tier-1 dynamics that involve crossing among the tier-1 free energy wells). Finally, by perturbing the water network (hydration shell) around the protein surface [i.e., the tier-0 dynamics that occur on the timescales of μs to ms and involve crossing among the tier-0 free energy wells). Therefore, the high mobility of the solvent, which arises from its high temperature, plays a crucial role in facilitating the cascade amplification of microscopic motions of atoms and atomic groups into the protein global collective motions. However, the low solvent mobility, which results from its low temperature, will inhibit such cascade amplification through suppressing the competitive interactions between atoms within the protein and between the protein and solvent, ultimately leading to insignificant anharmonic motions of the protein as observed in this study.Comparison between our constructed FELs reveals that elevating the solvent temperature increases the conformational substates and conformational entropy while reducing the thermostabilility of the protein. These results, in conjunction with the data presented in this work and available from the literature ,33,34,66 surface ,68 and i surface , the larwww.pdb.org) was used as an initial structure for MD simulations. The hetero atoms and crystallographic water molecules were removed while the protein atoms and the two bound Ca2+ cations were retained. The treated structure was soaked in a periodic cubic box filled with the single point charge (SPC) [The high-resolution X-ray crystal structure of proteinase K   obtainedge (SPC)  water moAll MD simulations were performed using the GROMACS software package  with the−1·nm−2). In the production MD runs, the solute (protein and Ca2+) and the solvent (water and counterions) were separately coupled to the v-rescale thermosat [Initially, the system was subject to energy minimizations (1000 steps of steepest descent followed by 1000 steps of conjugate gradient method) until no significant energy change could be detected. The molecular mechanics optimization was followed by four successive 200-ps position-restrained dynamics runs at 300 K with a gradual decrease in harmonic positional restraint force constant on the solute  relative to the starting structure. For each system of the temperature combinations, the equilibrated parts of each replica were concatenated together to obtain a single joined trajectory, representative of different sampling directions around the starting structure.i.e., close to 1), the corresponding largest scale motions in the protein dynamics resemble random diffusion, and thus the sampling is insufficient on the timescale of the simulation [The degree of the convergence of conformational sampling was evaluated by computing the cosine contents of the first few eigenvectors obtained from essential dynamics (see below for details of ED) analyses of the MD trajectories, including replicas 1–6 and the single joined trajectories at each combined temperature. Since the first few eigenvectors represent the most significant motions of the protein, their cosine similarity is a good indicator to assess whether the conformational sampling is converged or not . If the mulation . In contThe static structural properties of each frame extracted from the joined equilibrium trajectories, including the solvent accessible surface area (SASA), number of native contacts (NNC), radius of gyration (Rg), backbone RMSD with respect to the starting structure, and number of residues in secondary structural elements (SSE) were calculated using the programs g_sas, g_mindist, g_gyrate, g_rms, and do_dssp  within GThe protein internal HBs and the protein–solvent HBs were detected using the program g_hbond within GROMACS and the Hydrogen Bonds plugin within VMD , respectα atoms were built and diagonalized using the program g_covar within GROMACS; the projections of trajectories onto the eigenvectors were performed using the g_anaeig program within GROMACS.ED ,78, whici.e., the distribution, average value, and mean square displacement (MSD) of the projection) between different parts of the combined eigenvector can provide information on differences in equilibrium fluctuations (or average structures) and in conformational shifts between different simulations.A useful method for comparing the ED properties of two simulations is the combined ED . In thisThe Metadynamics ,81 is a The CV1 and CV2 used in metadynamics simulations were projection of MD trajectory onto the eigenvectors 1 and 2, respectively. The starting structure was the final snapshot from the standard MD simulation. The initial Gaussian height was set to 0.4 kJ/mol and was added every 2 ps; the Gaussian width was set to 0.35 nm; the bias factor was set to 10. Other simulation parameters and conditions are the same as those in the standard MD simulations. The well-tempered metadynamics simulations were run for 100 ns, followed by FEL constructions using the weighted histogram analysis method implemented in Plumed .By performing multiple, long MD simulations on the proteinase K-solvent system with the protein and solvent coupled to either 300 or 180 K, we demonstrate that the temperatures of the solvent, when compared to the temperatures of the protein itself, have an overwhelming effect on protein dynamics. The flexibility, mobility, global collective motions, and sampled conformational spaces of proteinase K are greatly enhanced (or suppressed) by elevating (or lowering) the solvent temperature, whereas the changes of the protein temperature exert relatively weak effect on the above properties. The constructed FELs reveal that proteinase K has more conformational substates, larger conformational entropy, and lower thermostabilility at the high than at the low solvent temperatures, but these thermodynamic properties are independent of the protein temperatures. Elevating the solvent temperature increases the mobility/kinetic energy of the water molecules. By comparing the dynamic HB numbers in the MD trajectories at different combined temperatures, we demonstrate that it is the competitive interactions between water molecules and surface-exposed protein atoms and between atoms within the protein that transmit the solvent kinetic energy over the entire protein structure, ultimately leading to enhanced conformational flexibility and collective motions of the protein at the high solvent temperatures. Finally, we propose a refined FEL model to explain the role of solvent mobility in facilitating the cascade amplification of microscopic motions of atoms and atomic groups into the protein global collective motions.