The objective of this study is to examine the effect of intumescent flame-retardants (IFR’s) on the spinnability of sheath/core bicomponent melt-spun fibers, produced from Polylactic acid (PLA) single polymer composites, as IFR’s have not been tested in bicomponent fibers so far. Highly crystalline PLA-containing IFR’s was used in the core component, while an amorphous PLA was tested in the sheath component of melt-spun bicomponent fibers. Ammonium polyphosphate and lignin powder were used as acid, and carbon source, respectively, together with PES as a plasticizing agent in the core component of bicomponent fibers. Multifilament fibers, with sheath/core configurations, were produced on a pilot-scale melt spinning machine, and the changes in fibers mechanical properties and crystallinity were recorded in response to varying process parameters. The crystallinity of the bicomponent fibers was studied by differential scanning calorimetry and thermal stabilities were analyzed by thermogravimetric analysis. Thermally bonded, non-woven fabric samples, from as prepared bicomponent fibers, were produced and their fire properties, such as limiting oxygen index and cone calorimetry values were measured. However, the ignitability of fabric samples was tested by a single-flame source test. Cone calorimetry showed a 46% decline in the heat release rate of nonwovens, produced from FR PLA bicomponent fibers, compared to pure PLA nonwovens. This indicated the development of an intumescent char by leaving a residual mass of 34% relative to the initial mass of the sample. It was found that the IFRs can be melt spun into bicomponent fibers by sheath/core configuration, and the enhanced functionality in the fibers can be achieved with suitable mechanical properties. The development in process technologies, for the value addition of commercially available polymers, has been steered in the synthetic fiber industry in the recent past ,2. AmongBicomponent spinning is used to impart diverse functionalities in the fibers, such as electrical conductivity, chemical resistance, tactile comfort, and fire resistance ,22,23. HAlthough, various aspects of bicomponent spinning process has been studied in different applications ,40,41,42−3 and average particle size of 17 μm, used as acid donor in intumescent formulations. The kraft lignin (KL) powder “UPM BioPiva 100” was purchased from UPM Biochemicals . A modified polyester based plasticizing agent (PES) in white granular form  was obtained from Preluna , to improve spinnability of IFR composites. PLA (Luminy L130), APP, PES and KL were vacuum dried at 100 °C for 4 h before compounding.Two different types of PLA pellets, i.e., highly crystalline PLA (Luminy L130) and amorphous PLA (Luminy LX930) were purchased from Total-Corbion . The physical properties of both types of PLA are presented in 18 twin-screw extruder  was used to prepare PLA/APP/PES/KL composites for the core component of bicomponent fibers. In the first feeding zone, highly crystalline PLA pellets (Luminy L130) were mixed together with plasticizing agent (PES) whereas in the second feeding zone, flame retardant (APP) was mixed with kraft lignin powder (KL). PLA/APP/PES/KL composites were prepared at screw rotation speed of 500 rpm to use as core component for bi-component fibers. The melt flow index (MFI) of the blends were in the range of 26–30 g/10 min. The formulations, with content of each component (w/w), as prepared composites are presented in Coperion ZSK McPLA/APP/PES/KL pellets to be used as the core component were vacuum dried at 100 °C for 4 h, whereas, PLA Luminy LX930 pellets, used as sheath components, were vacuum dried at 40 °C for 24 h before spinning, as per the instructions of the suppliers. Vacuum drying of the polymers is necessary to avoid possible filament breakage during melt spinning, due to lowered viscosity of the melt caused by the hydrolysis caused by excessive moisture in the polymers. The moisture content of the pellets was lower than 75 ppm, as determined by Karl Fischer Titrations method, according to ASTM D6869-03 . The bic−1 and then combined together to multifilaments, by applying a spin finish before they were collected by the suction gun. The multifilaments were passed through a take up roller followed by hot drawing at two set of heated rollers rotating at varying speeds. The multifilaments with sheath/core configuration were then wound on bobbins rotating on a winder for further analysis. The extruding sheath/core ratio for all bicomponent fibers was set to 1:2 (thin sheath and thick core) under constant melt flow.The monofilaments were passed through quenching section (quenching length = 1.4 m) where they were cooled down by maintaining the cool air velocity of 0.5 m·sTo further investigate the spinnability of bicomponent functional fibers in response to varying process parameters, a design of experiment was prepared using half factorial design in MINITAB 18 statistical software . Four factors/variables with two levels each were considered in this study and are shown in The design of experiment showing different spinning parameters used to produce bicomponent fibers is presented in −1) and elongation at break (%), were tested on Zwick Roell testing machine by using the EN ISO 5079 standard method [−1) were kept constant for all samples. Ten specimens were prepared from each sample and their average results with standard deviations were recorded.The mechanical properties of bicomponent fibers, such as tenacity . The specimens (15–20 mg) were heated at a constant rate of 10 °C·minThe surface morphology, cross sectional images and additives dispersion in bicomponent fibers were evaluated by using a Hitachi S-3200 scanning electron microscope . For better cross-sectional images, the bicomponent fibers were first immersed in liquid nitrogen to be freezed and then were cut with a razor blade. Standard specimen stubs with silver coating were used to attach the samples, which were then sputter coated with gold. Selective samples were inspected at a magnification of 1000 × and at an accelerating voltage of 20 kV. −1. The samples were heated at a constant rate of 10 °C·min−1 starting from 0 °C to 230 °C, and then cooled at the same rate. The degree of crystallinity (Xc) of bicomponent fibers was calculated by Equation (1),−1.Differential scanning calorimetry (DSC) analysis of bicomponent fibers was done using a DSC Q2000 (TA instruments). Nitrogen gas was used at a stream rate of 50 mL·min−2. Thermally bonded nonwoven fabrics were produced from flame retardant bicomponent fibers to be used as carpet backing. The process comprised the steps in laying down the continuous bicomponent filaments and entangling them together at filament crossover points by air pressure, which then were laid on the conveyer belt to form a nonwoven web. The nonwoven web was thermally bonded by calendaring, at a temperature of 135 °C, which is the melting temperature of the sheath material (low melting PLA) in the bicomponent yarn. After bonding the web thermally by passing over a rotating roller heated by a hot air; the web was passed through a cooling section to consolidate the fixation and then wound up on fabric rolls. The resultant nonwoven flame retardant fabric was dimensionally stable and had a width of around 50 cm as shown in 3, as required by ISO 4589-2 [The limiting oxygen index (LOI) of samples was tested. LOI is the fraction of oxygen that must be present to support burning, hence higher LOI values indicate lower flammability. The nonwoven samples . The cone calorimeter equipment operates on an oxygen consumption principle and measures the total heat of combustion, based on the amount of oxygen consumed. Peak heat release rate (PHRR), time to ignition (TTI), and residual mass (%) of thermally bonded nonwoven fabric samples, produced from FR bicomponent fibers, were recorded after the test. Cone calorimetry testing was also performed on pure PLA nonwoven fabric sample for the comparison purpose. Thermally bonded nonwoven fabric samples with dimensions of 100 × 100 × 3 mmISO 5660 , with heThe single-flame source test (ignitability test) as required by EN ISO 11925-2  was condThe spinning of bicomponent multifilament fibers was not only challenging, but it also took a while to find the right parameters to make the spinning process stable for continuous collection of fibers. The melt spinning process was stable without filament breakage for as spun fibers or for the fibers drawn at lower solid-state draw ratio (SSDR). However, an occasional filament breakage were observed when drawn at higher SSDR. This suggests that bicomponent fibers containing IFRs in the core component can be melt spun in to multifilaments if not drawn at too high SSDR. In our case, SSDR up to 1.4 was sufficient to produce bicomponent fibers without filament breakage. However, beyond this SSDR, filaments kept on breaking and continuous collection of the fibers was not possible. IFRs were used in the core component and not in the sheath component of the bicomponent fibers because previous studies ,40 sugge−1) as expected, since no crystallinity was induced in the fibers. However, it was significantly improved by increasing SSDR as a maximum tenacity of 12.6 cN·tex−1 containing 1 wt % KL was achieved at SSDR of 1.4. The tenacity was gradually reduced by increasing the wt % of KL in the core component at the same SSDR of 1.4. It can be seen in −1 respectively at the same SSDR of 1.4. Compared to as spun fibers, the fibers drawn at SSDR of 1.2, showed lower elongation at break (%), while the increasing SSDR of 1.4, the elongation at break further reduced, as shown in The summary of the test results are presented in Similar findings were reported by Hu et al.  and obseThe effect of IFR additives on thermal stability and thermal decomposition of bicomponent fibers was investigated by thermogravimetric analysis and the residual mass percentage of samples was determined at 700 °C. The thermal decomposition temperatures and mass residue percentage of the samples after TG analysis were compared to determine the influence of carbonization agent (KL) on thermal stabilities of bicomponent composite fibers. TG curves of bicomponent fibers and their respective data for the samples heated in a nitrogen atmosphere are presented in The temperatures corresponding to 5% and 50% mass loss for the bicomponent fibers are represented by T5 and T50 values in The thermogravimetric curves of bicomponent fibers containing 1, 3, and 5 wt % of KL are presented in The curves indicate that most of the thermal decomposition occurs between 300 °C and 400 °C. Whereas, all the bicomponent fibers decompose within a narrow temperature window, thereby, increasing the concentration of KL causes more residual mass to remain at temperatures between 375 °C and 700 °C. The thermal stabilities of bicomponent fibers, containing 5 wt % of KL are, therefore, better than those bicomponent fibers containing 1 and 3 wt % of KL. This behavior is mainly due to higher decomposition temperatures of these fibers for 5% and 50% mass loss  than other fibers. The other reasons could also be due to the greater char-forming ability of the polyhydric component (KL) and dehydration mechanism, established by acid source (APP). As the formation of phosphate compounds that further enhance the dehydration of KL result in higher char formation with compact structures.The dispersion characteristics of IFR additives and the microstructures of bicomponent fibers were examined by scanning electron microscopy. The cross-sectional images and surface morphology of bicomponent fibers are shown in Differential scanning calorimetry was used to investigate the crystallinity of the bicomponent fibers. DSC curves of the bicomponent fibers are presented in The peaks for the glass transition temperatures, cold crystallization temperatures, and melting temperatures can be clearly seen for all the three-bicomponent fibers in the DSC thermograms. However, all the three bicomponent fibers have different cold crystallization exotherms, which is attributed to the differently oriented and semi-crystallized core and sheath components. The thermal properties and crystallinity (%) of the bicomponent fibers, calculated by Equation (1), are presented in In melt spinning, the crystallinity of fibers changed by hot drawing them (stretching between two heated rollers). This attenuated and aligned the molecular chain within the fibers and, by doing so, their crystallinity increased. The bicomponent fibers containing 1, 3, and 5 wt % of KL in the core component  showed glass transition temperatures at 58.8 °C, 57.6 °C, and 58.5 °C, respectively, while, melting peaks were at 172.7 °C, 172.6 °C, and 172.8 °C, respectively. However, a major difference in their cold crystallization peaks was seen. Cone calorimetry gives useful insights on the burning behavior of materials by calculating parameters, such as peak heat release rate (PHRR), time to ignition (TTI), and residual mass (%) after burning of the material. The cone calorimetry data of pure PLA nonwoven fabric sample and nonwoven fabric samples, produced from FR PLA bicomponent fibers, are presented in When a material is decomposed, pyrolysis gases are released, and if the concentration of such gases is reached at certain threshold, the ignition of the material starts. Prolonged ignition time of a material is mainly due to slower decomposition of the material. The samples containing KL showed lower TTI than pure PLA, as it fits with common intumescent systems as the degradation of the intumescent samples has to begin prior charred layer is developed.−2. The fabric sample containing 1 wt % of KL after ignition presented PHRR of 368.2 kW·m−2. For the fabric sample containing 3 wt % of KL, PHRR declined to 337.1 kW·m−2, which was further reduced to 309.3 kW m−2 by the addition of 5 wt % of KL. These findings suggested that higher loading (%) of KL formed thicker and compact char layer on fabric surface which restricted further burning of the sample and helped in extinguishing the fire. The residual mass (%) of nonwoven fabric samples after cone calorimetry are also presented in The curves showing peak heat release rate (PHRR) of the fabric samples after cone calorimetry are presented in The intumescent system operates in the condensed phase where the burning of the material generates sponge-like multicellular structure (char), which protects the underlying material from further heat and mass transfer by acting as a physical barrier between the material and source of fire. The fabric samples containing 1 wt % of KL presented a thinner and porous char layer due to decreased viscosity of the substrate a very little pressure was built-up, which allowed gas bubbles and vapors to escape from the unclosed cells, resulting in reduced swelling of the char layer and increased heat release rate. On the other hand, more compact and uniform char presented by the sample, containing 5 wt % of KL, prevented the free escape of gas bubbles and vapor particles. This resulted in greater pressure build-up, due to closed cells, which further increased the melt viscosity of the condensed substrate hence, greater swelling of char can be seen. The limiting oxygen index (LOI) test is widely accepted to assess the flame retardancy of materials. We also tested the LOI values of nonwoven fabric samples, which are presented in The ignitability test under EN ISO 11925-2 method was used to determine the ignitability of nonwoven fabric samples in the vertical direction by direct flame impingement. Six specimens from each fabric sample were subjected to surface exposure to flame and their results are presented in The reason for no ignition of the specimen was an increase in the gap between flame and the specimen, due to the fleeing of heat flux. The temperature at the specimen surface decreased and heat flow was reduced hence, fuel availability was no more to continue ignition. Based on this test our fabric samples got the classification of E and Efl. To get E and Efl (for floorings) classification the flame should not reach the top marking (150 mm) within 20 s after 15 s of flame exposure and there should be no burning droplets and no ignition of the filter paper placed underneath. It can be seen in −2) of sample containing 5 wt % of KL was observed, which is 46% less than the PHRR of the nonwoven sample, which was made from pure PLA. TTI of KL samples decreased quite significantly since fabric sample containing 5 wt % of KL presented TTI of 57.1 (s) compared to 73.2 (s) for pure PLA, which fits best with common intumescent systems as degradation of the intumescent samples has to begin before the charred layer is developed. The nonwoven fabric sample, containing 5 wt % of KL, showed a residual mass of up to 34.5% relative to the initial mass of the sample, thereby indicating the development of an intumescent char. This protects the fabric sample from further burning. Limiting oxygen index of the fabric samples was also tested and the highest LOI (30.4%) was observed for the sample containing 5 wt % of KL. The ignitability test showed none of the fabric sample, produced from bicomponent fibers, was ignited after 15 (s) of flame exposure, and therefore, achieved classification of E and Efl as per the standards set for EN ISO 11925-2 method. This certifies that the product can be used commercially for floor coverings. Compared to our previously published PLA/IFR mono-component multifilament fibers with tenacity of up to 7.3 cN·tex−1, the tenacity of the bicomponent fibers was improved up to 12.6 cN·tex−1. Hence it was confirmed that IFRs can be melt spun into bicomponent fibers by sheath/core configuration, while flame retardant nonwoven fabrics can be produced for FR applications.In this study, novel bicomponent functional fibers, based on single polymers, consisting of highly-crystalline PLA with intumescent flame retardants in the core and low melting PLA were produced in the sheath. Thermogravimetric analysis confirmed that the thermal stabilities of bicomponent fibers increased quite significantly, and the fibers containing 5 wt % of KL presented the highest residual mass%. Thermally bonded nonwoven fabric samples produced from bicomponent fibers showed remarkable flame retardancy. A significantly low PHRR (309.3 kW·m


Microaspiration of gastric and oropharyngeal secretions is the main mechanism of entry of bacteria into the lower respiratory tract in intubated critically ill patients. The aim of this study is to determine the impact of enteral nutrition, as compared with parenteral nutrition, on abundant microaspiration of gastric contents and oropharyngeal secretions.Planned ancillary study of the randomized controlled multicenter NUTRIREA2 trial. Patients with shock receiving invasive mechanical ventilation were randomized to receive early enteral or parenteral nutrition. All tracheal aspirates were collected during the 48 h following randomization. Abundant microaspiration of gastric contents and oropharyngeal secretions was defined as the presence of significant levels of pepsin (> 200 ng/ml) and salivary amylase (> 1685 UI/ml) in > 30% of tracheal aspirates.p = 0.016), constipation rate was significantly lower  in patients with enteral than in patients with parenteral nutrition. No significant difference was found regarding other patient characteristics. The percentage of patients with abundant microaspiration of gastric contents was significantly lower in enteral than in parenteral nutrition groups , adjusted OR 0.79 ). The percentage of patients with abundant microaspiration of oropharyngeal secretions was significantly higher in enteral than in parenteral nutrition groups , adjusted OR 1.23 ). No significant difference was found in percentage of patients with ventilator-associated pneumonia between enteral (8%) and parenteral (10%) nutrition groups ).A total of 151 patients were included , and 1074 tracheal aspirates were quantitatively analyzed for pepsin and amylase. Although vomiting rate was significantly higher (31% vs 15%, Our results suggest that enteral and parenteral nutrition are associated with high rates of microaspiration, although oropharyngeal microaspiration was more common with enteral nutrition and gastric microaspiration was more common with parenteral nutrition.NCT03411447. Registered 18 July 2017. Retrospectively registered.ClinicalTrials.gov,  Microaspiration of contaminated oropharyngeal and gastric secretions is main route of entry of bacteria into the lower respiratory tract in patients receiving invasive mechanical ventilation , 2. The Enteral nutrition is associated with increased risk of vomiting . StudiesClinicalTrials.gov, identifier NCT03411447) of the randomized controlled multicenter open-label NUTRIREA2 study . The NUTRIREA-2 study was supported by the Programme Hospitalier de Recherche Clinique National 2012 of the French Ministry of Health (#PHRC-12-0184) and was designed to compare the effect of early enteral or parenteral nutrition on mortality in adult patients with shock requiring invasive mechanical ventilation [This was a planned ancillary study  (CHD085-13). According to French law, because the treatments and strategies used in the study were classified as standard care, there was no requirement for signed consent, but the patients or next of kin were informed about the study before enrolment and confirmed this fact in writing.Inclusion and exclusion criteria were similar to those of the NUTRIREA2 study. No additional criteria were required for the current study. In all participating ICUs, consecutive patients included in the NUTRIREA2 trial were included in the current ancillary study until the predetermined goal of sample size was met. Thus, adults (18 years or older) admitted to any of the participating ICUs were eligible if they were expected to require more than 48 h of invasive mechanical ventilation, concomitantly with vasoactive therapy  via a central venous catheter for shock, and to be started on nutritional support within 24 h after tracheal intubation (or within 24 h after ICU admission if intubation occurred before ICU admission). Exclusion criteria were invasive mechanical ventilation started more than 24 h earlier; surgery on the gastrointestinal tract within the past month; history of gastrectomy, oesophagectomy, duodeno-pancreatectomy, bypass surgery, gastric banding, or short bowel syndrome; gastrostomy or jejunostomy; specific nutritional needs, such as pre-existing long-term home enteral or parenteral nutrition; active gastrointestinal bleeding; treatment-limitation decisions; adult under legal guardianship; pregnancy; breastfeeding; current inclusion in a randomized trial designed to compare enteral nutrition to parenteral nutrition; contraindication to parenteral nutrition . Further details on methods are available in the published protocol of the NUTRIREA2 study .2O, three times a day. After randomization and starting enteral or parenteral nutrition, all tracheal aspirates were collected for 48 h for pepsin and alpha amylase measurements. Tracheal aspirates were performed according to the need of each individual patients, as determined by nurses at bedside. Saline instillation was not recommended during sampling for tracheal aspirates. All tracheal aspirates were stored at − 20 °C and sent to the central laboratory at Lille University Hospital, where all measurements were blindly performed .All study patients were positioned in semirecumbent position during their period of mechanical ventilation. Tracheal cuff pressure was monitored using a manometer and adjusted around 25 cmHAbundant microaspiration of gastric contents was defined by the presence of pepsin at significant concentration (> 200 ng/ml) in > 30% of tracheal aspirates. Abundant microaspiration of oropharyngeal secretions was defined by the presence of amylase at significant concentration (> 1685 IU/ml) in > 30% of tracheal aspirates , 15.The primary outcome was percentage of patients with abundant microaspiration of gastric contents. Secondary outcomes were the percentage of patients with abundant microaspiration of oropharyngeal secretions, the percentage of tracheal aspirates positive for pepsin, the percentage of tracheal aspirates positive for alpha amylase, and the percentage of patients with VAP.We calculated that a sample of 188 patients (94 patients per group) would provide a power of 80% to detect an absolute risk reduction in primary endpoint of 20% in the parenteral nutrition group, with a two-sided type I error of 0.05, assuming a primary endpoint rate of 50% in the enteral nutrition group.All analyses were performed in all randomized patients on the basis of their original group of randomization, according to the intention-to-treat principle. Qualitative variables were expressed as frequencies and percentages and compared using chi-square test. Quantitative variables were expressed as median (interquartile range) and compared between the two groups using Wilcoxon test. In order to take into account competition of some variables with the risk of death, survival analyses were performed using Fine and Gray’s method.p < 0.1 in the multivariable logistic regression model.The rate of patients with abundant microaspiration of gastric contents, or abundant microaspiration of oropharyngeal secretions, was compared between study groups using chi-square, and OR (95% CI) was calculated. Additional adjusted analysis was performed, including variables collected at ICU admission with Data were analyzed using SAS version 9.4  and R version 3.3.1.p = 0.862, in enteral and parenteral nutrition groups, respectively).One hundred eighty-two patients were included in the NUTRIREA2 study in the 13 centers participating in the ancillary study from February through June 2015, and 151 (84%) patients were included in the ancillary study. Among the 151 included and analyzed patients, 78 and 73 received enteral and parenteral nutrition, respectively  vs 22 of 73 patients (36%), unadjusted OR 0.8 (95% CI 0.69–0.93), and adjusted OR (0.79 (0.67–0.94)). The percentage of patients with abundant microaspiration of oropharyngeal secretions was significantly higher in enteral nutrition group, as compared with parenteral nutrition group (48 of 78 (74%) vs 33 of 73 patients (54%), unadjusted OR 1.21 (95% CI 1.03–1.44), and adjusted OR 1.23 (1.01–1.48)  was significantly lower in enteral, compared with parenteral nutrition groups. The median pepsin level in study patients  vs 97  ng/ml, p = 0.090) was not significantly different between enteral and parenteral nutrition groups. The median (IQR) percentage of tracheal aspirates positive for alpha amylase  vs 33 , p = 0.055), and the median alpha amylase level in study patients  vs 1916  IU/ml, p = 0.069) were not significantly different between enteral and parenteral nutrition groups, respectively.The median (IQR) percentage of tracheal aspirates positive for pepsin  vs 0 , p = 0.34), or between patients who received stress ulcer prophylaxis and those who did not  vs 104  IU/ml, p = 0.35). No significant difference was found in salivary amylase level between patients with vomiting and those with no vomiting  vs 3038  IU/ml, p = 0.17).No significant difference was found in pepsin level between patients with vomiting and those with no vomiting  vs 88  ng/ml, To our knowledge, our study is the first to evaluate the relationship between the route of nutrition and the risk for microaspiration in intubated and mechanically ventilated patients, using valuable biomarkers of gastric and oropharyngeal secretion microaspiration. Our results suggest that enteral nutrition, as compared with parenteral nutrition, is associated with reduced risk for abundant microaspiration of gastric contents and increased risk for abundant microaspiration of oropharyngeal secretions.The role of the stomach in the pathogenesis of VAP has been a matter for debate during the last decades. Although some studies suggested that the stomach played an important role in the occurrence of VAP , 11, 16,Some potential explanations could be suggested for this unexpected result. In patients receiving enteral nutrition, pepsin levels might have been artificially reduced because of pepsin dilution in the large quantity of liquid given for enteral nutrition. Further, enteral nutrition increases gastric pH  and coulOur results are in line with the NUTRIREA1 multicenter randomized trial, which showed that, compared to routine residual gastric volume (RGV) monitoring, absence of RGV monitoring in patients receiving invasive mechanical ventilation was associated with higher vomiting rate but no increased risk of VAP . Thus, iIn addition to the above-discussed limitations, pepsin and salivary amylase were only measured during 48 h and not during the whole period of mechanical ventilation. In addition, the study was not blinded. However, the measurement of pepsin and amylase was performed in a blinded manner. Further, our definition of abundant microaspiration was stringent, and one could argue that if a different cutoff had been used, our results might have been different. However, similar results were obtained regarding the percentage of tracheal aspirates positive for pepsin between the two groups. Strengths of our study include the randomized controlled multicenter design, the use of quantitative markers of microaspiration, and the relatively large number of patients and tracheal aspirates analyzed.Our results suggest that enteral nutrition is associated with a reduced risk for microaspiration of gastric contents and an increased risk for microaspiration of oropharyngeal secretions. The route for nutrition in mechanically ventilated patients with shock should probably not be determined based on the risk of microaspiration and VAP.


Cervus elaphus). Female red deer exhibited a higher mean and phenotypic variance in autosomal crossover counts (ACC). Animal models fitting genomic relatedness matrices showed that ACC was heritable in females (REC8 and RNF212B, with an additional region on linkage group 32 containing TOP2B approaching genome-wide significance. The REC8/RNF212B region and its paralogue RNF212 have been associated with recombination in cattle, mice, humans and sheep. Our findings suggest that mammalian recombination rates have a relatively conserved genetic architecture in both domesticated and wild systems, and provide a foundation for understanding the association between recombination loci and individual fitness within this population.Recombination is a fundamental feature of sexual reproduction, ensuring proper disjunction, preventing mutation accumulation and generating new allelic combinations upon which selection can act. However it is also mutagenic, and breaks up favorable allelic combinations previously built up by selection. Identifying the genetic drivers of recombination rate variation is a key step in understanding the causes and consequences of this variation, how loci associated with recombination are evolving and how they affect the potential of a population to respond to selection. However, to date, few studies have examined the genetic architecture of recombination rate variation in natural populations. Here, we use pedigree data from ∼ 2,600 individuals genotyped at ∼ 38,000 SNPs to investigate the genetic architecture of individual autosomal recombination rate in a wild population of red deer ( It has RNF212, REC8 and HEI10, among others, have been identified as candidates driving variation in rate, with PRDM9 driving recombination hotspot positioning in mammals  on the island of Rùm, Scotland  and has been subject to individual monitoring since 1971 . Individuals with high numbers of crossovers per gamete (>60) were assumed to have widespread phasing errors and were removed from the analysis; the maximum number of crossovers for an individual was 45 in the remaining dataset.A standardized sub-pedigree approach was used to identify the positions of meiotic crossovers  as there is substantial relatedness within the population, and initial models included parental effects and common environment which controls for effects of shared environments between relatives. ACC was modeled first using a univariate model:y is a vector of ACC; X is an incidence matrix relating individual measures to a vector of fixed effects, a and e is a vector of residual effects. The narrow-sense heritability i.e., permanent environment) to account for repeated measures in the same FID, maternal and paternal identity, and common environment effects of FID birth year and offspring birth year. A dominance GRM constructed in GCTA was also fitted but was not significant and explained <1% of the phenotypic variance, and was therefore not included in the final models. The significance of fixed effects was tested with a Wald test, and the significance of random effects was calculated using likelihood-ratio tests  was modeled as a trait of the FID. A restricted maximum-likelihood (REML) “animal model” approach  was usedBivariate models of male and female ACC were run to determine whether additive genetic variation was associated with sex-specific variation and the degree to which this was correlated between the sexes. The additive genetic correlation rGLS in the R library RepeatABEL v1.1 , rather than SNPs associated with recombination rate globally across the genome . Therefore, we repeated the GWAS modeling trans variation only, by examining associations between each SNP and ACC, minus the crossovers that occurred on the same chromosome as the SNP. For example, if the focal SNP occurred on linkage group 1, association was tested with ACC summed over linkage groups 2-33. Marker positions are known relative to the cattle genome vBTA_vUMD_3.1; in cases of significant associations with recombination rate, gene annotations and positions were obtained from Ensembl (Cattle gene build ID BTA_vUMD_3.1.89). LD was calculated between loci in significantly associated regions using the allelic correlation Genome-wide association studies (GWAS) of ACC were conducted using the function ffective  specifyii.e., a regional heritability approach) incorporates haplotype effects and determines the proportion of phenotypic variance explained by defined regions. The additive genetic variance was partitioned across all autosomes in sliding windows of 20 SNPs (with an overlap of 10 SNPs) as follows . Females had significantly higher phenotypic variance in ACC than males (P = 0.002) and within females only (P = 0.033), but was not heritable in males alone . Heritability estimates from the GRM were similar to those estimated using pedigree relatedness, which were 0.14 (SE = 0.052), 0.13 (SE = 0.064) and 0.10 (SE = 0.110) for both sexes, females and males, respectively. Bivariate models of ACC between the sexes indicated that the genetic correlation . Raw and quality controlled ACC counts and positions for each FID and offspring combination per chromosome are provided in Table S2.Autosomal crossover count (ACC) was significantly higher in females than in males, where females had 4.32 cela1_red_10_26005249 on deer linkage group 12 (CEL12), corresponding to position 26,005,249 on cattle chromosome 10 (BTA10). This marker was also the most highly associated SNP when considering recombination in trans, indicating that this region affects ACC across the genome (Table S3). The observed association was primarily driven by female ACC . Based on its position relative to the cattle genome, cela1_red_10_26005249 was OR5AU1 and TOX4, CHD8, SUPT16H and CCNB1IP1 (trans across the genome (Table S3).No SNPs were significantly associated with ACC at the genome-wide level . Detailed examination of this region in sliding windows of 6, 10 and 20 SNPs found the highest association at a 10 SNP window of REC8 . The total significant region after detailed examination was cela1_red_10_18871213 and cela1_red_10_21878407 , a homolog of RNF212, which has been directly implicated in synapsis and crossing-over during meiosis in mice ; inhibitors of this gene lead to defects in chromosome segregation and heterochromatin condensation during meiosis I in mice, Drosophila melanogaster and Caenorhabditis elegans  and 1.8 - 2.8 fewer crossovers per gamete in males  but not in males ; homozygotes for the haplotype were rare  and so the large effect size estimate was again likely to be subject to large sampling effects . Of these, two haplotypes, AGGAGAGAAG and AGAGAAGAGA, had a significant effect on ACC relative to all other haplotypes  is 1.2Using complementary trait mapping approaches, we identified a WAS hits ; associaAt present, it is not clear why the results of the two analyses occur in close vicinity, yet do not overlap. Assuming homology with humans, cattle, sheep and mice : inhibitors of this gene lead to defects in chromosome segregation and heterochromatin condensation during meiosis I in mice, Drosophila melanogaster and Caenorhabditis elegans , Chromodomain Helicase DNA Binding Protein 8 (CHD8) and SPT16 Homolog Facilitates Chromatin Remodelling Subunit (SUPT16H). These genes are involved in with chromatin binding and structure , histone binding , nucleosome organization (SP16H) and cell cycle transition (TOX4). One of these genes, SUPT16H, interacts with NIMA related kinase 9 (NEK9), which is involved with meiotic spindle organization, chromosome alignment and cell cycle progression in mice (cela1_red_10_26005249 was CCNB1IP1), also known as Human Enhancer Of Invasion 10 (HEI10), which interacts with RNF212 to allow recombination to progress into crossing-over in mice ; indeed, most phenotypic variance in recombination was attributed to residual effects. This suggests that despite some underlying genetic variation, recombination rate is mostly driven by stochastic effects, or otherwise unmeasured effects.We have shown that recombination rate is heritable in female red deer, and that it has a sexually dimorphic genetic architecture. Genomic regions associated with recombination rate in red deer are associated with this trait in other mammal species, supporting the idea that recombination rate variation has a conserved genetic architecture across distantly related taxa. A key motivation for this study is to compare how recombination rate and its genetic architecture is similar or different to that of model species that have experienced strong selection in their recent history, such as humans, cattle, mice and sheep. The heritability of recombination rate in deer was lower than that observed in other mammal systems (This represents one of the smallest datasets in which recombination rate has been investigated, and so it may be that the observed effects are underestimated due to the small sample size, sampling effects, or perhaps that other genetic variants present in this species do not segregate in the Rùm deer population. Nevertheless, identification of clear candidate genes and their effects on phenotype represents a valuable contribution to understanding the genetic architecture of recombination more broadly. Ultimately, our findings allow future investigation of the fitness consequences of variation in recombination rate and the relationship between identified variants and individual life-history variation, to address questions on the maintenance of genetic variation for recombination rates, and the relative roles of selection, sexually antagonistic effects and stochastic processes in contemporary natural populations.


Anopheles gambiae, caused by the widespread use of insecticides for malaria control, to investigate the role of CNVs in the evolution of insecticide resistance. Using the whole-genome sequencing data from 1142 samples in the An. gambiae 1000 genomes project, we identified 250 gene-containing CNVs, encompassing a total of 267 genes of which 28 were in gene families linked to metabolic insecticide resistance, representing significant enrichment of these families. The five major gene clusters for metabolic resistance all contained CNVs, with 44 different CNVs being found across these clusters and multiple CNVs frequently covering the same genes. These 44 CNVs are widespread  and have been spreading through positive selection, indicated by their high local frequencies and extended haplotype homozygosity. Our results demonstrate the importance of CNVs in the response to selection, highlighting the urgent need to identify the contribution of each CNV to insecticide resistance and to track their spread as the use of insecticides in malaria endemic countries intensifies and as the operational deployment of next-generation bed nets targeting metabolic resistance gathers pace. Our detailed descriptions of CNVs found across the species range provide the tools to do so.Polymorphisms in genetic copy number can influence gene expression, coding sequence, and zygosity, making them powerful actors in the evolutionary process. Copy number variants (CNVs) are however understudied, being more difficult to detect than single-nucleotide polymorphisms. We take advantage of the intense selective pressures on the major malaria vector  This cost is mitigated in heterozygotes, leading to the spread of heterogeneous Ace1 duplications in which the mutant and wild-type alleles co-occur (Copy number variants (CNVs) are a form of genetic variation that occur when a genomic sequence is deleted or duplicated, potentially affecting both the structure and expression levels of coding sequences and playing a crucial role in evolution and adaptation . Part ofcription . Alternacription . CNVs caco-occur .Although the importance of CNVs is widely recognized, they typically receive less attention than single-nucleotide polymorphisms (SNPs) in investigations of genetic variation, likely because they are harder to identify. Population-level genome-wide analyses of CNVs are thus rare, and the extent of their impact on evolution is poorly understood .An. gambiae and its close sister species An. coluzzii are excellent organisms in which to study the evolution of CNVs at a population level for three reasons. First, these species are the major vectors of malaria in Sub-Saharan Africa (SSA) and are highly anthropophilic (Anopheles gambiae 1000 Genomes Project (Ag1000G) has produced whole-genome sequencing data from 1142 individual An. gambiae and An. coluzzii from multiple locations in SSA, providing a unique opportunity to conduct genome-wide searches for CNVs from across the species’ distributions .The malaria mosquito pophilic . Consequpophilic . Despitepophilic . CNVs poAce1 (para voltage-gated sodium channel (Vgsc)  , 2017, al (Vgsc) , usually confers  or dimin confers .An. gambiae, up-regulation of the cytochrome P450s Cyp6m2 and Cyp6p3 is associated with resistance to pyrethroids and bendiocarb . The largest of these  was the well-documented duplication in the insecticide target site Ace1. No CNVs were found in the other common insecticide target sites Vgsc or Rdl.We first used short-read whole-genome sequencing data from the Ag1000G Phase 2 data set to find genomic regions with increased read coverage relative to the genomic average, controlling for GC richness and excluding 27 individual mosquitoes with unusually high coverage variance. Briefly, we filtered 300-bp genomic windows for mapping quality and rare levels of GC content  and identified regions covering at least five contiguous 300-bp windows of at least 1.5-fold increased coverage determined by a Hidden Markov Model (HMM). We then filtered this initial CNV set based on the relative likelihood of the observed read coverage in the absence of a CNV. After grouping identical CNVs between individuals according to their genomic locations and retaining only CNVs present in at least 5% of individuals in a population , we identified 1557 candidate CNVs  and good rates of recovery for CNVs spanning at least 10 windows , with a lower rate of recovery for CNVs spanning only five windows .P < 0.0002; from 10,000 simulations, the number of heterochromatic CNVs ranged from 44 to 112, with a mean of 77). CNVs in heterochromatin were also significantly larger than in euchromatin  .To determine the genomic factors associated with the presence of CNVs, we investigated how CNVs are distributed relative to chromatin type and gene content. We found that CNVs were particularly abundant in heterochromatic regions. Of the 1557 CNVs, 534 (34.3%) were found in the heterochromatin, which covers only 9.3% of the genome . Because euchromatin typically contains more genes than heterochromatin, we repeated the simulation, focusing on euchromatic CNVs and randomizing their position within the euchromatin. Of the 1023 euchromatic CNVs, 226 contained at least one gene, again representing a significant enrichment for genic regions . This enrichment was consistent for all sizes of euchromatic CNVs .CNVs were also more abundant in gene-containing regions. Of the 1557 CNVs, 250 contained at least one gene, significantly more than expected by chance . Of these 267 genes, 28 were candidate metabolic resistance genes . Because many related genes occur in clusters, and are therefore not independently included in CNV events, we counted the number of CNVs that included at least one metabolic detox gene. Of the 250 CNVs that contained any genes, 27 contained at least one metabolic detox gene, significantly more than expected by chance . The same result was found when only euchromatic CNVs were considered, for all CNV sizes . Although there was some variation between populations in the number of metabolic detox genes found in CNVs (P = 0.08).We next explored whether CNVs were enriched for genes potentially involved in metabolic insecticide resistance. Overall, out of 10,939 genes included in the analysis, 267 (2.4%) were within at least one CNV , primarily reflecting an enrichment for two classes of genes: cytochrome P450s  and proteases . This result also held when heterochromatic CNVs were excluded, with 12 of the 13 GO terms remaining significant . No GO terms from biological process or cellular compartment ontologies were significantly enriched.Genes found in CNVs were enriched for 13 molecular function Gene Ontology (GO) terms after multiple correction to a Supplemental Data S2), with 16 of the 28 genes being members of the glutathione S-transferase epsilon cluster (Gste on Chromosome 3R) or one of four cytochrome P450 clusters . This again indicates that genes involved in metabolic insecticide resistance have been the focus of amplification events. We therefore performed a detailed analysis of the CNVs around these five gene clusters , we extended the study region around Cyp6p3 to include these genes.The 28 metabolic detox genes found inside CNVs were predominantly from gene clusters that have previously been implicated in insecticide resistance (clusters A. BecausCyp9k1 (named Cyp9k1_Dup1–16) , 15 in the Cyp6aa1–Cyp6p2 cluster (Cyp6aap_Dup1–15) , 11 in the Gstu4–Gste3 cluster (Gstue_Dup1–11) , one in Cyp6m2 (Cyp6m_Dup1) , and one in Cyp6z3–Cyp6z1 (Cyp6z_Dup1) .To more accurately determine the number of CNVs in each of these five gene clusters, we used discordant read pairs and reads aligning to CNV breakpoints to distinguish different CNVs. Three of the five gene clusters showed high numbers of different CNVs. We identified 16 CNV alleles in An. coluzzii from Burkina Faso, Côte d'Ivoire, Ghana, and Guinea) , although none were found in all populations of either species . Furthermore, multiple CNV alleles covering the same genes could be found in the same population  . For the Cyp6aa/p cluster, CNVs were primarily found in An. coluzzii from Burkina Faso, Côte d'Ivoire, and Ghana  carried at least one of the CNVs described in these five gene clusters.Several CNV alleles were found across different populations , and two CNVs were supported by read pairs mapping in the same orientation, indicating a tandem inversion . The remaining CNVs were supported by reads whose mate or soft-clipped bases mapped elsewhere on the genome, often in multiple genomic locations, suggesting a role for transposable elements (TEs) in the duplication event . Two of these CNVs had flanking sequences that matched known transposable elements in An. gambiae. The soft-clipped bases of reads mapping to the breakpoints of Gstue_Dup7 and Gstue_Dup8, respectively, returned significant BLAST hits to a hAT DNA transposon  and a transposon bearing similarity to mariner/Tc1-like elements  (We used the patterns of discordant reads supporting each of the CNVs to putatively determine the type of duplication event that caused them . Of the = 10−34) , in the = 10−34) .Supplemental Data S4), making the real number of CNV events even greater than reported. Equally, a mutation that disrupts the breakpoint of a CNV could affect its pattern of discordant reads and lead to a single CNV event appearing as two different CNV alleles. Although we cannot exclude this possibility, analysis of the haplotype backgrounds of the different CNVs suggested that this is not the case for at least most of the CNV alleles .The CNVs were differentiated based on their patterns of discordant read pairs, which are influenced by the position of the CNV breakpoints and the orientation of the duplication . It is pSupplemental Data S9), suggesting that they are likely to be under positive selection. To investigate this possibility, we phased the CNV genotype calls onto the Ag1000G Phase 2 haplotype scaffold and calculated extended haplotype homozygosity (EHH) for the CNV alleles present in at least 5% of individuals in a population.Several CNV alleles were found at high local frequencies , supporting our contention that these alleles are reaching high frequency through positive selection. Furthermore, the median length of shared haplotypes was significantly higher between pairs of haplotypes carrying the same CNV allele than between wild-type haplotypes from the same population  .Rates of EHH decay around CNV alleles were consistently lower than for wild-type (WT) haplotypes  could not be phased. We therefore investigated whether this CNV was associated, at the sample level, with haplotypes under selection. Hierarchical clustering of the haplotypes in these three populations revealed two large cross-population haplotype clusters around Cyp9k1, indicating selective sweeps . Cluster 1 was very strongly associated with Cyp9k1_Dup11 in both males   and females  (P < 0.0001) .Phasing of CNV genotype calls was only possible for simple duplications, where the zygosity of the CNV alleles could be determined from the copy number estimates. For CNV alleles with higher copy numbers (triplications and above), this was not possible; thus the EHH decay could not be calculated. In the case of  0.0001) A. ClusteGstu4–Gste3 cluster included Gste2 . The presence of 114T homozygotes, together with the ratio of reads supporting the I114 and 114T alleles in heterozygotes (roughly 1:2), indicate that both copies of Gste2 in the Gstue_Dup1 CNV carry the 114T mutation.Ten of the 11 CNV alleles found in the ed Gste2 , perhapssistance  and coulCyp6aa1–Cyp6p2 cluster, five included Cyp6p3 but 13 included Cyp6aa1 (Cyp6p3 CNVs were found at high (>50%) frequency in one population (Côte d'Ivoire An. coluzzii: 90%), whereas Cyp6aa1 CNVs were found at high frequency in An. coluzzii from Burkina Faso (91%), Côte d'Ivoire (89%) and Guinea (75%), and in An. gambiae from Uganda (64%).Of the 15 CNV alleles found in the  Cyp6aa1 B. Cyp6p3An. gambiae and An. coluzzii, with CNVs being more abundant and larger in heterochromatin than in euchromatin. This parallels results in humans and rats, in which CNVs have been found to be particularly abundant in centromeres and telomeres , were all found to be amplified in at least one population. Furthermore, three of these genes showed evidence of repeated CNV events within and between populations, with a total of 44 different CNVs across the five clusters, and as many as 16 in Cyp9k1. Of 44 CNVs, the majority (33) were tandem duplications, two were tandem inversions, and two (both in the Gstu4–Gste3 cluster) showed evidence of association with known DNA transposons. The remaining seven could not be classified, but the presence of unmappable sequences around the breakpoints suggests that they may also be associated with repetitive elements.The genes found in CNVs were themselves enriched for families involved in metabolic insecticide resistance. These results mirror findings in  in CNVs . Similarulations . In our Gste2 in insecticide resistance comes from studies showing its up-regulation in DDT resistant An. gambiae , which interfere with cytochrome P450-mediated insecticide resistance, and which have been shown to be effective at reducing mosquito abundance and malaria incidence in at least some regions . Howevery region , need toGstu/e cluster included Gste2, indicating that this is the major target of gene amplification in this cluster. Given the body of evidence linking Gste2 to DDT and pyrethroid resistance across multiple species—An. gambiae  all show evidence of positive selection. Although Cyp6aa1 has received substantially less attention than Cyp6p3, it has previously been implicated in insecticide resistance. Expression of Cyp6aa1 is higher in populations of An. gambiae and An. coluzzii that are resistant to pyrethroids and DDT compared to susceptible laboratory colonies  . The specimens were collected from sites in 13 African countries  An. gambiae n = 9, Gabon An. gambiae n = 69, Ghana An. coluzzii n = 55, Ghana An. gambiae n = 12, Guinea An. coluzzii n = 4, Guinea An. gambiae n = 40, Guinea-Bissau (mixed ancestry) n = 91, Kenya (undetermined ancestry) n = 48, Mayotte An. gambiae n = 24, The Gambia (mixed ancestry) n = 65, Uganda An. gambiae n = 112). Individual specimens were sequenced using the Illumina HiSeq platform to obtain 100-bp paired-end reads with a target coverage of 30×. Further details of population sampling, sample preparation, sequencing, alignment, species identification, and data production are reported elsewhere  to count the number of aligned reads (coverage) in nonoverlapping 300-bp windows over the nuclear genome. The position of each read was considered to be its alignment start point; thus, each read was only counted once. Sequencing coverage can be biased by variation in local nucleotide composition. To account for this, we computed a normalized coverage from the read counts based on the expected coverage of each window given its GC content . The normalized coverage values were then multiplied by a factor of 2, so that genome regions with a normal diploid copy number should have an expected normalized coverage of 2.For each individual, we used the  content . For eacSupplemental Fig. S13), which indicates that a read is mapped ambiguously and could be mapped equally well to a different genomic location. This filter removed 159,587 (20.8%) of 768,225 windows. The second filter removed windows for which the percentage (G + C) content was extreme and rarely represented within the accessible reference sequence, that is, fewer than 100 accessible windows with the same (G + C) percentage, because the small number of windows makes the calculation of a (G + C) normalizing constant unreliable. This filter removed 13,484 (2.2%) of the 608,638 remaining windows. Windows retained for analysis were referred to as “filtered windows.”Before examining the normalized coverage data for evidence of copy number variation, we applied two filters to exclude windows for which coverage may be an unreliable indicator of copy number. The first filter removed windows in which >2% of reads were aligned with mapping quality 0 . Because we are primarily interested in amplifications rather than deletions, we obtained a raw set of CNV calls for each sample by locating contiguous runs of at least five windows with amplified CNS .To detect the most likely copy number state (CNS) at each window in each individual, we applied a Gaussian HMM to the individual's normalized windowed coverage data, following a similar approach to Supplemental Fig. S14), retaining 1115 samples for further analysis.From the raw CNV call set, we created a quality-filtered list of CNV calls. We first removed samples with very high coverage variance, because high variance could lead to erratic CNV calls. We therefore removed 27 samples for which the variance in normalized coverage was greater than 0.2 . For the second filter, we removed CNVs with low population frequencies. To do this, the raw CNV calls needed to be matched so that the same CNV in different individuals could be identified. We classed any two CNVs as identical if the breakpoints predicted by their copy number state transitions were within one window of each other. We then removed CNVs that were not found in at least 5% of individuals in at least one population .We then applied two filters to the raw CNV calls from these 1115 samples. For the first filter, we computed likelihoods for each raw CNV call for both the copy number state predicted by the HMM and for a null model of copy number = 2, and removed CNV calls for which the likelihood ratio was <1000 . The start/end points of each CNV were calculated as the median of the start/end points of all the raw CNV calls that were matched to it. To keep only genes for which good coverage data were available, we retained only genes containing at least 50% filtered windows. We classed a retained gene as copied by a CNV if all the filtered windows within the gene were inside the CNV. To determine whether the CNVs that we detected contained more genes than expected by chance, we performed simulations as described for investigating enrichment for chromatin types, this time counting the number of CNVs that contained at least one gene. We also repeated these simulations after excluding the heterochromatic regions by only randomizing the euchromatic CNVs and preventing their randomized positions from being in the heterochromatin.To determine the genes contained within each CNV, we compared the start and end points of the CNVs to the start and end points of all genes listed in the AgamP4.2 gene annotations (Anopheles-gambiae-PEST_ TRANSCRIPTS_AgamP4.2.fa). We performed simulations to determine whether genes copied by CNVs were enriched for metabolic detox genes. For each run of the simulation, we randomized each genic CNV by reallocating the genes encompassed by the CNV, keeping the number of consecutive genes covered by each CNV unchanged, and calculated the number of CNVs that included at least one metabolic detox gene. This simulation was run 10,000 times to obtain the distribution of the null model. Two-tailed P-values were obtained by calculating the proportion of simulations that produced at least as many genic CNVs containing detox genes as observed in the real data and multiplying this by two.We identified genes that could potentially be involved in metabolic resistance through detoxification (“metabolic detox genes”) by finding genes whose annotations contained the terms “P450,” “glutathione S-transferase,” or “carboxylesterase” in the AgamP4 transcript annotations . False discovery rates were calculated from the P-values using the R package fdrtool (https://cran.r-project.org/web/packages/fdrtool/index.html).GO term analysis of genes included in CNVs was performed using the Cyp6aa1–Cyp6p2, Gstu4–Gste3, Cyp6m2–Cyp6m4, Cyp6z3–Cyp6z1, Cyp9k1) using their unique patterns of discordant read pairs and reads crossing the CNV breakpoint . We manually inspected the five regions of interest in all 1142 samples to identify patterns of discordant and breakpoint reads (“diagnostic reads”) consistently associated with changes in coverage . The start and end point of each CNV allele could usually be precisely determined by the breakpoint reads and was otherwise determined by discordant read pairs or the point of change in coverage . Once the diagnostic reads were identified for a CNV allele, we recorded the presence of that allele in all samples with at least two supporting diagnostic reads.We characterized in detail the different duplication events  at five gene clusters of particular interest  of repbase/ ).scikit-allel Python package (https://zenodo.org/record/3238280).We used the phased haplotypes to calculate the pairwise shared haplotype length and the EHH for each CNV allele , using oCyp9k1 region were obtained with scikit-allel, using the first 1000 SNPs on the centromeric side of Cyp9k1 (the telomeric side of this gene has low levels of accessibility). A distance matrix between haplotypes was computed using the proportion of accessible SNPs that differed between pairwise haplotype combinations. This was used to perform hierarchical clustering, with haplotype clusters defined using a cutoff of 0.001.Haplotype clusters in the P-values were obtained using the “simulated.p.value” option with 106 replicates.Statistical analysis was performed in R . ContingSupplemental Fig. S15; Supplemental Methods SM5). Thus, even when overlapping CNV alleles were present in a single sample, we were usually able to determine the number of copies of each allele.To determine the allele-specific copy number in a sample, we estimated the change in coverage associated with each CNV allele . In one case (Cyp6m_Dup1), all individuals were found to have a copy number of 2, indicating that the CNV is a triplication, with no duplications present in the population. This CNV was therefore retained, with all samples carrying the CNV classed as heterozygotic.For single-copy CNVs, it is possible to determine the genotype of a sample from the copy numbers . For higher-order CNVs, this is not possible because a heterozygote triplication cannot be distinguished from a homozygote duplication. We therefore applied two filters to retain only single-copy CNV alleles. The first filter removed CNV alleles for which the allele-specific copy number was found to rise above 2 in the data . This filter removed five CNV alleles . For the second filter, we classed each sample as homozygote wild-type, heterozygote or homozygote CNV based on their copy numbers, and then removed CNV alleles that were inconsistent with Hardy-Weinberg expectations within the populations in which they were found. This filter removed four CNV alleles . Three CNV alleles  were also excluded because of difficulties in calling allele-specific copy number (λ = 0.1 to ensure that none of the input CNV genotype calls were changed during phasing. For each of the five gene clusters, phasing was performed using the 200 nonsingleton SNPs either side of the region in which CNVs were found, thus avoiding the inclusion of SNPs found inside any of the CNVs. Haplotypes that contained more than one CNV allele were rare and therefore excluded from subsequent calculations of haplotype homozygosity.CNV alleles that passed both filters were phased onto the Ag1000G Phase 2 haplotype scaffolds using the MVNcall software v1.0 , using d


The experimental solubility was determined in five different media using a validated HPLC method. Although the experimental solubility was lower than the predicted one, a good linear relationship was observed. The results also indicated a minimal impact of endogenous tensioactives on solubility, suggesting dissolution rate limited absorption. The in silico experiments were focused on identification of molecular determinants of solubility, evaluation of drug-likeness, prediction of in vivo absorption based on mechanistic models, as well as identification of the main factors that could impact on the oral bioavailability. The results suggested that dose, solubility and particle size are the main determinants of absorption, whereas permeability has little effect, confirming the BCS Class II behavior of the compounds. The present investigation was able to rank the tested compounds in terms of biopharmaceutical behavior, and indicated the B3 series compounds as having a more favorable absorption profile making them the main candidates for advance to the pre-clinical in vivo studies.Several new pyrazole derivatives have demonstrated promising antiproliferative and cytotoxic effects, but their poor solubility raised concerns over possible biopharmaceutical limitations. In order to improve their pharmaceutical potential we performed the biopharmaceutical profiling for nine pyrazole compounds using  Acanthamoeba parasites -thiourea presented logCI50 values under −4 in 43 of the NCI 60 cancer lines, values ranging from −4.12 to −5.75, with an average of −5.02. The best results were recorded on ovarian and the renal cancer cells [Triticum aestivum embryonic root length test, correlated with a low general toxicity [In our previous studies ,13 we syin vitro experiments raised concerns over their possible biopharmaceutical limitations.Although the preliminary cytotoxicity results suggested comparable activity for all the selected compounds, their poor solubility exhibited during the Discovery and development of new drugs is a very lengthy and costly process, with extremely high failure rates . A numbein vivo performance of a drug product.Some systematic approaches to biopharmaceutical profiling were introduced based on Biopharmaceutics Classification System (BCS) , which pin vivo fluids from different segments of the gastrointestinal tract by taking into consideration the solubilizing effect of physiological surface active agents. More recently, the concepts of “drugability” [in vitro approaches were used to assess molecular properties correlated with oral absorption and bioavailability [Since evaluation of solubility in aqueous buffer media is proved to have rather poor prediction power , dissoluability”  and “devability”  substantability” . Differelability ,27.in silico tools capable to forecast drug absorption and to identify critical factors influencing bioavailability based on different mechanistic absorption models i.e., GastroPlusTM which is based on the advanced compartmental absorption and transit ACAT model, SimcypTM, which is based on the advanced dissolution absorption and metabolism (ADAM) model or PK-SIMTM [The growing interest in the biopharmaceutical characterization of both drug candidates and pharmaceutical products was reflected in the development of several PK-SIMTM ,30,31,32in vivo studies.Since the pyrazolyl thiourea derivatives taken into the present study are poorly soluble compounds, their biopharmaceutical profiling arises as a necessity and the current paper presents their screening. The obtained profile was used to rank the selected compounds in terms of developability. This research also suggests a pharmaceutical risk assessment strategy that goes beyond the usual characterization of a clinical candidate molecule, in order to prioritize candidates for advance to the pre-clinical The values for the calculated descriptors are presented in Special emphasis was put on evaluation of the drug-like indices, since they can act as useful filters to identify good drug candidates from large collections of compounds. The “drug-like” indices calculated by E-Dragon are dummy variables taking value equal to 1 when all the criteria of the consensus definition of a drug-like molecule are satisfied and 0 otherwise. Two different classes of indices were evaluated:(a) A biopharmaceutical predictor -Lipinski Alert Index (LAI)- based on the “Lipinski Rule of 5” , which pi.e., antiinflammatory-like index (Inflammat), antidepressant-like index (Depressant), antipsychotic-like index (Psychotic), antihypertensive-like index (Hypertens), hypnotic-like index (Hypnotic), antineoplastic-like index (Neoplasic) and antiinfective-like index (Infective), were evaluated. Two different ranges have been proposed: the qualifying range covering approximately 80% of the active studied drugs and the preferred range defined as the smallest range within the qualifying range containing approximately 50% of the drugs [(b) A group of eight drug-like indices were proposed by Ghose-Viswanadhan-Wendoloski , derivedhe drugs .For the particular case of the Neoplasic-50 index, a compound with antineoplasic activity has AlogP between 0.0 and 3.7, AMR is between 60 and 107, MW between 258 and 388, the total number of atoms 30 to 55, and contains at least one of the structural characteristics mentioned above. The values obtained for the evaluated indices as well as values of the molecular descriptors used for their assessment are presented in Since LAI is an “alert index” a 0 value suggests that none of the poor absorption or permeation criteria are met, therefore all the compounds are likely to have an adequate biopharmaceutical profile.BC5, BM5 and BT5 are not drug-like structures at all in terms of GVWI. The other compounds have structure and molecular properties favorable for both antineoplastic and antiinfective activity.In terms of drug class likeliness, the indices based on the more restrictive preferred range (50%) were used. The compounds don’t seem to be active on the Central Nervous System, whereas 2 value >0.999 for all the tested compounds (BM2 to 0.024 µg/mL for BT3).The HPLC method employed method for the pyrazole derivatives quantitative analysis was found to be linear in the range 0.1–10 μg/mL, with a mean with a mean Rompounds . The limRepeatability and intermediate reproducibility experiments resulted in RSD values ranging from 0.24% to 4.2%. The bias (%) between the experimental and nominal concentrations of the quality control samples did not exceed 4.5%. All these results indicated that precision and accuracy of the assay are satisfactory. Representative chromatograms obtained for the analyzed pyrazole derivatives depicted in Solubility is a crucial parameter for successful drug development, as poor solubility could compromise the pharmacokinetic and pharmacodynamic profile of drug candidate molecules ,36.The equilibrium solubility experiments for the selected pyrazole derivatives were performed in five media simulating different segments of the GI tract  and two biorelevant media containing physiological surface active agents (FaSSIF and FeSSIF).BT3 > BM3 > BC3 > BT2 > BT5 > BM5 > BM2 > BC5 ≈ BC2. Solubility appeared to be slightly higher in the simulated gastric fluid , and decreased as the pH increased.The experimental solubility of the tested compounds in the above media is presented in Solubility in biorelevant media is generally higher compared to solubility measured in corresponding aqueous buffers ,38 due tw) of any organic non-electrolyte or weak electrolyte was found to be directly correlated with two molecular properties: octanol-water partition coefficient (logP) and melting point  of the compound [et al. [In order to explain this somewhat “strange” behavior we have to take into account the fact that aqueous solubility  could provide an explanation for that behavior.At pH 6.5, the theoretical distribution of the ionic species for the analyzed pyrazole derivatives suggested about 80% contribution of the neutral and 20% contribution of the negatively charged form.As lecithin is zwitterionic and taurocholic acid is fully deprotonated at pH 6.5, FaSSIF contains a net negative charge, and this could explain the different behavior observed for acids and bases, it is very likely that the negative charge of the compounds is detrimental for their solubility because of electrostatic repulsion with the media components .in silico predictions of solubility performed with the GastroPlusTM software  is in the range reported in literature to correspond to sodium taurocholate critical micellar concentration (CMC) (1–12 mM) [The FaSSIF media generated the highest discrepancies between the estimated and 1–12 mM) , and in 1–12 mM) .Two other important aspects can be observed. Firstly, the simulation software predicts an important contribution of solvation on solubility , whereas the experimental results prove a solid-state limited solubility behavior. As a result, solubility in biorelevant media is significantly overestimated by the software.BM2, BC2 and BC5) the solubility is systematically underestimated in all dissolution media. It is to note that BM2 and BC2 have typical “brick dust” characteristics , size and polarizability  had a significantly negative impact on solubility . The resin silico is presented in A selective presentation of the main physicochemical and pharmacokinetic parameters evaluated It is noteworthy that for all analyzed molecules the predicted effective human jejunal permeability values are higher than Peff of metoprolol, which is generally accepted as borderline between low- and high-permeability behavior ,52, therBC3, BM3 and BT3 (2.5 up to 9 times higher than the rest), as they also are the only compounds with complete absorption under the simulation conditions .The MAD values are significantly higher for TM the Human-Physiological-Fasted physiological model with the default Opt logD SA/V 6.1 Absorption Scale Factor (ASF) parameters resulted in the simulated absorption profiles presented in BC3, BM3 and BT3, for the rest of the compounds absorption being slow and incomplete  the simulations suggested that absorption will occur almost completely in the small intestine. This is in accordance with the fact that small intestine represents the optimal absorption site.As expected, for the higher solubility compounds  showed the influences of the various input variables on the absorbed fraction . The resIn terms of the effective particle radius, a variation from 2.5 μm to 250 μm was considered in the simulation. Oral exposure was found to be very sensitive to the particle size for the B5 and B2 series, and much more “inert” for B3 series compounds.i.e., Fa > 85%) could be achieved with particle radius up to about 10 μm for 2 and 5 series, 90 µm for BC3 and BM3 and up to 150 µm for BT3. Under this condition, particle size reduction can be an effective method to improve the dissolution rate, and therefore increase bioavailability.Nearly complete oral absorption , therefore its impact on bioavailability is of the utmost importance. The PSA on solubility impact on absorption provided results consistent with the other simulations: small changes in solubility led to significant bioavailability variations for the 2 and 5 series, whereas for the 3 series absorption was less sensitive to solubility variations.Physiological compounds—granular lecithin  and sodium taurocholate 97%  were used. HPLC grade acetonitrile and methanol was purchased from Merck KGaA . Trifluoroacetic acid for HPLC was purchased from Sigma-Aldrich. Water for chromatography (resistivity minimum 18.2 MΩ and TOC maximum 30 ppb) was produced within the laboratory by means of a TKA GenPure system and used during the experiments. The pyrazole compounds were synthesized according to the procedures described in previous research ,13. All The quantitative analysis of the selected compounds was carried out using a Waters liquid chromatographic system  consisting of consisting of a 600 E Multisolvent Delivery System, Waters AF in line degasser, 486 UV tunable absorbance detector and Waters 717 plus autosampler. Empower Pro software (Waters) was used to control the instrument, acquire and process data. The chromatographic separation was achieved on a Hypersil Gold, 5-μm 150 × 4 mm column  maintained under constant temperature (30 °C). The mobile phase consisted of an isocratic mixture of 0.1% trifluoroacetic acid-acetonitrile (50:50 v/v), delivered at 1.0 mL/min flow rate. The detector was set at the wavelength corresponding to the maximum absorbance of each compound in UV spectra.The HPLC method was subjected to validation in accordance with the International Conference on Harmonization (ICH) regulations Q2(R1)  in termsAssay specificity was examined in relation to interference from matrix components in the drug-free media used for determination of solubility.2) were calculated by least squares linear regression analysis.The linearity assessment, designed to measure the capability of the method to produce results related in a linear way to the concentrations of the analytes, was performed for each compound by using seven concentration levels, in the range 0.1–10 µg/mL. All analyses were performed in triplicate. Calibration curves and corresponding determination coefficients  and quantitation limit (LOQ) were determined based on the signal-to-noise ratio. The concentrations yielding to signal-to-noise ratios of 3:1 and 10:1 were taken as LOD and LOQ, respectively.low, QCmedium and QChigh). Precision was assessed by means of RSD% values computed for absolute peak areas resulting from interpolation on the corresponding calibration curves. Repeatability study was achieved by injection of five replicates from a single prepared spiked plasma sample within a single day experimental session, whereas intermediate reproducibility was tested by means of five different samples processed in different experimental sessions for each concentration level. The bias (%) between the concentration values determined for the QC samples and their nominal values was used as accuracy indicator.Precision was evaluated for repeatability and intermediate reproducibility on spiked samples, at three different concentration levels (QCThermodynamic (equilibrium) solubility experiments were performed by using the saturation shake-flask method. Since our study was focused on oral administration route, solubility was tested in different media simulating gastric and intestinal conditions of the fasted and fed state. Hence, the USP Simulated Gastric Fluid , Simulated Intestinal Fluid  and 0.05 M acetate buffer pH = 4.5 were used in the experiments .in vivo solubility in the GI tract can be significantly higher than expected from in vitro solubility tests performed in buffer solutions due to presence in the intestinal lumen of natural lipids and bile salts with surface-active properties [i.e., Fasted State Simulated Intestinal Fluid (FaSSIF) containing 3 mM sodium taurocholate at a pH of 6.5 and Fed State Simulated Intestinal Fluid (FeSSIF) with pH 5.0 and 15 mM sodium taurocholate concentration [et al., [However, the operties . Therefontration . Prepara[et al., , withoutFor each experiment, excess of substance was carefully added to 1.5 mL of medium, in 2 mL Eppendorf polypropylene microtubes. The vials were capped, stirred for 30 s at 2500 rpm on an IKA Genius 3 vortex mixer  and maintained under mild agitation (250 rpm) for 24 h at 25 °C on a IKA HS 260 orbital shaker (IKA Werke).The resulting samples were centrifuged at 12,000 rpm, for 10 min on a Hettich Mikro 220R centrifuge . Aliquots of 0.25 mL were collected from the supernatant and diluted with mobile phase to a final volume of 1 mL. A volume of 10 µL from the resulting sample was injected into the HPLC system. All experiments were performed in triplicate.More than 100 constitutional and topological descriptors, functional group counts, molecular properties were calculated by the E-Dragon Java based online platform ,58. HoweTM, Version 11.2  software . The importance of descriptors was evaluated by the Variable Importance Plot (VIP), values which summarizes the importance of the x variables in the model. VIP values higher than 0.8 were considered.The influence of the different descriptors on solubility was performed by means of multivariate data analysis applying partial least-squares projection to latent structures (PLS). The analysis of how molecular features are related to the the drug solubility in all selected media was performed using JMPSince the dataset was quite small, the obtained data were not regarded as predictive model, but were used as a tool to further investigate the properties of importance for poor solubility.Further, a biopharmaceutical evaluation as to which physicochemical and formulation factors could have an impact on the oral bioavailability of the selected compounds was performed based on computer simulations performed with GastroPlus™  software.TM module was used to generate estimates for many GastroPlusTM inputs: MW (g/mol), molecular radius (Å), acid and base ionization constants, (pKa or pKb), octanol/ water partition coefficient (logP); distribution coefficient at physiological pH = 7.4 (logD) aqueous solubility , solubility in biorelevant media (mg/mL), diffusion coefficient , effective human jejunal permeability , as well as some pharmacokinetic parameters, such as percent unbound in plasma , Blood/Plasma Ratio (RBP) and volume of distribution . Additionally, the software allowed calculation of Maximum Absorbable Dose , an important parameter in assessment of drug “developability” [The ADMET Predictorability” .vs. pH and logD vs. pH profiles (used in pH dependent dissolution and absorption models).The pKa information was further used to evaluate solubility TM was used to predict the rate and extent of oral absorption. Simulation of oral absorption was performed in the GastroPlusTM Single Simulation Mode with the default Absorption Scale Factor (ASF) values in the Human-Physiological-Fasted mode. The ACAT model of the human gastrointestinal tract used for the simulations comprises nine successive compartments, the first one representing the stomach and subsequent compartments corresponding to duodenum, jejunum (two compartments), ileum (three compartments), cecum, and ascending colon.The Advanced Compartmental Absorption and Transit Model (ACAT) model  implemeni.e., dose, reference solubility, particle radius as well as effective human jejunal permeability.Parameter sensitivity analysis (PSA) was performed in order to identify critical parameters for oral absorption. PSA was performed using the GastroPlusTM build-in PSA simulation mode, in order to identify the critical parameters for oral absorption. PSA was performed with range from one tenth to ten-fold the tested input values, in silico prediction and modeling approaches. Based on the results from both in silico and in vitro experiments, the present investigation was able to rank the tested compounds in terms of biopharmaceutical behavior. The in silico simulations highlighted that only 1-ethyl-1H-pyrazol-5-yl derivatives  are completely absorbed from the GI tract and their absorption is not sensitive to the main critical formulation variables i.e., dose and particle size, leading to low variability of their pharmacokinetic profile. The experimental in vitro studies also pointed out that the B3 series compounds are having the highest overall solubility in simulated gastric and intestinal media. Therefore, all results indicated that these compounds are having a more favorable absorption profile, making them the main candidates for advance to the pre-clinical in vivo studies. In addition, the results indicate that biopharmaceutical profiling represents a reliable method to improve the future prediction for similar compounds.This paper evaluated the biopharmaceutical profile of several thiourea derivatives with antiproliferative and cytotoxic potential based on experimental equilibrium solubility studies and