Women's empowerment in terms of both involvements in employment activities and with decision-making about household activities is the most evident factors that can affect the nutritional and health status of their children. This paper investigates the effect of women's empowerment (WE) on children's nutritional (CN) status in Pakistan. The Pakistan demographic health survey (PDHS 2012-13) cross-sectional data was used to analyze the impacts of WE on child malnutrition. The composite index of anthropometric failure (CIAF) was used as a dependent variable to measure the children's nutritional status, and the wealth index household size. The number of children in a house and indicators of women empowerment, which included the mother's education, employment status, and the household decision-making, were used as the independent variables. The method of binary logistic regression with marginal effects was used for the empirical analysis of the results. The results of the study showed the indicators of women's empowerment, such as the education of the mother and employment status had a negative relationship with child malnutrition. Women's decision-making about the visits to family, which is an indicator of WE, had an insignificant effect on CN. Similarly, socioeconomic status, which included the wealth index, also caused a reduction in child malnutrition. In addition, an increase in household size had a positive and significant relation to child malnutrition. Women are the primary caretakers of children in the household, and their intra-household dynamics affect the well-being of individuals. The empowerment of women acts as a means to enhance children's nutritional status, which causes important developmental outcomes.

High blood pressure is an important public health concern and the leading risk factor for global mortality and morbidity. To assess the implications of this condition, we aimed to review the existing literature and study the factors that are significantly associated with hypertension in the Pakistani population.

The primary goal of cattle genomics is the identification of genome-wide polymorphism associated with economically important traits. The bovine genome sequencing project was completed in 2009. Since then, using massively parallel sequencing technologies, a large number of Bos taurus cattle breeds have been resequenced and scanned for genome-wide polymorphisms. As a result, a substantial number of single nucleotide polymorphisms (SNPs) have been discovered across European Bos taurus genomes, whereas extremely less number of SNPs are cataloged for Bos indicus breeds. In this study, we performed whole-genome resequencing, reference-based mapping, functional annotation and gene enrichment analysis of 20 sires representing eleven important Bos indicus (indicine) breeds of Pakistan. The breeds sequenced here include: Sahiwal, Red Sindhi, Tharparkar and Cholistani (tropically adapted dairy and dual purpose breeds), Achai, Bhagnari, Dajal and Lohani (high altitude adapted dual and drought purpose breeds); Dhanni, Hisar Haryana and Gabrali (dairy and light drought purpose breeds). A total of 17.4 billion QC passed reads were produced using BGISEQ-500 next generation sequencing platform to generate 9 to 27-fold genome coverage (average ~16×) for each of the 20 sequenced sires. A total of 67,303,469 SNPs were identified, of which 3,850,365 were found novel and 1,083,842 insertions-deletions (InDels) were detected across the whole sequenced genomes (491,247 novel). Comparative analysis using coding region SNPs revealed a close relationship between the best milking indicine breeds; Red Sindhi and Sahiwal. On the other hand, Bhagnari and Tharparkar being popular for their adaptation to dry and extremely hot climates were found to share the highest number of SNPs. Functional annotation identified a total of 3,194 high-impact (disruptive) SNPs and 745 disruptive InDels (in 275 genes) that may possibly affect economically important dairy and beef traits. Functional enrichment analysis was performed and revealed that high or moderate impact variants in wingless-related integration site (Wnt) and vascular smooth muscle contraction (VSMC) signaling pathways were significantly over-represented in tropically adapted heat tolerant Pakistani-indicine breeds. On the other hand, vascular endothelial growth factor (VEGF) and hypoxia-inducible factor 1 (HIF-1) signaling pathways were found over-represented in highland adapted Pakistani-indicine breeds. Similarly, the ECM-receptor interaction and Jak-STAT signaling pathway were significantly enriched in dairy and beef purpose Pakistani-indicine cattle breeds. The Toll-like receptor signaling pathway was significantly enriched in most of the Pakistani-indicine cattle. Therefore, this study provides baseline data for further research to investigate the molecular mechanisms of major traits and to develop potential genomic markers associated with economically important breeding traits, particularly in indicine cattle.

We aimed to determine the prevalence of pulmonary tuberculosis (TB) amongst the adult population in 2010-2011 in Pakistan.

Pathogenic germline mutations in BRCA1/2 constitute the majority of hereditary breast and/or ovarian cancers worldwide. Incidence and mortality rate of breast and ovarian cancers in Pakistani women is high. Thus, to establish the diagnosis for targeted therapy in Pakistan, we conducted Next-generation sequencing-based germline testing for the detection of BRCA1/2 oncogenic variants associated with breast and ovarian cancer subtype.

Anaplasma centrale (A. centrale) is an obligate red blood cell residing tick transmitted rickettsiae that has not been studied extensively for its prevalence in cattle along with its epidemiology. Aim of this investigation was to report the seasonal prevalence, phylogeny and epidemiological parameters associated with the prevalence of A. centrale in cattle breeds enrolled from District Layyah in Punjab, Pakistan. A total of 844 blood samples [Cross breed = 300, Holstein Friesian = 244, Sahiwal breed = 300)] were collected from apparently healthy cattle along with epidemiological data during 2017-18. PCR amplified 426 base pair fragment from 16S rRNA gene of A. centrale in 14.4% (122/844) of cattle. Amplified 16S rRNA partial gene sequence of A. centrale were confirmed by DNA sequencing and deposited to GenBank. Highest A. centrale prevalence was observed in spring (24%) followed by autumn (12.4%) summer (10%) and winter (7.1%) seasons. Sahiwal breed (18.3%) was most susceptible to A. centrale infection followed by cross (12.3%) and Holstein Friesian breed (12.3%). 69/844 (8.2%) of Giemsa stained cattle blood smears were also found positive for Anaplasma spp. Farms where animal use to drink pool water and farms where dogs and other dairy animals were living with cattle had higher A. centrale prevalence. Female cattle and dogs having tick burden were found associated with A. centrale infection. Hematological profile was severely disturbed in A. centrale positive cattle. It is recommended that A. centrale should be screened in cattle, in addition to A. marginale, for the effective control of tick born diseases in Pakistan.

Live poultry retail stalls (LPRSs) are believed to be the source of human infection with avian influenza viruses (AIVs); however, little is known about epidemiology of these viruses in LPRSs of Pakistan.

Theileria annulata (T. annulata) and Anaplasma marginale (A. marginale) are among the most extensively reported tick borne pathogens and are associated with huge economic losses worldwide. A total of 298 cattle blood samples were screened to report the presence of these two pathogens. The samples were collected from apparently healthy cattle (Achai, n = 155, Jersy, n = 88 and crossbred, n = 55) in Bajaur district of Khyber Pakhtunkhwa (KPK) during June and July of 2022. A total of 31 out of 298 cattle (10.4%) were found infected with T. annulata as PCR amplified a 156 base pair fragment from Tams-1 gene of T. annulata from their blood. While 16/298 animals (5.4%) were found infected with A. marginale as they amplified a 382 base pair fragment specific for msp5 gene of this bacterium. Three animals (1%) were found co infected. Cattle susceptibility to T. annulata infection was significantly higher than A. marginale infection (P < 0.001). Phylogenetic analysis revealed that Pakistani isolates of both detected pathogen clustered together and were closely related isolates from worldwide countries. Prevalence of T. annulata varied significantly among the sampling sites (P = 0.05) while no such association was observed for A. marginale among the tested cattle. Epidemiological data analysis revealed that none of the studied risk factors was found associated either with the prevalence of T. annulata or A. marginale (P > 0.05) among enrolled cattle. In conclusion, our study has revealed a relatively higher prevalence of T. annulata than A. marginale in cattle from the Bajaur district in KPK. This information is important for improving the productivity of the livestock sector, which is one of the main sources of income in the country. It is recommended that this data be taken into account for the development and implementation of effective tick control programs, as well as for the improvement of livestock management practices to prevent and manage TBDs in Pakistan.

Caprine theileriosis, caused by Theileria ovis is a serious production issue, especially in the areas that depend on goats and sheep for milk, meat, and other economic benefits. Pakistan has a large goat population, but few reports have been documented from this country regarding PCR-based detection of T. ovis. The molecular prevalence of T. ovis, on a seasonal basis, in various goat breeds enrolled from Muzaffar Garh district of Punjab in Pakistan was determined from October 2018 to September 2019. In this study, 1084 goat blood samples were screened for the detection of T. ovis DNA through PCR-based amplification of 18S rRNA gene. Out of 1084 goats, 12 (1.11%) were infected with T. ovis. The parasite prevalence varied with the sampling seasons (Chi square test, P = 0.008), and the parasite prevalence was highest in goat blood samples collected in summer (2.39%) followed by winter (1.88%). DNA sequencing and BLAST analysis confirmed the presence of T. ovis, and the amplified isolates from the 18S rRNA gene of T. ovis were found to be highly conserved during phylogenetic analysis. Young goats (Fischer exact test, P = 0.022) were found more infected with T. ovis during the winter season. Infected goats had elevated white blood cell counts (Two-sample t-test, P = 0.04), blood urea nitrogen to Creatinine ratio (Two-sample t-test, P = 0.02) and decreased serum Creatinine (Two-sample t-test, P = 0.001) as compared to T. ovis negative goats. We report a relatively low molecular prevalence of T. ovis in goats from the Muzaffar Garh district. However, it is recommended that control measures to eradicate T. ovis infection in goats in this area should be taken.

Anaplasma marginale (A. marginale), Anaplasma ovis (A. ovis) and Theileria ovis (T. ovis) are among the most commonly reported intracellular tick borne pathogens that infect ruminants across the globe causing huge economic losses. This study aims to report the prevalence and phylogenetic evaluation of these three pathogens infecting sheep and goats (n = 333) that were enrolled from Fort Munro region in Pakistan by using msp1b, msp4 and 18S rRNA genes for A. marginale, A. ovis and T. ovis respectively. Results revealed almost similar infection rates in sheep and goats with an overall prevalence of 11% for A. marginale, 28% for A. ovis and 3% for T. ovis. Concurrent infection was also recorded, however, the number of animals infected with two pathogens (n = 24; 7.2%) was higher than infection with three pathogens (n = 2; 0.6%). Risk factor analysis revealed that sheep reared in small herds had higher A. marginale (P = 0.03) and A. ovis (P = 0.04) infection rates compared to those from large herds. In addition, it was observed that bucks (P ≤ 0.05) and tick-free goats (P ≤ 0.05) exhibited higher A. ovis infection rates than nannies. Phylogenetic analysis of all three pathogens showed that Pakistani isolates were clustered together and were closely related to previously deposited Pakistani isolates as well as with those that were reported from worldwide countries. In conclusion, we are reporting that Pakistani sheep and goats have A. marginale, A. ovis and T. ovis mediated infections and control measures should be taken against them to improve the productivity of the livestock sector.

Oxidative stress, lipid peroxidation, and transaminase reactions are some of the mechanisms that can lead to liver dysfunction. A time-dependent study was designed to evaluate the ability of silymarin (SLN) and glycyrrhizin (GLN) in different dosage regimens to lessen oxidative stress in the rats with hepatic injury caused by the hepatotoxin carbon tetrachloride. Wistar male albino rats (n = 60) were randomly assigned to six groups. Group A served as a positive control while groups B, C, D, E, and F received a dose of CCl4 (50% solution of CCl4 in liquid paraffin, 2 mL/kg, intraperitoneally) twice a week to induce hepatic injury. Additionally, the animals received SLN and GLN in different doses for a period of six weeks. CCl4 was found to induce hepatic injury by significantly increasing serum alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and thiobarbituric acid reactive substances while decreasing total protein and the activities of reduced glutathione, superoxide dismutase, and catalase. Treatment with various doses of SLN and GLN significantly reduced ALT, AST, ALP, and TBARS levels and increased GSH, SOD, and CAT levels. Our findings indicated that SLN and GLN have hepatoprotective effects against oxidative stress of the liver.

Bambusa arundinacea (RETZ.) Willd. is distributed in tropical regions of Pakistan, India, and China. It has been used for a long time as a folk remedy for cirrhosis, urinary tract ailments, and various other abdominal cavity disorders. It has antioxidant, free-radical-scavenging, and anti-inflammatory effects. The aims and objectives of this study were to validate the folkloric uses of Bambusa arundinacea and to evaluate its nephroprotective potential on scientific grounds. Gentamycin (G.M, 40 mg/kg) was used to induce nephrotoxicity in the animal model. Two doses of the methanolic extract of Bambusa arundinacea (MEBA; 300 and 500 mg/kg) were utilized in addition to silymarin (200 mg/kg/d). Treatments were administered once daily for 14 days. After 14 days, the blood was collected and the kidneys were removed. The antioxidant potential of the standardized MEBA was evaluated using the total phenolic content, the total flavonoid content, and the DPPH scavenging activity. The plant extract was rich with flavonoid content. The DPPH scavenging activity was 65% as compared to butylated hydroxy toluene (96%), with IC50 values 31.65 and 7.80 μg/mL, respectively. The phytochemical analysis was performed using HPLC, and MEBA was found to contain various phytoconstituents such as quercetin, caffeic acid, vanillic acid, gallic acid, chlorogenic acid, and cinnamic acid. Antioxidant enzymes such as superoxide dismutase and catalase were assayed, and MEBA exhibited significantly improved CAT and SOD levels. The levels of renal function markers such as serum creatinine, serum urea, blood urea nitrogen, serum urea, and serum uric acid levels also evaluated, and a significant retrieval was found in a dose-dependent fashion. Good improvement was also made in various hematological parameters. Statistical analysis was done using analysis of variance to determine the significance of differences among the data. In conclusion, the standardized methanolic extract of Bambusa arundinacea was able to alleviate gentamicin-induced nephrotoxicity by enhancing the antioxidant defensive mechanisms of renal tubular cells.

Background and Objectives: Dyslipidemia is gaining much attention among healthcare professionals because of its high association with the malfunctioning of a number of normal physiological and metabolic processes in the body. Obesity is directly interconnected with dyslipidemia and is said to be a denouement of hyperlipidemia and, if left untreated, may lead to intense damage to organs that are directly involved in fat metabolism. The objective of this study was to investigate the synergistic antiobesity and anti-hyperlipidemic activities along with hepato- and renoprotective potential of nanoemulsomes (NES) of lovastatin (LTN)-loaded ginger (GR) and garlic (GL) oils. Materials and Methods: LTN nanoemulsomes co-encapsulated with GR oil and GL oil were prepared by a thin hydration technique. Eight-week-old male Wistar rats weighing 200-250 g were induced with hyperlipidemia via a high-fat diet (HFD) comprising 40% beef tallow. Body weight, serum biochemical lipid parameters, and those for liver and kidney functions, serum TC, LDL-C, vLDL-C, HDL-C, TG, atherogenic index (AI), ALT, AFT, ALP, γ-GT, total protein (TP), serum albumin and globulin ratio (A/G), serum creatinine, blood urea nitrogen (BUN) and blood urea, and histopathology of hematoxylin and eosin (H&E) stained liver and kidney sections of all aforementioned groups were examined in the treated animals. Results: Nanoemulsomes of LTN-loaded GR and GL oils provided synergistic effects with LTN, exerted better ameliorative actions in reducing serum TC, LDL-C, vLDL-C, triglycerides, and AI, and improved serum HDL-C levels. Serum ALT, AST, ALP, and γ-GT levels were in the normal range for nanoemulsome groups. H&E stained liver and kidney sections of these animals confirmed better hepatoprotective and renoprotective effects than LTN alone. Serum biochemical parameters for renal functions also claimed to be in the moderate range for nanoemulsome-treated groups. Conclusion: This study demonstrated that nanoemulsomes of LTN-loaded GR and GL oils synergistically provided better antihyperlipidemic, hepatoprotective, and renoprotective effects as compared to LTN alone.

Inflammation is the core contributor in the pathogenesis of various acute and chronic illness including appendicitis, bronchitis, arthritis, cancer and neurological diseases. NSAIDs, commonly used medications for inflammatory diseases, on prolonged use cause GI bleeding, ulcers and many more issues. Plant-based therapeutic agents including essential oils in combination with low-dose synthetic drugs have been shown to produce synergistic effects and reduce complications of synthetic drugs. This study was designed to evaluate the anti-inflammatory, analgesic and anti-pyretic properties of Eucalyptus globulus essential oil alone and in combination with flurbiprofen. GC-MS analysis was performed to screen chemical composition of oil. In vitro anti-inflammatory assay (membrane stabilization assay) and in vivo inflammatory acute (carrageenan and histamine-induced paw oedema) and chronic (cotton pellet-induced granuloma and Complete Freund's adjuvant-induced arthritis) models were performed to check anti-inflammatory properties. Acetic acid-induced algesia and yeast-induced pyrexia models were performed to check analgesic and anti-pyretic properties. qRT-PCR was performed to study the effect of treatments on the expression of inflammatory biomarkers. GC-MS analysis of E. globulus essential oil showed the presence of eucalyptol along with other active biomolecules. 500 + 10 mg/kg of oil-drug combination showed significantly (p < 0.05) better in vitro membrane stabilization effects as compared with groups treated with 500 mg/kg of E. globulus oil and 10 mg/kg of Flurbiprofen alone. 500 + 10 mg/kg of oil-drug combination showed significantly (p < 0.05) better anti-inflammatory, analgesic and antipyretic effects as compared to 500 mg/kg of E. globulus oil alone in all in vivo models. When comparison was done between 500 + 10 mg/kg of oil-drug combination-treated and 10 mg/kg Flurbiprofen-treated group, the former group showed significantly (p < 0.05) better anti-inflammatory and anti-pyretic effects, but there were non-significant differences in the analgesic model. Animal group treated with 10 mg/kg of Flurbiprofen showed significantly (p < 0.05) better anti-inflammatory and analgesic effects than group treated with 500 mg/kg of oil alone while, there were non-significant differences in anti-pyretic effects. qRT-PCR analysis showed significant (p < 0.05) down-regulation in the expression of IL-4 and TNF-α in serum samples of animals treated with 500 + 10 mg/kg of oil-drug combination as compared to the diseased control (arthritic) group. Overall, the current research demonstrates that Eucalyptus globulus essential oil in combination with flurbiprofen showed better anti-inflammatory, analgesic and anti-pyretic effects than oil and flurbiprofen alone which is attributed to the down-regulation of pro-inflammatory biomarkers (IL-4 and TNF-α). Further studies are required to formulate a stable dosage form and to check the anti-inflammatory efficacy in different inflammatory disorders.

Insect pest complex, cotton leaf curl disease and weeds pose major threat to crop production worldwide, including Pakistan. To address these problems, in the present study a triple gene construct harboring Cry1Ac, Cry2Ab, and EPSPS cassettes has been developed for plant specifically in cotton transformation against lepidopteron insect-pests and weeds. Nicotiana benthamiana (tobacco) was used as a model system for characterization of this triple gene construct. The construct has been assembled in plant expression vector and transformed in N. benthamiana. In six transgenic tobacco lines the integration of Cry1Ac-Cry2Ab-EPSPS in tobacco genome was checked by PCR, while successful protein expression of all the three genes was confirmed through immunostrip assay. Efficacy of Cry1Ac and Cry2Ab was evaluated through insect bioassay using armyworm (Spodoptera littoralis). These transgenic tobacco plants showed significant insect mortality as compared to control plants during insect bioassay. Three out of six tested transgenic lines L3, L5, and L9 exhibited 100% mortality of armyworm, while three other lines L1, L10, and L7 showed 86, 80, and 40% mortality, respectively. This construct can readily be used with confidence to transform cotton and other crops for the development of insect resistant and herbicide tolerant transgenic plants. The transgenic crop plants developed using this triple gene construct will provide an excellent germplasm resource for the breeders to improve their efficiency in developing stable homozygous lines as all the three genes being in a single T-DNA border will inherit together.

Bt cotton expressing Cry1Ac is being cultivated in Pakistan. It has been observed that pink bollworm may have developed resistance against single Bt gene (Cry1Ac). For durable resistance, insect resistant NIBGE-1601 cotton harboring double gene Cry1Ac-Cry2Ab construct was developed. There was a need to characterize NIBGE-1601 event for intellectual property rights protection. The Presence of NIBGE Cry1Ac and NIBGE Cry2Ab genes was checked in NIBGE-1601 cotton plants through PCR, while there was no amplification using primers specific for Monsanto events (MON531, MON15985, MON1445). Using genome walking technology, NIBGE-601 event has been characterized. Event-specific primers of NIBGE-1601 were designed and evaluated to differentiate it from other cotton events mentioned above. NIBGE-1601 event detection primers are highly specific, therefore, can detect NIBGE 1601 event at different conditions using single or multiplex PCR. In the qualitative PCR, using NIBGE-1601 event specific primers, 0.05 ng was the limit of detection for NIBGE-1601double gene cotton genomic DNA. Thus event characterization and development of event-specific diagnostics will help in breeding new cotton varieties resistant to cotton bollworms.

Herein, we report co-encapsulation of ofloxacin with tea tree or lavender oil in gellan gum based hydrogel films by solvent casting ionotropic gelation method as wound dressing. Prepared films were transparent, flexible, and displayed antioxidant activity with superior antibacterial response against common inhabitants of wound i.e. gram positive and negative bacteria. Solid-state characterization of optimized formulation (OL3 and OT3) revealed successful incorporation of drug and oils in hydrogel structure without any noticeable interaction. In vitro release studies showed an initial burst release but remaining portion released in controlled manner over 48 h from the films and furthermore, presence of oils did not affected the ofloxacin release. Optimized formulation containing ofloxacin and 25% w/w lavender/tea tree oil showed 98% wound contraction in rats after ten days of treatment. Histological images displayed completely healed epidermis. Taken together, our prepared hydrogel films demonstrated favorable features with appreciable antibacterial, wound healing activity and could be useful for the treatment of full thickness wounds.

DNA barcoding is highly effective for identifying specimens once a reference sequence library is available for the species assemblage targeted for analysis. Despite the great need for an improved capacity to identify the insect pests of crops, the use of DNA barcoding is constrained by the lack of a well-parameterized reference library. The current study begins to address this limitation by developing a DNA barcode reference library for the pest aphids of Pakistan. It also examines the affinities of these species with conspecific populations from other geographic regions based on both conventional taxonomy and Barcode Index Numbers (BINs). A total of 809 aphids were collected from a range of plant species at sites across Pakistan. Morphological study and DNA barcoding allowed 774 specimens to be identified to one of 42 species while the others were placed to a genus or subfamily. Sequences obtained from these specimens were assigned to 52 BINs whose monophyly were supported by neighbor-joining (NJ) clustering and Bayesian inference. The 42 species were assigned to 41 BINs with 38 showing BIN concordance. These species were represented on BOLD by 7,870 records from 69 countries. Combining these records with those from Pakistan produced 60 BINs with 12 species showing a BIN split and three a BIN merger. Geo-distance correlations showed that intraspecific divergence values for 49% of the species were not affected by the distance between populations. Forty four of the 52 BINs from Pakistan had counterparts in 73 countries across six continents, documenting the broad distributions of pest aphids.

The limited aqueous solubility of many active pharmaceutical ingredients (APIs) is responsible for their poor performance and low drug levels in blood and at target sites. Various approaches have been adopted to tackle this issue. Most recently, mesoporous silica nanoparticles (MSN) have gained attention of pharmaceutical scientists for bio-imaging, bio-sensing, gene delivery, drug solubility enhancement, and controlled and targeted drug release. Here, we have successfully incorporated the poorly water soluble antiviral drug velpatasvir (VLP) in MSN. These spherical particles were 186 nm in diameter with polydispersity index of 0.244. Blank MSN have specific surface area and pore diameter of 602.5 ± 0.7 m2/g and 5.9 nm, respectively, which reduced after successful incorporation of drug. Drug was in amorphous form in synthesized VLP-loaded silica particles (VLP-MSN) with no significant interaction with carrier. Pure VLP showed poor dissolution with progressive increment in pH of dissolution media which could limit its availability in systemic circulation after oral administration. After VLP loading in silica carriers, drug released rapidly over a wide range of pH values, i.e., 1.2 to 6.8, thus indicating an improvement in the solubility profile of VLP. These particles were biocompatible, with an LD50 of 448 µg/mL, and in-vivo pharmacokinetic results demonstrated that VLP-MSN significantly enhanced the bioavailability as compared to pure drug. The above results clearly demonstrate satisfactory in-vitro performance, biocompatibility, non-toxicity and in-vivo bioavailability enhancement with VLP-MSN.

Ω-3 fatty acids perform several therapeutic functions in the body, however, their applications are limited due to the inferior oxidative stability. To improve the oxidative stability and release properties of Ω-3 fatty acids, microencapsulation is performed. Butter is a good source of fat-soluble vitamins and antioxidant systems however, it is not a good source of Ω-3 fatty acids. Supplementation of butter with microcapsules of vegetable oils rich in Ω-3 fatty acids is not reported in literature.