Preimplantation genetic testing for human blastocysts with potential parental contamination using a quantitative parental contamination test (qPCT): an evidence-based study.

RESEARCH QUESTION: Is it possible to develop a quantitative method for detecting parental DNA contamination in conventional IVF using preimplantation genetic testing for aneuploidy (PGT-A)? DESIGN: In this study, a quantification method was established for the parental contamination test (qPCT), which ensured more reliable results, and then verified its effectiveness for vitrified conventional IVF embryos. A total of 120 surplus vitrified blastocysts from patients who underwent prior routine IVF cycles were available for study. RESULTS: The results of the prospective clinical study of qPCT-PGT-A showed that the maternal contamination rate was 0.83% (1/120) and that the risk of paternal contamination was negligible. The 24 frozen embryo transfer cycles resulted in 16 clinical pregnancies, including 13 live births, one late inevitable miscarriage and two ongoing pregnancies. CONCLUSIONS: The risk of PGT in embryos with potential parental contamination is relatively low, and PGT-A is applicable for vitrified conventional IVF embryos.

In vitro maturation of human oocytes maintaining good development potential for rescue intracytoplasmic sperm injection with fresh sperm.

BACKGROUND: The outcomes of the use of commercial in vitro maturation (IVM) medium to culture immature oocytes obtained from conventional ovulation induction, followed by rescue intracytoplasmic sperm injection (RICSI), are not ideal. It is thus difficult to widely adopt this approach in clinical practice. Therefore, it is necessary to explore methods for improving the clinical outcome of IVM. AIM: To study the effect of sperm on the developmental potential of in vitro-matured oocytes in conventional culture. METHODS: This was a retrospective study of patients whose immature oocytes were harvested from conventional oocyte stimulation cycles and underwent ICSI at our hospital between June 2018 and August 2020. RICSI was performed using sperm collected on the day of oocyte harvest (old) and sperm collected on the day of RICSI (fresh) and oocytes matured in vitro after 24 h of culture in conventional medium. The rates of in vitro oocyte maturation, normal fertilization, normal cleavage, day-3 top-quality embryos, and useful blastocyst formation were compared between the two groups. RESULTS: In total, 102 germinal vesicle (GV)-stage immature oocytes were cultured in the old sperm group. In the fresh sperm group, 122 GV-stage immature oocytes were collected and cultured in vitro for 24 h. There were no significant differences in the general conditions of males and females between the two groups (P > 0.05). The oocyte maturation, normal fertilization, and normal cleavage rates of the old and fresh groups were 51.0% vs 55.7%, 61.5% vs 64.7%, and 93.8% vs 93.2%, respectively. None of the rates differed significantly (P > 0.05) between the two groups. However, the day-3 top-quality embryo and useful blastocyst rates of the old and fresh sperm groups were 16.6% vs 63.4%; 6.67% vs 34.6%, respectively. The day-3 top-quality embryos and useful blastocyst rates of the old sperm group were significantly lower than those of the fresh group (P < 0.05). CONCLUSION: In vitro maturation with conventional culture medium combined with the use of fresh sperm collected on the day of RICSI is an easy-to-implement strategy for patients whose oocytes are completely or mostly immature.

[Preimplantation Genetic Diagnosis of α/ß Complex Thalassemia by Next Generation Sequencing].

OBJECTIVE: To explore the application value of next generation sequencing (NGS) in preimplantation genetic diagnosis of α/ß complex thalassemia couple. METHODS: The coding regions of α-globin genes (HBA1, HBA2) and ß-globin gene (HBB) were selected as the target regions. The high-density and closely linked single nucleotide polymorphism (SNP) sites were selected as the genetic linkage markers in the upstream and downstream 2M regions of the gene. After NGS, the effective SNP sites were selected to construct the haplotype of the couple, and the risk chromosome of the mutation carried by the couple was determined. The NGS technology was used to sequence the variations of HBA1, HBA2 and HBB directly and construct haplotype linkage analysis for preimplantation genetic diagnosis. RESULTS: Direct sequencing and haplotype linkage analysis of HBA1, HBA2 and HBB showed that two of the six blastocysts were α/ß complex thalassemia, one was ß-thalassemia heterozygote, two were α-thalassemias heterozygotes, and one was intermediate α-thalassemia. A well-developed embryo underwent preimplantation genetic diagnosis was implanted into the mother's uterus, and a healthy infant was born at term. CONCLUSION: Preimplantation genetic diagnosis can be carried out by NGS technology in α/ß complex thalassemia couples, and abortion caused by aneuploid embryo selection can be avoided.

Successful birth after preimplantation genetic testing for a couple with two different reciprocal translocations and review of the literature.

BACKGROUND: Preimplantation genetic testing for chromosomal structural rearrangements (PGT-SR) is widely applied in couples with single reciprocal translocation to increase the chance for a healthy live birth. However, limited knowledge is known on the data of PGT-SR when both parents have a reciprocal translocation. Here, we for the first time present a rare instance of PGT-SR for a non-consanguineous couple in which both parents carried an independent balanced reciprocal translocation and show how relevant genetic counseling data can be generated. METHODS: The precise translocation breakpoints were identified by whole genome low-coverage sequencing (WGLCS) and Sanger sequencing. Next-generation sequencing (NGS) combining with breakpoint-specific polymerase chain reaction (PCR) was used to define 24-chromosome and the carrier status of the euploid embryos. RESULTS: Surprisingly, 2 out of 3 day-5 blastocysts were found to be balanced for maternal reciprocal translocation while being normal for paternal translocation and thus transferable. The transferable embryo rate was significantly higher than that which would be expected theoretically. Transfer of one balanced embryo resulted in the birth of a healthy boy. CONCLUSION(S): Our data of PGT-SR together with a systematic review of the literature should help in providing couples carrying two different reciprocal translocations undergoing PGT-SR with more appropriate genetic counseling.

Alternative splicing patterns of doublesex reveal a missing link between Nix and doublesex in the sex determination cascade of Aedes albopictus.

Sexual development in insects is regulated by a complicated hierarchical cascade of sex determination. The primary signals are diverse, whereas the central nexus doublesex (dsx) gene is relatively conserved within the pathway. Aedes (Stegomyia) albopictus is an important vector with an extensive worldwide distribution. We previously reported that Ae. albopictus dsx (Aalbdsx) yields one male- (AalbdsxM ) and three female-specific isoforms (AalbdsxF1-3 ); however, the spatiotemporal expression profiles and mechanisms regulating sex-specific alternative splicing require further investigation. In this study, we demonstrated that the AalbdsxM messenger RNA (mRNA) represents the default pattern when analyzed in human foreskin fibroblasts and HeLa cells. We combined reverse transcription polymerase chain reaction with RNA immunoprecipitation using specific antibodies against tagged Ae. albopictus male-determining factor AalNix and confirmed that AalNix indirectly regulates dsx pre-mRNA and regulates its alternative splicing. During the early embryo stage (0-2 and 4-8 h), maternal dsxF and default splicing dsxM were detected in both sexes; the expression of dsxM then decreased until sufficient AalNix transcripts accumulated in male embryos at 20-24 h. These findings suggest that one or more potential dsx splicing enhancers can shift dsxM to dsxF in both sexes; however, the presence of Nix influences the function of this unknown splicing enhancer and ultimately leads to the formation of dsxM in males. Finally, our results provide important insight into the regulatory mechanism of dsx alternative splicing in the mosquito.

Differentiation of Long Non-Coding RNA and mRNA Expression Profiles in Male and Female Aedes albopictus.

The Asia tiger mosquito (Aedes albopictus) is an important vector of arboviruses, and females can transmit pathogens such as the dengue, zika, and chikungunya viruses. Understanding sex-related differences in this mosquito is fundamental for vector control. However, there are no reports of systematic characterization of long non-coding RNAs (lncRNAs) in male and female Ae. albopictus. To investigate the roles of coding and non-coding RNAs in both sexes of Ae. albopictus, RNA sequencing was performed on male and female samples. The results showed 305 differentially expressed protein-coding genes (DEGs) between males and females, of which 198 were highly expressed in males and 125 were highly expressed in females. Sex-associated gene ontology terms were enriched. Analysis with the FEELnc software identified 2,623 novel lncRNAs, of which 26 showed male high expression and 11 showed female high expression. Quantitative real-time PCR of randomly selected DEGs and lncRNAs supported the validity of the RNA sequencing results. Knocking down male high-expressed gene AALF000433 in male adults reduced the egg hatching rate. This study provides valuable data on sex-specific expression of protein-coding genes and lncRNAs in adult Ae. albopictus, which will guide further studies aimed at understanding sex development and determination mechanisms in this species.

A Novel Densovirus Isolated From the Asian Tiger Mosquito Displays Varied Pathogenicity Depending on Its Host Species.

Mosquito-borne viral diseases (MBVDs) continue to pose a significant global public health burden. Mosquito control remains a core intervention strategy in integrated mosquito management (IMM) programs to reduce the transmission of MBVDs. Mosquito densoviruses (MDVs) are mosquito-specific entomopathogenic viruses, and their attractive biological and pathogenic characteristics make MDVs potential biological control agents as alternatives to traditional chemical pesticides. However, different viral strains vary greatly in their pathogenicity against different mosquito species, which poses an obstacle for the wide application of MDVs in vector control. In this study, a novel MDV, Aedes albopictus densovirus-7 (AalDV-7), was isolated from field-collected Aedes albopictus in the dengue-endemic area of Guangzhou, China. The complete 4,048 nt genome of AalDV-7 was cloned and sequenced, and the transcription and translation of three open reading frames (ORFs) were characterized. Phylogenetic analysis indicated that AalDV-7 clustered with MDVs mostly isolated from indigenous mosquitoes. The pathogenicity of AalDV-7 to A. albopictus, Aedes aegypti, and Culex quinquefasciatus larvae was completely different, and the median lethal dose (LD50) of AalDV-7 in A. albopictus which was 109.48 genome equivalents per ml (geq/ml) was 12 and 46 times lower than those in A. aegypti (1010.56 geq/ml) and C. quinquefasciatus (1011.15 geq/ml). Furthermore, the median lethal time (LT50) value in A. albopictus (7.72 days) was 25% and 26% shorter than those in A. aegypti (10.24 days) and C. quinquefasciatus (10.42 days) at a titer of 1011 geq/ml. Furthermore, the mortality of AalDV-7-infected mosquitoes increased in a dose-dependent manner, and the highest mortality was found in A. albopictus larvae exposed to 1011 geq/ml AalDV-7 (82.00%). Sublethal effects analysis also showed that AalDV-7 infection significantly decreased pupation and emergence rates. The 1st-2nd instar larvae of all three mosquito species showed a near 100% infection rate, and the highest relative vial titer (305.97 ± 67.57 geq/ng) was observed in the 1st-2nd instar larvae of C. quinquefasciatus. These pathogenic characteristics make AalDV-7 a potential MBVDs control agent in China, whereas its negligible pathogenicity and high infection rate and viral dose in vivo make it a good candidate for gene delivery vectors in C. quinquefasciatus gene function analysis. In conclusion, the continuous discovery and isolation of new MDVs enrich the pool of mosquito entomopathogenic viruses and provide a variety of choices for optimal MDVs or combinations of MDVs to target certain mosquitoes.

Development of large-scale mosquito densovirus production by in vivo methods.

BACKGROUND: Mosquito-borne diseases (MBDs) cause a significant proportion of the global infectious disease burden. Vector control remains the primary strategy available to reduce the transmission of MBDs. However, long-term, wide-scale and large-scale traditional chemical pesticide application has caused significant and increased negative effects on ecosystems and broader emerging insecticide resistance in vectors; therefore, the development of a novel alternative approach is urgently needed. Mosquito densoviruses (MDVs) are entomopathogenic viruses that exhibit a narrow host range and multiple transmission patterns, making MDVs a great potential bioinsecticide. However, the application process has been relatively stagnant over the past three decades. The major obstacle has been that viruses must be produced in mosquito cell lines; therefore, the production process is both expensive and time-consuming. METHODS: In our study, two wild-type (wt) MDVs, AaeDV and AalDV-3, and a recombinant rAaeDV-210 were used to infect the Aag2 and C6/36 mosquito cell lines and the 1st-2nd-instar and 3rd-4th-instar larvae of Ae. albopictus, Ae. aegypti and Cx. quinquefasciatus. Viral titers and yields in cells, media, larvae and rearing water and total viral yield were evaluated. Three kinds of virus displayed higher maximum virus titers in vivo than in vitro, and they displayed higher maximum viral yields in rearing water. RESULTS: The three viruses displayed higher total maximum viral yields in C6/36 cells than in Aag2 cells. The three viruses displayed higher total maximum viral yields in Aedes mosquitoes than in Culex mosquitoes. Higher viral yields were produced by 1st-2nd-instar larvae compared to 3rd-4th-instar larvae. The recombinant viruses did not display significantly lower yields than wt viruses in nearly all samples. In summary, by using 100 1st-2nd-instar Aedes mosquito larvae in 200 ml of rearing water, more than 1013 genome equivalents (geq) MDV yield can be obtained. CONCLUSIONS: Considering the lower production cost, this in vivo method has great potential for the large-scale production of MDVs. MDVs exhibit promising prospects and great potential for mosquito control in the future.

Two of the three Transformer-2 genes are required for ovarian development in Aedes albopictus.

In Drosophila melanogaster, transformer-2 (tra2) plays an essential role in the sex-specific splicing of doublesex (dsx) and fruitless (fru), two key transcription factor genes that program sexual differentiation and regulate sexual behavior. In the present study, the sequences and expression profiles of three tra2 (Aalbtra2) genes in the Asian tiger mosquito, Aedes albopictus (Ae. albopictus) were characterized. Phylogenetic analysis revealed that these paralogs resulted from two duplication events. The first occurred in the common ancestor of Culicidae, giving rise to the tra2-α and tra2-ß clades that are found across divergent mosquito genera, including Aedes, Culex, and Anopheles. The second occurred within the tra2-α clade, giving rise to tra2-γ in Ae. albopictus. In addition to the conserved RNA recognition motif (RRM), arginine-rich/serine-rich regions (RS domains) and a linker region, a glycine-rich region located between the RRM and RS2 was observed in Tra2-α and Tra2-γ of Ae. albopictus that has not yet been described in the Tra2 proteins of dipteran insects. Quantitative real-time PCR detected relatively high levels of transcripts from all three tra2 paralogs in 0-2 h embryos, suggesting maternal deposition of these transcripts. All three Aalbtra2 genes were highly expressed in the ovary, while Aalbtra2-ß was also highly expressed in the testis. RNAi-mediated knockdown of any or all Aalbtra2 genes did not result in an obvious switch of the sex-specificity in dsx and fru splicing in the whole-body samples. However, knockdown of transcripts from all three tra2 genes significantly reduced the female isoform of dsx mRNA and increased the male isoform of the dsx mRNA in both the ovary and the fat body in adult females. Furthermore, knockdown of either Aalbtra2-α or Aalbtra2-γ or all three Aalbtra2 led to a decrease in ovariole number and ovary size after a blood meal. Taken together, these results indicate that two of the three tra2 genes affect female ovarian development.

Use of a Recombinant Mosquito Densovirus As a Gene Delivery Vector for the Functional Analysis of Genes in Mosquito Larvae.

In vivo microinjection is the most commonly used gene transfer technique for analyzing the gene functions in individual mosquitoes. However, this method requires a more technically demanding operation and involves complicated procedures, especially when used in larvae due to their small size, relatively thin and fragile cuticle, and high mortality, which limit its application. In contrast, viral vectors for gene delivery have been developed to surmount extracellular and intracellular barriers. These systems have the advantages of easy manipulation, high gene transduction efficiency, long-term maintenance of gene expression, and the ability to produce persistent effects in vivo. Mosquito densoviruses (MDVs) are mosquito-specific, small single-stranded DNA viruses that can effectively deliver foreign nucleic acids into mosquito cells; however, the replacement or insertion of foreign genes to create recombinant viruses typically causes a loss of packaging and/or replication abilities, which is a barrier to the development of these viruses as delivery vectors. Herein, we report using an artificial intronic small-RNA expression strategy to develop a non-defective recombinant Aedes aegypti densovirus (AaeDV) in vivo delivery system. Detailed procedures for the construction, packaging and quantitative analysis of the rAaeDV vectors, and for larval infection are described. This study demonstrates, for the first time, the feasibility of developing a non-defective recombinant MDV micro RNA (miRNA) expression system, and thus providing a powerful tool for the functional analysis of genes in mosquito and establishing a basis for the application of viral paratransgenesis for controlling mosquito-borne diseases. We demonstrated that Aedes albopictus 1st instar larvae could be easily and effectively infected by introducing the virus into the water body of the larvae breeding site and that the developed rAaeDVs could be used to overexpress or knock down the expression of a specific target gene in larvae, providing a tool for the functional analysis of mosquito genes.

Developmental piRNA profiles of the invasive vector mosquito Aedes albopictus.

BACKGROUND: In eukaryotic organisms, Piwi-interacting RNAs (piRNAs) control the activities of mobile genetic elements and ensure genome maintenance. Recent evidence indicates that piRNAs are involved in multiple biological pathways, including transcriptional regulation of protein-coding genes, sex determination and even interactions between host and pathogens. Aedes albopictus is a major invasive species that transmits a number of viral diseases in humans. Ae. albopictus has the largest genome and the highest abundance of repetitive sequences when compared with members that belong to Culicidae with a published genome. Analysis of piRNA profiles will provide a developmental and evolutionary perspective on piRNAs in Ae. albopictus. METHODS: piRNAs were identified and characterized during the development of Ae. albopictus, and piRNA expression patterns in adult males and females as well as sugar-fed females and blood-fed females were compared. RESULTS: Our results reveal that, despite the large genome size of Ae. albopictus, the piRNA pool of Ae. albopictus (1.2 × 107) is smaller than those of Aedes aegypti (1.7 × 107) and Drosophila melanogaster (1.6 × 107). In Ae. albopictus, piRNAs displayed the highest abundance at the embryo stage and the lowest abundance at the pupal stage. Approximately 50 % of the piRNAs mapped to intergenic regions with no known functions. Approximately 30 % of the piRNAs mapped to repetitive elements, and 77.69 % of these repeat-derived piRNAs mapped to Class I TEs; 45.42 % of the observed piRNA reads originated from piRNA clusters, and most of the top 10 highest expressed piRNA clusters and 100 highest expressed piRNAs from each stage displayed biased expression patterns across the developmental stages. All anti-sense-derived piRNAs displayed a preference for uridine at the 5' end; however, the sense-derived piRNAs showed adenine bias at the tenth nucleotide position and a typical ping-pong signature, suggesting that the biogenesis of piRNAs was conserved throughout development. Our results also show that 962 piRNAs displayed sex-biased expression, and 522 piRNAs showed higher expression in the blood-fed females than in the sugar-fed females. CONCLUSIONS: Our results suggest that piRNAs, aside from silencing transposable elements in Ae. albopictus, may have a role in other biological pathways.

Development of non-defective recombinant densovirus vectors for microRNA delivery in the invasive vector mosquito, Aedes albopictus.

We previously reported that mosquito densoviruses (MDVs) are potential vectors for delivering foreign nucleic acids into mosquito cells. However, considering existing expression strategies, recombinant viruses would inevitably become replication-defective viruses and lose their ability for secondary transmission. The packaging limitations of the virion represent a barrier for the development of MDVs for viral paratransgenesis or as high-efficiency bioinsecticides. Herein, we report the development of a non-defective recombinant Aedes aegypti densovirus (AaeDV) miRNA expression system, mediated by an artificial intron, using an intronic miRNA expression strategy. We demonstrated that this recombinant vector could be used to overexpress endogenous miRNAs or to decrease endogenous miRNAs by generating antisense sponges to explore the biological functions of miRNAs. In addition, the vector could express antisense-miRNAs to induce efficient gene silencing in vivo and in vitro. The recombinant virus effectively self-replicated and retained its secondary transmission ability, similar to the wild-type virus. The recombinant virus was also genetically stable. This study demonstrated the first construction of a non-defective recombinant MDV miRNA expression system, which represents a tool for the functional analysis of mosquito genes and lays the foundation for the application of viral paratransgenesis for dengue virus control.

High-throughput screening identifies CHMP4A associated with hypoxia-inducible factor 1.

AIMS: Tumor hypoxia is a common phenomenon and hypoxia-inducible factor-1 is the transcription factor that is most closely associated with hypoxia. Hypoxia-inducible factor-1 is overexpressed in most solid tumors and plays a vital role in hypoxic acclimatization, energy metabolism, tumor angiogenesis, tumor invasion, and drug tolerance in cancer cells. We aimed to identify novel human genes associated with the stability and transcriptional activity of hypoxia-inducible factor-1. MAIN METHODS: A cell-based dual luciferase reporter system based on a hypoxia responsive element luciferase reporter gene was constructed to screen 409 novel human genes cloned in our lab. Western blot analysis was used to examine the changes in the expression level of hypoxia-inducible factor-1 α, and RT-PCR analysis was used to detect the transcription level of adenylate kinase 3. KEY FINDINGS: Our results demonstrated that chromatin-modifying protein 4A could significantly up-regulate the hypoxia responsive element luciferase activity under both normoxic and cobalt chloride-induced hypoxic environment in HeLa cells. Moreover, Chromatin-modifying protein 4A could increase the expression of hypoxia-inducible factor-1 α protein under normoxic condition, and enhance the transcription level of adenylate kinase 3, which is one of the target genes of hypoxia-inducible factor-1. SIGNIFICANCE: The functional screening platform therefore can be applied for the high-throughput screening of hypoxia-inducible factor-1-related genes, which would provide new insights into underlying molecular mechanisms that may regulate hypoxia in mammalian cells.

A recombinant AeDNA containing the insect-specific toxin, BmK IT1, displayed an increasing pathogenicity on Aedes albopictus.

The Aedes aegypti densovirus (AeDNV) has previously shown potential in mosquito control. To improve its efficacy as a biopesticide, the gene for an excitatory insect-specific toxin from Buthus martensii Karsch (BmK IT1) was inserted into the AeDNV genome and cloned into pUCA plasmid. The coding sequence for green fluorescent protein was ligated to the C-terminus of the BmK IT1 gene as a screening marker. Recombinant and helper plasmids were cotransfected into C6/36 cells; wild-type viruses were the controls. The recombinant viruses were identified and quantified by real-time polymerase chain reaction and exposed to Ae. albopictus larvae for the evaluation of its bioinsecticidal activity. LT(50) and LD(50) bioassays showed that the recombinant AeDNV had stronger and faster pathogenic effects on Ae. albopictus than the wild-type virus. This is the first report on the recombinant AeDNA containing the insect-specific toxin, BmK IT1, which may be used to develop a novel type of insecticide.

[Effect of cinobufagin on nuclear factor-kappaB pathway in HepG2 cells].

OBJECTIVE: To investigate the effect of cinobufagin on nuclear factor-kappaB (NF-kappaB) pathway of liver cancer cell line HepG2. METHODS: Dual-luciferase cis-reporting system was used to detect the relative value of pNF-kappaB-TA-luc upon tumor necrosis factor-alpha (TNF-alpha) stimulation of NF-kappaB pathway. Western blotting was used to detect the protein level of NF-kappaB p65, and RT-PCR was used to detect the gene transcription level of intercellular adhesion molecule-1 (ICAM-1), a target downstream gene of NF-kappaB. RESULTS: At the concentration of 0.25 and 0.5 microg/ml, cinobufagin significantly lowered the relative value of luciferase (P<0.05). The results of Western blotting showed that cinobufagin significantly suppressed the protein expression of NF-kappaB p65. The transcription level of ICAM-1 was reduced by different doses of cinobufagin. CONCLUSION: The anti-cancer effect of cinobufagin may be related to its activity in inhibiting the activation of NF-kappaB pathway.

[Bioinformatics analysis of mosquito densovirus nostructure protein NS1].

OBJECTIVE: To analyze and predict the structure and function of mosquito densovirus (MDV) nostructual protein1 (NS1). METHODS: Using different bioinformatics software, the EXPASY pmtparam tool, ClustalX1.83, Bioedit, MEGA3.1, ScanProsite, and Motifscan, respectively to comparatively analyze and predict the physic-chemical parameters, homology, evolutionary relation, secondary structure and main functional motifs of NS1. RESULTS: MDV NS1 protein was a unstable hydrophilic protein and the amino acid sequence was highly conserved which had a relatively closer evolutionary distance with infectious hypodermal and hematopoietic necrosis virus (IHHNV). MDV NS1 has a specific domain of superfamily 3 helicase of small DNA viruses. This domain contains the NTP-binding region with a metal ion-dependent ATPase activity. A virus replication roller rolling-circle replication(RCR) initiation domain was found near the N terminal of this protein. This protien has the biological function of single stranded incision enzyme. CONCLUSION: The bioinformatics prediction results suggest that MDV NS1 protein plays a key role in viral replication, packaging, and the other stages of viral life.