Females with autism spectrum disorders show stronger DNA methylation signatures than males in perinatal tissues.

Autism spectrum disorder (ASD) comprises a group of neurodevelopmental conditions currently diagnosed by behavioral assessment in childhood, with reported underdiagnosis in females. Though diagnosis in early life is linked to improved outcomes, we currently lack objective screening tools for newborns. To address this gap, we sought to identify a sex-specific DNA methylation signature for ASD using perinatal tissues that reflect dysregulation in the brain. DNA methylation was assayed from ASD and typically developing (TD) newborn blood, umbilical cord blood, placenta, and post-mortem cortex samples using whole genome bisulfite sequencing (WGBS) in a total of 511 samples. We found that methylation levels of differentially methylated regions (DMRs) differentiated samples by ASD diagnosis in females more than males across the perinatal tissues. We tested three theories for ASD sex differences in newborn blood, finding epigenetic support for an X chromosome-related female protective effect, as well as a high replication rate of DMRs (48.1%) in females across two independent cohorts. In our pan-tissue analysis, three genes (X-linked BCOR , GALNT9 , OPCML ) mapped to ASD DMRs replicated in all four female tissues. ASD DMRs from all tissues were enriched for neuro-related processes (females) and SFARI ASD-risk genes (females and males). Overall, we found a highly replicated methylation signature of ASD in females across perinatal tissues that reflected dysregulation in the brain and involvement of X chromosome epigenetics. This comparative study of perinatal tissues shows the promise of newborn blood DNA methylation biomarkers for early detection of females at risk for ASD and emphasizes the importance of sex-stratification in ASD studies.

Sex-specific single cell-level transcriptomic signatures of Rett syndrome disease progression.

Dominant X-linked diseases are uncommon due to female X chromosome inactivation (XCI). While random XCI usually protects females against X-linked mutations, Rett syndrome (RTT) is a female neurodevelopmental disorder caused by heterozygous MECP2 mutation. After 6-18 months of typical neurodevelopment, RTT girls undergo poorly understood regression. We performed longitudinal snRNA-seq on cerebral cortex in a construct-relevant Mecp2e1 mutant mouse model of RTT, revealing transcriptional effects of cell type, mosaicism, and sex on progressive disease phenotypes. Across cell types, we observed sex differences in the number of differentially expressed genes (DEGs) with 6x more DEGs in mutant females than males. Unlike males, female DEGs emerged prior to symptoms, were enriched for homeostatic gene pathways in distinct cell types over time, and correlated with disease phenotypes and human RTT cortical cell transcriptomes. Non-cell-autonomous effects were prominent and dynamic across disease progression of Mecp2e1 mutant females, indicating wild-type-expressing cells normalizing transcriptional homeostasis. These results improve understanding of RTT progression and treatment.

Assessment of myofascial medialization following intraoperative fascial traction (IFT) in a cadaveric model.

PURPOSE: Intraoperative fascial traction (IFT) for the treatment of large ventral hernias and loss of domain (LOD) hernias is a promising tool in abdominal wall surgery. However, little is known about the extent of gain in myofascial advancement especially for the anterior rectus sheath. We, therefore, used a cadaveric model to determine the medialization during IFT. METHODS: 4 fresh frozen specimens were used. Retromuscular preparation was carried out followed by IFT with diagonal vertical traction for 30 min. Medial advancement of the anterior rectus sheath was measured after 15 and 30 min as well as traction forces. RESULTS: Total medialization for anterior rectus sheath after 30 min of IFT was 10.5 cm (mean). The mean traction force was 16.28 kg. Total medialization was significantly higher during the first 15 min of vertical fascial traction (p < 0.05). CONCLUSIONS: IFT provides significant medialization for the anterior rectus sheath in the cadaveric model. The findings align with results from a retrospective case study. Therefore, we see IFT as a beneficial tool in abdominal wall surgery.

Integration of CTCF Loops, Methylome, and Transcriptome in Differentiating LUHMES as a Model for Imprinting Dynamics of the 15q11-q13 Locus in Human Neurons.

Human cell line models, including the neuronal precursor line LUHMES, are important for investigating developmental transcriptional dynamics within imprinted regions, particularly the 15q11-q13 Angelman (AS) and Prader-Willi (PWS) syndrome locus. AS results from loss of maternal UBE3A in neurons, where the paternal allele is silenced by a convergent antisense transcript UBE3A-ATS, a lncRNA that normally terminates at PWAR1 in non-neurons. qRTPCR analysis confirmed the exclusive and progressive increase in UBE3A-ATS in differentiating LUHMES neurons, validating their use for studying UBE3A silencing. Genome-wide transcriptome analyses revealed changes to 11,834 genes during neuronal differentiation, including the upregulation of most genes within the 15q11-q13 locus. To identify dynamic changes in chromatin loops linked to transcriptional activity, we performed a HiChIP validated by 4C, which identified two neuron-specific CTCF loops between MAGEL2-SNRPN and PWAR1-UBE3A. To determine if allele-specific differentially methylated regions (DMR) may be associated with CTCF loop anchors, whole genome long-read nanopore sequencing was performed. We identified a paternally hypomethylated DMR near the SNRPN upstream loop anchor exclusive to neurons and a paternally hypermethylated DMR near the PWAR1 CTCF anchor exclusive to undifferentiated cells, consistent with increases in neuronal transcription. Additionally, DMRs near CTCF loop anchors were observed in both cell types, indicative of allele-specific differences in chromatin loops regulating imprinted transcription. These results provide an integrated view of the 15q11-q13 epigenetic landscape during LUHMES neuronal differentiation, underscoring the complex interplay of transcription, chromatin looping, and DNA methylation. They also provide insights for future therapeutic approaches for AS and PWS.

Placenta and fetal brain share a neurodevelopmental disorder DNA methylation profile in a mouse model of prenatal PCB exposure.

Polychlorinated biphenyls (PCBs) are developmental neurotoxicants implicated as environmental risk factors for neurodevelopmental disorders (NDDs). Here, we report the effects of prenatal exposure to a human-relevant mixture of PCBs on the DNA methylation profiles of mouse placenta and fetal brain. Thousands of differentially methylated regions (DMRs) distinguish placenta and fetal brain from PCB-exposed mice from sex-matched vehicle controls. In both placenta and fetal brain, PCB-associated DMRs are enriched for functions related to neurodevelopment and cellular signaling and enriched within regions of bivalent chromatin. The placenta and brain PCB DMRs overlap significantly and map to a shared subset of genes enriched for Wnt signaling, Slit/Robo signaling, and genes differentially expressed in NDD models. The consensus PCB DMRs also significantly overlap with DMRs from human NDD brain and placenta. These results demonstrate that PCB-exposed placenta contains a subset of DMRs that overlap fetal brain DMRs relevant to an NDD.

Placental methylome reveals a 22q13.33 brain regulatory gene locus associated with autism.

BACKGROUND: Autism spectrum disorder (ASD) involves complex genetics interacting with the perinatal environment, complicating the discovery of common genetic risk. The epigenetic layer of DNA methylation shows dynamic developmental changes and molecular memory of in utero experiences, particularly in placenta, a fetal tissue discarded at birth. However, current array-based methods to identify novel ASD risk genes lack coverage of the most structurally and epigenetically variable regions of the human genome. RESULTS: We use whole genome bisulfite sequencing in placenta samples from prospective ASD studies to discover a previously uncharacterized ASD risk gene, LOC105373085, renamed NHIP. Out of 134 differentially methylated regions associated with ASD in placental samples, a cluster at 22q13.33 corresponds to a 118-kb hypomethylated block that replicates in two additional cohorts. Within this locus, NHIP is functionally characterized as a nuclear peptide-encoding transcript with high expression in brain, and increased expression following neuronal differentiation or hypoxia, but decreased expression in ASD placenta and brain. NHIP overexpression increases cellular proliferation and alters expression of genes regulating synapses and neurogenesis, overlapping significantly with known ASD risk genes and NHIP-associated genes in ASD brain. A common structural variant disrupting the proximity of NHIP to a fetal brain enhancer is associated with NHIP expression and methylation levels and ASD risk, demonstrating a common genetic influence. CONCLUSIONS: Together, these results identify and initially characterize a novel environmentally responsive ASD risk gene relevant to brain development in a hitherto under-characterized region of the human genome.

Sex disparate gut microbiome and metabolome perturbations precede disease progression in a mouse model of Rett syndrome.

Rett syndrome (RTT) is a regressive neurodevelopmental disorder in girls, characterized by multisystem complications including gut dysbiosis and altered metabolism. While RTT is known to be caused by mutations in the X-linked gene MECP2, the intermediate molecular pathways of progressive disease phenotypes are unknown. Mecp2 deficient rodents used to model RTT pathophysiology in most prior studies have been male. Thus, we utilized a patient-relevant mouse model of RTT to longitudinally profile the gut microbiome and metabolome across disease progression in both sexes. Fecal metabolites were altered in Mecp2e1 mutant females before onset of neuromotor phenotypes and correlated with lipid deficiencies in brain, results not observed in males. Females also displayed altered gut microbial communities and an inflammatory profile that were more consistent with RTT patients than males. These findings identify new molecular pathways of RTT disease progression and demonstrate the relevance of further study in female Mecp2 animal models.

Knowledge and attitudes towards oral and dental health among seventh and eighth grade students compared with their teeth examination.

AIM: Oral and dental heath are important aspects of general health and impact the quality of life and well-being. In this study, we aimed to assess the level of knowledge and attitudes towards oral and dental health and to examine the relative effect of social-behavioural risk factors on caries and other teeth problems among seventh and eighth grade students. METHODS: Study Design: The study population consisted of all seventh and eighth graders who started the 2011 school year in Sarigol village of the Manisa city, Turkey (n = 377). Data were gathered by questionnaires which were carried out before tooth examination. Associations between sociodemographic characteristics, knowledge items, attitudinal factors and number of dental caries, dental fillings and teeth loss were studied. RESULTS: Oral health was worse among females (p=0.002). As the number of children under care increased in the house, the number of dental filling and teeth loss increased in the students (p= 0.001 and p=0.021). Education of mothers of students who had worse dental health was significantly lower compared to the others (p=0.029). Among students who had four and more dental caries and dental fillings, frequency of dentist visit was lower (p= 0.034 and p= 0.005). Among students who had more dental caries, the knowledge that consumption of acidic beverages is not good for oral health was less prevalent (p=0.011). STATISTICS: Associations between sociodemographic characteristics, knowledge items, attitudinal factors and number of dental caries, dental fillings and teeth loss were studied using chi-square test. CONCLUSIONS: Health authorities should strengthen the implementation of oral disease prevention and oral health promotion programs rather than traditional curative care. Community-oriented education programs for students and their families are crucial for this purpose.

The Molecular Functions of MeCP2 in Rett Syndrome Pathology.

MeCP2 protein, encoded by the MECP2 gene, binds to DNA and affects transcription. Outside of this activity the true range of MeCP2 function is still not entirely clear. As MECP2 gene mutations cause the neurodevelopmental disorder Rett syndrome in 1 in 10,000 female births, much of what is known about the biologic function of MeCP2 comes from studying human cell culture models and rodent models with Mecp2 gene mutations. In this review, the full scope of MeCP2 research available in the NIH Pubmed (https://pubmed.ncbi.nlm.nih.gov/) data base to date is considered. While not all original research can be mentioned due to space limitations, the main aspects of MeCP2 and Rett syndrome research are discussed while highlighting the work of individual researchers and research groups. First, the primary functions of MeCP2 relevant to Rett syndrome are summarized and explored. Second, the conflicting evidence and controversies surrounding emerging aspects of MeCP2 biology are examined. Next, the most obvious gaps in MeCP2 research studies are noted. Finally, the most recent discoveries in MeCP2 and Rett syndrome research are explored with a focus on the potential and pitfalls of novel treatments and therapies.

The association between cagL and cagA, vacAs-m, babA genes in patients with gastric cancer, duodenal ulcer, and non-ulcer dyspepsia related to Helicobacter pylori.

INTRODUCTION: As a component of the cag T4SS, the cagL gene is involved in the translocation of CagA into host cells and is essential for the formation of cag PAI-associated pili between H. pylori and gastric epithelial cells. AIM: We aimed to investigate the clinical association of the cagL gene with other virulence factors (VacA, CagA, EPIYA-C, and BabA protein) of H. pylori strains isolated from GC, duodenal ulcer (DU), and non-ulcer dyspepsia (NUD) cases. METHODS: The patient group (PG), including 47 patients (22 GC and 25 DU) and a 25 control group (CG= NUD) were included. Amplification of the H. pylori cagL, cagA, vacA, and babA2 genes and typing of EPIYA motifs were performed by PCR methods. RESULTS: Sixty-one (84.7%) H. pylori strains were detected with cagL (93.6% in SG, 68% in CG). We detected a significant difference between SG and CG for the presence of cagL (p=0.012) but no statistical comparison was done for (≥2) EPIYA-C repeats In the comparison of H. pylori strains with cagA/vacAs1m1 and cagA/ vacAs1m2 and babA2 for the presence of cagL, we could not detect a significant difference (p=1). CONCLUSION: We detected a significant difference between groups for the presence of cagL genotype (p=0.012). The vacAs1m1 (OR: 2.829), genotypes increased the GC and DU risk by 2.8 times, while multiple (≥2) EPIYA-C repeats incresed the GC and DU risk by 3.524 times. Gender (to be female) (OR: 0.454) decreased the GC and DU risk by inversly decreased in the multivariate analysis.

Effect of Feed Additives as Alternatives to In-feed Antimicrobials on Production Performance and Intestinal Clostridium perfringens Counts in Broiler Chickens.

Numerous non-antibiotic feed additives (alternatives to antibiotics, ATAs) have been marketed, but few have been evaluated under uniform testing conditions modelling commercial flocks. We compared 24 ATA treatments and the ionophorous coccidiostat narasin against a diet without any feed additives. Feed conversion ratio and body weight gain were registered from day 0 to 28 in Ross 308 chickens housed on litter floor. The chickens were challenged with Eimeria spp., and cecal Clostridium perfringens (CP) counts were investigated. Active components from all ATA classes had a positive impact on intestinal health or production performance. Whereas narasin had a strong CP-reducing effect in combination with performance-promoting impact, only two ATA treatments achieved significantly beneficial effects on CP counts as well as feed conversion during the time span following Eimeria challenge. Active components present in these two treatments include a Bacillus subtilis probiotic strain, short- and medium-chain fatty acids and Saccharomyces cerevisiae components. Different ATA classes had beneficial impact during distinct rearing phases and on specific performance targets, suggesting that optimizing combinations and use of active components can make ATAs even more useful tools in broiler rearing without the use of in-feed antimicrobials. Further studies of promising ATAs and ATA combinations are required.

Predictors and Outcomes of Early Intubation in Infants Born at 28-36 Weeks of Gestation Receiving Noninvasive Respiratory Support.

OBJECTIVE: To identify predictors and outcomes of early intubation in preterm infants with respiratory distress, and predictors of need for brief respiratory support (≤1 day). STUDY DESIGN: Secondary analysis of data from a randomized trial comparing nasal high-flow with continuous positive airway pressure as primary respiratory support in preterm infants born at 28-36 weeks of gestation. Intubation was assessed within 72 hours of randomization. RESULTS: There were 564 included infants with a mean (SD) gestational age of 32.0 (2.2) weeks and birth weight 1744 (589) g; 76 infants (13.5%) received early intubation. On multivariable analysis, lower gestational age and higher pre-randomization fraction of inspired oxygen (FiO2) predicted intubation. A test based on gestational age of <30 weeks and an FiO2 of ≥0.30 produced a likelihood ratio of 9.1. Intubation was associated with prolonged duration of respiratory support and supplemental oxygen, with pneumothorax and nasal trauma, and in infants born at <32 weeks of gestational, with bronchopulmonary dysplasia and patent ductus arteriosus requiring treatment. Greater gestational age and lower FiO2 predicted the need for ≤1 day of respiratory support. A test based on a gestational age of ≥34 weeks and an FiO2 of 0.21 produced a likelihood ratio of 4.7. CONCLUSIONS: In preterm infants 28-36 week of gestation receiving primary noninvasive respiratory support, lower gestational age, and higher FiO2 predicted need for intubation within 72 hours. Intubation was associated with adverse respiratory outcomes. Greater gestational age and lower FiO2 predicted need for ≤1 day of respiratory support. It may be reasonable to defer the use of respiratory support in more mature infants with low FiO2 requirements. TRIAL REGISTRATION AUSTRALIAN NEW ZEALAND CLINICAL TRIALS REGISTRY: ACTRN12613000303741.

Effect of Reaction Conditions on Catalytic and Noncatalytic Lignin Solvolysis in Water Media Investigated for a 5 L Reactor.

The high content of oxygen in the lignin polymer and the prevalence of phenolic functional groups make the conversion of lignin to fuels and value-added products with well-defined chemical properties challenging. The lignin-to-liquid process using a water/formic acid reaction medium has been shown to convert the lignin polymer to monomers with a molecular weight range of 300-600 Da. The bio-oil comprises a complex mixture of monomeric phenols, aromatics, and aliphatic hydrocarbons with a high H/C and low O/C ratio. This study investigates the effect of the stirring rate, level of loading, and catalyst at 305 and 350 °C in a 5 L pilot scale reactor. The oil yields are found to be highest for experiments conducted using the maximum stirring rate, maximum level of loading, and Ru/Al2O3 catalyst with yields of more than 69 wt % on lignin intake. Goethite as a catalyst does not show good conversion efficiency at either reaction temperatures. The carbon recovery is highest for products produced at 305 °C. Furthermore, results from solid phase extraction on a DSC-CN solid phase show that 65-92 wt % the bio-oils can be recovered as fractions separated based on polarity.

MicroRNAs in Daphnia magna identified and characterized by deep sequencing, genome mapping and manual curation.

MicroRNAs (miRNAs) are small non-coding RNAs that function in RNA silencing and post-transcriptional regulation of gene expression in most organisms. The water flea, Daphnia magna is a key model to study phenotypic, physiological and genomic responses to environmental cues and miRNAs can potentially mediate these responses. By using deep sequencing, genome mapping and manual curations, we have characterised the miRNAome of D. magna. We identified 66 conserved miRNAs and 13 novel miRNAs; all of these were found in the three studied life stages of D. magna (juveniles, subadults, adults), but with variation in expression levels between stages. Forty-one of the miRNAs were clustered into 13 genome clusters also present in the D. pulex genome. Most miRNAs contained sequence variants (isomiRs). The highest expressed isomiRs were 3' template variants with one nucleotide deletion or 3' non-template variants with addition of A or U at the 3' end. We also identified offset RNAs (moRs) and loop RNAs (loRs). Our work extends the base for further work on all species (miRNA, isomiRs, moRNAs, loRNAs) of the miRNAome of Daphnia as biomarkers in response to chemical substances and environment cues, and underline age dependency.

Placental DNA methylation levels at CYP2E1 and IRS2 are associated with child outcome in a prospective autism study.

DNA methylation acts at the interface of genetic and environmental factors relevant for autism spectrum disorder (ASD). Placenta, normally discarded at birth, is a potentially rich source of DNA methylation patterns predictive of ASD in the child. Here, we performed whole methylome analyses of placentas from a prospective study MARBLES (Markers of Autism Risk in Babies-Learning Early Signs) of high-risk pregnancies. A total of 400 differentially methylated regions (DMRs) discriminated placentas stored from children later diagnosed with ASD compared to typically developing controls. These ASD DMRs were significantly enriched at promoters, mapped to 596 genes functionally enriched in neuronal development, and overlapped genetic ASD risk. ASD DMRs at CYP2E1 and IRS2 reached genome-wide significance, replicated by pyrosequencing and correlated with expression differences in brain. Methylation at CYP2E1 associated with both ASD diagnosis and genotype within the DMR. In contrast, methylation at IRS2 was unaffected by within DMR genotype but modified by preconceptional maternal prenatal vitamin use. This study therefore identified two potentially useful early epigenetic markers for ASD in placenta.

Cognitive deficits in the Snord116 deletion mouse model for Prader-Willi syndrome.

Prader-Willi syndrome (PWS) is an imprinted neurodevelopmental disease caused by a loss of paternal genes on chromosome 15q11-q13. It is characterized by cognitive impairments, developmental delay, sleep abnormalities, and hyperphagia often leading to obesity. Clinical research has shown that a lack of expression of SNORD116, a paternally expressed imprinted gene cluster that encodes multiple copies of a small nucleolar RNA (snoRNA) in both humans and mice, is most likely responsible for many PWS symptoms seen in humans. The majority of previous research using PWS preclinical models focused on characterization of the hyperphagic and metabolic phenotypes. However, a crucial understudied clinical phenotype is cognitive impairments and thus we investigated the learning and memory abilities using a model of PWS, with a heterozygous deletion in Snord116. We utilized the novel object recognition task, which doesn't require external motivation, or exhaustive swim training. Automated findings were further confirmed with manual scoring by a highly trained blinded investigator. We discovered deficits in Snord116+/- mutant mice in the novel object recognition, location memory and tone cue fear conditioning assays when compared to age-, sex- matched, littermate control Snord116+/+ mice. Further, we confirmed that despite physical neo-natal developmental delays, Snord116+/- mice had normal exploratory and motor abilities. These results show that the Snord116+/- deletion murine model is a valuable preclinical model for investigating learning and memory impairments in individuals with PWS without common confounding phenotypes.

Prader-Willi locus Snord116 RNA processing requires an active endogenous allele and neuron-specific splicing by Rbfox3/NeuN.

Prader-Willi syndrome (PWS), an imprinted neurodevelopmental disorder characterized by metabolic, sleep and neuropsychiatric features, is caused by the loss of paternal SNORD116, containing only non-coding RNAs (ncRNAs). The primary SNORD116 transcript is processed into small nucleolar RNAs (snoRNAs), which localize to nucleoli, and their spliced host gene 116HG, which is retained at its site of transcription. While functional complementation of the SNORD116 ncRNAs is a desirable goal for treating PWS, the mechanistic requirements of SNORD116 RNA processing are poorly understood. Here we developed and tested a novel transgenic mouse which ubiquitously expresses Snord116 on both a wild-type and a Snord116 paternal deletion (Snord116+/-) background. Interestingly, while the Snord116 transgene was ubiquitously expressed in multiple tissues, splicing of the transgene and production of snoRNAs was limited to brain tissues. Knockdown of Rbfox3, encoding neuron-specific splicing factor neuronal nuclei (NeuN) in Snord116+/--derived neurons, reduced splicing of the transgene in neurons. RNA fluorescence in situ hybridization for 116HG revealed a single significantly larger signal in transgenic mice, demonstrating colocalization of transgenic and endogenous 116HG RNAs. Similarly, significantly increased snoRNA levels were detected in transgenic neuronal nucleoli, indicating that transgenic Snord116 snoRNAs were effectively processed and localized. In contrast, neither transgenic 116HG nor snoRNAs were detectable in either non-neuronal tissues or Snord116+/- neurons. Together, these results demonstrate that exogenous expression and neuron-specific splicing of the Snord116 locus are insufficient to rescue the genetic deficiency of Snord116 paternal deletion. Elucidating the mechanisms regulating Snord116 processing and localization is essential to develop effective gene replacement therapies for PWS.

MeCP2 isoform e1 mutant mice recapitulate motor and metabolic phenotypes of Rett syndrome.

Mutations in the X-linked gene MECP2 cause the majority of Rett syndrome (RTT) cases. Two differentially spliced isoforms of exons 1 and 2 (MeCP2-e1 and MeCP2-e2) contribute to the diverse functions of MeCP2, but only mutations in exon 1, not exon 2, are observed in RTT. We previously described an isoform-specific MeCP2-e1-deficient male mouse model of a human RTT mutation that lacks MeCP2-e1 while preserving expression of MeCP2-e2. However, RTT patients are heterozygous females that exhibit delayed and progressive symptom onset beginning in late infancy, including neurologic as well as metabolic, immune, respiratory and gastrointestinal phenotypes. Consequently, we conducted a longitudinal assessment of symptom development in MeCP2-e1 mutant females and males. A delayed and progressive onset of motor impairments was observed in both female and male MeCP2-e1 mutant mice, including hind limb clasping and motor deficits in gait and balance. Because these motor impairments were significantly impacted by age-dependent increases in body weight, we also investigated metabolic phenotypes at an early stage of disease progression. Both male and female MeCP2-e1 mutants exhibited significantly increased body fat compared to sex-matched wild-type littermates prior to weight differences. Mecp2e1-/y males exhibited significant metabolic phenotypes of hypoactivity, decreased energy expenditure, increased respiratory exchange ratio, but decreased food intake compared to wild-type. Untargeted analysis of lipid metabolites demonstrated a distinguishable profile in MeCP2-e1 female mutant liver characterized by increased triglycerides. Together, these results demonstrate that MeCP2-e1 mutation in mice of both sexes recapitulates early and progressive metabolic and motor phenotypes of human RTT.