The purpose of this study was to investigate the effects of treadmill exercise on oxidative stress in the hippocampal tissue and mitochondrial dynamic-related proteins in rats fed a long-term high-fat diet (HFD).Obesity was induced in experimental animals using high fat feed, and the experimental groups were divided into a normal diet-control , a high fat diet-control  and a high fat diet-treadmill exercise  group. The rats were subsequently subjected to treadmill exercise (progressively increasing load intensity) for 8 weeks . We assessed weight, triglyceride (TG) concentration, total cholesterol (TC), area under the curve, homeostatic model assessment of insulin resistance, and AVF/body weight. Western blotting was used to examine expression of proteins related to oxidative stress and mitochondrial dynamics, and immunohistochemistry was performed to examine the immunoreactivity of gp91phox.Treadmill exercise effectively improved the oxidative stress in the hippocampal tissue, expression of mitochondrial dynamic-related proteins, and activation of NADPH oxidase (gp91phox) and induced weight, blood profile, and abdominal fat loss.Twenty weeks of high fat diet induced obesity, which was shown to inhibit normal mitochondria fusion and fission functions in hippocampal tissues. However, treadmill exercise was shown to have positive effects on these pathophysiological phenomena. Therefore, treadmill exercise should be considered during prevention and treatment of obesity-induced metabolic diseases. Nonetheless, the continuous production of ROS due to obesity increases oxidative stress in mitochondria and causes mitochondrial dysfunction. Notably, the oxidative stress generated in the mitochondria is produced by NADPH oxidase (NOX), a membrane bound enzyme complex. NOX2, an isoform of NOX, in particular, has been shown to play a key role in metabolic and neurodegenerative disorders5. NOX2 expression has been reported in various cell types including neurons and endothelial cells, and its largest expression is known to occur in microglial cells involved in immune/inflammatory reactions6,7.In energy metabolism, reactive oxygen species (ROS) are inevitably generated, but the hyperglycemia or insulin resistance induced by obesity leads to continuous production of ROS, which is a major cause of increased oxidative stress. The accumulation of ROS within mitochondria is generally known to be reduced by antioxidant enzymes such as catalase and superoxide dismutase (SOD) as part of homeostasis maintenance8,9. The maintenance of homeostasis by regulating changes in mitochondrial form is very important in maintaining normal mitochondrial function and requires control of both the quantity and quality of proteins associated with mitochondrial dynamics10. Several studies have identified the key proteins regulating the shift in mitochondrial form. Dynamin-related protein 1 (Drp1), mitochondrial fission 1 (Fis1), mitochondrial fission factor (MFF), mitochondrial dynamics 49 (MiD49), and MiD51 are known to be involved in facilitating mitochondrial fission, while mitofusin 1 (Mfn1), mitofusin 2 (Mfn2), and optic atrophy protein 1 (OPA1) are involved in mitochondrial fusion8,11.Mitochondria are vital intracellular organelles responsible for biological oxidation in most eukaryotic cells. As cells forms constantly change, mitochondrial dynamics are regulated through repeated fusion (where the length of mitochondria increases) and fission (where mitochondria split into two)12. A recent study reported that, an imbalance in mitochondrial dynamics may be associated with imbalances in energy metabolism such as diabetes and obesity13. This suggests the possibility that mitochondrial dysfunction might play a role in the pathogenesis of several metabolic disorders14–16. In other words, unbalanced mitochondrial dynamics in the tissues of the pancreas, liver, and muscle, caused by obesity, may increase the production of ROS that induces oxidative stress, which inhibits insulin signaling pathways and ultimately increases insulin resistance leading to diabetes17-19. Moreover, unbalanced mitochondrial dynamics have been observed in neurodegenerative disorders such as Alzheimer’s and Parkinson’s disease, where it was reported to aggravate conditions20,21. In other words, the slowdown in mitochondrial dynamics induced by obesity was shown to contribute to the pathogenesis of various metabolic disorders, and from this, it can be seen that solving the problem of obesity is the key for recovering normal mitochondrial dynamics.Since mitochondria play a pivotal role in intracellular energy metabolism, its form and function are regulated through continuous cycles of fusion and fission22, in particular, reported alleviation of insulin resistance when exercise was used to partially restore the increase in fission and decrease in fusion of mitochondria in the human skeletal muscle where insulin resistance occurs. Treadmill exercise has also been reported to have a positive influence on recovering cognitive function by restoring the balance of mitochondrial dynamics based on an increase in Drp1, a protein involved in mitochondrial fission present in neurodegenerative disorders23,24. Nonetheless, contrasting results have also been reported, where exercise led to increased or decreased expression of proteins related to mitochondrial fission or fusion25,26. This may be attributed to the changes caused by exercise intensity, disease pathology, tissue type, and various experimental conditions. In particular, previous studies that examined the effects of exercise on unbalanced mitochondrial dynamics have mostly focused on skeletal muscles, while there is a lack of studies on brain tissues. Notably, brain hippocampal tissue plays a central role in human cognition such as learning and memory and has a deep connection with neurodegenerative disorders such as Alzheimer's, which supports the significance of studies on the hippocampus.Physical activity has recently been recognized as an effective way to enhance mitochondrial function, and exercise has been reported to improve damaged mitochondrial dynamics in various disorders. Fealy et al.As previously mentioned, reducing obesity through exercise has been reported to have a positive effect on energy metabolism, and numerous studies have investigated the role of exercise in improving pathological physiology in peripheral tissues. However, there are currently an extremely limited number of studies on the role of exercise and obesity in nerve cell mitochondrial dynamics. Thus, this study aims to examine the changes in the factors related to nerve cell mitochondrial dynamics in the brain, when long-term, high-fat diet obesity-induced lab animals perform treadmill exercise.This study was approved by the Institutional Animal Care and Use Committee at H University prior to performing the experiments (KNSU-IACUC-2017-01). The lab animals were 8-week old male Sprague Dawley rats purchased from KOATECH, Co., Ltd and reared until 24 weeks in an animal lab at H University . When the rats reached 24 weeks of age, obesity was induced in some rats (n = 24) with high-fat feed  purchased from the Central Lab, Animal Inc., for 20 weeks, while they were allowed liberal intake and supply of water. The lab animals were categorized into three groups: normal diet-control , high fat diet-control , and high fat diet-treadmill exercise .27.The HFD-TE group was acclimatized with pre-training for 30 min a day for five days, on a rodent treadmill  fixed at a 0 % gradient. After pre-training was complete, the main exercise was carried out five days a week for eight weeks. Fixing the gradient at 0 %, the animals were given the Maximal Graded Moderate to Intensive Exercise Program suggested in Kang et al. Twelve hours following the 8-week treadmill exercise, plasma glucose was measured from the tail of the rats in fasting state. Next, 1 mL triple distilled water and 0.3 mL glucose were mixed to prepare a 30 % glucose concentrate, which was administered to the rats at 1 mL∙kg-1. Blood was then taken from the rat tail five times  and using a blood glucose monitoring device , plasma glucose was measured and the reaction area for total glucose secretion  was determined.Twenty four hours after the 8-week’s treadmill exercise, the rats were anesthetized by intraperitoneal injection of Rompun/Zoletil mixture , and to estimate protein expression, brain tissue, blood, and abdominal adipose tissue were removed from seven rats. The extracted brain tissue was separated into the cerebral cortex and hippocampus, which were rapidly cooled in liquid nitrogen and stored in a -80 ℃. After collection, blood from the heart was centrifuged to obtain serum for analyzing glucose and insulin. The abdominal adipose tissue was removed by incision and washed with cold physiological saline, and after removing the moisture with gauze, was weighed. For immunohistochemical staining, five rats were anesthetized, their thoracic cavities were opened, and 50 mM phosphate-buffered saline was injected through the left ventricle for 10 min, and perfused with 4 % paraformaldehyde (PFA) fixative dissolved in 100 mM phosphate buffer. After perfusion, the brain was removed and placed in 4 % PFA for 4-h fixation at 4 ℃. The fixed brain tissue was left to settle for two days in 30 % sucrose solution. Rodent Brain Matrix  was used to extract regions of the cortex and hippocampus (from –4.88 mm to –1.76 mm from bregma) from the fixed brain tissue (whole brain). A freezing microtome  was then used to prepare a 40 μm thick serial coronal section.The blood collected by cardiac perforation was centrifuged  and the serum was collected. The glucose, insulin, triglyceride (TG), and total cholesterol (TC) concentrations were analyzed by Green Cross Corp. The HOMA-IR index was calculated from the concentrations of glucose and insulin according to the following equation: HOMA-IR = fasting serum glucose (mmol/L) × fasting serum insulin (μU/Ml)/22.5.The Mitochondria Extraction Kit  was used to isolate mitochondria. One mL homogenizing buffer was added per 100 mg of hippocampal tissue, followed by 10-min centrifugation at 4 °C and 3,000 rpm, the supernatant was removed and centrifuged again for 30 min at 4 °C at 12,000 rpm. The centrifuged supernatant (cytosolic fraction) was isolated as cytosol, while the remaining pellet was mixed thoroughly with 1 mL suspension buffer and centrifuged again for 10 min at 4 °C and 12,000 rpm. The supernatant was removed, the pellet was resuspended in 1 mL suspension buffer, and after another 10-min centrifugation at 4 °C and 12,000 rpm, the supernatant was removed and the resulting pellet was dissolved in 1 mL Complete Mitochondrial lysis buffer for 30 min at 4 °C. The separated mitochondrial extract was centrifuged for 5 min at 4 °C and 12,000 rpm, and the supernatant (mitochondria fraction) was collected.Hippocampal tissue from isolated brain tissues was homogenized using lysis buffer and a homogenizer. It was then centrifuged for 15 min at 4 ℃ and 13,000 g, and the total protein content in the supernatant was quantified by the Bradford method. Protein (30 μg) was separated by electrophoresis in SDS-Polyacrylamide gel , transferred to PVDF membrane , blocked in 1× TBS-T solution containing 3 % BSA for 1 h at room temperature. The proteins and the corresponding primary antibody  were lefFor the five rats to be analyzed by immunofluorescence assay, the brain tissue samples from each group (5 rats) were washed three times for 10 min using 0.01 M PBS according to the free-floating method. Then, each sample was placed in a beaker containing 0.01 M sodium citrate for 60 min for incubation at 90 °C, followed by blocking with 10 % normal donkey serum for 60 min. After blocking, the primary antibody (gp91phox) was added and the sample was left overnight for 12 h at 4 ℃, then washed three times for 5 min using 0.01 M PBS. The sample was then reacted with the secondary antibody  for 2 h and washed four times, and each sample was moved to a slide and sealed using mounting solution . The slides were imaged using an immunofluorescence microscope .All data obtained in this study were used to generate descriptive statistics (mean ± SEM) using the 18.0 SPSS program for Windows. Intergroup differences were analyzed by one way ANOVA, and when a significant intergroup difference was found, the Bonferroni method was used as the post hoc test. Here, the statistical level of significance was set as α=0.05.The changes in weight based on high-fat diet and treadmill exercise were measured at weeks 24, 43, 44, and 51, and the results are presented in The area under the curve of glucose (AUC), HOMA-IR, and AVF/bodyweight, obtained from testing the plasma lipids TG, TC, and OGTT, is given in The effect of treadmill exercise on CS, gp91phox, SOD-2, and Catalase expression is presented in The effect of exercise on mitochondrial fusion factors  is given in The effect of treadmill exercise on mitochondrial fission factors (Drp1 and Fis1) is given in This study investigated the effects of treadmill exercise on oxidative stress and the fusion and fission of mitochondria in the hippocampal tissue of lab animals, where obesity was induced by a long-term, high-fat diet. The 20-week high-fat diet increased the weight and abdominal visceral fat in the animals while decreasing the level of HOMA-IR, an indicator of insulin resistance, and their ability to control plasma glucose (AUC from OGTT data). It also increased the serum concentrations of TG and TC. However, treadmill exercise was shown to improve these pathological symptoms associated with obesity. The long-term, high-fat diet was also shown to promote the fusion of mitochondria in the hippocampal tissue while reducing their fission, thereby partially disrupting mitochondrial dynamics. Moreover, the high-fat diet led to an increase in oxidative stress due to increased activity of NADPH gp91phox and decreased expression of CS, an indicator of mitochondrial energy metabolism activity. However, treadmill exercise suppressed the oxidative stress by enhancing the expression of antioxidant enzymes, and this improvement alleviated the imbalance in mitochondrial dynamics. A detailed discussion is given below.28,29. In other words, treadmill exercise may contribute to increasing the utilization of glucose and triglycerides and enhance insulin sensitivity, which suggests that it may be an effective intervention for improving insulin resistance and the ability to control plasma glucose30.The long-term, high-fat diet increased the weight, abdominal visceral fat, and serum TG and TC concentrations in the lab animals. The level of HOMA-IR and its AUC also increased. While conditions like impaired glucose tolerance, hyperlipidemia, or type II diabetes cannot be conclusively demonstrated, their negative influence on energy metabolism can be seen, such as the decrease in the ability to control plasma glucose or the increased incidence of hyperinsulinemia compared to that seen in rats fed a normal diet. However, treadmill exercise reduced the increase in weight due to high-fat diet and improved AUC, HOMA-IR, and abdominal fat content while reducing the concentrations of plasma TG and TC. These results are in concordance with previous studies that reported the effect of exercise on improving the negative influence of obesity on energy metabolism31. This study analyzed gp91phox, a membrane subunit of the NOX isoform NOX2. All groups that were fed a long-term, high-fat diet showed increased activation of gp91phox in the hippocampus, compared to the normal diet group. Immunohistochemical assays also found increased immune reaction from gp91phox in the hippocampus (DG). Nevertheless, treadmill exercise downregulated the activation of gp91phox; i.e., treadmill exercise effectively inhibited the activation of NADPH oxidase, which can produce ROS. This finding agreed with the result of a previous study that reported an increase in NOX activation due to a high-fat diet although the analyzed tissue (aorta) was different32. It was also consistent with NOX activity being downregulated by treadmill exercise in the aorta of a high-fat diet-induced obesity model33. In addition, expression of catalase and SOD-2, the mitochondrial antioxidant enzymes that protect the cell from oxidative stress, was reduced in both the HD-Con and ND-Con groups, which was increased by treadmill exercise. Likewise, expression of citrate synthase, an enzyme that is part of the Krebs cycle in mitochondria and is an indicator of oxidative capacity, was reduced in the high-fat diet group, while treadmill exercise led to increased expression. The antioxidant enzymes SOD-2 and catalase are the major defense against ROS generated during exercise, so their expression increases as a response to exercise34,35. Treadmill exercise in this study also led to increased SOD-2 and catalase activities, and notably, the increased activity of citrate synthase, an enzyme specialized for mitochondrial substrates and the most important enzyme in the Krebs cycle, confirmed the improvement of mitochondrial dysfunction.According to a recent study, oxidative stress plays a critical role in linking obesity to cognitive dysfunction and brain function is sensitive to oxidative stress, which was reported to increase in obesity36. In particular, Mfn2 inhibition is related to substrate oxidation of the ETC complex, cellular metabolism, and a decrease in membrane potential in obesity37. Despite being focused on skeletal muscle, a study on a long-term high-fat diet reported decreased levels of Mfn1 and Mfn2 protein and increased levels of Fis1 and Drp1 protein38. On the other hand, treadmill exercise was shown to decrease the expression of mitochondrial fission proteins, Drp1 and Fis1, while increasing the expression of mitochondrial fusion proteins, Mfn1, Mfn2, and Opa1. Although a direct comparison is difficult due to the lack of studies on the link between obesity and mitochondrial dynamics in the brain, Axelrod et al.39 reported that exercise may control mitochondrial fusion and fission in human muscle and thus induce post-translational modifications in mitochondria, and Konopka et al.40 reported that 12 weeks of aerobic exercise training led to a similar increase in the expressions of MFN1 and MFN2 in skeletal muscle of both the young and elderly subjects. Zampieri et al.41 reported increased expression of OPA1 upon 12-week electrical stimulation of skeletal muscle. Considering that these studies examined mitochondrial dynamics in skeletal muscle cells, exercise can be seen to be an effective way to improve the imbalance in mitochondrial dynamics in the brain.Mitochondrial dynamics are a crucial mechanism in the maintenance of mitochondrial homeostasis, and oxidative stress was reported to increase due to unbalanced mitochondrial dynamics in obesity. This study also showed that, in the HFD-Con group, where obesity was induced, the expression of proteins related to mitochondrial fission, Drp1 and Fis1, increased, whereas the expression of proteins related to mitochondrial fusion, Mfn1, Mfn2, and Opa1, decreased. This can be explained by an excessive increase in mitochondrial fusion partially reducing mitochondrial function. Obesity causes an imbalance in mitochondrial fusion and fission, which reduces the mitochondria content. It is also associated with defects in neuronal development, plasticity, and functionTo conclude, a long-term high-fat diet induced increased weight in lab animals, a decrease in the ability to control plasma glucose, and hematopathological changes that elevate the oxidative stress in the mitochondria of the brain hippocampal tissue to cause an imbalance in mitochondrial dynamics. However, treadmill exercise improved the pathological changes induced by obesity, so that it may contribute to functional improvement in mitochondria in the hippocampal tissue. Thus, this study suggests the need to consider treadmill exercise in the prevention and treatment of mitochondrial dysfunction in the brain hippocampal tissue and in the alleviation of the pathological changes induced by obesity.


The clinical and genetic characteristics of nephrogenic diabetes insipidus (NDI) were described via assessing 2 cases of NDI patients from a Chinese family.Two patients who manifest polyuria and polydipsia were admitted to hospital for definite diagnosis.AVPR2) was related to the disease of NDI.Water deprivation-vasopressin tests showed that the patients may possess renal-origin diabetes insipidus. All the levels of thyroid-stimulating hormone, luteinizing hormone, follicle stimulation hormone, adrenocorticotropic hormone, prolactin, and growth hormone in both patients were normal. These results were certified that both patients possess a nephropathy-type diabetes insipidus. B-mode ultrasonography and urinalysis test demonstrated that the patient's diabetes insipidus is unlikely to originate from renal organic disease. Remarkably, by nucleotide sequencing, we found a novel mutation c.414_418del in arginine-vasopressin receptor 2 (Two patients were treated with oral hydrochlorothiazide and indomethacin. In addition, low salt diet and potassium supplementation throughout the patients’ treatment.The clinical symptoms of 2 patients were significantly reduced after targeted therapy.AVPR2 was discovered to be associated with NID. It provides a new target for molecular diagnosis of NDI, enabling families to undergo genetic counseling and obtain prenatal diagnoses.A mutation in  Approximately 90% of patients with an X-linked form of NDI (OMIM 304800), which affect male patients, are unable to concentrate their urine in response to the antidiuretic hormone, arginine-vasopressin (AVP). This is caused by mutations in the AVP receptor 2 gene (AVPR2), while the remaining 10% of patients have an autosomal form of NDI (OMIM 125800), caused by mutations in the aquaporin 2 gene (AQP2).–8Nephrogenic diabetes insipidus (NDI) is a human kidney disease affecting the urine-concentrating ability of the kidney. This stems from an inability to respond to the antidiuretic hormone, arginine vasopressin, resulting in a massive excretion of diluted urine. NDI patient manifest polyuria and polydipsia.AVPR2 mutations had been published, comprising missense, nonsense, small insertions and deletions, large deletions, and complex rearrangements. Those mutations have been identified in more than 300 congenital NDI families, about 56% of which were missense mutations. Molecular diagnosis is the best method for the diagnosis of NDI and studying its genetic mechanisms. Due to the heterogeneity of NDI, we performed exon capture sequencing using next-generation sequencing (NGS), to identify the genetic features of 2 brothers with NDI clinical characteristics. We discovered a novel mutation in AVPR2, and performed the Sanger sequencing for verification. Identification of the patient's clinical characteristics combined with the pedigree verification leads us to believe that we have found 2 patients who conform to X-linked NDI. To the best of our knowledge, this was the first report of the mutation, both nationally and internationally, presenting great significance for the epidemiological study of NDI in China. Owing to the progressive development of techniques for molecular diagnosis, the disease is now diagnosed in most patients at an early age, which is beneficial to the treatment of congenital NDI.,12 Meanwhile, our findings will further supplement the databases of genetic diseases in China, contribute to the diagnosis of diseases, and promote the development of precision medicine, such as preimplantation genetic screening, preimplantation genetic diagnosis, prenatal screening, as well as prenatal diagnosis.According to other scientific findings, over 200 disease-causing 22.1This study enrolled 2 patients; an 18-year-old male (Patient 1) and his 9-year-old brother (Patient 2), whose were suspected to be afflicted with NDI, from the 7th Pediatric Department and Outpatient Department of Pediatrics of Hunan Provincial People's Hospital, in February 2017. The medical history, physical examination data, as well as related auxiliary inspection data, were collected after obtaining informed consent from the parents and approval from the Ethics Committee of the hospital and Hunan Normal University. Written informed consent for molecular analysis was obtained from the patient or in the case of children, from their parent or legal guardian. The study was approved by the Clinical Research Ethics Committee, People's Hospital of Hunan province and Hunan Normal University (NM: 2017–16).2.22.2.1On the provision of informed consent, 4-mL whole blood samples were collected from the patient, his brother, and his parents, using an EDTA anticoagulant; thereafter, genomic DNA of the patient was extracted using the BloodGen Midi Kit , as per manufacturer's instructions and as previously described.2.2.2For whole-exome capture of the target genes, the NimbleGen capture probe  was prepared for genomic exon regions relevant to >4000 genetic diseases based on information from existing literature and the OMIM and Orphanet databases Table .2.2.2.1Genome fragmentation: Using Cavoris auxiliary reagent for Illumina, genes were fragmented into approximately 200-bp fragments.Free-end filling-in and repair: This was performed for the DNA fragments using the Klenow fragment, T4 DNA polymerase, and T4PNK.3′-terminal adenosine addition: In the polymerase system, an A base was added to the 3′-terminal of the repaired products obtained in the preceding step to prepare them for connection in the subsequent step.Connector addition: The T4DNA ligase reaction system was configured, while adapter and supplementary “A” products were connected after reaction at the appropriate temperature for a certain period in the ThermoMixer.Amplification: The ligation products were subjected to 4 to 6 rounds of ligation-mediated polymerase chain reaction (LM-PCR) amplification.Hybridization: Library and probes were mixed in a hybridization system at 65°C for 60 to 68 hours.Bead washing and DNA elution: Streptomycin beads were incubated with the hybridized samples, following which elution was performed.Eluted product amplification: Eluted products were amplified by 10 rounds of LM-PCR.2.2.2.2(1)Sequencing: The operational process of sequencing was standardized by using the Illumina hiseq2500 platform.(2)Raw image data obtained by sequencing were analyzed using the Illumina official base call analysis software, BclToFastq.2.2.2.3Analysis of base data:(1)Optimization of raw data production: connector contamination and low-quality data were excised.(2)Comparison: Comparative statistics were applied to the data and reference sequence (Burrows–Wheeler Alignment software was used); the hg19 genome was used as reference.(3)SNP detection and annotation were performed using the Samtools software.(4)Indel detection and annotation were performed using the Pindel software.(5)False-positive mutation filter: to obtain high quality and reliable mutations, the detected SNPs and indels were filtered and screened based on sequencing depth and mutation quality.(6)Mutation annotation: SNPs and indels were analyzed to obtain the impact of amino-acid changes, shear effects, UTR, and intron mutations, according to gene location.(7)Prediction of effects of screened mutations on protein function: The impact of screened mutations on protein was predicted by SIFT, the homology alignment algorithm, and protein structure conservation.(8)The splicing hazard was predicted for mutations near splice sites.In-depth data analysis was conducted to associate mutations in related genes with their genetic patterns and clinical symptoms matching those in Patient 1.2.2.3AVPR2. Amplification was performed using PCR, and sequencing was conducted with the ABI 3730XL sequencing device. The original PCR primers were used for sequencing. Genetic sequence analysis and alignment were performed using DNASTAR software, and the messenger RNA alignment template was NM_003560. Samples from Patient 1, Patient 2, and their parents were validated using first-generation sequencing.Primers were designed according to the sequences of sites, validated by 33.1Two patients were admitted to hospital due to their manifest polyuria and polydipsia. Patient 1 had an input water quantity of 4770 mL/d, and an output urine volume of 5200 mL/d, which is within diabetes insipidus diagnostic criteria. At the same time, Patient 2 had an input water quantity of 5850 mL/d, and an output urine volume of 6750 mL/d, which also met the diagnostic criteria of diabetes insipidus. They were born without asphyxia at birth and their mother had no special medical history of pregnancy.3.2The parents of the patients were healthy without consanguineous marriage. There was no consultable history of special genetic diseases within this family. Both patients presented similar clinical characteristics Fig. .3.3+ levels and blood osmotic pressure showed an increase. The patient showed little change in urine output, and urine osmolality was still maintained at a very low level. Based on these characteristics, we concluded that the patient could be distinguished from psychogenic polydipsia and the normal population. Furthermore, Patient 1 showed a stable level of urine output, urine gravity, urine osmotic pressure, and blood osmolality after injected pituitrin. It was, therefore, demonstrated that the patient had no response to pituitary vasopressin, so was considered to possess renal-origin diabetes insipidus. The results for Patient 2 were found to be similar to those of Patient 1 . The results showed that Patient 1 displayed a microadenoma at the pituitary body Fig. A and B, 3.5In order to confirm whether both patients’ posterior pituitary glands display normal levels of secretion, we tested their pituitary hormone levels. The results showed that the levels of thyroid-stimulating hormone, luteinizing hormone, follicle stimulation hormone, adrenocorticotropic hormone, prolactin, and growth hormone in both patients were all normal Table . This co3.6In order to identify whether the patients’ diabetes insipidus originated from a renal disease or a genetic defect, both patients were subjected to a urinary B-mode ultrasonography and urinalysis test. The B-mode ultrasonography results showed that there were no obvious abnormalities in the kidneys of either patient Fig. , while t3.7It was noted from the clinical data that both patients suffered from polyuria and polydipsia, in line with the clinical diagnostic criteria of NDI. In addition, the patients presented with the clinical characteristics of hypophyseal adenoma and normal pituitary hormone levels. It is therefore difficult to confirm the patients’ phenotype, according to classical clinical knowledge, and the differential diagnosis of this case required further molecular genetic analysis.4AVPR2 in the pediatric patient, by using high-throughput, exon-capture sequencing, bioinformatic analysis, and clinical database analysis. Validation using first-generation sequencing and pedigree verification were also performed. The results were as follows; A novel mutation c.414_418del was found in AVPR2 in Patient 1 . The whole genomic locus of AVPR2 is 2,193 bp in length, consisting of 3 exons, within which mutations have been found to be responsible for congenital NDI.–20A number of families of patients suffering from the X-linked form of NDI, have been studied genetically. The gene responsible for congenital NDI has been mapped to the long arm of the X chromosome, near the Xq28 marker, where it colocalizes with  The subfamily that includes the V2 receptor as well as the V1a and V1b vasopressin receptors, the oxytocin receptor, and isotocin and mesotocin receptors in nonmammals, is well conserved, though several members carry out signaling via other G proteins. The V2 receptor is expressed in the kidney tubule, mainly in the distal convoluted tubule and collecting ducts, where its primary function is to respond to the pituitary hormone, arginine vasopressin, by stimulating mechanisms that concentrate urine and maintain water homeostasis in the organism. Loss-of-function of this gene results in NDI disease. X-linked congenital NDI is caused by the loss of, or decreased function of, AVPR2, and large deletions that lead to the complete loss of AVPR2 have been reported comprehensively. Furthermore, mutations to AQP2, ARHGAP4, and L1CAM have also been reported to result in NDI.–24 However, previous studies have indicated that variations in different genes lead to distinct clinical phenotypes. Therefore, clinical differential diagnosis and the detection of variation are very necessary for the diagnosis of NDI.The gene encoding the vasopressin type-2 receptor, also known as the V2 receptor, belongs to the 7-transmembrane-domain G protein-coupled receptor superfamily, and couples to G proteins to stimulate adenylate cyclase activity.AVPR2 in 2 Chinese patients with NDI from the same family who display similar clinical characterization and symptoms. The parents of the patients were healthy and there was no known history of special genetic diseases in this family. The patients manifested polyuria and polydipsia, and met the criteria for a diagnosis of diabetes insipidus. The results of a water deprivation – vasopressin test excluded the possibility of psychogenic polydipsia. The MRI results showed that both patients displayed a normal pituitary signal, and provided powerful evidence to demonstrate that their clinical features did not originate from central nervous problems. The water deprivation – vasopressin test results and the normal pituitary hormone levels of 2 patients verified these conclusions. The novel mutation of the patients is a 5-bp deletion in AVPR2, located in the second-transmembrane-domain. The deletion results in a frameshift mutation, and may contribute to the loss of protein function, leading to abnormal cell function. Finally, we affirmed that the children suffered from X-linked NDI, through performing NGS and family lineage verification.In this study, we have identified a novel type of 5-bp deletion in AVPR2 is the cause of NDI in the great majority of congenital NDI families. In families in which an AVPR2 mutation has been identified, NDI is caused by the mutation in about 90% of cases. There remain a few NDI families for which a mutation has not been identified. Based on recent improvements in NGS techniques, mass DNA sequencing (including full exon sequencing or whole genome sequencing) is progressively being used in the antidiastole of NDI. Currently, genetic testing is used for precise diagnosis, such as; Sanger sequencing, amplification refractory mutation system-polymerase chain reaction, and Droplet Digital PCR are all used to identify the sequences of certain genes. However, the number of genes is very high, and the above methods are costly, with lengthy run times. This makes them very difficult to use in clinical practice, especially for diagnosing diseases with similar characteristics. NGS could provide high-throughput sequencing of multiple genes, including all genes associated with a given disease, thus reducing costs and run times. Therefore, NGS is suitable for screening genes associated with hereditary diseases showing similar clinical characteristics.We have demonstrated that a mutation in AVPR2, which has a certain value for the epidemiological investigation of NDI in China. In addition, the identification of this gene site could further complement the Hereditary Disease Library of China. Currently, dozens of monogenic diseases could be screened in China, which would effectively reduce the risk of birth defects. In addition, precise classification and diagnosis provides the foundation for disease assessment, and a basis for prenatal screening, prenatal diagnosis, and genetic counseling. We believe that with the accumulation of cases and genetic data in China, birth defects, including NDI, could be effectively avoided in the future.In the present study, we used a chip capturing high-throughput sequencing method for whole-exome sequencing of monogenic pulmonary disease-related genes, and successfully identified a virulence gene site in 1 pedigree. To our knowledge, this novel mutation is the first report about Data curation: Xuan Xu, Ying Dai, Danxia Peng.Formal analysis: Xuan Xu, Danxia Peng.Funding acquisition: Xuan Xu.Methodology: Xuan Xu.Project administration: Xuan Xu, Ying Dai.Software: Ying Dai.Writing – original draft: Xuan Xu, Ying Dai.Writing – review & editing: Ying Dai.Xuan Xu orcid: 0000-0001-5543-2650.


Oreochromis mossambicus) larvae along 12 days of development, from pre-hatching to the completion of yolk sac absorption. Gene expression analysis revealed differential and complimentary time-dependent expression of the PepT1 variants and PepT2 along the larval development period. Immunofluorescence analysis showed differential protein localization of the three peptide transporters (PepTs) along the gastrointestinal tract, in a similar pattern to the adult. In addition, PepT1a was localized in mucosal cells in the larvae esophagus, in much higher abundance than in the adults. The results of this study demonstrate specialization of intestinal sections and absorbance potential of the enterocytes prior to the onset of active exogenous feeding, thus pointing to an uncharacterized function and role of the gastrointestinal tract and its transporters during the larval period.Fish larvae differ greatly from the adult form in their morphology and organ functionality. The functionality of the gastrointestinal tract depends on the expression of various pumps, transporters, and channels responsible for feed digestion and nutrients absorption. During the larval period, the gastrointestinal tract develops from a simple closed tube, into its complex form with differentiated segments, crypts and villi, as found in the adult. In this study, we characterized the expression of three peptide transporters  in the gastrointestinal tract of Mozambique tilapia ( Dicentrarchus labrax)  (Oreochromis mossambicus) (Oreochromis niloticus) (Sparus aurata) (Gadus morhua)   (Danio rerio) has been established as a model organism for various developmental studies. However, when addressing feeding and nutritional physiology, this species is not an ideal model  , Senegalalensis) , Mozambiambicus) , Nile tiloticus) , 2002, g aurata) , Atlanti morhua) , Califorornicus) , and Sumentatus) . In the entatus) . The zebal model . Unlike Tilapia is an important group in the cichlid family with increasing research interest in its physiology, genetics, and regulatory processes . FujimurProtein is an important nutrient in fish diets, as it supports energetic supply for physiological processes and growth, in addition to tissue and protein construction . In addislc15a genes. These transporters are mainly known for their important role in di- and tri-peptides absorption into the enterocytes – the epithelial cells of the intestine. In mammalians, two peptide transporters have been found, PepT1 and PepT2, but in fish, there are three transporters, with two PepT1 paralogs . PepTs are solute carriers-proton-dependent transporters, members of the POT family and coded by the paralogs . All thrparalogs .In recent years, there has been increased interest and research on PepTs’ participation in protein absorption in fish under different environmental and dietary conditions . HoweverMozambique tilapia fish used in this study were derived from a stock maintained at the aquaculture facility of the Agricultural Research Organization (ARO). This stock originated from Natal, South Africa, and was brought to Israel in the 1970s.This study was approved by the Agricultural Research Organization Committee for Ethics in Experimental Animal Use, and was carried out in compliance with the current laws governing biological research in Israel .Tissue samples of gills, esophagus, stomach, liver, spleen, muscle, heart, intestine, kidney, skin, brain, pituitary, and fat were taken from four males Mozambique tilapia (54 ± 3 g). The intestine from each fish was divided into three segments; anterior intestine (AI), middle intestine (MI), and posterior intestine (PI). The tissues were kept in 1 ml of RNA-save buffer  at −20°C until RNA extraction procedure.Breeding families consisting of one male and 4–6 females, in 200 L aquaria, were constantly monitored to observe spawning. Two days after spawning and fertilization (2 dpf), eggs were removed from the females’ buccal cavities and transferred to hatching jars. Each experiment was conducted on full-sibs from a single spawn.In order to determine the main tissues expressing the peptide transporters in the larvae, 60 larvae (full sibs from a single spawn) were sampled at 9 dpf and dissected using microsurgery under binocular. The yolk sac was removed and the gastrointestinal tract was separated from the larvae body. Tissue samples (GI tract and Larvae body) from 10 larvae were pooled (six replicas per tissue), and stored in 1 ml of RNA-save buffer at −20°C until RNA extraction procedure.In order to track the expression of the PepT transcripts along the embryonic period, six embryos from an additional spawn were sampled daily, commencing from 3 dpf to 14 dpf. Each larva served as an individual biological replica. The larvae were kept in 1 ml of RNA-save buffer at −20°C until RNA extraction procedure.For immunofluorescence staining, a second time-course experiment was conducted. Larvae from a single spawn were sampled each day, between 6 to14 dpf. The larvae were fixed in 4% PFA for 10 min and then dried on paper. The yolk sac was removed using microsurgery under binocular while making sure that the gastrointestinal tract remained untouched. Following the yolk sac removal, the larvae were incubated in 4% PFA for 24 h at 4°C, followed by two washes in PBS, 50% ethanol, and stored in 70% ethanol at 4°C. The larvae were then dehydrated through a series of graded ethanol baths to displace water . Samples were then embedded in paraffin and 5-μm sections were cut using a microtome and placed on microscope slides. The slides were incubated overnight on a 39°C heated plate and were stored at 4°C until staining.Total RNA was extracted using Trizol reagent, purified from DNA contamination using TURBO DNA-free Kit (Ambion), quantified with Nano-Drop spectrophotometer (Thermo Scientific), and then reversed transcribed into cDNA using Verso cDNA Synthesis Kit (Thermo Scientific).For each gene, forward and reverse primers for qPCR analysis  were tesTissues sections slides from larvae and adults were stained according to the protocol described by Post hoc comparisons among groups were performed using the Tukey-Kramer HSD for tissue distribution and larvae time-course real time results, and Student’s t-test for the ratio between the GI tract and the larvae body (α = 0.05). Data are presented as means ± SEM.Statistical analyses were conducted separately, for each experiment and each expressed gene, using one-way analysis of variance (ANOVA). p < 0.0001), while PepT2 expression was detected in the MI and PI segments. PepT2 was also found to express in the kidney but in lower level compare to the MI segment (p < 0.0001). The expression level in the rest of the tissues was significantly lower (p < 0.0001) than in the intestine (p < 0.0001) of the PepT variants in comparison to the larvae body. The ratio between expression levels in the GI tract and the larvae body was approximately 565 for PepT2, 46,000 for PepT1a, and 140,000 for PepT1b transcripts  (Fundulus heteroclitus)  , killifioclitus) , and Euroclitus) . These toclitus) . The conImmunofluorescence localization of the PepT1a in the esophagus was unexpected, as PepT1 expression was reported mainly in the intestine , while tIn fish, the esophagus has an important role in maintaining salt and water balance. It has been shown that the desalination of seawater starts at the esophagus, which exhibit a low membrane permeability to water together with active and passive absorption of ions [reviewed by The localization of PepTs on the apical membrane of the enterocytes, as seen in the immunofluorescence staining, demonstrates that the intestine can uptake peptides even in the pre-feeding larvae. Our results indicate that in addition to the yolk sac absorption there might be another source of nutrients supplementation, even at early developmental stages. These nutrients might be of exogenous , endogenous, or of microbial source. This hypothesis, as well as the nutritional and developmental significance of intestinal nutrient absorbance for the larva, needs further study.Cyprinus carpio), the addition of small peptides to the feed, increased the expression of PepT transcript and the abundance of cholecystokinin (CCK) secreting cells in the intestine .PC and AC conceived and designed the experiments. PC, TN, and TS bred, sampled and handled the fish. PC performed the microsurgery, gene expression, and immunofluorescence analyses. AC and SH secured funding and supervised the project. PC and AC wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The choice of melting technique is crucial for controlling the purity of a superalloy, which is especially important because purity has come to limit progress in the superalloy field. In this study, double- and triple-melting techniques were used to refine the GH4738 superalloy. Elemental analyses, inductively coupled plasma-atomic emission spectroscopy, X-ray diffraction analysis, scanning electron microscopy with energy-dispersive spectroscopy, high-temperature cupping machine, high-temperature fatigue testing machine, and Image-Pro Plus software were used to analyze and compare the contents of specific elements, the types and sizes of inclusions, the mechanical properties, and the probabilities of white spot formation using the two melting techniques. The effects of the different melting processes on the purity of the superalloy were systematically studied. In terms of controlling the presence of impurities, the triple-melting process resulted in lower levels of harmful N, S, and O impurities in the superalloy, the triple-melted superalloy also contained fewer types of inclusion of smaller sizes and in smaller amounts than the double-melted alloy. Triple melting also promotes tensile strength and fatigue life, and minimizes the probability of forming defects in the superalloy. Superalloys, owing to their excellent high-temperature strengths and good resistance to oxidation, fatigue, and creep deformation ,2,3, havAs aerospace and aviation engines become more reliable, develop higher thrust-to-weight ratios, and become larger in size, the presence of various types of inclusions in superalloys, and high percentages of harmful elements that reduce the purities of these materials, pose threats to their operating safety and stability ,8,9,10. Presently, little enhancement in superalloy purity can be achieved by changing the chemical composition of the superalloy, adjusting the forging process, or improving the thermal processing technique ,16,17. ATo summarize, researchers have substantially improved superalloy purity by adjusting the conditions of the melting technique ,19,20. HIn this experiment, electrolytic Ni plates, high-purity Cr and Mo, revert GH4738, and alloys of other elements were used as raw materials. The GH4738 superalloy was melted by VIM . The vacuum was maintained at around 2 Pa during the entire melting process. Argon was injected prior to the addition of trace elements to prevent volatilization and its effect on alloy composition accuracy. Two alloy ingots of diameters of 460 mm (#1) and 360 mm (#2) were cast at 1400 °C. Their main chemical compositions are listed in 2, Al2O3, CaO, MgO, and TiO2 as the slag components. Slag-melting of the ingot required about 80 min. The melt rate in the steady melting stage was maintained at 3–4 kg/min. Argon was injected at a flow rate of 100 L/min as the protective gas during the entire melting and refining process. A Ф 460-mm (#2) alloy ingot was obtained as the product.Risers were removed from the two ingots and each ingot surface was polished to prevent secondary contamination during ingot melting by refractory and surface oxide coatings. Ingot #2 was then re-melted in a protective gas ESR  furnace with CaFThe ESR-refined ingot #2 was turned to avoid surface pollution by the slag during subsequent melting and refining. Ingots #1 and #2 were both re-melted by VAR . The melt rate was maintained at 7–9 kg/min to form the molten pool and was steadily and gradually lowered at a constant rate of 4–5 kg/min during the steady melting stage. Towards the end of the process, the voltage and current were adjusted to gradually reduce the melt rate and provide a controlled hot top. A vacuum of less than 1 Pa was used during the entire refining process. Two Ф 508-mm alloy ingots were produced. The melting process is shown in The contents of Al, Ti, C, N, S, and O elements in ingots #1 and #2 during the intermediate stages of melting under various process conditions were determined by elemental analysis using an oxygen/nitrogen/hydrogen analyzer  and a carbon/sulfur analyzer , and by inductively coupled plasma optical emission spectroscopy (ICP-OES) . The contents of these elements at various stages of melting were recorded and analyzed.Ten tensile samples of dimensions Φ 12 mm × 66 mm and five tensile samples of dimensions Ф 12 mm × 110 mm were extracted from the two Ф 508-mm alloy bars, respectively. Metallographic specimens of dimensions 15 mm × 15 mm × 15 mm were extracted from the centers, 1/2-R positions (R = radius), and edges of the longitudinal and transverse sections of the two Ф 508-mm alloy bars . The mecThe sizes, morphologies, and compositions of the inclusions were characterized by scanning electron microscopy with energy dispersive spectroscopy (SEM-EDS) . To obtain accurate inclusion data, each metallographic specimen was divided into four zones, with 25 different fields of view selected for the analysis of each inclusion and for recording purposes. The Image-Pro Plus software  was used to analyze the average size and number of inclusions.The percentage contents of Al, Ti, C, N, S, and O in ingots #1 and #2 at various stages of melting and refining were statistically analyzed by the oxygen/nitrogen/hydrogen analyzer, the carbon/sulfur analyzer, and ICP-OES, and are listed in 2) +The XRD spectra of samples collected at different positions of the ingots formed by the two different melting processes exhibited almost identical spectral features ; hence, The XRD patterns exhibited no diffraction peaks attributed to inclusions at any sampling position. Only Ni diffraction peaks were observed, and they were shifted to smaller angles compared with those of the standard (PDF 65-2865 Ni), indicating that the Ni lattice had changed. Since most elements  form solid solutions in superalloy matrices, the Ni-lattice spacings and lattice constants increase, shifting the Ni peaks to values lower than those of the standard . NeverthAnalyses of the types, amounts, and sizes of the inclusions in all the superalloy samples can determine the influence of the smelting process. Inclusions at different sampling positions only exhibit minor variations in amount and size when the same smelting method was used. As such, only the types, amounts, and sizes of the inclusions formed using different melting modes were discussed in detail, and the relevant results are shown in As shown in Further analyses of the Ce-MoS and SiC inclusions revealed that, although Ce was not present in the original GH4738 superalloy, it was introduced during the VIM process by the addition of revert GH4738 or impure raw materials to give Ce-MoS inclusions. SiC inclusions, on the other hand, were formed by the addition of C during VIM into the furnace as a deoxidizer for the removal of oxygen from the alloy solution through the formation of carbon monoxide in a carbon-oxygen reaction . Since tOther types of inclusions were also present in small amounts in the ingots refined by either smelting method, as shown in The tensile samples of the ingots, formed by the different melting processes, were separately subjected to tensile tests at room temperature (25 °C) and high temperature (535 °C), and fatigue life test at high temperature (650 °C). The relevant results  reveal tThe amounts and sizes of the inclusions greatly influence the performance of the superalloy. Since the inclusions had different properties compared to the matrix, it would hinder the dislocation motion in the matrix during deformation, causing dislocation pile-up in front of the inclusions, resulting in local stress concentration . As defoThe above analyses show that, in terms of controlling the presence of harmful elements, the triple melting process maintained the O and S contents in ingot #2 at low levels. Ingot #2 contained fewer types and smaller amounts of inclusions than ingot #1. In terms of mechanical properties, the tensile strength and fatigue life of the ingot #2 samples were higher than those of the ingot #1 samples. Hence, triple melting provides a superalloy of higher purity and mechanical properties than double melting. However, the fact that white spots were also formed in superalloys refined by different melting techniques , which tThe formation of white spots is closely related to the characteristics of the VAR procedure. In VAR, uneven heating of the electrode (ingot) may cause it to unexpectedly fall into the molten metal pool. Since the electrode has high density, it sinks to the bottom of the molten pool without fully melting and remains in the solidified ingot to form white spots . Uneven Double melting reduced the levels of N, S, and O in the GH4738 superalloy to 0.0057 wt.%, 0.002 wt.%, and 0.0012 wt.%, respectively, while triple melting reduced the contents of these elements to 0.0056 wt.%, 0.0007 wt.%, and 0.0008 wt.%, respectively. Hence, triple melting maintains lower levels of these harmful elements in the superalloy.The superalloy samples refined by both techniques exhibited only Ni diffraction peaks in their XRD spectra, which were shifted to lower values compared with the standard peak positions (PDF 65-2865 Ni).Four primary types of inclusion were present in the double-melted GH4738 superalloy samples, the number of inclusions was 5763 and the average inclusion size was 3.1 μm. Two dominant types of inclusion were present in the triple-melted superalloy samples; the number of inclusions was 3412 and the average inclusion size was 2.5 μm. Hence, triple melting generates fewer types and amounts of inclusion and the average inclusion size is also smaller.The average tensile strengths of the double-melted GH4738 superalloy samples were 1051 and 902 MPa at 25 °C and 535 °C, respectively, and its average cycle number was 1223. The average tensile strengths of the triple-melted GH4738 superalloy samples were 1164 and 1053 MPa at 25 °C and 535 °C, respectively, and its average cycle number was 1417. Hence, triple melting produces higher mechanical properties in the superalloy.Superalloy purity has come to limit progress in the superalloy field. The influence of the different melting techniques on superalloy purity had thus far been unclear. It is difficult to precisely determine the melting technique best suited for the preparation of a high-purity superalloy, which limits further improvements in superalloy performance. Strict control of superalloy purity is of particular importance. Therefore, herein we analyzed the effects of two melting techniques, viz. double melting, and triple melting, on chemical composition (various stages of melting and refining), inclusion characteristics and their formation mechanism, mechanical properties and white-spot formation probability in GH4738 superalloy. This study could serve as a guide for the selection of appropriate melting techniques for improving superalloy purity; in this sense, it also provides a theoretical foundation. The following conclusions were drawn from this study:


Escalator-related incidents (EIs) have recently resulted in serious injuries and even deaths. Given the frequency and severity of EIs, a systematic exploration of factors influencing EIs is critical in order to identify preventive measures. Twenty-two factors influencing EIs were identified by analyzing 213 EI cases in China and related literatures. A combination of the Interpretive Structural Modeling (ISM) and Decision Making Trial and Evaluation Laboratory (DEMATEL) methods were utilized to establish a hierarchical structure of the influencing factors and to distinguish cause factors and effect factors. The results show: (i) behavior, emergency plan, safety rules, safety supervision, information exchange, safety culture, and safety education are the most important factors influencing EIs; (ii) safety education is the cause factor imposing the greatest influence on other factors while behavior is the effect factor that is the most influenced; and (iii) the structure of influencing factors has five hierarchies, and factors in the root cause layer are settings and components, safety rules, safety supervision, safety culture, and safety education. Management priority should be given according to the hierarchy level, and the interaction of factors should be considered when taking preventive measures. The corresponding five-layer countermeasures are proposed to reduce escalator-related injuries. An escalator is a type of vertical transportation in the form of moving staircases. Along with common transportation instruments like subways and trains, escalators are a special and important part of the public transportation system that enable people to travel to their destinations. However, the dynamic interaction of escalator-to-passenger, passenger-to-passenger, passenger-to-environment, and passenger-to-management forms a complex escalator–passenger–environment–management (EPEM) system. Safety risks in this system are unpredictable, hard to identify and difficult to control, increasing the likelihood of EIs. The recent emergence of EIs in various countries has brought many negative effects on public safety and social sustainability. On 23 October 2018, an escalator incident in a subway station in Rome, Italy injured more than 20 passengers. According to General Administration of Quality Supervision Inspection and Quarantine (AQSIQ) statistics, 56 serious EIs occurred in China in 2017 and 31 occurred in 2018. EIs have consistently been the leading cause of injuries and deaths related to special equipment from 2008 to 2018 in China. A number of serious EIs were reported by mainstream media in recent years and are listed in Statistical studies on EIs were conducted to analyze the frequency distribution of factors such as cause, hazard pattern, injured body part, gender, and age. Xing et al. identified the main causes of EIs in subway stations in Guangzhou, China as failing to keep balance, carrying out other tasks, not holding the handrail and unhealthy passengers . O’Neil Simulation methods were utilized to study the evolution process of EIs in a virtual world . Li et aIn the EPEM system, however, EIs are much more likely to be caused by multiple influencing factors rather than one single factor. The dynamic interaction of factors usually constitutes multiple influencing loops to form a complex structure. Influencing factors of EIs have not been adequately analyzed through a traditional linear analysis or simulation method mentioned above. Further in-depth understanding of the influencing mechanism of the factors is crucial for EPEM system safety.ISM is an analytical method exploring complex socio-economic system problems. It is also an important structural modeling technique for system analysis ,13,14. DThis paper systematically analyzed influencing factors of EIs in China and accordingly proposed preventive measures, providing theoretical support for safety management of escalator passengers. The main aims of this paper were: (a) to identify the influencing factors of EIs and distinguish the importance and degree of influence of each factor; (b) to establish the hierarchical structure of the factors and analyze the influencing mechanism; and (c) to correspondingly propose preventive measures to reduce escalator-related injuries.News is an original record with authoritative description, which provides the latest statistics and the detailed information . A Web cThirty keywords which represent the core topic of EI cases, occurring at least 10 times, were chosen as samples for the descriptive analysis, as listed in According to the analysis of 213 cases in China, each EI was the result of multiple factors, not a single factor. The multiple factors make the EPEM system uncertain and opaque overall, reducing the ability of managers to reduce risk and exacerbating the consequences . Based oBasir et al. conducted research on escalator incidents and found that the main factors in contributing to incidents were passenger factors which are dynamic and complicated . Traditi−2 in order to eliminate the risk of passenger falls caused by unplanned stoppages [Escalator factors refer to the factors related to the operation of escalators which might have encouraged, facilitated, or enabled the incident to happen, including type, size, materials, service term, labeling, safety devices, malfunctions and maintenance. Al-Sharif et al. found that escalator braking should be set at 1.16 m·stoppages . EscalatEnvironmental factors are the factors related to the surroundings of escalators which might have contributed to EIs. The operating environment of escalator is rather complex and has many aspects, such as location, surrounding object, ground, lighting and visibility, and signage. By analyzing previous research results, installation location, size of space, signage, and physical environment were selected as the main environmental factors to be analyzed.When implementing complex system safety control, human factors, management factors, and organizational factors should be fully considered. Organizational management, safety culture, and employee participation are important to prevent safety incidents in companies . ManagemISM-DEMATEL is a systems analysis approach utilizing matrix and graph theory to analyze the relationship between system factors. The combination of ISM and DEMATEL can not only identify the influence degree and importance of factors in the EPEM system, but also comprehensively analyze the hierarchical structure of the system. The framework of the ISM-DEMATEL method is illustrated in The Equations (1)–(12) for calculating the matrices are listed below. The detailed steps of ISM-DEMATEL method can be found from the related literatures ,18.(1)XBased on case study and literature analysis, 22 factors influencing EIs, denoted as Influence relationships between these factors are determined through Delphi method. A 3-point Likert-type scale was designed. Twenty questionnaires were collected from safety management experts in universities, safety managers in subway stations and escalator manufacturers in Wuhan. Questionnaire data were averaged, and the average values were rounded-off to determine the measure of the direct relationship between every two factors. The direct influence matrix A direct influence matrix reflects the direct relationship between influencing factors. However, considering just the direct relationship is inadequate, since multiple factors affect the safety risk in the EPEM system. A comprehensive influence matrix reflects the comprehensive relationship among system factors, including both direct and indirect relations. The comprehensive influence matrix Based on the comprehensive influence matrix, influencing degree, influenced degree, centrality and causality of each factor were calculated via Equations (4)–(7). The results are listed in x-axis and y-axis, as shown in The cause-and-effect relation diagram of the influencing factors is drawn using 22, safety rules S17, settings and components S9, safety supervision S18, safety culture S20, safety awareness S6, information exchange S19, and knowledge about escalators S5. These factors are not clearly visible but are very important, as they can affect escalator passenger safety by influencing other factors. Actions should be taken with respect to these factors so as to make a positive change in other factors and eventually lead to a higher safety level of the EPEM system. In the influenced degree ranking, behavior S4, emergency plan S21, maintenance S12, safety cognition S7, safety devices S11, and operating status S10 are the top six factors, indicating that enhancing their influence has a direct impact on improving passenger safety.The influencing degree and influenced degree of the factors clearly seen from 4 is the most important factor, followed by emergency plan S21, safety rules S17, safety supervision S18, information exchange S19, safety culture S20, safety education S22, and safety awareness S6. The centrality value of these factors is greater than 2.10 in Centrality 2, settings and components S9, safety rules S17, safety supervision S18, installation location S13, size of space S14, safety culture S20, knowledge about escalators S5, physical environment S16, safety awareness S6, and information exchange S19, while the rest are effect factors. The cause factors are of great importance for system safety, and investment in them tends to create compound returns.The causality value of the cause factors is greater than 0, and these factors are on the right region of The reachability matrix Reachable set 3, S4, S10 and S11, i = 3, 4, 10 and 11), S3, S4, S10, and S11 were classified into L1. The i-th  rows and columns were deleted to create a new reachability matrix. For factors S15 and S16 in this new matrix, i = 15 and 16), then S15 and S16 were classified into L2. This step was repeated until all influencing factors in the EPEM system were classified into 5 layers: L1, L2, L3, L4, and L5. L1 denotes the direct cause layer with four factors such as outfits and belongings S3, behavior S4, operating status S10 and safety devices S11. Signage S15 and physical environment S16 are in the indirect cause layer L2. L3 and L4 are the transition cause layers. L3 has eight influencing factors including age and gender S1, mental or physical condition S2, safety cognition S7, safety skill S8, maintenance S12, installation location S13, size of space S14 and emergency plan S21. L4 consists of three factors including knowledge about escalators S5, safety awareness S6 and information exchange S19. L5 is the root cause layer, including five factors that are settings and components S9, safety rules S17, safety supervision S18, safety culture S20, and safety education S22.In Finally, a hierarchical structure model was established and is illustrated in 5 are the most fundamental and important factors in the EPEM system. Accordingly, optimization of escalator settings and components, strengthening of safety supervision, and implementation of safety education must be emphasized as the top priority for creating long-term system safety. Transition factors in L4 and L3 connect the root cause layer and the direct cause layer in the system. Enhancing these factors helps to form a positive loop for higher safety levels. Outfits and belongings S3, behavior S4, operating status S10 and safety devices S11 are the direct causes of EIs. These factors should be closely monitored and promptly identified with effective and timely responses so as to prevent the occurrence of EIs.(1) Management priority and preventive measures should be arranged according to the hierarchy level. The rule is that the higher the hierarchy level, the more attention deserved. Root cause factors in L2 and L3 include installation location S13, size of space S14, signage S15, and physical environment S16 influence the factors in L1 but are less influenced by other factors. Special attentions should be paid to these factors, and targeted measures should be conducted to mitigate the environment risk.(2) Factors influencing system safety are interactive, and the interaction should be considered when implementing preventive measures. Factors directly or indirectly influence the factors in lower levels. Factors in the same level also influence each other. In particular, four environmental factors in L17, safety supervision S18, safety culture S20, and safety education S22 are in L5, and information exchange S19 and emergency plan S21 are in L4 and L3, respectively. Passenger characteristic factors are distributed in L4 and L3, and passenger behavior factors in L1. Environmental factors are located in L3 and L2. The exception is escalator factors. One is distributed in L4, two in L3, and two in L1. A general rule is that management factors affect passenger characteristic factors and environmental factors, and characteristic factors and environmental factors affect passenger behavior factors and escalator factors.(3) Factors belonging to the same category are distributed on the same or adjacent hierarchies. Four of management factors include safety rules SThe hierarchical structure of factors influencing EIs were deduced using the proposed method. With DEMATEL, influencing factors were divided into cause factors and effect factors, and the importance of all factors is identified. With ISM, the nonlinear and complicated EPEN system was decomposed into five hierarchies according to the relationships of influencing factors.The distinguishing features of the ISM-DEMATEL method are that it is systematic and quantitative. First, the method stems from system theory, and the analysis is summarized in a hierarchical structure model. The five hierarchies of factors provide a holistic scenario for understanding the EPEN system. Second, the analysis is based solely on mathematical equations.Additionally, the influencing factors in this paper were determined based on both news reports cases and related literatures. The combination of two data sources to identify factors is more objective and authentic than reliance on single data source.The hierarchical structure provides a visualization of interrelationships and interdependences among the influencing factors of EIs. It can serve as a useful reference for escalator incident prevention.The corresponding five-layer countermeasures are proposed to reduce escalator-related injuries.(1) The top layer measures are to implement safety education and to create a safety climate. A prime example is a more-than-ten-year-old education program called the “Safe-T rider program” in the U.S. that educates school children about how to behave on elevators and escalators. Government management departments of safety in China can consider conducting a similar national escalator safety education program utilizing multi-media and field education modules. Additionally, the public can be educated through publicity campaigns and educational videos. Educational videos and public service announcements on escalator safety can be filmed and broadcasted on mainstream media and social media such as TV channels and Weibo. These efforts should target children and elders. Escalator safety knowledge should be compiled into the safety education textbook for primary students. For elders, watching safety education performances organized by communities is a relaxing and effective way to learn about escalator-related knowledge.(2) The fourth layer measures are standardizing escalator safety rules and strengthening safety supervision. New standard rules of riding escalators should be formulated and implemented. Escalator safety rule-makers should consist of professionals with escalator safety knowledge and practice, such as escalator technicians, safety managers, safety experts and academics. The formulation of safety rules should refer to the internationally established safety rules for escalators and, more importantly, should be based on the characteristics of passengers and incidents found in safety management practice in China. For example, the rule of walking on the left and standing to the right that originated in the U.K. has been accepted and practiced by escalator passengers worldwide. However, this rule has recently been banned in some cities in China, like Shanghai, Nanjing and Guangzhou, as the practice proven to be unsafe for passengers and damaging to the escalator. Under these circumstances, prohibition of walking on escalators should be written into the safety rules and implemented nationwide. Ensure passengers understand and follow safety rules through communication and supervision.(3) The third layer measures are to optimize escalator settings and components and to strengthen maintenance. For escalator manufacturers, the construction and installation of escalators must strictly accord with the relevant international standards. The investment in research and development of safety devices should be encouraged since escalator safety could be much improved through the use of high-quality safety devices and equipment. Narrowing the gap between the step and balustrade skirt is another improvement worthy of investment that can reduce the occurrence of the common injury of entrapment. For escalator owners, maintenance on escalators should occur frequently. Quality escalator combined with a robust maintenance program should enable the equipment to last for thirty years. Reliable maintenance helps to reduce overall operating costs and reduce the risk to escalator passengers.(4) The second layer measures focus on enhancing the management of the environment around escalators. When installing escalators, the specific location of the escalator should be optimized to provide a comfortable and safe environment for passengers. Wet floors and obstacles should be cleaned up in a timely manner. Signage with safety rules should be placed at the entrance to every escalator. Broadcasting escalator safety messages can alert passengers and enhance their safety.(5) The first layer measures focus on correcting passenger behavior. Passengers are supposed to actively participate in safety education program, learn more escalator safety knowledge and behave properly on escalators. Some of the escalator injuries, such as stumbles and entrapments, are age-specific. Hence, the preventive measures should also be age-specific. Young children should be supervised by parents while riding escalators. They also need to be informed that escalators are dangerous and are not places for jumping or playing. Elders, unhealthy passengers and passengers carrying large luggage or strollers should take elevators instead of escalators. Passengers who wear high-heel shoes, Crocs, long scarves, and long shirts should be on high alert to avoid being entrapped.This study revealed the five-hierarchy structure of factors influencing EIs in China. Importantly, root cause factors, including settings and components, safety rules, safety supervision, safety culture, and safety education, were identified and should be given priority when taking preventive measures. The hierarchical structure model suggests that occurrence of EIs can be reduced in the short-term by correcting passenger behavior and in the long-term by enhancing safety education. The proposed five-layer preventive measures provide both theoretical and practical support for managing the safety of escalator passengers. The findings contribute to passenger safety research by offering a deeper understanding of interactive influencing mechanisms of the EPEM system.


Their biological affinity remains unknown, but most commonly, they are interpreted as eggs of marine metazoans. Here, we provide new insights into their palaeobiology from three lines of inquiry. We examine morphological variation of a new, well-preserved Late Ordovician species, Hercochitina violana; analyse a compiled dataset of measurements on 378 species representing all known chitinozoan genera; and compare these data with the size variation of eggs of both extinct and extant aquatic invertebrates. The results indicate that the magnitude of size variation within chitinozoan species is larger than observed in fossil and modern eggs. Additionally, delicate morphological structures of chitinozoans, such as prosome and complex ornamentation, are inconsistent with the egg hypothesis. Distinct and continuous morphological variation in H. violana is more plausibly interpreted as an ontogenetic series of individual microorganisms, rather than as eggs.Chitinozoans are organic-walled microfossils widely recorded in Ordovician to Devonian ( They are characterized by a radially symmetrical shell, known as vesicle, which is commonly jar-, vase- or tube-shaped and has an opening in one end sealed with a plug (prosome) or lid (operculum). The vesicle length varies from ca 50 to 2000 µm, with a newly reported largest one of 2700 µm [Chitinozoans are an extinct group of cryptic organic-walled microfossils widely distributed in Ordovician to Devonian marine sedimentary rocks  and divHercochitina violana sp. nov. is conducted. Additionally, we compiled a dataset of measurements of 378 randomly selected species representing all 57 chitinozoan genera currently known, as well as a dataset of coefficients of variation (CV) in eggs from 45 extant aquatic metazoan species. For the first time, this allowed a reliable comparison of the magnitude of size variation between chitinozoans and undisputed eggs to further assess their possible analogy.Here, we re-evaluate the egg hypothesis from the perspective of morphological variation of chitinozoans. In order to allow for a more quantitative analysis, a detailed case study focusing on single highly variable species 2.H. violana sp. nov. were collected from six limestone samples in the upper part of the Viola Springs Formation at the auxiliary Global Stratotype Section and Point section for the base of the Katian Stage (Upper Ordovician) in Oklahoma, USA [H. violana sp. nov. obtained from two of the six samples were measured for quantitative analysis . All specimens are stored at the Nanjing Institute of Geology and Palaeontology, Chinese Academy of Sciences.Well-preserved specimens of oma, USA . Rock saoma, USA  or digesA compiled chitinozoan dataset includes 593 records of all 57 type species and additional 321 species randomly selected, without previous sorting or emendation, from the literature, has been established and presented in the electronic supplementary material, S2. The CV of egg sizes representing six phyla and 45 species of extant aquatic (mostly marine) metazoan species was assembled and the criteria for selecting the CV data are detailed illustrated in the electronic supplementary material, S3. Multivariate statistical analysis ) and locally weighted scatterplot smoothing (LOESS) regression were carried out using R .3.(a)Incertae sedis group CHITINOZOA Eisenack, 1931Order PROSOMATIFERA Eisenack, 1972Family CONOCHITINIDAE Eisenack, 1931, emend. Paris, 1981Subfamily BELONECHITININAE Paris, 1981Hercochitina Jansonius, 1964Genus Hercochitina violana sp. nov. Nõlvak and Lianga–k)a–kFigurHercochitina repsinata  n. comb; Melchin & Legault, p. 208, pl. 1, fig. 1.?1985 Clathrochitina? sp. n. 1; Goldman et al., p. 267, fig. 13: 1, 4.2007 Etymology. In reference to the type stratum, the Viola Springs Formation of Oklahoma.Diagnosis. This species is characterized by its distinct ornamentation, low and multi-rooted longitudinal spines developed in the lower part of the chamber, and jungle-like complex ones on the basal margin. A constriction near the base and a mucron on the base are also evident.Types. Holotype, sample HWY99-43 (43 m above the base of the formation), figure 1i (repository number NIGPAS19-28); Paratype, sample HWY99-37.8, figure 1a (repository number NIGPAS19-20).Type locality. Section D in the west side of State Highway 99, 3.3 miles south of Fittstown, Arbuckle Mountains, south-central Oklahoma, USA [oma, USA .Type stratum. The upper part of the Viola Springs Formation, ranging from sample HWY99-37.8 to HWY99-51, Katian, Late Ordovician.Material. Seventy-three specimens from 50 g of sample HWY99-37.8, 258 specimens from ca 300 g of sample HWY99-43 were measured.Dimensions. See Description. The vesicle size ranges from small to medium and the outline from stout to slender. Chamber subcylindrical to subconical with a short cylindrical neck developing a short flaring collar. The flexure is so broad that it is difficult to identify the boundary between the neck and the chamber. Flanks are straight to slightly convex. Basal margin sharp to blunt. Base is flat with a mucron. Constrictions are inconspicuous or weak in smaller specimens and become stronger and more distinct in larger, always slender, specimens. The constriction diameter is about three-fifths to nine-tenths of the maximum diameter at the base of the vesicle. The vesicle wall is covered by multiple kinds of spines: simple or multi-rooted spines distributed near the collar, low and multi-rooted longitudinal spines developed in the lower part of the chamber around the constriction, much stronger and more complex ones on the basal margin, and simple and multi-rooted spines developed concentrically in the base around the mucron.Remarks. The new species, H. violana sp. nov., is easily distinguished from other Hercochitina species by its less-developed longitudinally distributed spines in the upper and middle parts of the chamber. The distinct ornamentations more likely represent a transitional form between the typical Belonechitina and Hercochitina, but more closely resemble the ornamentation of Hercochitina by occupying well-developed vertical rows of spines or distinct crests, even if these distinguished features are confined within the lower chamber of the vesicle, which is the main feature of typical Hercochitina.Hercochitina repsinata  Melchin & Legault [Belonechitina repsinata Schallreuter, 1981 [B. repsinata is considered to be a badly preserved Belonechitina robusta  [B. repsinata might be an invalid name and the specimens reported by Melchin & Legault [H. violana sp. nov. Additionally, the specimens previously identified as Gen. et sp. n. 1 (Clathrochitina?) [Hercochitina because the spines on the margin are multi-rooted rather than processes and the flexures are inconspicuous.The species resembles  Legault  both in er, 1981  in develk, 1959) . Thus, B Legault  could pohitina?)  are from(b)Dp) and the diameter of the constriction (Dcons) vary from 54 to 125 µm and 40 to 94 µm, respectively. All the measured parameters are normally distributed and the specimens on the PCA are well grouped, which support the idea that all the specimens belong to a single population.The morphology of the new species shows variation both in vesicle size and outline figures  and 2. Tg). The vesicle outline changes from a stout shape to a slender form with an indistinct but longer neck. The constriction also becomes more distinct as the specimens become larger and slenderer. Taking into consideration the normally distributed parameters, the substantial but continuous variation suggests a longitudinal growth trend rather than intraspecific variation. If the specimens are exhibiting population variation, it is plausible to argue that the predominant length, from ca 150 to 210 µm, potentially represent the mature stage of the species. Individuals longer than 210 µm and shorter than 130 µm show significantly decreased proportionality and may represent the largest grown and juvenile specimens, respectively. Notably, except for some small-chambered taxa of the family Desmochitinidae [Desmochitina, Calpichitina and Eisenackitina, no specimens smaller than the smallest H. violana sp. nov have been reported from the same sample.Overall, the length variation is much greater than the diameter variation g. The veitinidae , such as4.(a)a); however, in most species, only a relatively small number of specimens have been measured. When using data with more than 40 specimens measured, the ratio increases: 74% of the 116 valid data range from 1.5 to 2.5 and 19% with an Lmax/Lmin ratio more than 2.5 (b). Furthermore, 60% of the data share a ratio around 2.5 and only 15% are less than 2 but still larger than 1.5 when the ratios are based on more than 100 specimen measurements .According to the compiled dataset, including 574 valid records , the ratio between the longest and shortest specimens within the measured assemblage ranges from 1.03 to 4.43, three-quarters of which range from 1.0 to 2.0 a; howevethan 2.5 b. Furthed). The variability range in the family Desmochitinidae (ratio of ca 2.7) has no evident difference from the Conochitinidae (ca 2.8) and Lagenochitinidae (ca 2.5) in the expected data by LOESS regression (e–g), even though the size of the dataset within each family varies significantly. These results indicate that Desmochitinidae, characterized by the key structure of operculum and with the only record of cocoon-like preservation [The LOESS regression of 498 valid data (complete records with measured specimen numbers) indicates that variation estimates increase when at least 40 specimens are measured and an expected ratio around 2.7 is estimated d. The vagression e–g, evenervation ,28—severDesmochitina specimens, which are characterized by an ovoid vesicle and absence of neck, and sealed with the most typical operculum, indicates a smaller morphological variation. Except for the record of Desmochitina ornensis, some specimens of which occupying a short but distinct neck as shown in the type locality [Lmax/Lmin ratio limited to 2 (a) and two-thirds range from 1.2 to 1.6. The LOESS regression, which might be less reliable owing to the limited data, shows an estimated value around 1.9 and 1.5, respectively, with or without D. ornensis (b). The variation of Desmochitina minor, within which recorded the only cocoon-like preservation, ranges from 1.1 to 1.9 with an average value of 1.47 based on 11 datasets in which the number of measured specimens varied from 2 to 50. Although the data of Desmochitina are limited for statistical analysis, a plausible trend is that taxa within Desmochitina, especially the D. minor, sharing an Lmax/Lmin ratio of less than 2 can be estimated, which is smaller than the variation in other taxa occupying a neck.However, an analysis focusing on the data gathered on locality , all theted to 2 a and twoornensis b. The vaDesmochitina, on average, the intraspecific chitinozoan vesicle length variation is around 2.7 and can be more than 3 when a sufficient number of specimens are measured. This new analysis of variation is important because the size limit is among the main diagnostic characters used in species definitions and identifications. However, the biological variation in fossil populations has always been difficult to document because of preservation, time-averaging and inability to assess geographical variation. In addition, chitinozoan specimens are commonly compressed and deformed, the diagnostic ornamentations are easily removed or destroyed and have a relatively simple anatomical outline. All of this has contributed to the fact that the range of variability in chitinozoans has often been underestimated, and promoted a general hesitancy for workers to consider a group of specimens with great size variation to belong to a single species. In other words, size variation is potentially even larger within most groups in chitinozoans.Except for the group of (b)Lmax/Lmin is generally below 2 [Desmochitina. Overall, the fossil egg-size variation is poorly known and has thus little significance in the analogous study owing to the limited number of case studies as well as limited number of eggs in each individual case.Fossils of aquatic invertebrate eggs are generally rare –35 and l below 2 ,36,37, wW-test; W = 0.916863, p < 0.0018; However, the size variation of eggs is well studied among a wide phylogenetic range of extant animals –42. TherH. violana is 20.68, with an Lmax/Lmin ratio of 3.4. A one-tailed, one-sample t-test [H. violana is significantly different from the above distribution . This allows us to conclude that extant aquatic invertebrates in our broad phylogenetic survey exhibit smaller variation in egg size than exists in chitinozoans. The t-test rejected the hypothesis that the CV of H. violana reported here is part of the same distribution with a high level of probability. In other words, data on the egg-size variation of both fossils and extant invertebrates are incompatible with the hypothesis that chitinozoans are eggs.However, the CV of size variation on e t-test  indicate(c)Apart from the distinct size variation of chitinozoans, the predominant egg hypothesis does not explain well the complex structure of prosome, which is the primary character for subdividing chitinozoans at the order level. For example, systematic studies of prosome morphologies have documented diverse forms and structures among chitinozoan species ,45. AnotDesmochitina [In the history of exploring the biological affinity of chitinozoans, three main stages can be summarized: (i) relationship with various groups of Protozoa mainly based on the vesicle outline from the 1930s to 1980s; (ii) eggs of unknown metazoans, ignited by the discovery of the cocoon-like clusters  during tochitina . FurtherDesmochitina, there are linear or coiled catenary forms, which are common in Desmochitina, Bursachitina, Armoricochitina and Linochitina [In addition to the cocoon-like clusters in ochitina ,50. Thosochitina ,23,51. Hochitina .5.H. violana sp. nov. potentially indicate an ontogenetic series rather than an intraspecific variation. Additionally, there is no analogue in extant metazoan eggs which have such complicated and delicate structures as developed in chitinozoans, not to mention the rapid evolutionary trend of ornamentations, which is also inconsistent with the prevailing egg hypothesis. It is more plausible to argue that most chitinozoans, with the exception of the unique Desmochitina, represent independent organic-walled microorganisms rather than eggs of metazoans.The results show that the range of variation in most chitinozoans is much larger than is typical of eggs of extant marine invertebrates or the hitherto reported undisputed fossil eggs. The range and type of size variation shown in Finally, we note that because chitinozoans are an extinct group, there remains the unlikely possibility that they represent the eggs of an extinct group of marine organisms that displayed a uniquely large size variation in addition to exhibiting very unusual structures for eggs. We think that this is an extremely unlikely hypothesis and is contrary to the basic concepts of uniformitarianism. Unless demonstrated to the contrary, it is reasonable to assume that the range of variation exhibited in the eggs of modern and available fossil invertebrate metazoans is also representative of the rest of Earth's past biota.Conochitina ex aff. campanulaeformis [Conochitina sp. [Ancyrochitina cf. brevis [Furthermore, some of the previously reported abnormal specimens, such as the aeformis , Conochitina sp.  and Ancy. brevis , may pro. brevis ,20 in oc. brevis . However