Evolution of the gene regulatory network of body axis by enhancer hijacking in amphioxus.

A central goal of evolutionary developmental biology is to decipher the evolutionary pattern of gene regulatory networks (GRNs) that control embryonic development, and the mechanism underlying GRNs evolution. The Nodal signaling that governs the body axes of deuterostomes exhibits a conserved GRN orchestrated principally by Nodal, Gdf1/3, and Lefty. Here we show that this GRN has been rewired in cephalochordate amphioxus. We found that while the amphioxus Gdf1/3 ortholog exhibited nearly no embryonic expression, its duplicate Gdf1/3-like, linked to Lefty, was zygotically expressed in a similar pattern as Lefty. Consistent with this, while Gdf1/3-like mutants showed defects in axial development, Gdf1/3 mutants did not. Further transgenic analyses showed that the intergenic region between Gdf1/3-like and Lefty could drive reporter gene expression as that of the two genes. These results indicated that Gdf1/3-like has taken over the axial development role of Gdf1/3 in amphioxus, possibly through hijacking Lefty enhancers. We finally demonstrated that, to compensate for the loss of maternal Gdf1/3 expression, Nodal has become an indispensable maternal factor in amphioxus and its maternal mutants caused axial defects as Gdf1/3-like mutants. We therefore demonstrated a case that the evolution of GRNs could be triggered by enhancer hijacking events. This pivotal event has allowed the emergence of a new GRN in extant amphioxus, presumably through a stepwise process. In addition, the co-expression of Gdf1/3-like and Lefty achieved by a shared regulatory region may have provided robustness during body axis formation, which provides a selection-based hypothesis for the phenomena called developmental system drift.

Three amphioxus reference genomes reveal gene and chromosome evolution of chordates.

The slow-evolving invertebrate amphioxus has an irreplaceable role in advancing our understanding of the vertebrate origin and innovations. Here we resolve the nearly complete chromosomal genomes of three amphioxus species, one of which best recapitulates the 17 chordate ancestor linkage groups. We reconstruct the fusions, retention, or rearrangements between descendants of whole-genome duplications, which gave rise to the extant microchromosomes likely existed in the vertebrate ancestor. Similar to vertebrates, the amphioxus genome gradually establishes its three-dimensional chromatin architecture at the onset of zygotic activation and forms two topologically associated domains at the Hox gene cluster. We find that all three amphioxus species have ZW sex chromosomes with little sequence differentiation, and their putative sex-determining regions are nonhomologous to each other. Our results illuminate the unappreciated interspecific diversity and developmental dynamics of amphioxus genomes and provide high-quality references for understanding the mechanisms of chordate functional genome evolution.

Joint profiling of gene expression and chromatin accessibility during amphioxus development at single-cell resolution.

Vertebrate evolution was accompanied by two rounds of whole-genome duplication followed by functional divergence in terms of regulatory circuits and gene expression patterns. As a basal and slow-evolving chordate species, amphioxus is an ideal paradigm for exploring the origin and evolution of vertebrates. Single-cell sequencing has been widely used to construct the developmental cell atlas of several representative species of vertebrates (human, mouse, zebrafish, and frog) and tunicates (sea squirts). Here, we perform single-nucleus RNA sequencing (snRNA-seq) and single-cell assay for transposase accessible chromatin sequencing (scATAC-seq) for different stages of amphioxus (covering embryogenesis and adult tissues). With the datasets generated, we constructed a developmental tree for amphioxus cell fate commitment and lineage specification and characterize the underlying key regulators and genetic regulatory networks. The data are publicly available on the online platform AmphioxusAtlas.

A Potential Method for Rapid Screening of Amphioxus Founder Harboring Germline Mutation and Transgene.

Amphioxus is a promising model organism for understanding the origin and evolution of vertebrates due to its basal phylogenetic position among chordates. We here compared the mutation efficacy and mutation type of tail tips and gametes of amphioxus founders injected with Cas9 protein and six different sgRNAs targeting five distinct genes, and revealed a strong correlation for mutation efficacy and a mild correlation for mutation type among the two tissues. In addition, we also observed a positive relationship between gene insertions observed in tail tips and gametes of amphioxus founders injected with Tol2 transposase and two different transgenic constructs. Finally, we showed that amphioxus larvae which had their tail tips cut at the 3-4 gill-slit stage were able to recover within 6 days and developed a normal number of gonads at the adult stage, and that F0 larvae carry similar mutation efficacy and type in the posterior end to that in the tail tips after their metamorphosis. Together, these findings suggest a great potential for obtaining valid amphioxus founders with desired mutations and transgenes at as early as the early larval stage, which will certainly speed up the generation of amphioxus mutants and transgenes and make it more cost- and labor-effective.

Application of CRISPR/Cas9 Nuclease in Amphioxus Genome Editing.

The cephalochordate amphioxus is a promising animal model for studying the origin of vertebrates due to its key phylogenetic position among chordates. Although transcription activator-like effector nucleases (TALENs) have been adopted in amphioxus genome editing, its labor-intensive construction of TALEN proteins limits its usage in many laboratories. Here we reported an application of the CRISPR/Cas9 system, a more amenable genome editing method, in this group of animals. Our data showed that while co-injection of Cas9 mRNAs and sgRNAs into amphioxus unfertilized eggs caused no detectable mutations at targeted loci, injections of Cas9 mRNAs and sgRNAs at the two-cell stage, or of Cas9 protein and sgRNAs before fertilization, can execute efficient disruptions of targeted genes. Among the nine tested sgRNAs (targeting five genes) co-injected with Cas9 protein, seven introduced mutations with efficiency ranging from 18.4% to 90% and four caused specific phenotypes in the injected embryos. We also demonstrated that monomerization of sgRNAs via thermal treatment or modifying the sgRNA structure could increase mutation efficacies. Our study will not only promote application of genome editing method in amphioxus research, but also provide valuable experiences for other organisms in which the CRISPR/Cas9 system has not been successfully applied.

A ZZ/ZW Sex Chromosome System in Cephalochordate Amphioxus.

Sex determination is remarkably variable among animals with examples of environmental sex determination, male heterogametic (XX/XY) and female heterogametic (ZZ/ZW) chromosomal sex determination, and other genetic mechanisms. The cephalochordate amphioxus occupies a key phylogenetic position as a basal chordate and outgroup to vertebrates, but its sex determination mechanism is unknown. During the course of generating Nodal mutants with transcription activator-like effector nucleases (TALENs) in amphioxus Branchiostoma floridae, serendipitously, we generated three mutant strains that reveal the sex determination mechanism of this animal. In one mutant strain, all heterozygous mutant offspring over three generations were female and all wild-type descendants were male. This pattern suggests the Nodal allele targeted is on a female-specific W chromosome. A second mutant showed the same W-linked inheritance pattern, with a female heterozygote passing the mutation only to daughters. In a third mutant strain, both male and female offspring could be heterozygous, but a female heterozygote passed the mutation only to sons. This pattern is consistent with the targeted allele being on a Z chromosome. We found an indel polymorphism linked to a Nodal allele present in most females, but no males in our cultured population. Together, these results indicate that Nodal is sex chromosome-linked in B. floridae, and that B. floridae has a ZZ/ZW sex chromosome system.

Cilia-driven asymmetric Hedgehog signalling determines the amphioxus left-right axis by controlling Dand5 expression.

Cilia rotation-driven nodal flow is crucial for the left-right (L-R) break in symmetry in most vertebrates. However, the mechanism by which the flow signal is translated to asymmetric gene expression has been insufficiently addressed. Here, we show that Hedgehog (Hh) signalling is asymmetrically activated (L

Ribosylation-Derived Advanced Glycation End Products Induce Tau Hyperphosphorylation Through Brain-Derived Neurotrophic Factor Reduction.

Advanced glycation end products (AGEs) have been implicated in the disease process of diabetes mellitus. They have also been found in senile plaques and neurofibrillary tangles in the brains of Alzheimer's disease patients. Furthermore, abnormally high levels of D-ribose and D-glucose were found in the urine of patients with type 2 diabetes mellitus, suggesting that diabetic patients suffer from dysmetabolism of not only D-glucose but also D-ribose. In the present study, intravenous tail injections of ribosylated rat serum albumin (RRSA) were found to impair memory in rats, but they did not markedly impair learning, as measured by the Morris water maze test. Injections of RRSA were found to trigger tau hyperphosphorylation in the rat hippocampus via GSK-3ß activation. Tau hyperphosphorylation and GSK-3ß activation were also observed in N2a cells in the presence of ribosylation-derived AGEs. Furthermore, the administration of ribosylation-derived AGEs induced the suppression of brain-derived neurotrophic factor (BDNF) and tropomyosin-related kinase B (TrkB). Both GSK-3ß inhibition and BDNF treatment decreased the levels of phosphorylated tau in N2a cells. In particular, the administration of BDNF could rescue memory failure in ribosylated AGE-injected rats. Ribosylation-derived AGEs downregulated the BDNF-TrkB pathway in rat brains and N2a cells, leading to GSK-3ß activation-mediated tau hyperphosphorylation, which was involved in the observed rat memory loss. Targeting ribosylation may be a promising therapeutic strategy to prevent Alzheimer's disease and diabetic encephalopathies.

An Effective Microinjection Method and TALEN-Mediated Genome Editing in Pacific Abalone.

Pacific abalone, Haliotis discus hannai, is an economically important marine mollusk species and an important model animal for studies on ecological, fertilization and developmental biology. While embryonic injection and genome editing have been wildly used in gene function study and trait improvement in many species, they have not been developed in abalones. In this study, we reported an effective method to inject exogenous materials in H. discus hannai unfertilized eggs. The injected eggs could be fertilized at a ratio of 52.6% ± 5.9% and hatch at a ratio of 14.6% ± 1.6%. On the base of this, we further developed an efficient genome editing approach in this species with the transcription activator-like effector nuclease (TALEN) technique. Two TALEN pairs targeting the coding sequence of the abalone nodal gene were assembled and tested. While one of the TALEN pairs showed no detectable mutation efficacy, the other one generated mutations in 50% of the targeted loci. The mutation includes small insertions and deletions and base pair replacements like that reported in other species when the TALEN method was applied. Overall, this is the first study to demonstrate site-specific genome editing in abalone. This work can serve as a reference for future studies focusing on the functional genomics in mollusks.

Formaldehyde produced from d-ribose under neutral and alkaline conditions.

Formaldehyde is toxic and has been implicated in the pathologies of various diseases, such as cognitive impairment and cancer. Though d-ribose is widely studied and provided as a supplement to food such as flavor and drinks, no laboratories have reported that d-ribose is involved in the formaldehyde production. Here, we show that formaldehyde is produced from d-ribose in lysine or glycine solution and Tris-HCl buffer under neutral and alkaline conditions. Intraperitoneal injection of C57BL/6J mice with d-ribose significantly increased the concentration of brain formaldehyde, compared to the injection with d-glucose or saline. These data suggest that formaldehyde levels should be monitored for the people who take d-ribose as a supplement.

[Effect of polydatin on miR-214 expression and liver function in ApoE-/- mice].

OBJECTIVE: To study the effect of polydatin on the expression level of miR-214 and liver function in atherosclerotic mice. METHODS: Forty male ApoE(-/-) mice were randomly allocated into 4 groups (n=10), namely the model group, low- and high-dose polydatin groups, and simvastin group, with 10 male C57BL/6J mice serving as the normal control group. Mouse models of atherosclerosis were established by feeding the ApoE(-/-) mice with a high-fat diet. After 12 weeks of treatment, blood levels of glucose, lipids, AST, and ALT and the contents of T-SOD and MDA in the liver tissue were detected. The pathologies of the liver were examined with HE staining, and miR-214 expression in the liver was detected using quantitative real-time PCR. RESULTS: Compared with the normal control mice, the mice in the model group showed significantly increased blood glucose, serum TC, TG, LDL-C, ALT, and AST levels, and MDA contents in the liver (P<0.01), with significantly decreased serum HDL-C level and SOD and miR-214 levels in liver (P<0.01). Polydatin treatment significantly ameliorated such changes in blood glucose, serum ALT, AST, TC, TG, LDL-C, and HDL-C levels, and MDA, SOD, and miR-214 contents in liver tissue (P<0.05). CONCLUSION: s Polydatin can reduce blood glucose and lipid levels and protect the liver function in atherosclerotic mice possibly by up-regulating the expression of miR-214 and T-SOD and down-regulating MDA in the liver.

Dressings for Preventing Pressure Ulcers: A Meta-analysis.

The purpose of this analysis is to determine the effectiveness of dressing material in the prevention of pressure ulcers. Results showed that hydrocolloid, foam, and film were more effective than a standard care protocol in patients at risk for pressure ulcers.

Nestin(+) kidney resident mesenchymal stem cells for the treatment of acute kidney ischemia injury.

Renal resident mesenchymal stem cells (MSCs) are important regulators of kidney homeostasis, repair or regeneration. However, natural distribution and the starting population properties of these cells remain elusive because of the lack of specific markers. Here, we identified post-natal kidney derived Nestin(+) cells that fulfilled all of the criteria as a mesenchymal stem cell. These isolated Nestin(+) cells expressed the typical cell-surface marker of MSC, including Sca-1, CD44, CD106, NG2 and PDGFR-α. They were capable of self-renewal, possessed high clonogenic potential and extensive proliferation for more than 30 passages. Under appropriate differentiation conditions, these cells could differentiate into adipocytes, osteocytes, chondrocytes and podocytes. After intravenous injection into acute kidney injury mice, Nestin(+) cells contributed to functional improvement by significantly decreasing the peak level of serum creatinine and BUN, and reducing the damaged cell apoptosis. Furthermore, conditioned medium from Nestin(+) cells could protect against ischemic acute renal failure partially through paracrine factor VEGF. Taken together, our findings indicate that renal resident Nestin(+) MSCs can be derived, propagated, differentiated, and repair the acute kidney injury, which may shed new light on understanding MSCs biology and developing cell replacement therapies for kidney disease.

Correlation between functional status and quality of life after surgery in patients with primary malignant bone tumor of the lower extremities.

PURPOSE: This study aims to explore the correlation between functional status and quality of life after surgery in patients with primary malignant bone tumor of the lower extremities. METHODS: A total of 94 patients with primary malignant bone tumor of the lower extremities were enrolled. Correlations between their functional status and quality of life after surgery were descriptively analyzed through functional mobility assessment, Toronto extremity salvage scaling, reintegration to normal life index, and the 36-item Short Form Health Survey. RESULTS: All patients presented decreased physical function, activities of daily life (ADL), and social participation capability. Their quality of life was significantly lower than the norm. Scores under all items of functional status significantly correlated with the quality-of-life score (r = .265-.427; p < .01). The postoperative functional status of patients with primary malignant bone tumor of the lower extremities greatly influences quality of life. Lower levels of physical function, ADL, and social participation indicate poorer quality of life. CONCLUSION: To improve quality of life, necessary nursing measures should be adopted to intervene with postoperative functional rehabilitation processes.

Modified glomerular filtration rate-estimating equations developed in asiatic population for chinese patients with type 2 diabetes.

Objectives. To evaluate eight modified equations developed in Asiatic populations in type 2 diabetic patients in China. Methods. A total of 209 Chinese patients with type 2 diabetes were recruited. Using the technetium-99m diethylenetriaminepentaacetic acid-glomerular filtration rate (GFR) to act as the reference, comparisons of their efficiency to estimate GFR in the subjects were made between various equations. Results. Median of difference of the Chinese equation 1 was the lowest (median of difference, 0.51 mL/min/1.73 m(2)). Median percent of absolute difference of the Chinese equation 2 was less than those of the other equations (26.97 versus ranged from 32.54 to 37.61 mL/min/1.73 m(2), [P < 0.001 for all]). Precision of the simplified reexpressed MDRD equation was the best (92.9 mL/min/1.73 m(2)). Accuracies of the Chinese equation 2 were greater (P < 0.05 for all). There was also an improvement in chronic kidney disease (CKD) stage misclassification of the Chinese equation 2 (55.0 versus ranged from 61.2 to 64.6%, [P < 0.001 for all]). However, the 30% accuracies of all the equations were less than 70%. Conclusions. Our study highlighted a limitation in the use of the above equations in the majority of Chinese diabetic subjects. A better equation is needed in order to give an accurate estimation of GFR in type 2 diabetic patients in China.

Advanced glycation end-products reduce podocyte adhesion by activating the renin-angiotensin system and increasing integrin-linked kinase.

The aim of this study was to investigate the effects of advanced glycation end-products (AGEs) on podocyte adhesion and the underlying mechanisms. Immortalized mouse podocytes were exposed to various conditions and podocyte adhesion was evaluated using a hexosaminidase assay. The expression levels of integrin-linked kinase (ILK) were measured by quantitative polymerase chain reaction (qPCR) and western blotting. Treatment with AGEs resulted in a significant, concentration-dependent reduction in podocyte adhesion (P<0.05) and an incremental rise in ILK expression up to a maximum of 100%. Pretreatment with losartan significantly prevented the upregulation of ILK and attenuated the loss of podocyte adhesion observed in podocytes exposed to AGEs (P<0.05). However, the adhesion of losartan-treated podocytes remained lower than that of the podocytes exposed to bovine serum albumin. The results indicate that AGEs reduce podocyte adhesion via the upregulation of ILK expression, which occurs partly through activation of the renin-angiotensin system in podocytes.

A new equation to estimate glomerular filtration rate in Chinese elderly population.

BACKGROUND: We sought to develop a new equation to estimate glomerular filtration rate (GFR) in Chinese elderly population. METHODS: A total of 668 Chinese elderly participants, including the development cohort (n = 433), the validation cohort (n = 235) were enrolled. The new equation using the generalized additive model, and age, gender, serum creatinine as predictor variables was developed and the performances was compared with the CKD-EPI equation. RESULTS: In the validation data set, both bias and precision were improved with the new equation, as compared with the CKD-EPI equation (median difference, -1.5 ml/min/1.73 m(2) vs. 7.4 ml/min/1.73 m(2) for the new equation and the CKD-EPI equation, [P<0.001]; interquartile range [IQR] for the difference, 16.2 ml/min/1.73 m(2) vs. 19.0 ml/min/1.73 m(2) [P<0.001]), as were accuracies (15% accuracy, 40.4% vs. 30.6% [P = 0.02]; 30% accuracy, 71.1% vs. 47.2%, [P<0.001]; 50% accuracy, 90.2% vs. 75.7%, [P<0.001]), allowing improvement in GFR categorization (GFR category misclassification rate, 37.4% vs. 53.2% [P = <0.001]). CONCLUSIONS: A new equation was developed in Chinese elderly population. In the validation data set, the new equation performed better than the original CKD-EPI equation. The new equation needs further external validations. Calibration of the GFR referent standard to a more accurate one should be an useful way to improve the performance of GFR estimating equations.

Estimating glomerular filtration rates in elderly Chinese patients with chronic kidney disease: performance of six modified formulae developed in Asian populations.

OBJECTIVES: The aim of the present study was to evaluate modified glomerular filtration rate (GFR) prediction formulae in an elderly Chinese population with chronic kidney disease (CKD). METHODS: A total of 378 elderly Chinese patients with CKD were enrolled. The GFR was estimated with six modified GFR prediction formulae. The performances of the estimated GFRs were compared with those of the standard GFRs measured by technetium-99m diethyl-enetraminepentaacetic acid. RESULTS: Biases were similar for Chinese formula 1, the Asian formula, and Chinese formula 2 (median difference, 2.22 mL/min/1.73 m(2) and 2.59 mL/min/1.73 m(2) for Chinese formula 1 and the Asian formula, respectively, versus (vs) 3.69 mL/min/1.73 m(2) for Chinese formula 2 [P = 0.298 and P = 0.913, respectively]). Precision was improved with the Japanese formula (interquartile range of the difference, 3.14 mL/min/1.73 m(2) of the Japanese formula versus 15.53-23.06 mL/min/1.73 m(2) of the other formulae). The accuracy of Chinese formula 2 was the highest (30% accuracy, 59.3% vs range 37.8-54.0% [P < 0.05 for all comparisons]). However, none of the modified formulae surpassed the acceptable tolerance (>70%), and the GFR category misclassification rates for all the formulae exceeded 50%. CONCLUSION: Our findings suggest that all six modified formulae developed in Asian populations may show great bias in elderly Chinese patients with CKD. Also, our study suggests the need for uniform measures for the assessment of CKD in the elderly to guarantee better sensitivity and specificity.