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.

Downregulated BMP-Smad1/5/8 signaling causes emphysema via dysfunction of alveolar type II epithelial cells.

Bone morphogenetic protein (BMP)-Smad1/5/8 signaling plays a crucial regulatory role in lung development and adult lung homeostasis. However, it remains elusive whether BMP-Smad1/5/8 signaling is involved in the pathogenesis of emphysema. In this study, we downregulated BMP-Smad1/5/8 signaling by overexpressing its antagonist Noggin in adult mouse alveolar type II epithelial cells (AT2s), resulting in an emphysematous phenotype mimicking the typical pathological features of human emphysema, including distal airspace enlargement, pulmonary inflammation, extracellular matrix remodeling, and impaired lung function. Dysregulation of BMP-Smad1/5/8 signaling in AT2s leads to inflammatory destruction dominated by macrophage infiltration, associated with reduced secretion of surfactant proteins and inhibition of AT2 proliferation and differentiation. Reactivation of BMP-Smad1/5/8 signaling by genetics or chemotherapy significantly attenuated the morphology and pathophysiology of emphysema and improved the lung function in Noggin-overexpressing lungs. We also found that BMP-Smad1/5/8 signaling was downregulated in cigarette smoke-induced emphysema, and that enhancing its activity in AT2s prevented or even reversed emphysema in the mouse model. Our data suggest that BMP-Smad1/5/8 signaling, located at the top of the signaling cascade that regulates lung homeostasis, represents a key molecular regulator of alveolar stem cell secretory and regenerative function, and could serve as a potential target for future prevention and treatment of pulmonary emphysema. © 2023 The Pathological Society of Great Britain and Ireland.

The evolutionary origin and domestication history of goldfish (Carassius auratus).

Goldfish have been subjected to over 1,000 y of intensive domestication and selective breeding. In this report, we describe a high-quality goldfish genome (2n = 100), anchoring 95.75% of contigs into 50 pseudochromosomes. Comparative genomics enabled us to disentangle the two subgenomes that resulted from an ancient hybridization event. Resequencing 185 representative goldfish variants and 16 wild crucian carp revealed the origin of goldfish and identified genomic regions that have been shaped by selective sweeps linked to its domestication. Our comprehensive collection of goldfish varieties enabled us to associate genetic variations with a number of well-known anatomical features, including features that distinguish traditional goldfish clades. Additionally, we identified a tyrosine-protein kinase receptor as a candidate causal gene for the first well-known case of Mendelian inheritance in goldfish-the transparent mutant. The goldfish genome and diversity data offer unique resources to make goldfish a promising model for functional genomics, as well as domestication.

The protective role of curcumin in human dental pulp stem cells stimulated by lipopolysaccharide via inhibiting NF-κB p65 phosphorylation to suppress NLRP3 inflammasome activation.

OBJECTIVES: This study aims to investigate the anti-inflammatory effect of curcumin and underlying mechanisms regarding the modulation of the nod-like receptor pyrin domain containing 3 (NLRP3) inflammasome in human dental pulp stem cells (hDPSCs). MATERIALS AND METHODS: The impact of curcumin on the viability of hDPSCs was evaluated. The effect of curcumin on the expression of IL-1ß and NLRP3 in hDPSCs stimulated by lipopolysaccharide (LPS) was assessed. Then, LPS-primed hDPSCs were pre-treated with curcumin before ATP triggering NLRP3 inflammasome activation, and NLRP3 inflammasome-related mediators were assessed. The mechanism of curcumin inactivation of LPS plus ATP-induced inflammasome associated with NF-κB pathway was explored. The NF-κB pathway related pro-inflammatory mediators at mRNA and protein levels were evaluated. The expression of NF-κB p65 and phosphorylation p65 was visualized after curcumin or NF-κB inhibitor administrating respectively in hDPSCs with an activated NLRP3 inflammasome. Statistical analysis was performed. RESULTS: While curcumin at the concentration of 0.5-5 µM showed no obvious impact on the viability of hDPSCs, it significantly decreased IL-1ß and NLRP3 mRNA expression in LPS-induced hDPSCs in a dose-dependent manner. Curcumin significantly inhibited the LPS plus ATP-primed NLRP3 inflammasome activation in hDPSCs (NLRP3, ASC, caspase-1, and IL-1ß). Curcumin evidently attenuated the LPS plus ATP-induced expression of NF-κB pathway-related pro-inflammatory mediators (IL-6, IL-8, TNF-α, and COX-2). Furthermore, curcumin effectively reduced p65 phosphorylation, which acts as an NF-κB inhibitor in hDPSCs with an activated NLRP3 inflammasome. CONCLUSIONS: Curcumin pre-treatment may exert an anti-inflammatory role via inactivation of the NLRP3 inflammasome by inhibiting NF-κB p65 phosphorylation in cultured hDPSCs. CLINICAL RELEVANCE: Curcumin may have therapeutic potential in pulp inflammation.

Nkx2-5 defines a subpopulation of pacemaker cells and is essential for the physiological function of the sinoatrial node in mice.

The sinoatrial node (SAN), the primary cardiac pacemaker, consists of a head domain and a junction/tail domain that exhibit different functional properties. However, the underlying molecular mechanism defining these two pacemaker domains remains elusive. Nkx2-5 is a key transcription factor essential for the formation of the working myocardium, but it was generally thought to be detrimental to SAN development. However, Nkx2-5 is expressed in the developing SAN junction, suggesting a role for Nkx2-5 in SAN junction development and function. In this study, we present unambiguous evidence that SAN junction cells exhibit unique action potential configurations intermediate to those manifested by the SAN head and the surrounding atrial cells, suggesting a specific role for the junction cells in impulse generation and in SAN-atrial exit conduction. Single-cell RNA-seq analyses support this concept. Although Nkx2-5 inactivation in the SAN junction did not cause a malformed SAN at birth, the mutant mice manifested sinus node dysfunction. Thus, Nkx2-5 defines a population of pacemaker cells in the transitional zone. Despite Nkx2-5 being dispensable for SAN morphogenesis during embryogenesis, its deletion hampers atrial activation by the pacemaker.

Overexpression of Fgf8 in the epidermis inhibits hair follicle development.

The hair follicle is a classical model for studying epithelial-mesenchymal interactions. Given the critical role of fibroblast growth factor 8 (Fgf8) in embryonic development, we generated a mouse model that overexpresses Fgf8 specifically in the epidermis. Interestingly, these mutant mice exhibited stunted, smaller bodies and severe hypotrichosis. Histological analysis showed that the hair follicles in the mutants were arrested at stage 2 of hair development. The density of hair follicles in the mutant mice was also lower compared to that in the control mice. Overexpression of Fgf8 inhibited the proliferation of epidermal cells and simultaneously promoted apoptosis, leading to the arrest of hair follicle development. Further analysis showed that sonic hedgehog (Shh) and bone morphogenetic protein 4 (Bmp4) were downregulated and upregulated, respectively. To summarize, our study demonstrates that FGF signalling plays an important role in the regulation of hair follicle development.

Tissue interactions are indispensable for cavity formation and disc separation in the temporomandibular joint.

Purpose: Our previous study found that in the temporomandibular joint (TMJ) of the K14-cre; Ctnnb1ex3f mouse embryo, the morphogenesis of glenoid fossa was interrupted by the dislocated condyle. This observation suggested that the formation of the glenoid fossa required tissue interactions with condylar mesenchyme. The purpose of this study was to clarify if the interactions between other components are essential for TMJ morphogenesis.Materials and methods: We examined the gross morphology, histology, cell proliferation, and gene expression in the developing TMJ of K14-cre; Ctnnb1ex3f mice by whole-mount bone and cartilage staining, Azon staining, BrdU labeling, and in situ hybridization, respectively.Results: In K14-cre; Ctnnb1ex3f mice, the zygomatic arch was misconnected to the mandibular bone by ectopic bone formation, which disrupted the attachment of temporalis to the mandibular bone and joint capsule formation. Although the initiation and differentiation of the condylar cartilage were slightly impacted, the K14-cre; Ctnnb1ex3f TMJ lacked joint cavities and separated disc, suggesting that the tissue interactions between the joint capsule and the TMJ were indispensable for the cavity formation and disc separation. The ectopic activation of Gli2 in the cells occupying the cavities, and the enhanced PTHrP transcription in the condylar perichondrium of the K14-cre; Ctnnb1ex3f TMJ suggested that the disrupted interactions between the joint capsule and the TMJ impaired cavity formation and disc separation by altering Hh signaling.Conclusion: Joint capsule formation was essential for cavity formation and disc separation during TMJ development.

Shox2 regulates osteogenic differentiation and pattern formation during hard palate development in mice.

During mammalian palatogenesis, cranial neural crest-derived mesenchymal cells undergo osteogenic differentiation and form the hard palate, which is divided into palatine process of the maxilla and the palatine. However, it remains unknown whether these bony structures originate from the same cell lineage and how the hard palate is patterned at the molecular level. Using mice, here we report that deficiency in Shox2 (short stature homeobox 2), a transcriptional regulator whose expression is restricted to the anterior palatal mesenchyme, leads to a defective palatine process of the maxilla but does not affect the palatine. Shox2 overexpression in palatal mesenchyme resulted in a hyperplastic palatine process of the maxilla and a hypoplastic palatine. RNA sequencing and assay for transposase-accessible chromatin-sequencing analyses revealed that Shox2 controls the expression of pattern specification and skeletogenic genes associated with accessible chromatin in the anterior palate. This highlighted a lineage-autonomous function of Shox2 in patterning and osteogenesis of the hard palate. H3K27ac ChIP-Seq and transient transgenic enhancer assays revealed that Shox2 binds distal-acting cis-regulatory elements in an anterior palate-specific manner. Our results suggest that the palatine process of the maxilla and palatine arise from different cell lineages and differ in ossification mechanisms. Shox2 evidently controls osteogenesis of a cell lineage and contributes to the palatine process of the maxilla by interacting with distal cis-regulatory elements to regulate skeletogenic gene expression and to pattern the hard palate. Genome-wide Shox2 occupancy in the developing palate may provide a marker for identifying active anterior palate-specific gene enhancers.

A unique stylopod patterning mechanism by Shox2-controlled osteogenesis.

Vertebrate appendage patterning is programmed by Hox-TALE factor-bound regulatory elements. However, it remains unclear which cell lineages are commissioned by Hox-TALE factors to generate regional specific patterns and whether other Hox-TALE co-factors exist. In this study, we investigated the transcriptional mechanisms controlled by the Shox2 transcriptional regulator in limb patterning. Harnessing an osteogenic lineage-specific Shox2 inactivation approach we show that despite widespread Shox2 expression in multiple cell lineages, lack of the stylopod observed upon Shox2 deficiency is a specific result of Shox2 loss of function in the osteogenic lineage. ChIP-Seq revealed robust interaction of Shox2 with cis-regulatory enhancers clustering around skeletogenic genes that are also bound by Hox-TALE factors, supporting a lineage autonomous function of Shox2 in osteogenic lineage fate determination and skeleton patterning. Pbx ChIP-Seq further allowed the genome-wide identification of cis-regulatory modules exhibiting co-occupancy of Pbx, Meis and Shox2 transcriptional regulators. Integrative analysis of ChIP-Seq and RNA-Seq data and transgenic enhancer assays indicate that Shox2 patterns the stylopod as a repressor via interaction with enhancers active in the proximal limb mesenchyme and antagonizes the repressive function of TALE factors in osteogenesis.

Conditional deletion of Bmp2 in cranial neural crest cells recapitulates Pierre Robin sequence in mice.

Bone morphogenetic protein (BMP) signaling plays a crucial role in the development of craniofacial organs. Mutations in numerous members of the BMP signaling pathway lead to several severe human syndromes, including Pierre Robin sequence (PRS) caused by heterozygous loss of BMP2. In this study, we generate mice carrying Bmp2-specific deletion in cranial neural crest cells using floxed Bmp2 and Wnt1-Cre alleles to mimic PRS in humans. Mutant mice exhibit severe PRS with a significantly reduced size of craniofacial bones, cleft palate, malformed tongue and micrognathia. Palate clefting is caused by the undescended tongue that prevents palatal shelf elevation. However, the tongue in Wnt1-Cre;Bmp2f/f mice does not exhibit altered rates of cell proliferation and apoptosis, suggesting contribution of extrinsic defects to the failure of tongue descent. Further studies revealed obvious reduction in cell proliferation and differentiation of osteogenic progenitors in the mandible of the mutants, attributing to the micrognathia phenotype. Our study illustrates the pathogenesis of PRS caused by Bmp2 mutation, highlights the crucial role of BMP2 in the development of craniofacial bones and emphasizes precise coordination in the morphogenesis of palate, tongue and mandible during embryonic development.

Overexpression of acetyl-CoA carboxylase increases fatty acid production in the green alga Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii is a photosynthetic unicellular model algae with multiple biotechnological advantages, and its fatty acids can be used to produce biofuels. Numerous studies suggest that acetyl-coA carboxylase (ACCa) catalyzes the first committed and rate-limiting step of fatty acid biosynthesis, thereby playing a central role in oil accumulation. Here, we cloned and overexpressed ACCa in C. reinhardtii to directly evaluate its effect on fatty acid synthesis. GC-MS analysis found that the unsaturated FAs contents of the CW15-24 and CW15-85 strains were 55.45% and 56.15%, which were significantly enriched compared to the wild type CW15 (48.39%). Under the optimized conditions, the content of lipid by overexpressed the ACCa gene in the mutant CW15-85 (0.46 g/l) was 1.16-fold greater than control through optimization of N and P sources. Altogether, our data clearly demonstrate that ACCa overexpression in C. reinhardtii can directly increase the synthesis of fatty acids.

A common Shox2-Nkx2-5 antagonistic mechanism primes the pacemaker cell fate in the pulmonary vein myocardium and sinoatrial node.

In humans, atrial fibrillation is often triggered by ectopic pacemaking activity in the myocardium sleeves of the pulmonary vein (PV) and systemic venous return. The genetic programs that abnormally reinforce pacemaker properties at these sites and how this relates to normal sinoatrial node (SAN) development remain uncharacterized. It was noted previously that Nkx2-5, which is expressed in the PV myocardium and reinforces a chamber-like myocardial identity in the PV, is lacking in the SAN. Here we present evidence that in mice Shox2 antagonizes the transcriptional output of Nkx2-5 in the PV myocardium and in a functional Nkx2-5(+) domain within the SAN to determine cell fate. Shox2 deletion in the Nkx2-5(+) domain of the SAN caused sick sinus syndrome, associated with the loss of the pacemaker program. Explanted Shox2(+) cells from the embryonic PV myocardium exhibited pacemaker characteristics including node-like electrophysiological properties and the capability to pace surrounding Shox2(-) cells. Shox2 deletion led to Hcn4 ablation in the developing PV myocardium. Nkx2-5 hypomorphism rescued the requirement for Shox2 for the expression of genes essential for SAN development in Shox2 mutants. Similarly, the pacemaker-like phenotype induced in the PV myocardium in Nkx2-5 hypomorphs reverted back to a working myocardial phenotype when Shox2 was simultaneously deleted. A similar mechanism is also adopted in differentiated embryoid bodies. We found that Shox2 interacts with Nkx2-5 directly, and discovered a substantial genome-wide co-occupancy of Shox2, Nkx2-5 and Tbx5, further supporting a pivotal role for Shox2 in the core myogenic program orchestrating venous pole and pacemaker development.

FGF signaling sustains the odontogenic fate of dental mesenchyme by suppressing ß-catenin signaling.

Odontoblasts and osteoblasts develop from multipotent craniofacial neural crest cells during tooth and jawbone development, but the mechanisms that specify and sustain their respective fates remain largely unknown. In this study we used early mouse molar and incisor tooth germs that possess distinct tooth-forming capability after dissociation and reaggregation in vitro to investigate the mechanism that sustains odontogenic fate of dental mesenchyme during tooth development. We found that after dissociation and reaggregation, incisor, but not molar, mesenchyme exhibits a strong osteogenic potency associated with robustly elevated ß-catenin signaling activity in a cell-autonomous manner, leading to failed tooth formation in the reaggregates. Application of FGF3 to incisor reaggregates inhibits ß-catenin signaling activity and rescues tooth formation. The lack of FGF retention on the cell surface of incisor mesenchyme appears to account for the differential osteogenic potency between incisor and molar, which can be further attributed to the differential expression of syndecan 1 and NDST genes. We further demonstrate that FGF signaling inhibits intracellular ß-catenin signaling by activating the PI3K/Akt pathway to regulate the subcellular localization of active GSK3ß in dental mesenchymal cells. Our results reveal a novel function for FGF signaling in ensuring the proper fate of dental mesenchyme by regulating ß-catenin signaling activity during tooth development.

Directed Bmp4 expression in neural crest cells generates a genetic model for the rare human bony syngnathia birth defect.

Congenital bony syngnathia, a rare but severe human birth defect, is characterized by bony fusion of the mandible to the maxilla. However, the genetic mechanisms underlying this birth defect are poorly understood, largely due to limitation of available animal models. Here we present evidence that transgenic expression of Bmp4 in neural crest cells causes a series of craniofacial malformations in mice, including a bony fusion between the maxilla and hypoplastic mandible, resembling the bony syngnathia syndrome in humans. In addition, the anterior portion of the palatal shelves emerged from the mandibular arch instead of the maxilla in the mutants. Gene expression assays showed an altered expression of several facial patterning genes, including Hand2, Dlx2, Msx1, Barx1, Foxc2 and Fgf8, in the maxillary and mandibular processes of the mutants, indicating mis-patterned cranial neural crest (CNC) derived cells in the facial region. However, despite of formation of cleft palate and ectopic cartilage, forced expression of a constitutively active form of BMP receptor-Ia (caBmprIa) in CNC lineage did not produce the syngnathia phenotype, suggesting a non-cell autonomous effect of the augmented BMP4 signaling. Our studies demonstrate that aberrant BMP4-mediated signaling in CNC cells leads to mis-patterned facial skeleton and congenital bony syngnathia, and suggest an implication of mutations in BMP signaling pathway in human bony syngnathia.

Expression profile of critical genes involved in FGF signaling pathway in the developing human primary dentition.

Mammalian tooth development is regulated by paracrine signal molecules of several conserved family interactions between epithelium and mesenchyme. The expression patterns and regulative roles of FGF signaling have been extensively studied in the mouse odontogenesis; however, that is not well known in human tooth development. In order to unveil the molecular mechanisms that regulate human tooth morphogenesis, we examined the expression patterns of the critical molecules involved in FGF signaling pathway in the developing human tooth germ by in situ hybridization, immunohistochemistry, and real-time RT-PCR, including FGF ligands, receptors, and intracellular transducer. We found overlapping but distinct expression pattern of FGF ligands and receptors in the different stages and components. Expression of FGF4, FGF7, FGF8, and FGF9 persists widespread in human tooth mesenchyme, which is quite different to that of in mouse. FGFR1 may be the major receptor in regulate mechanisms of FGF signals in human tooth development. Real-time RT-PCR indeed confirmed the results of in situ hybridization. Results of K-Ras, p-ERK1/2, p-p38, p-JNK, and p-PDK1 expression reveal spatial and temporal patterns of FGF signaling during morphogenesis and organogenesis of human tooth germ. Activity of the FGF signaling transducer protein in human tooth germ was much higher than that of in mouse. Our results provided important FGF singling information in the developing process, pinpoint to the domains where the downstream target genes of FGF signaling can be sought, and enlightened our knowledge about the nature of FGF signaling in human tooth germ.

Spleen Transcriptome Profile of Muscovy Ducklings in Response to Infection With Muscovy Duck Reovirus.

Muscovy duck reovirus (MDRV) causes high morbidity and mortality in ducklings. However, the molecular basis for pathogenesis of this virus remains poorly understood, and the complete genome sequence of Muscovy duck is lacking. Here we report the transcriptome profile of Muscovy ducks in response to MDRV infection. RNA sequencing technology was employed to obtain a representative complement of transcripts from the spleen of ducklings, and then differential gene expression was analyzed between MDRV-YB strain infected ducks and noninfected ducks. This analysis generated 65,536 unigenes. Of these, 6458 genes were found to be significantly differentially expressed between the infected and noninfected groups. The symptom and pathology of ducks infected with MDRV-YB was correlated with the greater proportion of decreased expression genes (4906) than increased expression (1552) level. Gene ontology analysis assigned differentially expressed genes to the categories: "biological processes," "cellular functions," and "molecular functions." Differentially expressed genes involved in the innate immune system were analyzed further, and 128 of these genes showed decreased expression and 86 showed increased expression between the infected and noninfected groups. These genes represented the Janus kinase-signal transducer and activator of transcription signaling pathway, and the retinoic acid-inducible gene I (RIG-I)-like and Toll-like receptor (TLR) signaling pathways and included interferon (IFN) α, IFNγ, interleukin 6, RIG-I, and TLR4. The data were verified by SYBR fluorescence quantitative polymerase chain reaction (SYBR-qPCR). Our findings offer new insight into the host immune response to MDRV infection.

Precise chronology of differentiation of developing human primary dentition.

While correlation of developmental stage with embryonic age of the human primary dentition has been well documented, the available information regarding the differentiation timing of the primary teeth was largely based on the observation of initial mineralization and varies significantly. In this study, we aimed to document precise differentiation timing of the developing human primary dentition. We systematically examined the expression of odontogenic differentiation markers along with the formation of mineralized tissue in each developing maxillary and mandibular teeth from human embryos with well-defined embryonic age. We show that, despite that all primary teeth initiate development at the same time, odontogenic differentiation begins in the maxillary incisors at the 15th week and in the mandibular incisors at the 16th week of gestation, followed by the canine, the first primary premolar, and the second primary premolar at a week interval sequentially. Despite that the mandibular primary incisors erupt earlier than the maxillary incisors, this distal to proximal sequential differentiation of the human primary dentition coincides in general with the sequence of tooth eruption. Our results provide an accurate chronology of odontogenic differentiation of the developing human primary dentition, which could be used as reference for future studies of human tooth development.

Expression patterns of genes critical for BMP signaling pathway in developing human primary tooth germs.

The developing murine tooth has been used as an excellent model system to study the molecular mechanism of organ development and regeneration. While the expression patterns of numerous regulatory genes have been examined and their roles have begun to be revealed in the developing murine tooth, little is known about gene expression and function in human tooth development. In order to unveil the molecular mechanisms that regulate human tooth morphogenesis, we examined the expression patterns of the major BMP signaling pathway molecules in the developing human tooth germ at the cap and bell stages by in situ hybridization, immunohistochemistry, and real-time RT-PCR. Expression of BMP ligands and antagonist, including BMP2, BMP3, BMP4, BMP7, and NOOGGIN, exhibited uniform patterns in the tooth germs of incisor and molar at the cap and bell stages with stronger expression in the inner dental epithelium than that in the dental mesenchyme. Both type I and type II BMP receptors were present in widespread expression pattern in the whole-enamel organ and the dental mesenchyme with the strongest expression in inner dental epithelium at the cap and bell stages. SMAD4 and SMAD1/5/8 showed an expression pattern similar to that of BMP ligands with more intensive signals in the inner dental epithelium. Despite some unique and distinct patterns as compared to the mouse, the intensive expression of BMP signaling pathway molecules in the developing human tooth strongly suggests conserved functions of BMP signaling during human odontogenesis, such as in mediating tissue interactions and regulating differentiation and organization of odontogenic tissues. Our results provide an important set of documents for studying molecular regulatory mechanisms underlying tooth development and regeneration in humans.

Expression of codon optimized human bone morphogenetic protein 4 in Pichia pastoris.

Bone morphogenetic proteins (BMPs) are TGF-ß family member proteins that have therapeutic potential. The amount of BMPs from natural resources is limited, and the production of biologically active BMPs in heterologous protein expression systems remains an obstacle for their clinical application. In this study, the DNA sequence of human BMP4 mature domain (hBMP4) was optimized according to the codon relative synonymous codon usage values in Pichia pastoris, and the A+T content in the sequence after optimization was within the range of 30% to 55%. In Pichia pastoris cultured in shake-flask, the expression level of hBMP4 protein from the optimized sequence (48 mg/L) increased fourfold in comparison with that from the native sequence (12 mg/L). Recombinant hBMP4 protein was purified by SP Sepharose and heparin affinity chromatography. The biological activities of recombinant hBMP4 were examined by measuring proliferation stimulation in cells and induction of ectopic cartilage formation in mouse models. Our results demonstrated that the optimized DNA sequence could significantly enhance hBMP4 protein expression in Pichia pastoris compared with the native sequence and produce biologically active recombinant hBMP4; this indicates the potential of this optimized sequence for bulk production of hBMP4 protein in future clinical applications.

Evidence for A1 and A 3 receptors mediating adenosine-induced intracellular calcium release in the dorsal root ganglion neurons by using confocal microscopy imaging.

Adenosine exerts a key role in analgesia. In the present study, adenosine-induced Ca(2+) responses were revealed by using confocal microscopy imaging in the rat dorsal root ganglia (DRG) neurons in vitro. Our results showed that adenosine could evoke increases in the intracellular Ca(2+) concentration in the DRG neurons. In addition, by application of selective receptor antagonists, two types of receptors, A1R and A3R, were identified to be involved in the adenosine-induced Ca(2+) release from intracellular stores in neurons. Altogether, these results suggest that confocal microscopy imaging combined with fluorescent dyes could help to detect the analgesic-induced ion signaling in single cell.