Piezo1-mediated mechanosensation in bone marrow macrophages promotes vascular niche regeneration after irradiation injury.

Background: Irradiation disrupts the vascular niche where hematopoietic stem cells (HSCs) reside, causing delayed hematopoietic reconstruction. The subsequent recovery of sinusoidal vessels is key to vascular niche regeneration and a prerequisite for hematopoietic reconstruction. We hypothesize that resident bone marrow macrophages (BM-Mφs) are responsible for repairing the HSC niche upon irradiation injury. Methods: We examined the survival and activation of BM-Mφs in C57BL/6 mice upon total body irradiation. After BM-Mφ depletion via injected clodronate-containing liposomes and irradiation injury, hematopoietic reconstruction and sinusoidal vascular regeneration were assessed with immunofluorescence and flow cytometry. Then enzyme-linked immunosorbent assay (ELISA) and flow cytometry were performed to analyze the contribution of VEGF-A released by BM-Mφs to the vascular restructuring of the HSC niche. VEGF-A-mediated signal transduction was assessed with transcriptome sequencing, flow cytometry, and pharmacology (agonists and antagonists) to determine the molecular mechanisms of Piezo1-mediated responses to structural changes in the HSC niche. Results: The depletion of BM-Mφs aggravated the post-irradiation injury, delaying the recovery of sinusoidal endothelial cells and HSCs. A fraction of the BM-Mφ population persisted after irradiation, with residual BM-Mφ exhibiting an activated M2-like phenotype. The expression of VEGF-A, which is essential for sinusoidal regeneration, was upregulated in BM-Mφs post-irradiation, especially CD206+ BM-Mφs. The expression of mechanosensory ion channel Piezo1, a response to mechanical environmental changes induced by bone marrow ablation, was upregulated in BM-Mφs, especially CD206+ BM-Mφs. Piezo1 upregulation was mediated by the effects of irradiation, the activation of Piezo1 itself, and the M2-like polarization induced by the phagocytosis of apoptotic cells. Piezo1 activation was associated with increased expression of VEGF-A and increased accumulation of NFATC1, NFATC2, and HIF-1α. The Piezo1-mediated upregulation in VEGF-A was suppressed by inhibiting the calcineurin/NFAT/HIF-1α signaling pathway. Conclusion: These findings reveal that BM-Mφs play a critical role in promoting vascular niche regeneration by sensing and responding to structural changes after irradiation injury, offering a potential target for therapeutic efforts to enhance hematopoietic reconstruction.

EloA promotes HEL polyploidization upon PMA stimulation through enhanced ERK1/2 activity.

Megakaryocytes (MKs) are the unique non-pathological cells that undergo polyploidization in mammals. The polyploid formation is critical for understanding the MK biology, and transcriptional regulation is involved in the differentiation and maturation of MKs. However, little is known about the functions of transcriptional elongation factors in the MK polyploidization. In this study, we investigated the role of transcription elongation factor EloA in the polyploidy formation during the MK differentiation. We found that EloA was highly expressed in the erythroleukemia cell lines HEL and K562. Knockdown of EloA in HEL cell line was shown to impair the phorbol myristate acetate (PMA) induced polyploidization process, which was used extensively to model megakaryocytic differentiation. Selective over-expression of EloA mutants with Pol II elongation activity partially restored the polyploidization. RNA-sequencing revealed that knockdown of EloA decelerated the transcription of genes enriched in the ERK1/2 cascade pathway. The phosphorylation activity of ERK1/2 decreased upon the EloA inhibition, and the polyploidization process of HEL was hindered when ERK1/2 phosphorylation was inhibited by PD0325901 or SCH772984. This study evidenced a positive role of EloA in HEL polyploidization upon PMA stimulation through enhanced ERK1/2 activity.

Phylogeography of Schizopygopsis malacanthus Herzenstein (Cypriniformes, Cyprinidae) in relation to the tectonic events and Quaternary climatic oscillations in the Shaluli Mountains Region.

The tectonic events and Quaternary climatic oscillations in the Shaluli Mountains (the margin of the southeastern Tibetan Plateau) had an extensive effect on the genetic structure and distribution patterns of this region's terrestrial fauna and flora. It is not yet clear whether similar mechanisms influence this region's fish fauna. Schizopygopsis malacanthus is limited to high-elevation rivers and lakes, and exhibits distinct adaptations to the mountains of, and near to, the Qinghai-Tibet Plateau (QTP). Therefore, this species is a good candidate for investigating patterns of genetic variation resulting from palaeoenvironmental fluctuations in the Shaluli Mountains (China). Here, we used microsatellite and mitochondrial DNA control region sequences to analyze six populations of S. malacanthus collected from the Jinsha and Yalong River drainages. Genealogical analyses identified four maternal lineages and perhaps even four putative species, of which the Ouqu River lineage played a pivotal role during the course of the species' evolution. Two lineages from the Yalong River drainage did not cluster together, whereas those from different drainages grouped together, suggesting tectonic event impacts that possibly altered regional river drainages have been highly influential in population connectivity and gene flow. Population genetic analysis indicated that the geographic barriers and this species preference for higher elevations both played key roles in the divergence of S. malacanthus populations. Demographic tests suggested large-scale spatial synchrony in population fluctuations of S. malacanthus, accompanying dramatic Pleistocene climatic oscillations. It appears that palaeoenvironmental changes in the Shaluli Mountains influenced the distribution and evolution of studied S. malacanthus populations, which provide important information about the factors that influenced the phylogeographic history of this region's fish fauna. Additionally, our study also has implications for ongoing conservation of this vulnerable fish.

Long noncoding RNA TUG1 inhibits osteogenesis of bone marrow mesenchymal stem cells via Smad5 after irradiation.

Irradiation can greatly inhibit osteogenesis of bone marrow mesenchymal stem cells (BM-MSCs). However, the mechanism remains unclear. Methods: We analyzed the expression profile of long noncoding RNAs (lncRNAs) in BM-MSCs using microarray data. LncRNA TUG1 (Taurine Upregulated Gene 1) was selected and tested in radiated BM-MSCs and non-radiated BM-MSCs. Functional analyses (in vitro) were performed to confirm the role of TUG1 in the osteogenic inhibition induced by irradiation. A RIP (RNA immunoprecipitation) assay was performed to detect the interaction of TUG1 and Smad5. Smad5 and the phosphorylated Smad5 (p-Smad5) were tested by western blot. The nuclear translocation of p-Smad5 were tested by immunofluorescence analysis. Furthermore, a series of Smad5 deletions was constructed to identify the TUG1 binding site of Smad5. Results: We found that numerous lncRNAs, including TUG1, exhibit significant expression differences after irradiation. After irradiation TUG1 was significantly increased in BM-MSCs and inhibited osteogenesis. Furthermore, TUG1 directly bound to Smad5, an osteogenic enhancer. Although the phosphorylation level of Smad5 was increased following irradiation, osteogenesis of BM-MSCs was decreased. Mechanistically, TUG1 interacting with the 50-90 aa region of Smad5 and blocks the nuclear translocation of p-Smad5, abolishing osteogenic signalling after irradiation. Conclusion: These results indicate that TUG1 is a negative regulator of Smad5 signalling and suppresses osteogenesis of BM-MSCs after irradiation.

Identification of Triplophysa species from the Qinghai-Tibetan Plateau (QTP) and its adjacent regions through DNA barcodes.

The genus Triplophysa is the largest and most difficult to identity morphologically fish group of superfamily Cobitoidea with 140 currently valid species, and is mainly distributed in the Qinghai-Tibetan Plateau (QTP) and adjacent regions. Most species within this genus possess highly similar morphological characteristics for adaption to the highland environment and are very difficult to be identified only based on morphology. The traditional species identification, mainly based on external morphological diagnostic characters, leads to inconsistent results in many cases. Herein, we provided a molecular method based on mitochondrial cytochrome c subunit I (COI) for the identification of Triplophysa fishes. Thirty-three Triplophysa species, 244 individuals, were used to determine whether barcoding was effective in discriminating species for this genus. The mean intraspecific and interspecific K2P distances ranged from 0 to 14.9% (mean, 2.9%) and 0 to 23.4% (mean, 9.7%), respectively. The tree-based analysis displayed most of species formed discrete clusters with strong bootstrap support values (>90%). The results showed that most of Triplophysa species could be identified by DNA barcode and indicated DNA barcode could be used as a molecular marker for these species.

Complete mitochondrial genome of Triplophysa bleekeri (Cypriniformes: Balitoridae: Nemacheilinae), and analysis of mitochondrial genetic variability among Triplophysa species.

The complete mitochondrial genome of the stone loach Triplophysa bleekeri collected from the Anning River, a tributary of the Yalong River, in Sichuan Province was sequenced. The genome is 16,573 bp in length, consisting of 13 protein-coding genes, 22 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes, and a noncoding control region. Most of the genes are encoded on the heavy strand, except for ND6 and eight tRNAs. The overall base composition of T. bleekeri is 28.1% for A, 28.4% for T, 25.5% for C, 18.0% for G, with a slight A+T bias of 56.5%. The complete mitochondrial genetic variability is high among different Triplophysa species and between the two T. bleekeri individuals from different rivers.

Complete mitochondrial genome of Triplophysa robusta (Teleostei: Cypriniformes: Balitoridae).

The complete mitochondrial genome of the Triplophysa robusta has been determined in this study. It is 16,572 bp in size and consists of 13 protein-coding genes, 22 tRNA genes, two rRNA genes, and one non-coding control region (D-loop). The overall base composition of the heavy strand of the T. robusta mitochondrial genome was A: 28.20%, T: 28.27%, C: 25.37%, and G: 18.16%. The total length of the 13 protein-coding genes was 11,428 bp. Analysis of the genes indicated the high genetic variability among Triplophysa species.

Complete mitochondrial genome of Ptychobarbus kaznakovi (Teleostei: Cypriniformes: Cyprinidae), and repetitive sequences in the D-loop.

The complete mitochondrial DNA genome of Ptychobarbus kaznakovi was sequenced and characterized. The genome is 16,842 bp in length. Similar with most teleosts, it has two ribosomal RNA (rRNA) genes, 13 protein-coding genes, 22 transfer RNA (tRNA) genes, and one displacement loop (D-loop) region. Conserved sequence blocks, including ETAS, CSB-B, D, E, F, and CSB1-3, were identified in the D-loop, which is similar to other species in Cypriniformes. Nevertheless, a 55 bp tandem repeat array was also identified at 3' end of the D-loop, which is the first finding in Schizothoracinae. Phylogenetic analysis showed that the species of Ptychobarbus (P. dipogon and P. kaznakovi) formed a monophyletic group and represented close relationship to the species without scales in Schizothoracinae.

Molecular cloning and expression of two heat-shock protein genes (HSC70/HSP70) from Prenant's schizothoracin (Schizothorax prenanti).

Through the RT-PCR and rapid amplification of cDNA ends, two complementary deoxyribonucleic acid (cDNA) clones encoding heat-shock cognate 70 (HSC70, designated Sp-HSC70) and inducible heat-shock protein 70 (HSP70, designated Sp-HSP70) were isolated from the liver of Prenant's schizothoracin (Schizothorax prenanti). The cDNAs were 2344- and 2292-bp in length and contained 1950- and 1932-bp open reading frames, encoded proteins of 649 and 643 amino acids, respectively. Amino acid sequence analysis indicated that both Sp-HSC70 and Sp-HSP70 contained three signature sequences of HSP70 family, two partial overlapping bipartite nuclear localization signal sequences (an ATP-binding site motif, a bipartite nuclear targeting signal), and a cytoplasmic characteristic motif EEVD. Homology analysis revealed that Sp-HSC70 and Sp-HSP70 shared 77.5% identity and Sp-HSC70 shared more than 81.1% identity with the known HSC70s of other vertebrates, while Sp-HSP70 shared more than 77.5 % identity with the known HSP70s of other vertebrates. Fluorescent real-time quantitative RT-PCR showed that Sp-HSC70 and Sp-HSP70 mRNAs were found in all tested tissues, including blood, brain, heart, liver, spleen, head kidney, white muscle, skin, gonad, hypophysis, red muscle, and gill. The Sp-HSC70 and Sp-HSP70 mRNA expression level in blood and head kidney displayed a significant increase in vibrio-challenged group with the bacterium Aeromonas hydrophila at 24 h post-infection compared to a control group. Temporally, there was a clear time-dependent expression pattern of Sp-HSC70 or Sp-HSP70 gene after bacterial challenge, and the expression of Sp-HSC70 and Sp-HSP70 mRNAs reached a maximum level at 12 and 6 h post-challenge, respectively. Both returned to control level after 7 × 24 h. The results suggest that Sp-HSC70 and Sp-HSP70 genes may play important roles in mediating the immune responses of A. hydrophila-related diseases in the Prenant's schizothoracin.

Complete mitochondrial genome of the stone loach, Triplophysa stoliczkae (Teleostei: Cypriniformes: Balitoridae).

The complete mitochondrial genome of the stone loach Triplophysa stoliczkae is 16,571 bp in size, consisting of 13 protein-coding genes, 22 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes, and a noncoding control region. As in other vertebrates, most mitochondrial genes are encoded on the heavy strand, except for nd6 and eight tRNA genes which are encoded on the light strand. The overall base composition of the heavy strand of the T. stoliczkae mitochondrial genome is A: 28.1%, T: 29.0%, C: 25.0%, and G: 17.9%. The alignment of the Triplophysa species control regions exhibited high genetic variability and rich A+T content.