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Leech, a classical traditional Chinese medicine for promoting blood circulation and removing blood stasis, is mainly used in the clinical treatment of cardiovascular and cerebrovascular diseases. The discovery of activity proteins or peptides in the dead and dried medicinal leech is an important task with great challenges. The aim of this study was to provide a basic proteome profile and help further discover active proteins and quality control for medicinal leeches, which would also provide insight into the research of animal medicines. Seventeen batches of dried medicinal leeches covering three species were collected from medicinal markets, which were authenticated by DNA barcoding. Then the proteome of different species leeches was profiled to reveal the significantly different proteins using label-free proteomics. The characteristic peptides were screened out based on biological pathways analysis, which were further absolutely quantified using the developed stable isotope-labeled based parallel reaction monitoring method. Seventeen batches of leech materials were Whitmania pigra Whitman (WP), Whitmania laevis Whitman (WL) and Poecilobdella manillensis Lesson (PM), respectively. A total of 1,035 proteins (452 in WP, 425 in WL and 158 in PM) were identified. Among them, 90 overlapping proteins were mainly concentrated in diverse metabolic pathways and primarily localized in the cytoplasm and mitochondrial inner membrane, which mainly related to ATP binding, catalytic activity and structural molecular activity. In total of 51 uniquely expressed proteins (21 in WP, 23 in WL and 7 in PM), associated with multiple key signaling pathways, including Rap1, cGMP-PKG, PI3K-Akt, Wnt and HIF-1, etc., relevant to treating cardiovascular diseases, diabetes, cancer and even a variety of neurodegenerative diseases. Three proteins with potential bioactivities, including Neurohemerythrin, Hirudin and Eglin C, were selected as the quality makers and then quantified based on the characteristic peptides. This work profiled the proteome of three species of leeches, and addressed potential active proteins of the medicinal leech, which would help to provide the potential molecular mechanisms involved in disease treatment. The proteomics-based approach developed in this work is not only useful for the discovery of proteins with potential bioactivities but also helpful for the bioactivity relevant quality control of animal medicines. [Display omitted] • 17 batches of commercial leeches were identified using DNA barcoding based on COI gene. • A total of 1035 proteins were identified from 3 species leeches. • 90 co-existed proteins and 51 uniquely expressed proteins were characterized. • A characteristic peptides-based parallel reaction monitoring quantitative approach was developed. • Proteome profiling helps to discover active proteins of medicinal leeches. [ABSTRACT FROM AUTHOR]

Fgfr1 is a fibroblast growth factor receptor involved in regulating cell growth, proliferation, differentiation and migration. Here, we report the isolation and characterization of duplicated fgfr1 genes in blunt snout bream ( Megalobrama amblycephala ). Blunt snout bream fgfr1a and - 1b cDNAs were found to share a relatively high sequence identity of 82%. During embryogenesis, both fgfr1a and - 1b mRNAs were highly detected at zygotes but gradually decreased and then constantly expressed after 16 hpf, besides a strong expression for the fgfr1b mRNA at 12 hpf. Whole-mount in situ hybridization demonstrated that fgfr1a mRNA was transcribed at the eyes, mid-hindbrain boundary (MHB), brain, posterior somites and tailbud at 16 hpf, while the fgfr1b mRNA was only detected at the eyes and posterior somites at the same period. At 28 hpf embryos, both fgfr1a and - 1b mRNAs were expressed in the eyes, brain, pharyngeal arches and tailbud, and in the eyes, brain, pharyngeal arches and notochord at 55 hpf. In adult fish, fgfr1a mRNA was strongly expressed in the gill, gonad, brain and midgut, but examined relatively low in the skin and kidney. In contrast, the fgfr1b mRNA was highly detected in the brain and liver and quite low in the skin, gill and kidney. During starvation, both fgfr1a and - 1b mRNAs were significantly up-regulated in the intestine and liver, but down-regulated in the brain. Moreover, duplicated fgfr1 mRNAs were differentially inhibited in tissues with exogenous recombinant hGH. Our results suggest that two fgfr1 genes play important roles in regulating growth and development in blunt snout bream. [ABSTRACT FROM AUTHOR]

Connective tissue growth factor (CTGF) is a peptide involved in tissue growth and development, and can be regulated by hypoxia stress. This study aimed to isolate and characterize duplicate Ctgf genes in blunt snout bream Megalobrama amblycephala , and determine their expression patterns and response to hypoxia. The blunt snout bream Ctgfa and Ctgfb were found to be highly divergent, sharing a relatively low sequence identity of 57%. During embryogenesis, Ctgfa mRNA expression levels were low, gradually decreased from zygotes to 12 h post-fertilization (hpf), markedly increased from 16 hpf, and then stabilized from 32 to 40 hpf. Ctgfb expression levels were constant but low from zygotes to 20 hpf, then gradually increased from 24 to 40 hpf. Ctgfa mRNA was expressed in the adaxial cells of the somites, floor plate, and tailbud at 24 hpf, and in the notochord and ethmoid plate at 36 hpf, whereas Ctgfb mRNA was weakly expressed in the adaxial cells and floor plate at 24 hpf, and in the notochord at 36 hpf. In adult fish, Ctgfa mRNA was strongly expressed in the kidney, brain, intestine, muscles, and skin, while Ctgfb mRNA was detected in all examined tissues. During hypoxic treatment, the mRNA levels of both Ctgfa and - b were significantly upregulated in the gill and liver, whereas Ctgfa mRNAs in the brain and kidney and Ctgfb mRNAs in the kidney significantly decreased. These results provide new insights into the functional conservation and divergence of Ctgf genes and reveal their responses to hypoxia. [ABSTRACT FROM AUTHOR]

Abstract: Mesenchymal stem cells (MSCs) are the common progenitors of osteoblasts and adipocytes. A reciprocal relationship exists between osteogenesis and adipogenesis in the bone marrow, and the identification of signaling pathways that stimulate MSC osteogenesis at the expense of adipogenesis is of great importance from the viewpoint of developing new therapeutic treatments for bone loss. The adipogenic transcription factor peroxisome proliferator-activated receptor γ (PPARγ) has been reported to play a vital role in modulating mesenchymal lineage allocation within the bone marrow compartment, stimulating adipocyte development at the expense of osteoblast differentiation. Hence, PPARγ may be a valuable target for drugs intended to enhance bone mass. However, little direct evidence is available for the role played by PPARγ in human mesenchymal lineage allocation. In this study, using human MSCs as an in vitro model, we showed that the two isoforms of PPARγ, PPARγ1 and PPARγ2, were differentially induced during hMSC adipogenesis, whereas only PPARγ1 was detected during osteogenesis. BADGE and GW9662, two potential antagonists of PPARγ, as well as lentivirus-mediated knockdown of PPARγ, inhibited hMSC adipogenesis but did not significantly affect osteogenesis. PPARγ knockdown did not significantly influence the expression level of the osteogenic transcription factor Runx2. Together, these results suggest that PPARγ is not the master factor regulating mesenchymal lineage determination in human bone marrow. [Copyright &y& Elsevier]

In this study, we found that blunt snout bream ( Megalobrama amblycephala ) had a relatively high critical oxygen tension at which it lost its equilibrium (LOE crit ), confirming that it is a hypoxia sensitive fish. Blunt snout bream has the ability to remodel its gill structure in response to oxygen levels. When blunt snout bream were exposed to 4- or 7-days of hypoxia, the average protruding lamella heights and mean lamellar area of gills were significantly ( P < 0.01) larger than those of normoxic controls. These changes resulted in reduced average interlamellar cell mass (ILCM) height and volume under hypoxia. After 1 week of normoxic recovery, gill lamellae were reversibly embedded with ILCM cells. Irrespective of dissolved oxygen concentration, the average protruding lamella height of fish gills at 25 °C was significantly ( P < 0.01) larger than that of fish cultured at 10 °C, suggesting that blunt snout bream also have the ability to modify gill structure in response to water temperature. In response to hypoxia, blood erythrocyte count and haemoglobin (Hb) concentration increased significantly ( P < 0.01) under hypoxia. Furthermore, the plasma chloride concentration ([Cl − ]) was significantly ( P < 0.01) reduced. Our results are the first to show that blunt snout bream, as a hypoxia sensitive fish, have the capacity to alter respiratory surface area in response to hypoxia and temperature. [ABSTRACT FROM AUTHOR]