Optimal conditions for cryopreservation by vitrification of largemouth bass (Micropterus salmoides) embryos.

The largemouth bass (Micropterus salmoides) is one of the important freshwater aquaculture species in the world. However, due to limitations on introduction scale, high-density farming, inbreeding, and species hybridization, the germplasm resources of largemouth bass face threats such as degradation and susceptibility to diseases. Therefore, it is urgent to conduct research on the conservation of its original and good germplasm resources. We optimized the conditions of cryopreservation to vitrify and revive largemouth bass embryos, including the mixing ratio of cryoprotectants, embryo stage, equilibration step and temperature, and washing regent. The results showed that the least toxic single, binary, and ternary mixed permeating cryoprotectants were PG, PM (PG: MeOH = 2:1), and PMD (PM: DMSO = 3:1), respectively. The least toxic non-permeating cryoprotectant was 5 % glucose. The optimal vitrification solution selected was PMDG (30 % PMD + 5 % glucose) with an 80.67 % survival rate of embryos. Embryos at the heartbeat stage exhibited strong tolerance to the PMDG solution, which is the optimal embryo stage for cryopreservation. During the equilibration process, either the five-step equilibration method or pre-cooling the cryoprotectant to 4°C could reduce its toxicity. During the washing process, a 0.125 mol·L-1 sucrose solution yielded the best results. Based on the optimized conditions, 650 embryos at the heartbeat stage were subjected to cryopreservation by vitrification, resulting in a total of 350 intact transparent eggs, two of which hatched successfully. The results provide a reference for further improving the efficiency of cryopreservation by vitrification of largemouth bass and other fish species.

Three-dimensional printing and decellularized-extracellular-matrix based methods for advances in artificial blood vessel fabrication: A review.

Blood vessels are the tubes through which blood flows and are divided into three types: millimeter-scale arteries, veins, and capillaries as well as micrometer-scale capillaries. Arteries and veins are the conduits that carry blood, while capillaries are where blood exchanges substances with tissues. Blood vessels are mainly composed of collagen fibers, elastic fibers, glycosaminoglycans and other macromolecular substances. There are about 19 feet of blood vessels per square inch of skin in the human body, which shows how important blood vessels are to the human body. Because cardiovascular disease and vascular trauma are common in the population, a great number of researches have been carried out in recent years by simulating the structures and functions of the person's own blood vessels to create different levels of tissue-engineered blood vessels that can replace damaged blood vessels in the human body. However, due to the lack of effective oxygen and nutrient delivery mechanisms, these tissue-engineered vessels have not been used clinically. Therefore, in order to achieve better vascularization of engineered vascular tissue, researchers have widely explored the design methods of vascular systems of various sizes. In the near future, these carefully designed and constructed tissue engineered blood vessels are expected to have practical clinical applications. Exploring how to form multi-scale vascular networks and improve their compatibility with the host vascular system will be very beneficial in achieving this goal. Among them, 3D printing has the advantages of high precision and design flexibility, and the decellularized matrix retains active ingredients such as collagen, elastin, and glycosaminoglycan, while removing the immunogenic substance DNA. In this review, technologies and advances in 3D printing and decellularization-based artificial blood vessel manufacturing methods are systematically discussed. Recent examples of vascular systems designed are introduced in details, the main problems and challenges in the clinical application of vascular tissue restriction are discussed and pointed out, and the future development trends in the field of tissue engineered blood vessels are also prospected.

Preparation and Characterization of Polylactic Acid/Nano Hydroxyapatite/Nano Hydroxyapatite/Human Acellular Amniotic Membrane (PLA/nHAp/HAAM) Hybrid Scaffold for Bone Tissue Defect Repair.

Bone tissue engineering is a novel and efficient repair method for bone tissue defects, and the key step of the bone tissue engineering repair strategy is to prepare non-toxic, metabolizable, biocompatible, bone-induced tissue engineering scaffolds of suitable mechanical strength. Human acellular amniotic membrane (HAAM) is mainly composed of collagen and mucopolysaccharide; it has a natural three-dimensional structure and no immunogenicity. In this study, a polylactic acid (PLA)/Hydroxyapatite (nHAp)/Human acellular amniotic membrane (HAAM) composite scaffold was prepared and the porosity, water absorption and elastic modulus of the composite scaffold were characterized. After that, the cell-scaffold composite was constructed using newborn Sprague Dawley (SD) rat osteoblasts to characterize the biological properties of the composite. In conclusion, the scaffolds have a composite structure of large and small holes with a large pore diameter of 200 µm and a small pore diameter of 30 µm. After adding HAAM, the contact angle of the composite decreases to 38.7°, and the water absorption reaches 249.7%. The addition of nHAp can improve the scaffold's mechanical strength. The degradation rate of the PLA+nHAp+HAAM group was the highest, reaching 39.48% after 12 weeks. Fluorescence staining showed that the cells were evenly distributed and had good activity on the composite scaffold; the PLA+nHAp+HAAM scaffold has the highest cell viability. The adhesion rate to HAAM was the highest, and the addition of nHAp and HAAM could promote the rapid adhesion of cells to scaffolds. The addition of HAAM and nHAp can significantly promote the secretion of ALP. Therefore, the PLA/nHAp/HAAM composite scaffold can support the adhesion, proliferation and differentiation of osteoblasts in vitro which provide sufficient space for cell proliferation, and is suitable for the formation and development of solid bone tissue.

Adolescents experienced more treatment failure than children with chronic myeloid leukemia receiving imatinib as frontline therapy: a retrospective multicenter study.

To explore the differences in the clinical features, treatment responses, and outcomes among children, adolescents, and adults with chronic myeloid leukemia in the chronic phase (CML-CP) receiving imatinib as first-line therapy. Data from children (0-8 years for girls and 0-10 years for boys), adolescents (9-19 years for girls and 11-19 years for boys), and adults (age ≥ 20 years) with newly diagnosed CML-CP receiving imatinib as first-line therapy between 2006 and 2019 were retrospectively reviewed. In total, 135 children (cohort 1), 189 adolescents (cohort 2), and 658 adults (cohort 3: age 20-39 years, n = 305; cohort 4: age 40-59 years, n = 270; and cohort 5: age 60-83 years, n = 83) were included in this study. When compared with children, adolescents showed a significantly higher white blood cell count (P = 0.033) and basophil percentage in peripheral blood (P = 0.002) and a significantly higher prevalence of splenomegaly (P = 0.004). Both children and adolescents presented with more aggressive clinical features than adults. During median follow-ups of 28 months (range, 3-161 months) in children, 33 months (range, 3-152 months) in adolescents, and 48 months (range, 3-157 months) in adults, multivariate analysis showed that children and adolescents had higher probabilities of achieving complete cytogenetic response, major molecular response, and molecular response4.5. Notably, compared with not only adults (cohort 3 vs. cohort 1: HR = 2.03 [1.03, 3.98], P = 0.040; cohort 4 vs. cohort 1: HR = 2.15 [1.07, 4.33], P = 0.033; cohort 5 vs. cohort 1: HR = 4.22 [1.94, 9.15], P < 0.001) but also adolescents (cohort 2 vs. cohort 1: HR = 2.36 [1.18, 4.72], P = 0.015), children had significantly longer failure-free survival. Age was not associated with progression-free survival or overall survival. Although they exhibited more aggressive clinical features at diagnosis, both children and adolescents achieved superior treatment responses than adults. Adolescents showed even more adverse features and a poor FFS than children.

Identification of a novel TNRC18-RARA fusion in acute promyelocytic leukemia lacking t(15;17)(q24;q12)/PML-RARA.

Acute promyelocytic leukemia (APL) is a unique disease entity in acute myeloid leukemia, characterized by PML-RARA fusion gene, which is generated by chromosomal translocation t(15;17)(q24;q21). We identified TNRC18-RARA as novel RARA fusion in resembling APL. Our study highlights the importance of combining multiple molecular techniques to characterize and optimally manage APL lacking classic t(15;17)(q24;q12)/PML-RARA fusion.

Paeoniflorin ameliorates rheumatoid arthritis in rat models through oxidative stress, inflammation and cyclooxygenase 2.

Paeoniflorin has anti-inflammatory, anti-allergy, immune regulatory and pain-relieving effects, amongst other roles. However, the mechanisms underlying the protective effects of paeoniflorin on rheumatoid arthritis (RA) remain under investigation; the objective of the current study was to evaluate these protective effects in the context of an RA model. Rats were randomly divided into 5 groups, as follows: The control group, the RA rat model group, and the paeoniflorin groups, in which paeoniflorin was administered at concentrations of 5, 10 and 20 mg/kg for 3 weeks. The pain thresholds and arthritic symptoms of the RA rats were measured. Oxidative stress and inflammatory cytokines were also analyzed and western blot analysis was used to evaluate cyclooxygenase-2 (COX-2) protein expression levels. Paeoniflorin significantly increased the pain threshold and decreased the arthritic symptoms in the RA rat model. Notably, paeoniflorin reduced the malondialdehyde concentration and increased the activity of superoxide dismutase, catalase and glutathione peroxidase. Furthermore, paeoniflorin attenuated the activity of nuclear factor-κB p65 unit, tumor necrosis factor-α, interleukin (IL)-1ß and IL-6, and reduced the COX-2 protein expression level. The present study indicates that paeoniflorin ameliorates disease in rat models of RA through oxidative stress, inflammation and alterations to COX-2 expression.

Changes of glucocorticoid receptor isoforms expression in acute lymphoblastic leukemia correlate with glucocorticoid resistance.

Alternative splicing of the glucocorticoid receptor (GR) gene results in several GR isoforms, we examined their expression (GRα, GRß, GRγ and GR-P) by real-time RT-PCR in glucocorticoid (GC) sensitive (CEM-C7), GC resistant (CEM-C1) cells and adult acute lymphoblastic leukemia (ALL) patients, to determine the association of GR isoform expression profiles and GC resistance in adult ALL patients. With GC treatment, GR levels in C1 cells showed no obvious changes. In C7 cells, the mRNA levels of GRα, GRß and GRγ first increased and then decreased, whereas GR-P mRNA had a continued rising trend. C7 cells had a higher GRα/GRγ, lower GRα/GR-P and GRγ/GR-P ratios than C1 cells (P < 0.01). In adult ALL patients, GRγ mRNA varied in different ALL stages (complete remission CR 15.82 vs. relapsed 8.21 vs. initial 1.93 P < 0.05). It also did in the ratios between GR isoforms that GRα/GRγ and GRα/GR-P in initial patients were higher than relapsed and CR (P < 0.05), while GRγ/GR-P in CR was higher than initial and relapsed patients (P < 0.05). GR-P mRNA in T-ALL patients was much higher than that in B-ALL patients (P < 0.05). Peripheral blood hemoglobin (HB) was positively correlated with GRα mRNA and GR-P mRNA (P < 0.05), while white blood cells (WBC) negatively correlated with GRγ mRNA (P < 0.05). The present study demonstrates that GR autoinduction is more important to GC sensitivity than its basal level expression. GC sensitivity is also significantly correlated with GRα mRNA and mildly associated with GRß mRNA expression. Both GRγ mRNA and the ratios between GR isoforms (GRα/GRγ, GRα/GR-P and GRγ/GR-P) are correlated with ALL stages. The changes of mRNA expression levels of GRα, GR-P and GRγ may provide valuable information for GC resistance. Peripheral blood HB and WBC affect GR isoform expression.

Low expression of glucocorticoid receptor alpha isoform in adult immune thrombocytopenia correlates with glucocorticoid resistance.

The expression of glucocorticoid receptor (GR) isoforms has been linked to glucocorticoid (GC) resistance in various diseases treated with GC. However, existing data are conflicting in these diseases, and little information is available regarding immune thrombocytopenia (ITP). To further investigate the role of GR isoforms in GC resistance in adult ITP patients, we measured the mRNA expression of GR isoforms (GRα, GRß, GRγ, GRp) in peripheral blood mononuclear cells (PBMC) from 54 newly diagnosed ITP patients, including GC-sensitive (GCS) and GC-resistant (GCR) patients and 35 healthy volunteers. The GRα and GRß proteins in PBMC, nuclear factor-κB (NF-κB), and activator protein-1 (AP-1) in the nucleus were detected by Western blotting. Compared to normal subjects, both GRα and GRß mRNAs were significantly increased in ITP patients (p < 0.05), while there was no significant difference in the mRNA expression of GRγ and GRp. Compared to GCR patients, the expressions of GRα mRNA and GRα protein were significantly higher in GCS patients (p < 0.05). Moreover, no significant difference in the mRNA expression of the GRß, GRγ, and GRp isoforms was observed between GCS and GCR patients and the GRß protein could not be detected. Compared to GCS group, the expression of p65/NF-κB was significantly higher in the GCR group (p < 0.05). Overall, we did not find differences in c-Jun/AP-1 protein expression between GCS and GCR patients. In summary, GC resistance in adult ITP patients is associated with a reduced expression of GRα, which may be related with increased NF-κB. GRß was very low and may not be involved in GC resistance in adult ITP, warranting further exploration.

[Identification of acute leukemia-specific genes from leukemia recipient/sibling donor pairs by distinguishing study with oligonucleotide microarrays].

To explore differentially expressed genes in leukemia gene expression profile and identify main related genes in acute leukemia, gene expression profiles were analyzed in bone marrow/leucopheresis peripheral blood stem cells samples from 9 acute leukemia patients and their sibling donors with the use of oligonucleotide microarrays. 163 reported leukemia-related genes were involved in the study. The oligonucleotide primers were designed, synthesized and spotted on the chemical-material-coated-glass plates in array. The total RNAs were isolated from nine patients' bone marrow or leucopheresis peripheral blood cells and from nine their sibling donors peripheral blood stem cells treated by G-CSF, then collected by CS-3000 cell selection machine, and were reversely transcribed to cDNAs with the incorporations of fluorescent dUTP. The mixed probes were then hybridized to the oligonucleotide microarray. The results showed that in four patient/donor pairs with B-ALL, 5 up-regulated (RIZ, STK-1, T-cell leukemia/lymphoma 1A, Cbp/p300, Op18) and 1 down-regulated genes (hematopoietic proteoglycan core protein) were identified; In five patient/donor pairs with AML-M(4) and AML-M(5), 6 up-regulated (STAT5B, ligand p62 for the Lck SH2, CST3, LTC4S, myeloid leukemia factor 2 and epb72) and 1 down-regulated genes (CCR5) were identified. In conclusion, on the basis of distinguishing study of specific genetic related recipient/sibling donor pairs, screening leukemia-related genes with oligonucleotide microarrays, a set of 13 up-regulated or down-regulated genes among 163 leukemia-related genes has been identified. The result has further confirmed that above genes play critical role in the molecular mechanism of acute leukemia.