Bone marrow macrophages are involved in the ineffective hematopoiesis of myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) are a group of heterogeneous myeloid clonal disorders characterized by ineffective hematopoiesis. Accumulating evidence has shown that macrophages (MΦs) are important components in the regulation of tumor progression and hematopoietic stem cells (HSCs). However, the roles of bone marrow (BM) MΦs in regulating normal and malignant hematopoiesis in different clinical stages of MDS are largely unknown. Age-paired patients with lower-risk MDS (N = 15), higher-risk MDS (N = 15), de novo acute myeloid leukemia (AML) (N = 15), and healthy donors (HDs) (N = 15) were enrolled. Flow cytometry analysis showed increased pro-inflammatory monocyte subsets and a decreased classically activated (M1) MΦs/alternatively activated (M2) MΦs ratio in the BM of patients with higher-risk MDS compared to lower-risk MDS. BM MФs from patients with higher-risk MDS and AML showed impaired phagocytosis activity but increased migration compared with lower-risk MDS group. AML BM MΦs showed markedly higher S100A8/A9 levels than lower-risk MDS BM MΦs. More importantly, coculture experiments suggested that the HSC supporting abilities of BM MΦs from patients with higher-risk MDS decreased, whereas the malignant cell supporting abilities increased compared with lower-risk MDS. Gene Ontology enrichment comparing BM MΦs from lower-risk MDS and higher-risk MDS for genes was involved in hematopoiesis- and immunity-related pathways. Our results suggest that BM MΦs are involved in ineffective hematopoiesis in patients with MDS, which indicates that repairing aberrant BM MΦs may represent a promising therapeutic approach for patients with MDS.

Continuous constant potential model for describing the potential-dependent energetics of CO2RR on single atom catalysts.

In this work, we have proposed a Continuous Constant Potential Model (CCPM) based on grand canonical density functional theory for describing the electrocatalytic thermodynamics on single atom electrocatalysts dispersed on graphene support. The linearly potential-dependent capacitance is introduced to account for the net charge variation of the electrode surface and to evaluate the free energetics. We have chosen the CO2 electro-reduction reaction on single-copper atom catalysts, dispersed by nitrogen-doped graphene [CuNX@Gra (X = 2, 4)], as an example to show how our model can predict the potential-dependent free energetics. We have demonstrated that the net charges of both catalyst models are quadratically correlated with the applied potentials and, thus, the quantum capacitance is linearly dependent on the applied potentials, which allows us to continuously quantify the potential effect on the free energetics during the carbon dioxide reduction reaction instead of confining it to a specific potential. On the CuN4@Gra model, it is suggested that CO2 adsorption, coupled with an electron transfer, is a potential determining step that is energetically unfavorable even under high overpotentials. Interestingly, the hydrogen adsorption on CuN4@Gra is extremely easy to occur at both the Cu and N sites, which probably results in the reconstruction of the CuN4@Gra catalyst, as reported by many experimental observations. On CuN2@Gra, the CO2RR is found to exhibit a higher activity at the adjacent C site, and the potential determining step is shifted to the *CO formation step at a wide potential range. In general, CCPM provides a simple method for studying the free energetics for the electrocatalytic reactions under constant potential.

Prophylactic NAC promoted hematopoietic reconstitution by improving endothelial cells after haploidentical HSCT: a phase 3, open-label randomized trial.

BACKGROUND: Poor graft function (PGF) or prolonged isolated thrombocytopenia (PT), which are characterized by pancytopenia or thrombocytopenia, have become serious complications after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Our previous single-arm trial suggests that N-acetyl-L-cysteine (NAC) prophylaxis reduced PGF or PT after allo-HSCT. Therefore, an open-label, randomized, phase 3 trial was performed to investigate the efficacy and tolerability of NAC prophylaxis to reduce PGF or PT after allo-HSCT. METHODS: A phase 3, open-label randomized trial was performed. Based on the percentage of CD34+VEGFR2 (CD309)+ endothelial cells (ECs) in bone marrow (BM) detected by flow cytometry at 14 days before conditioning, patients aged 15 to 60 years with acute leukemia undergoing haploidentical HSCT were categorized as low-risk (EC ≥ 0.1%) or high-risk (EC < 0.1%); patients at high risk were randomly assigned (2:1) to receive NAC prophylaxis or nonprophylaxis. The primary endpoint was PGF and PT incidence at +60 days post-HSCT. RESULTS: Between April 18, 2019, and June 24, 2021, 120 patients with BM EC <0.1% were randomly assigned for NAC (group A, N = 80) or nonprophylaxis (group B, N = 40), and 105 patients with EC≥0.1% (group C) were also analyzed. The +60 days incidence of PGF and PT was 7.5% (95% CI, 1.7 to 13.3%) and 22.5% (95% CI, 9.1 to 35.9%) in group A and group B (hazard ratio, 0.317; 95% CI, 0.113 to 0.890; P = 0.021) and 11.4% (95% CI, 5.2 to 17.6%) in group C (hazard ratio, 0.643; 95% CI, 0.242 to 1.715; P = 0.373). Consistently, NAC prophylaxis gradually improved BM ECs and CD34+ cells in group A, whereas reduced their reactive oxygen species (ROS) levels post-HSCT. Within 60 days post-HSCT, the most common grade 3 to 5 adverse events for the NAC and control groups were infections (19/80 [24%] vs. 10/40 [25%]) and gastrointestinal adverse events (16/80 [20%] vs. 7/40 [18%]). There were no treatment-related deaths. CONCLUSIONS: N-Acetyl-L-cysteine prophylaxis can prevent the occurrence of poor hematopoietic function and is well tolerated in haploidentical HSCT. It may offer a potential pathogenesis-oriented therapeutic approach for patients with poor hematopoietic function. TRIAL REGISTRATION: This trial was registered at ClinicalTrials.gov as #NCT03967665.

Dysfunctional bone marrow endothelial progenitor cells are involved in patients with myelodysplastic syndromes.

BACKGROUND: Myelodysplastic syndromes (MDS) are a group of heterogeneous myeloid clonal disorders characterized by ineffective haematopoiesis and immune deregulation. Emerging evidence has shown the effect of bone marrow (BM) endothelial progenitor cells (EPCs) in regulating haematopoiesis and immune balance. However, the number and functions of BM EPCs in patients with different stages of MDS remain largely unknown. METHODS: Patients with MDS (N = 30), de novo acute myeloid leukaemia (AML) (N = 15), and healthy donors (HDs) (N = 15) were enrolled. MDS patients were divided into lower-risk MDS (N = 15) and higher-risk MDS (N = 15) groups according to the dichotomization of the Revised International Prognostic Scoring System. Flow cytometry was performed to analyse the number of BM EPCs. Tube formation and migration assays were performed to evaluate the functions of BM EPCs. In order to assess the gene expression profiles of BM EPCs, RNA sequencing (RNA-seq) were performed. BM EPC supporting abilities of haematopoietic stem cells (HSCs), leukaemia cells and T cells were assessed by in vitro coculture experiments. RESULTS: Increased but dysfunctional BM EPCs were found in MDS patients compared with HDs, especially in patients with higher-risk MDS. RNA-seq indicated the progressive change and differences of haematopoiesis- and immune-related pathways and genes in MDS BM EPCs. In vitro coculture experiments verified that BM EPCs from HDs, lower-risk MDS, and higher-risk MDS to AML exhibited a progressively decreased ability to support HSCs, manifested as elevated apoptosis rates and intracellular reactive oxygen species (ROS) levels and decreased colony-forming unit plating efficiencies of HSCs. Moreover, BM EPCs from higher-risk MDS patients demonstrated an increased ability to support leukaemia cells, characterized by increased proliferation, leukaemia colony-forming unit plating efficiencies, decreased apoptosis rates and apoptosis-related genes. Furthermore, BM EPCs induced T cell differentiation towards more immune-tolerant cells in higher-risk MDS patients in vitro. In addition, the levels of intracellular ROS and the apoptosis ratios were increased in BM EPCs from MDS patients, especially in higher-risk MDS patients, which may be therapeutic candidates for MDS patients. CONCLUSION: Our results suggest that dysfunctional BM EPCs are involved in MDS patients, which indicates that improving haematopoiesis supporting ability and immuneregulation ability of BM EPCs may represent a promising therapeutic approach for MDS patients.

Major osteoporosis fracture prediction in type 2 diabetes: a derivation and comparison study.

The widely recommended fracture prediction tool FRAX was developed based on and for the general population. Although several adjusted FRAX methods were suggested for type 2 diabetes (T2DM), they still need to be evaluated in T2DM cohort. INTRODUCTION: This study was undertaken to develop a prediction model for Chinese diabetes fracture risk (CDFR) and compare its performance with those of FRAX. METHODS: In this retrospective cohort study, 1730 patients with T2DM were enrolled from 2009.08 to 2013.07. Major osteoporotic fractures (MOFs) during follow-up were collected from Electronic Health Records (EHRs) and telephone interviews. Multivariate Cox regression with backward stepwise selection was used to fit the model. The performances of the CDFR model, FRAX, and adjusted FRAX were compared in the aspects of discrimination and calibration. RESULTS: 6.3% of participants experienced MOF during a median follow-up of 10 years. The final model (CDFR) included 8 predictors: age, gender, previous fracture, insulin use, diabetic peripheral neuropathy (DPN), total cholesterol, triglycerides, and apolipoprotein A. This model had a C statistic of 0.803 (95%CI 0.761-0.844) and calibration χ2 of 4.63 (p = 0.86). The unadjusted FRAX underestimated the MOF risk (calibration χ2 134.5, p < 0.001; observed/predicted ratio 2.62, 95%CI 2.17-3.08), and there was still significant underestimation after diabetes adjustments. Comparing FRAX, the CDFR had a higher AUC, lower calibration χ2, and better reclassification of MOF. CONCLUSION: The CDFR model has good performance in 10-year MOF risk prediction in T2DM, especially in patients with insulin use or DPN. Future work is needed to validate our model in external cohort(s).

Venom-gland transcriptomic, venomic, and antivenomic profiles of the spine-bellied sea snake (Hydrophis curtus) from the South China Sea.

BACKGROUND: A comprehensive evaluation of the -omic profiles of venom is important for understanding the potential function and evolution of snake venom. Here, we conducted an integrated multi-omics-analysis to unveil the venom-transcriptomic and venomic profiles in a same group of spine-bellied sea snakes (Hydrophis curtus) from the South China Sea, where the snake is a widespread species and might generate regionally-specific venom potentially harmful to human activities. The capacity of two heterologous antivenoms to immunocapture the H. curtus venom was determined for an in-depth evaluation of their rationality in treatment of H. curtus envenomation. In addition, a phylogenetic analysis by maximum likelihood was used to detect the adaptive molecular evolution of full-length toxin-coding unigenes. RESULTS: A total of 90,909,384 pairs of clean reads were generated via Illumina sequencing from a pooled cDNA library of six specimens, and yielding 148,121 unigenes through de novo assembly. Sequence similarity searching harvested 63,845 valid annotations, including 63,789 non-toxin-coding and 56 toxin-coding unigenes belonging to 22 protein families. Three protein families, three-finger toxins (3-FTx), phospholipase A2 (PLA2), and cysteine-rich secretory protein, were detected in the venom proteome. 3-FTx (27.15% in the transcriptome/41.94% in the proteome) and PLA2 (59.71%/49.36%) were identified as the most abundant families in the venom-gland transcriptome and venom proteome. In addition, 24 unigenes from 11 protein families were shown to have experienced positive selection in their evolutionary history, whereas four were relatively conserved throughout evolution. Commercial Naja atra antivenom exhibited a stronger capacity than Bungarus multicinctus antivenom to immunocapture H. curtus venom components, especially short neurotoxins, with the capacity of both antivenoms to immunocapture short neurotoxins being weaker than that for PLA2s. CONCLUSIONS: Our study clarified the venom-gland transcriptomic and venomic profiles along with the within-group divergence of a H. curtus population from the South China Sea. Adaptive evolution of most venom components driven by natural selection appeared to occur rapidly during evolutionary history. Notably, the utility of commercial N. atra and B. multicinctus antivenoms against H. curtus toxins was not comprehensive; thus, the development of species-specific antivenom is urgently needed.

Improved function and balance in T cell modulation by endothelial cells in young people.

Elderly individuals exhibit unbalanced bone marrow (BM) effector T cell subset differentiation, such as increased T helper type 1 (Th1) and T cytotoxic type 1 (Tc1) cell frequencies, but the underlying mechanism is still unclear. Endothelial cells (ECs), which are instructive components of the BM microenvironment, exhibit the phenotype of semi-professional antigen-presenting cells and regulate T cell recruitment and activation. Thus, we compared the frequency and function of BM ECs, especially their capacity to regulate effector T cell subsets, between young and elderly healthy individuals, and explored the underlying mechanism of this immunomodulatory discrepancy. Although the young and elderly EC percentages were comparable, young ECs showed fewer reactive oxygen species and better migratory and tube-forming abilities than elderly ECs. Notably, increased T cell activation molecules and inflammatory cytokines were found in elderly ECs which regulated T cells to differentiate into more proinflammatory T cells, including Th1 and Tc1 cells, than young ECs.

Induced defense and its cost in two bryophyte species.

PREMISE: Current knowledge about defense strategies in plants under herbivore pressure is predominantly based on vascular plants. Bryophytes are rarely consumed by herbivores since they have ample secondary metabolites. However, it is unknown whether bryophytes have induced defenses against herbivory and whether there is a trade-off between growth and defense in bryophytes. METHODS: In an experiment with two peatland bryophytes, Sphagnum magellanicum Brid. and S. fuscum (Schimp.) H. Klinggr., two kinds of herbivory, clipping with scissors and grazing by mealworms (Tenebrio molitor L.) were simulated. At the end of the experiment, we measured growth traits, carbon-based defense compounds (total phenolics and cellulose) and storage compounds (total nonstructural carbohydrates) of these two Sphagnum species. RESULTS: Grazing but not clipping increased total phenolics and C:N ratio and reduced biomass production and height increment. A negative relationship between biomass production and total phenolics was found in S. magellanicum but not in S. fuscum, indicating a growth-defense trade-off that is species-specific. Grazing reduced the sugar starch content of S. magellanicum and the sugar of S. fuscum. Either clipping or grazing had no effect on chlorophyll fluorescence (including actual and maximum photochemical efficiency of photosystem II) except that a significant effect of clipping on actual photochemical efficiency in S. fuscum was observed. CONCLUSIONS: Our results suggest that Sphagnum can have induced defense against herbivory and that this defense can come at a cost of growth. These findings advance our knowledge about induced defense in bryophytes, the earliest land plants.

Monocyte subsets in bone marrow grafts may contribute to a low incidence of acute graft-vs-host disease for young donors.

Young donors are associated with a lower cumulative incidence of acute graft-vs-host disease (aGVHD) after allogenic haematopoietic stem cell transplantation (allo-HSCT) than old donors. Although grafts are harvested from healthy donors, it is unclear whether donor age is associated with aGVHD occurrence owing to its effect on cell compositions in grafts. Moreover, the differences in monocyte subsets in grafts between young and old donors and the association between monocyte subsets in bone marrow (BM) grafts and aGVHD remain to be elucidated. In the current study, non-classical monocytes and the CD4+ /CD8+ T cell ratio were remarkably decreased in BM grafts in donors <30 years old. Multivariate analysis further revealed that the level of non-classical monocytes in BM grafts (≥0.31 × 106 /kg) was an independent risk factor for the occurrence of II-IV aGVHD. In summary, our data indicate that non-classical monocytes in BM grafts may help identify patients at high risk for aGVHD after allo-HSCT. Although further validation is required, our results suggest that the low level of non-classical monocytes and a low ratio of CD4+ /CD8+ T cell in BM grafts may be correlated with the lower incidence of aGVHD in young donors.

Investigation of the molecular characteristics of Brucella isolates from Guangxi Province, China.

BACKGROUND: Human brucellosis has become a severe public health problem in China's Guangxi Province, and there has been higher prevalence of brucellosis in this region after 2010. Both multiple locus variable-number tandem repeat analysis (MLVA) and multilocus sequence typing (MLST) assay schedules were used to genotype isolates and determine relationships among isolates. RESULTS: A total of 40 isolates of Brucella were obtained from humans, pigs, and dogs from 1961 to 2016. There were at least three species of Brucella detected in Guangxi Province, Brucella melitensis, Brucella suis, and Brucella canis, with 16, 17, and 7 isolates, respectively. Of which B. suis biovar 3 was the predominant species resulting in pig brucellosis in the area examined before 2000s. Moreover, B. melitensis biovar 3 was found to be mainly responsible for human brucellosis during 2012-2016. All B. melitensis isolates in this study belonged to East Mediterranean lineage. MLVA-11 genotype 116 was the dominant genotype and represented 81.2% of the isolates. MLVA cluster analysis showed there to be 44% (7/16) brucellosis cases caused by B. melitensis with a profile of outbreak epidemic from 2012 to 2016. However, nearly 83.3% (20/24) of brucellosis cases resulting from both B. suis and B. canis showed no epidemiological links or sporadic characteristics. MLVA-16 analysis confirmed extensive genotype-sharing events between B. melitensis isolates from Guangxi and other northern provinces within China. These data revealed that there are potential epidemiology links among these strains. B. suis strains of this study showed a unique genetic lineage at the global level and may have existed historically in this area. However, present B. canis isolates were closely related to previously reported isolates in Korea, where they may have originated. MLST typing showed that the population structure of Brucella strains had changed considerably in this province; ST17 and ST21, two previously predominant populations appeared to have been replaced by recently emerging ST8 group. CONCLUSIONS: Our investigation data have inspired the hypothesis that Guangxi Province had been subject to an imported human brucellosis epidemic. Our data suggest that strains found in Northern regions of China are the principal source of infections in recent cases of human brucellosis in Guangxi Province. Comparative genomic analysis from more strains is necessary to confirm this hypothesis. This work will facilitate better understanding of the epidemiology and improve the effectiveness of control and prevention of brucellosis in this region.

The effects of autophagy on the replication of Nelson Bay orthoreovirus.

BACKGROUND: Nelson Bay orthoreovirus (NBV) was first isolated over 40 years ago from a fruit bat in Australia. Normally, NBV does not cause human diseases, but recently several NBV strains have been associated with human respiratory tract infections, thus attracting clinical attention. Autophagy, an evolutionarily conserved process in eukaryotic cells, degrades intracellular substrates, participates in multiple physiological processes, and maintains cellular homeostasis. In addition, autophagy is intimately involved in viral infection. METHODS: A new strain of NBV, isolated from a patient with a respiratory tract infection who returned to Japan from Bali, Indonesia, in 2007, was used in this study. NBV was rescued using a reverse genetics system involving cotransfection of BHK cells with 11 plasmids (pT7-L1 MB, pT7-L2 MB, pT7-L3 MB, pT7-M1 MB, pT7-M2 MB, pT7-M3 MB, pT7-S1 MB, pT7-S2 MB, pT7-S3 MB, pT7-S4 MB, and pcDNA3.1-T7), yielding NBV-MB. Recovered viruses were confirmed by immunofluorescence. The effect of NBV-MB on autophagy was evaluated by measuring the LC3-I/II proteins by immunoblot analysis after infection of BHK cells. Furthermore, after treatment with rapamycin (RAPA), 3-methyladenine (3-MA), chloroquine (CQ), or plasmid (GFP-LC3) transfection, the changes in expression of the LC3 gene and the amount of LC3-I/II protein were examined. In addition, variations in viral titer were assayed after treatment of BHK cells with drugs or after transfection with plasmids pCAGM3 and pCAGS3, which encode virus nonstructural proteins µNS and σNS, respectively. RESULTS: NBV-MB infection induced autophagy in host cells; however, the level of induction was dependent on viral replication. Induction of autophagy increased viral replication. By contrast, inhibiting autophagy suppressed NBV replication, albeit not significantly. The NBV-MB nonstructural protein µNS was involved in the induction of autophagy with viral infection. CONCLUSIONS: NBV-MB infection triggered autophagy. Also, the NBV nonstructural protein µNS may contribute to augmentation of autophagy upon viral infection.

PITUITARY STALK THICKENING IN A LARGE COHORT: TOWARD MORE ACCURATE PREDICTORS OF PITUITARY DYSFUNCTION AND ETIOLOGY.

Objective: To summarize the characteristics of patients with pituitary stalk thickening, analyze the association between pituitary stalk width and hypopituitarism, and develop a diagnostic model to differentiate neoplastic and inflammatory origins. Methods: A total of 325 patients with pituitary stalk thickening in a tertiary teaching hospital between January 2012 and February 2018 were enrolled. Basic characteristics and hormonal status were evaluated. Indicators to predict etiology in patients with histologic diagnoses were analyzed. Results: Of the 325 patients, 62.5% were female. Deficiency in gonadotropin was most common, followed by corticotropin, growth hormone, and thyrotropin. The increase in pituitary stalk width was associated with a risk of central diabetes insipidus (odds ratio [OR], 3.57; P<.001) and with a combination of central diabetes insipidus and anterior pituitary deficiency (OR, 2.28; P = .029). The cut-off pituitary stalk width of 4.75 mm had a sensitivity of 69.2% and a specificity of 71.4% for the presence of central diabetes insipidus together with anterior pituitary deficiency. Six indicators (central diabetes insipidus, pattern of pituitary stalk thickening, pituitary stalk width, neutrophilic granulocyte percentage, serum sodium level, and gender) were used to develop a model having an accuracy of 95.7% to differentiate neoplastic from inflammatory causes. Conclusion: Pituitary stalk width could indicate the presence of anterior pituitary dysfunction, especially in central diabetes insipidus patients. With the use of a diagnostic model, the neoplastic and inflammatory causes of pituitary stalk thickening could be preliminarily differentiated. Abbreviations: APD = anterior pituitary dysfunction; AUC = area under the curve; CDI = central diabetes insipidus; GH = growth hormone; MRI = magnetic resonance imaging; OR = odd ratio; PHBS = posterior hypophyseal bright spots; PST = pituitary stalk thickening; PSW = pituitary stalk width.

Dysfunctional Bone Marrow Mesenchymal Stem Cells in Patients with Poor Graft Function after Allogeneic Hematopoietic Stem Cell Transplantation.

Poor graft function (PGF) is a life-threatening complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT) and is characterized by defective hematopoiesis. Mesenchymal stem cells (MSCs) have been shown to support hematopoiesis, but little is known about the role of MSCs in the pathogenesis of PGF. In the current prospective case-control study, we evaluated whether the number and function of bone marrow (BM) MSCs in PGF patients differed from those in good graft function (GGF) patients. We found that BM MSCs from PGF patients expanded more slowly and appeared flattened and larger, exhibiting more apoptosis and senescence than MSCs from GGF patients. Furthermore, increased intracellular reactive oxygen species, p-p53, and p21 (but not p38) levels were detected in MSCs from PGF patients. Moreover, the ability of MSCs to sustain hematopoiesis was significantly reduced in PGF patients, as evaluated by cell number, apoptosis, and the colony-forming unit-plating efficiency of CD34+ cells. In summary, the biologic characteristics of PGF MSCs are different from those of GGF MSCs, and the in vitro hematopoiesis-supporting ability of PGF MSCs is significantly lower. Although requiring further validation, our study indicates that reduced and dysfunctional BM MSCs may contribute to deficient hematopoiesis in PGF patients. Therefore, improvement of BM MSCs may represent a promising therapeutic approach for PGF patients after allo-HSCT.

N-acetyl-L-cysteine improves mesenchymal stem cell function in prolonged isolated thrombocytopenia post-allotransplant.

Prolonged isolated thrombocytopenia (PT) is a serious complication of allogeneic haematopoietic stem cell transplantation (allo-HSCT). Murine studies and in vitro experiments suggest that mesenchymal stem cells (MSCs) can, not only to support haematopoiesis, but also preferentially support megakaryocytopoiesis in bone marrow (BM). However, little is known about the quantity and function of BM MSCs in PT patients. In a case-control study, we found that BM MSCs from PT patients exhibited significantly reduced proliferative capacities, increased reactive oxygen species and senescence. Antioxidant (N-acetyl-L-cysteine, NAC) treatment in vitro not only quantitatively and functionally improved BM MSCs derived from PT patients through down-regulation of the p38 (also termed MAPK14) and p53 (also termed TP53) pathways but also partially rescued the impaired ability of BM MSCs to support megakaryocytopoiesis. Subsequently, a pilot study showed that the overall response of NAC treatment was obtained in 7 of the enrolled PT patients (N = 10) without significant side effects. Taken together, the results indicated that dysfunctional BM MSCs played a role in the pathogenesis of PT and the impaired BM MSCs could be improved by NAC in vitro. Although requiring further validation, our data indicate that NAC might be a potential therapeutic approach for PT patients after allo-HSCT.

Impairment of bone marrow endothelial progenitor cells in acute graft-versus-host disease patients after allotransplant.

Graft-versus-host disease (GVHD) is a major complication after allogeneic haematopoietic stem cell transplantation (allo-HSCT) that is frequently associated with bone marrow (BM) suppression, and clinical management is challenging. BM endothelial progenitor cells (EPCs) play crucial roles in the regulation of haematopoiesis and thrombopoiesis. However, little is known regarding the functional roles of BM EPCs in acute GVHD (aGVHD) patients. In the current prospective case-control study, reduced and dysfunctional BM EPCs, characterized by decreased migration and angiogenesis capacities and increased levels of reactive oxygen species (ROS) and apoptosis, were found in aGVHD patients compared with those without aGVHD. Moreover, lower frequency and increased levels of ROS, apoptosis and DNA damage, but reduced colony-forming unit-plating efficiency were found in BM CD34+ cells of aGVHD patients compared with those without aGVHD. The severity of aGVHD and GVHD-mediated cytopenia was associated with BM EPC impairment in aGVHD patients. In addition, the EPC impairment positively correlated with ROS level. Taken together, our results suggest that reduced and dysfunctional BM EPCs may be involved in the pathogenesis of aGVHD. Although these findings require validation, our data indicate that improvement of BM EPCs may represent a promising therapeutic approach for aGVHD patients.

An unbalanced monocyte macrophage polarization in the bone marrow microenvironment of patients with poor graft function after allogeneic haematopoietic stem cell transplantation.

Poor graft function (PGF) is a severe complication of allogeneic haematopoietic stem cell transplantation (allo-HSCT). Murine studies have demonstrated that effective haematopoiesis depends on the specific bone marrow (BM) microenvironment. Increasing evidence shows that BM macrophages (MФs), which constitute an important component of BM immune microenvironment, are indispensable for the regulation of haematopoietic stem cells (HSCs) in the BM. However, little is known about the number and function of BM MФs or whether they directly interact with HSCs in PGF patients. In the current prospective case-control study, PGF patients showed a significant increase in classically activated inflammatory MФs (M1; 2·18 ± 0·11% vs. 0·82 ± 0·06%, P < 0·0001), a striking reduction in alternatively activated anti-inflammatory MФs (M2; 3·02 ± 0·31% vs. 21·89 ± 0·90%, P < 0·0001), resulting in a markedly increased M1/M2 ratio (0·82 ± 0·06 vs. 0·06 ± 0·002; P < 0·0001) in the BM compared with good graft function patients. Meanwhile, standard monocyte subsets were altered in PGF patients. Dysfunctional BM MФs, which were characterized by reduced proliferation, migration and phagocytosis, were evident in PGF patients. Furthermore, BM MФs from PGF patients with high tumour necrosis factor-α and interleukin 12 levels and low transforming growth factor-ß levels, led to impaired BM CD34+ cell function. In summary, our data indicate that an unbalanced BM M1/M2 ratio and dysfunctional MФs may contribute to the occurrence of PGF following allo-HSCT.

1-α,25-dihydroxyvitamin D3 potentiates avian osteoclast activation by increasing the formation of zipper-like structure via Src/Rac1 signaling.

Avian bone metabolism diseases affect the development and production of chickens, and many of these diseases can be prevented and controlled by balanced nutrition and hormone medicine. The steroid hormone 1α,25-dihydroxyvitamin D3 plays a key role in maintaining the balance of avian bone metabolism. Clinically, 1α,25-(OH)2D3 has been used to treat several bone diseases. Although several previous studies have investigated the effects of 1α,25-(OH)2D3 on osteoclastogenesis, the mechanisms underpinning osteoclast (OC) activity remain largely unknown. Herein, we used molecular and cell biology approaches to demonstrate that 1α,25-(OH)2D3 increases avian OC formation and activity, and upregulates bone resorption-related genes. Moreover, 1α,25-(OH)2D3 regulates the OC cytoskeleton by increasing the formation of zipper-like structure in OC precursor cells to potentiate OC activity via the Src/Rac1 signaling pathway. These findings provide new insight into the role of 1α,25-(OH)2D3 in OC activity.

Engineering Bacillus pumilus alkaline serine protease to increase its low-temperature proteolytic activity by directed evolution.

BACKGROUND: Mesophilic alkaline serine proteases from various bacteria have been commercially applied in a range of industries owing to their high catalytic efficiency and wide substrate specificity. However, these proteases have an optimal catalytic temperature of approximately 50 °C, and their activity decreases significantly at low temperature. Therefore, to enhance their cold activity, it is necessary to improve the catalytic performance of these proteases at low temperature. The alkaline serine protease (DHAP) from Bacillus pumilus BA06 is a typical mesophilic enzyme, which has demonstrated great potential in various industrial applications. Here we attempted to improve the cold activity of DHAP via directed evolution. RESULTS: Seven variants (P9S, A1G/K27Q, A38V, A116T, T162I, S182R, and T243S) of DHAP from B. pumilus were obtained via directed evolution. The results showed that all of the variants had increased proteolytic activity at 15 °C towards both the casein and synthetic peptide substrates. With the exception of variant T243S, the thermostability of these variants did not decrease in comparison with the wild-type enzyme. Kinetic analysis indicated that the increase in catalytic efficiency was largely attributed to the increase in turnover number (kcat). Furthermore, the combined variants generated by site-directed mutagenesis showed a further increase in specific caseinolytic activity and the kcat value for hydrolysis of the synthetic peptide. The combined variants of P9S/K27Q and P9S/T162I exhibited an approximate 5-fold increase in caseinolytic activity at 15 °C and almost no loss of thermostability. Finally, the possible mechanism responsible for the change in catalytic properties for these variants was interpreted based on structural modeling. CONCLUSIONS: Directed evolution and site-directed mutagenesis were combined to engineer variants of the DHAP from B. pumilus. All of the variants exhibited an increase in hydrolytic efficiency at low temperature towards both of the substrates, casein and synthetic peptide, without any loss of thermostability compared with the wild-type. These data suggest that engineering low-temperature activity for a bacterial protease is not always associated with the loss of thermostability. Furthermore, our findings demonstrate that enhanced cold activity and thermostability could be integrated into a single variant.

Genetic polymorphisms identify in species/biovars of Brucella isolated in China between 1953 and 2013 by MLST.

BACKGROUND: Brucellosis incidence in China is divided into three stages: high incidence (1950s-1960s), decline (1970s-1980s), and re-emergence (1990s-2010s). At the re-emergence stage, Brucellosis incidence grew exponentially and spread to all 32 provinces. We describe the magnitude and the etiological distribution changes in mainland China by genotyping data and emphasize its recent reemergence. We also provide the genetic diversity and molecular epidemiological characteristics of Brucella. RESULTS: From a total of 206 Brucella isolates, 19 MLST genotypes (STs) were identified and 13 new STs(ST71-83)were found. MLST grouped the population into three clusters. B. melitensis, B. abortus and B. suis were grouped into cluster 1, 2 and 3 respectively. The predominant genotype in the first cluster by MLST, remained unchanged during the three stages. However, the proportion of genotypes in the three stages had changed. More isolates were clustered in ST8 at the re-emergence stage. STs71-74, which were not found in the two former stages, appeared at the re-emergence stage. CONCLUSIONS: The changing molecular epidemiology of brucellosis improve our understanding of apparent geographic expansion from the historically affected north of China to southern provinces in recent reemergence.

Leukemia-propagating cells demonstrate distinctive gene expression profiles compared with other cell fractions from patients with de novo Philadelphia chromosome-positive ALL.

Relapse remains one of the major obstacles in Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL) even after allogeneic hematopoietic stem cell transplantation. The persistence of leukemia-propagating cells (LPCs) may lead to the recurrence of Ph+ALL. Using a xenograft assay, LPCs enrichment in the CD34+CD38-CD58- fraction in Ph+ALL was recently identified. A further cohort study indicated that the LPCs phenotype at diagnosis was an independent risk factor for relapse of Ph+ALL. However, little is known about the potential molecular mechanism of LPCs-mediated relapse. Therefore, the gene expression profiles of the sorted LPCs and other cell fractions from patients with de novo Ph+ALL were investigated using RNA sequencing (RNA-Seq). Most of the differentially expressed genes between the LPCs and other cell fractions were related to the regulation of the cell cycle and metabolism, as identified by the gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. Consistent with the RNA-Seq results, the mRNA levels of cell cycle-related genes, such as cyclin-dependent kinase 4, were significantly lower in the LPCs fraction than in other cell fractions. Moreover, the proportion of quiescent cells in LPCs was significantly higher than in other cell fractions. In summary, distinctive gene expression profiles and clusters, which were mostly related to the regulation of the cell cycle and metabolism, were demonstrated between LPCs and other cell fractions from patients with de novo Ph+ALL. Therefore, it would be beneficial to develop novel LPCs-based therapeutic strategies for Ph+ALL patients.