A new pathway for the transformation of AML in MDS: APA mechanism regulated by NUDT21 and RUNX1.

We have identified that NUDT21 plays a vital role in MDS transformations, while the transcription factor RUNX1 is essential for normal hematopoiesis, which is a high expression in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS), and we aim to explore the linkage between the two genes and new pathways for MDS transformation to AML. Prediction of RUNX1 expression levels and its relationship with NUDT21 in AML and MDS patients was performed using bioinformatics techniques and validated in patients. Using lentiviral packaging technology, NUDT21 knockdown and overexpression models were developed in AML and MDS cell lines. These models were validated using quantitative polymerase chain reaction (qPCR) and western blotting. The cell cycle, apoptosis, differentiation, and cytokines were examined by flow cytometry, CCK-8 analyzed proliferation, and the intracellular localization of NUDT21 and RUNX1 was examined by immunofluorescence. mRNA transcriptome sequencing was performed on THP-1, MUTZ-1, and Dapars analyzed SKM-1 cell lines and the sequencing data to observe the knockdown effect of NUDT21 on RUNX1. qPCR and western blot revealed a positive correlation between NUDT21 and RUNX1; both were located in the nucleus. Overexpression of NUDT21 reduced apoptosis, promoted cell proliferation, and possibly increased the invasive ability of cells. It also altered the APA site in the RUNX1 3'-UTRs region. NUDT21 regulates RUNX1 gene expression and promotes AML transformation in MDS through an APA mechanism.

[Impact of CSF3R Mutation on Treatment Response and Survival of Patients with Acute Myeloid Leukemia].

OBJECTIVE: To investigate the expression of CSF3R mutation in acute myeloid leukemia (AML) and analyze its clinical characteristics and prognosis. METHODS: A retrospective study was conducted in 212 patients with AML who were newly diagnosed in the Second Hospital of Shanxi Medical University from January 1th 2018 to June 30th 2021, including 22 patients with CSF3R mutations as mutation group and 190 patients with CSF3R wild type ï¼»66 cases of them were screened by propensity score matching (PSM), as control groupï¼½. The early efficacy and survival between the two groups were compared. RESULTS: The median age of patients in the mutation group was 50(17-73) years old, and the ratio of male to female was 1.2:1 The main types were AML with maturation (11 cases) and acute myelomonocytic leukemia (9 cases). Prognostic stratification was carried out according to the risk stratification system of the European leukemia network in 2017, with 16 cases (72.73%) in the middle and high-risk group. At the initial diagnosis, the median count of white blood cell (WBC) was 44.75(1.30-368.71)×109/L, among which 15 cases (68.18%) were >10×109/L, and the median count of platelet (PLT) was 24(4-55)×109/L. CSF3R T618I (68.18%) was a common mutation site, which had concomitant gene mutations, in which CEBPA mutation was the most common (10 cases, 45.45%), but only existed in CSF3R T618I mutation. The CR/CRi rate was 68.18% and 71.21% in the mutant group and the control group (P >0.05), the median over all survival time was 15 months and 9 months (P >0.05), and the median disease-free survival time was 8 months and 4 months (P >0.05), respectively. CONCLUSION: Most AML patients with CSF3R mutation are middle-aged patients, the main types are AML with maturation and acute myelomonocytic leukemia, and most of them have middle and high-risk prognosis. CSF3R mutation may not be an independent prognostic marker for newly diagnosed AML patients.

[Clinical Significance of Truncated Mutant ΔJBP of TET2 Gene in Patients with Acute Myeloid Leukemia].

OBJECTIVE: To the clinical characteristics and prognostic value of the patients with complete deletion of TET_JBP domain (ΔJBP) in TET2 acute myeloid leukemia (AML). METHODS: Next Generation Sequencing technology was used to determine the mutations of 34 AML-related genes (including TET2 gene). The I-TASSER tool was used to predict the tertiary structure of the full-length TET2 protein and TET_JBP structure deletion. RESULTS: Among 38 AML patients with TET2 mutations, 22(57.9%) showed truncation mutations, of which 16 (72.7%) produced TET2ΔJBP truncation mutants. Protein structure prediction showed that the deletion of TET_JBP domain lead to the significant changes of tertiary structure in TET2 protein. Compared with the patients in non-ΔJBP group, the age of patients in ΔJBP group were older (63 vs 54 years old, P=0.047), and the occurrence rate of CEBPA double mutation (CEBPAdm) were more frequency (31.3% vs 0, P=0.009), the complete remission (CR) rate after induction chemotherapy(37.5% vs 81.8%, P=0.008) were lower, the median EFS (5 vs 19 months, P=0.000) and median OS (16 vs 22 months, P=0.041) were shorter. Univariate analysis showed that platelets <50×109/L (P=0.004) and CEBPAdm (P=0.001) were related to the shorter OS of the patients. Further COX multivariate analysis showed that CEBPAdm is an independent prognostic factors of OS in TET2ΔJBP patients (P=0.010). In addition, ΔJBP patients with CEBPAdm showed lower hemoglobin levels (62 vs 75g/L, P=0.030) and lower median OS (9 months vs 18 months, P=0.000) than the patients without CEBPAdm. CONCLUSION: AML patients with TET2ΔJBP truncation mutant shows lower CR rate, shorter EFS and OS after induction chemotherapy, which may be related to the poor prognosis, and co-mutation with CEBPAdm, which is the independent prognostic factors of OS in AML patients with TET2ΔJBP.

[Quantitative Analysis on Immunophenotype of CD34+ Myeloid Precursor Cells in Myelodysplastic Syndrome and Its Correlation with Clinical Features].

OBJECTIVE: To investigate the quantitative expression of immunophenotype of CD34+ myeloid precursor cells in myelodysplastic syndrome (MDS) patients and its correlation with clinical characteristics, and understand the effect of quantitative expression of CD7 and CD117 on the prognosis of low-risk MDS patients. METHODS: Multi-parameter flow cytometry (FCM) was used to detect the proportion and mean fluorescence intensity (MFI) of each antigen of bone marrow CD34+ myeloid precursor cells in 79 MDS patients. The correlation between the expression level of each immune marker and clinical characteristics was compared. The effects of quantitative expressions of CD7 and CD117 on the overall survival rate of low-risk patients were explored. RESULTS: Bone marrow blast cell proportion (P<0.01), RBC level (P<0.01), and Hb level (P<0.05) of high-risk MDS patients were higher, while EPO level (P<0.05) was lower than those of low-risk patients. The proportion of CD34+ blast cells (P<0.01), the proportion of CD117 (P<0.05) and the MFI of CD7 (P<0.05) were higher in high-risk patients than those in low-risk patients, but the MFI of CD123 was lower (P<0.05). In high-risk MDS patients, CD15/CD34 (MFI) and CD19/CD34 (MFI) positively correlated with the proportion of total T cells (r=0.458; r=0.505), while CD19/CD34 (%) and CD19/CD34 (MFI) negatively correlated with WBC levels (r=-0.469; r=-0.503). In low-risk MDS patients, CD34+ (%) positively correlated with bone marrow erythrocyte proportion, PLT level and neutrophil level (r=0.426; r=0.486; r=0.495), but negatively correlated with LDH level (r=-0.421); WT1 expression level was positively correlated with CD10/CD34 (%), CD10/CD34 (MFI) and CD117/CD34 (MFI) (r=0.745; r=0.800; r=0.434), while negatively correlated with CD11b/CD34 (%)(r=-0.457); CD19/CD34 (%) and CD71/CD34 (MFI) negatively correlated with NK cell proportion (r=-0.786; r=-0.514); CD10/CD34 (%) positively correlated with Th/Ts, while CD7/CD34 (MFI) negatively correlated with the proportion of Th cells (r=0.738; r=-0.513); HLADR/CD34 (%) and HLADR/CD34 (MFI) negatively correlated with PLT level (r=-0.461; r=-0.445), while HLADR/CD34 (MFI) positively correlated with bone marrow NAP fraction (r=0.552). The quantitative expression of CD7 and CD117 had no significant effect on the overall survival rate of low-risk MDS patients. CONCLUSION: The immunophenotype of CD34+ myeloid precursor cell in different risk groups in MDS patients is related to clinical characteristics. Bone marrow cell morphology, clinical and laboratory features and immunophenotype will be of great significance to the diagnosis, clinical classification and prognosis evaluation of MDS patients.

Two new bioactive polyphenols from the soft coral-derived fungus Talaromyces sp. SCSIO 041201.

Two new polyphenols, talaversatilis A (1) and B (2), together with fifteen known compounds (3-17) were isolated from the extract of the culture broth of a soft coral-derived fungus Talaromyces sp. SCSIO 041201. The structures of these compounds were elucidated by the extensive analyses of spectroscopic data and by comparison with the reported literature. Antifouling and antibacterial activities of all purified compounds were tested and evaluated. Compounds 5 and 6 showed antifouling activity towards Bugula neritina larva, with LC50 values of 3.86 µg/mL and 3.05 µg/mL, respectively. Compounds 7, 8, 10 and 13 exhibited significant antibacterial activities against E. coli, MRSA, S. aureus and E. faecalis, with MIC values ranging from 0.45 to 15.6 µg/mL.

[Effect of Signal Transduction Pathway Gene Mutations on the One- course Induced Remission Rate and Analysis of Clinical Characteristics in Patients with CBF-AML].

OBJECTIVE: To investigate the effect of other gene mutations outside the fusion gene on the first complete remission (CR1) induced by one course of induction chemotherapy in patients with core binding factor-associated acute myeloid leukemia (CBF-AML). METHODS: DNA was extracted from bone marrow or peripheral blood samples of newly diagnosed CBF-AML patients admitted to the Hematology Department of the Second Hospital of Shanxi Medical University from January 2015 to January 2019. Next-generation sequencing was used for detection of 34 kinds of hematologic malignancy-related gene mutations in patients with CBF-AML, the effect of related gene mutations on the first complete remission (CR1) rate in one course of induction chemotherapy was analyzed by combineation with clinical characteristics. RESULTS: 34 kinds of genes in bone marrow or peripheral blood of 43 patients were detected by high throughput sequencing and the gene mutations were detected in 16 out of 34 genes. The mutation rate of KIT gene was the highest (48.8%), followed by NRAS (16.3%), ASXL1 (16.3%), TET2 (11.6%), CSF3R (9.3%), FLT3 (9.3%), KRAS (7.0%). The detection rates of mutations in different functional genes were as follows: genes related with signal transduction pathway (KIT, FLT3, CSF3R, KRAS, NRAS, JAK2, CALR, SH2B3, CBL) had the highest mutation frequency (72.1% (31/43); epigenetic modification gene mutation frequency was 30.2% (13/43), including ASXL1, TET2, BCOR); transcriptional regulation gene mutation frequency was 7.0% (3/43), including ETV6, RUNX1, GATA2). Splicing factor related gene mutation frequency was 2.3% (1/43), including ZRSR2). The CR1 rate was 74.4% after one course of induction chemotherapy. At first diagnosis, patients with low expression of WT1 (the median value of WT1 was 788.9) were more likely to get CR1 (P=0.032) and the RFS of patients who got CR1 after one course of induction chemotherapy was significantly longer than that of patients without CR1 [7.6 (2.2-44.1) versus 5.8 (1-19.4), (P=0.048)]. The rate of CR1 in the signal transduction pathway gene mutation group was significantly lower than that in non-mutation group (64.5% vs 100%) (P=0.045), while the level of serum hydroxybutyrate dehydrogenase (HBDH) was significantly higher than that in non-mutation group [(418 (154-2702) vs 246 (110-1068)] (P=0.032). There was no difference in CD56 expression between the two groups (P=0.053), which was limited to the difference between (≥20%) expression and non-expression. (P=0.048). CONCLUSION: CBF-AML patients with signal transduction pathway gene mutation are often accompanied by high HBDH level and CD56 expression, moreover, the remission rate induced by one course of treatment is low.

Predictors of primary patency after percutaneous balloon angioplasty for stenosis of Brescia-Cimino hemodialysis arteriovenous fistula.

OBJECTIVE: Percutaneous transluminal balloon angioplasty (PTA) is recommended as the first choice to treat stenosis of Brescia-Cimino arteriovenous fistulas (B-C AVFs). The ability to predict which B-C AVFs are at risk for recurrent stenosis post-PTA would allow closer monitoring of patients, and possibly result in surgical intervention rather than repeat PTA. The purpose of this study was to identify predictive factors of primary patency after PTA in B-C AVFs. METHODS: Patients diagnosed with B-C AVF primary stenosis and treated by PTA between November 2013 and March 2018 were included in the study. Patient and stenotic lesion characteristics and PTA procedure factors were included in the analysis. The Kaplan-Meier method was used to analyze the primary patency rate. Cox proportional hazard regression analysis was used to identify factors predictive of decreased primary patency. RESULTS: 74 patients (35 males, 39 females) with a mean age of 61.68 ± 11.44 years (range, 36-84 years) were included in the study. The mean B-C AVF age was 16.34 ± 12.93 months (range, 2-84 months), and the median primary patency time was 7.79 ± 0.48 months. Cox proportional hazard regression analysis revealed stenosis location at the inflow artery [hazard ratio (HR)=3.83, 95% confidence interval (CI): 1.46-10.09] or anastomosis (HR = 1.90, 95% CI: 1.09-3.32), dilation >2 times during PTA (HR = 2.30, 95% CI: 1.22-4.34), and residual stenosis >30% (HR = 2.42, 95% CI: 1.26-4.63) were significantly associated with decreased patency. CONCLUSION: In conclusion, the primary patency rate of PTA for B-C AVF dysfunction is reduced by dilation >2 times, residual stenosis >30%, and stenosis located at the inflow artery or anastomosis. These results may help in tailoring surveillance programs, multiple PTA, or a proximal re-anastomosis surgery in patients with AVF dysfunction. ADVANCES IN KNOWLEDGE: A number of studies have been conducted to examine the predictors of primary patency after PTA, however, no definitive conclusions have been reached. Our study revealed that stenosis location at the inflow artery or anastomosis, dilation >2 times during PTA, and residual stenosis >30% were the predictors of primary patency after PTA, which may help in tailoring surveillance programs, multiple PTA, or a proximal re-anastomosis surgery in patients with arteriovenous fistulas dysfunction.

Acute pro-B-Cell lymphoblastic leukemia transformed from myelodysplastic syndrome with an ASXL1 missense mutation: A case report with literature review.

The development of acute lymphoblastic leukemia (ALL) from myelodysplastic syndrome (MDS) is a very rare event. The current report presents a rare case of a 33-year-old man who was diagnosed with MDS with multiple-lineage dysplasia (MDS-MLD) that transformed into pro-B-ALL. A missense mutation (S1231F) of the additional sex combs like 1, transcriptional regulator gene was identified, which may have a substantial role in the progression, however does not act as an unfavorable prognostic marker. The patient died during induction chemotherapy. The present study further conducted an analysis on 30 patients to determine progression to ALL. Patients were predominantly male (76.7%, 23/30) with a median age of 56 years (3-90 years). The median time to transformation was 5.5 months (2-50 months). The most common type of MDS with ALL transformation comprised of MDS-excess blasts (MDS-EB; 40%, 12/30), MDS with single-lineage dysplasia (MDS-SLD; 30%, 9/30) and MDS with ring sideroblasts (MDS-RS; 16.7%, 5/30). The majority of the patients transformed to B-cell (66.7%, 16/24) followed by T-cell (33.3%, 8/24) ALL. From the 25 cases where data was available, the complete remission rate was 75% (15/20) with ALL-directed chemotherapy and the median remission duration was 15 months (range 4.5 to 51 months). However, the results indicated that ALL following MDS is characterized by a high rate of early death (20%, 5/25).

Droplet digital PCR for BCR/ABL(P210) detection of chronic myeloid leukemia: A high sensitive method of the minimal residual disease and disease progression.

OBJECTIVE: This study intended to establish a droplet digital PCR (dd-PCR) for monitoring minimal residual disease (MRD) in patients with BCR/ABL (P210)-positive chronic myeloid leukemia (CML), thereby achieving deep-level monitoring of tumor load and determining the efficacy for guided clinically individualized treatment. METHODS: Using dd-PCR and RT-qPCR, two cell suspensions were obtained from K562 cells and normal peripheral blood mononuclear cells by gradient dilution and were measured at the cellular level. At peripheral blood (PB) level, 61 cases with CML-chronic phase (CML-CP) were obtained after tyrosine kinase inhibitor (TKI) treatment and regular follow-ups. By RT-qPCR, BCR/ABL (P210) fusion gene was undetectable in PB after three successive analyses, which were performed once every 3 months. At the same time, dd-PCR was performed simultaneously with the last equal amount of cDNA. Ten CML patients with MR4.5 were followed up by the two methods. RESULTS: At the cellular level, consistency of results of dd-PCR and RT-qPCR reached R2  ≥ 0.99, with conversion equation of Y = 33.148X1.222 (Y: dd-PCR results; X: RT-qPCR results). In the dd-PCR test, 11 of the 61 patients with CML (18.03%) tested positive and showed statistically significant difference (P < .01). In the follow-up of 10 CML patients who were in MR4.5. All patients were loss of MR4.0, and 4 were tested positive by dd-PCR 3 months earlier than by RT-qPCR. CONCLUSION: In contrast with RT-qPCR, dd-PCR is more sensitive, thus enabling accurate conversion of dd-PCR results into internationally standard RT-qPCR results by conversion equation, to achieve a deeper molecular biology-based stratification of BCR/ABL(P210) MRD. It has some reference value to monitor disease progression in clinic.

Truncated Latrunculins as Actin Inhibitors Targeting Plasmodium falciparum Motility and Host Cell Invasion.

Polymerization of the cytosolic protein actin is critical to cell movement and host cell invasion by the malaria parasite, Plasmodium falciparum. Any disruption to actin polymerization dynamics will render the parasite incapable of invading a host cell and thereby unable to cause infection. Here, we explore the potential of using truncated latrunculins as potential chemotherapeutics for the treatment of malaria. Exploration of the binding interactions of the natural actin inhibitor latrunculins with actin revealed how a truncated core of the inhibitor could retain its key interaction features with actin. This truncated core was synthesized and subjected to preliminary structure-activity relationship studies to generate a focused set of analogues. Biochemical analyses of these analogues demonstrate their 6-fold increased activity compared with that of latrunculin B against P. falciparum and a 16-fold improved selectivity ex vivo. These data establish the latrunculin core as a potential focus for future structure-based drug design of chemotherapeutics against malaria.

Quantitative phospho-proteomics reveals the Plasmodium merozoite triggers pre-invasion host kinase modification of the red cell cytoskeleton.

The invasive blood-stage malaria parasite - the merozoite - induces rapid morphological changes to the target erythrocyte during entry. However, evidence for active molecular changes in the host cell that accompany merozoite invasion is lacking. Here, we use invasion inhibition assays, erythrocyte resealing and high-definition imaging to explore red cell responses during invasion. We show that although merozoite entry does not involve erythrocyte actin reorganisation, it does require ATP to complete the process. Towards dissecting the ATP requirement, we present an in depth quantitative phospho-proteomic analysis of the erythrocyte during each stage of invasion. Specifically, we demonstrate extensive increased phosphorylation of erythrocyte proteins on merozoite attachment, including modification of the cytoskeletal proteins beta-spectrin and PIEZO1. The association with merozoite contact but not active entry demonstrates that parasite-dependent phosphorylation is mediated by host-cell kinase activity. This provides the first evidence that the erythrocyte is stimulated to respond to early invasion events through molecular changes in its membrane architecture.

Disassembly activity of actin-depolymerizing factor (ADF) is associated with distinct cellular processes in apicomplexan parasites.

Proteins of the actin-depolymerizing factor (ADF)/cofilin family have been shown to be crucial for the motility and survival of apicomplexan parasites. However, the mechanisms by which ADF proteins fulfill their function remain poorly understood. In this study, we investigate the comparative activities of ADF proteins from Toxoplasma gondii and Plasmodium falciparum, the human malaria parasite, using a conditional T. gondii ADF-knockout line complemented with ADF variants from either species. We show that P. falciparum ADF1 can fully restore native TgADF activity, demonstrating functional conservation between parasites. Strikingly, mutation of a key basic residue (Lys-72), previously implicated in disassembly in PfADF1, had no detectable phenotypic effect on parasite growth, motility, or development. In contrast, organelle segregation was severely impaired when complementing with a TgADF mutant lacking the corresponding residue (Lys-68). Biochemical analyses of each ADF protein confirmed the reduced ability of lysine mutants to mediate actin depolymerization via filament disassembly although not severing, in contrast to previous reports. These data suggest that actin filament disassembly is essential for apicomplexan parasite development but not for motility, as well as pointing to genus-specific coevolution between ADF proteins and their native actin.

Cryo-EM structure of the Plasmodium falciparum 80S ribosome bound to the anti-protozoan drug emetine.

Malaria inflicts an enormous burden on global human health. The emergence of parasite resistance to front-line drugs has prompted a renewed focus on the repositioning of clinically approved drugs as potential anti-malarial therapies. Antibiotics that inhibit protein translation are promising candidates for repositioning. We have solved the cryo-EM structure of the cytoplasmic ribosome from the human malaria parasite, Plasmodium falciparum, in complex with emetine at 3.2 Å resolution. Emetine is an anti-protozoan drug used in the treatment of ameobiasis that also displays potent anti-malarial activity. Emetine interacts with the E-site of the ribosomal small subunit and shares a similar binding site with the antibiotic pactamycin, thereby delivering its therapeutic effect by blocking mRNA/tRNA translocation. As the first cryo-EM structure that visualizes an antibiotic bound to any ribosome at atomic resolution, this establishes cryo-EM as a powerful tool for screening and guiding the design of drugs that target parasite translation machinery.

Quantitative analysis of Plasmodium ookinete motion in three dimensions suggests a critical role for cell shape in the biomechanics of malaria parasite gliding motility.

Motility is a fundamental part of cellular life and survival, including for Plasmodium parasites--single-celled protozoan pathogens responsible for human malaria. The motile life cycle forms achieve motility, called gliding, via the activity of an internal actomyosin motor. Although gliding is based on the well-studied system of actin and myosin, its core biomechanics are not completely understood. Currently accepted models suggest it results from a specifically organized cellular motor that produces a rearward directional force. When linked to surface-bound adhesins, this force is passaged to the cell posterior, propelling the parasite forwards. Gliding motility is observed in all three life cycle stages of Plasmodium: sporozoites, merozoites and ookinetes. However, it is only the ookinetes--formed inside the midgut of infected mosquitoes--that display continuous gliding without the necessity of host cell entry. This makes them ideal candidates for invasion-free biomechanical analysis. Here we apply a plate-based imaging approach to study ookinete motion in three-dimensional (3D) space to understand Plasmodium cell motility and how movement facilitates midgut colonization. Using single-cell tracking and numerical analysis of parasite motion in 3D, our analysis demonstrates that ookinetes move with a conserved left-handed helical trajectory. Investigation of cell morphology suggests this trajectory may be based on the ookinete subpellicular cytoskeleton, with complementary whole and subcellular electron microscopy showing that, like their motion paths, ookinetes share a conserved left-handed corkscrew shape and underlying twisted microtubular architecture. Through comparisons of 3D movement between wild-type ookinetes and a cytoskeleton-knockout mutant we demonstrate that perturbation of cell shape changes motion from helical to broadly linear. Therefore, while the precise linkages between cellular architecture and actomyosin motor organization remain unknown, our analysis suggests that the molecular basis of cell shape may, in addition to motor force, be a key adaptive strategy for malaria parasite dissemination and, as such, transmission.

A mechanism for actin filament severing by malaria parasite actin depolymerizing factor 1 via a low affinity binding interface.

Actin depolymerizing factor (ADF)/cofilins are essential regulators of actin turnover in eukaryotic cells. These multifunctional proteins facilitate both stabilization and severing of filamentous (F)-actin in a concentration-dependent manner. At high concentrations ADF/cofilins bind stably to F-actin longitudinally between two adjacent actin protomers forming what is called a decorative interaction. Low densities of ADF/cofilins, in contrast, result in the optimal severing of the filament. To date, how these two contrasting modalities are achieved by the same protein remains uncertain. Here, we define the proximate amino acids between the actin filament and the malaria parasite ADF/cofilin, PfADF1 from Plasmodium falciparum. PfADF1 is unique among ADF/cofilins in being able to sever F-actin but do so without stable filament binding. Using chemical cross-linking and mass spectrometry (XL-MS) combined with structure reconstruction we describe a previously overlooked binding interface on the actin filament targeted by PfADF1. This site is distinct from the known binding site that defines decoration. Furthermore, total internal reflection fluorescence (TIRF) microscopy imaging of single actin filaments confirms that this novel low affinity site is required for F-actin severing. Exploring beyond malaria parasites, selective blocking of the decoration site with human cofilin (HsCOF1) using cytochalasin D increases its severing rate. HsCOF1 may therefore also use a decoration-independent site for filament severing. Thus our data suggest that a second, low affinity actin-binding site may be universally used by ADF/cofilins for actin filament severing.

[Effect of Nrf2 and TrxR on proliferation of chronic myeloid leukemia cell and its mechanism].

OBJECTIVE: To explore the effect of nuclear factor erythroid-2 related factor 2 (Nrf2) and thioredoxin reductase (TrxR) gene on proliferation of chronic myeloid leukemia (CML) line cells and its mechanism. METHODS: Four interfering sequences of Nrf2 and one negative control sequence were designed and synthesised based on the principle of target sequence of siRNA, then constructed lentivirus vectors, which were transfected into K562 cell lines. The transfection effect was observed by laser scanning confocal microscope (LSCM) and flow cytometer (FCM); The depressing effect of siRNA was analyzed by real-time PCR. The cell proliferation inhibiting rate was measured with CCK-8 assay, the apoptotic rate by Annexin V-PE/PI with FCM and the apoptotic morphology of cells by LSCM. RESULTS: The transfection efficiency of lentivirus was 65%. One cell line K562-C3 which significantly inhibited Nrf2 mRNA was obtained by real-time PCR, Nrf2 relative quantitation (RQ) expressions were 1.003±0.093 and 0.344±0.032 in the control group and K562-C3 respectively; TrxR expression also decreased with RQ as 1.090±0.549 and 0.395±0.029 respectively. The cellular proliferation inhibition rates of K562-C3 were (4.74±0.39)%, (6.13±1.78)% and (25.36±3.77)%, respectively at 24, 48 and 72 h. The apoptotic rate induced by K562-C3 (29.9%) at 72 hours was obviously higher than in the control group (7.9%). The Annexin V-PE positive K562-C3 cells presented the following apoptotic characteristics, such as karyopyknosis, nuclear fragmentation and apoptotic bodies observed by LSCM. CONCLUSION: Nrf2 specific siRNA could repress its expression at the cellular level and down-regulate the expression of its downstream antioxidant enzyme, such as TrxR, which lead to increased apoptotic rate and decreased cell proliferation.

[Establishment of diagnostic model to monitor minimal residual disease of acute promyelocytic leukemia by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry].

OBJECTIVE: To screen the potential protein biomarkers in minimal residual disease (MRD) of the acute promyelocytic leukemia (APL) by comparison of differentially expressed serum protein between APL patients at diagnosis and after complete remission (CR) and healthy controls, and to establish and verify a diagnostic model. METHODS: Serum proteins from 36 cases of primary APL, 29 cases of APL during complete remission and 32 healthy controls were purified by magnetic beads and then analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The spectra were analyzed statistically using FlexAnalysis(TM) and ClinProt(TM) software. RESULTS: Two prediction model of primary APL/healthy control, primary APL/APL CR were developed. Thirty four statistically significant peptide peaks were obtained with the m/z value ranging from 1000 to 10 000 (P < 0.001) in primary APL/healthy control model. Seven statistically significant peptide peaks were obtained in primary APL/APL CR model (P < 0.001). Comparison of the protein profiles between the two models, three peptides with m/z 4642, 7764 and 9289 were considered as the protein biomarker of APL MRD. A diagnostic pattern for APL CR using m/z 4642 and 9289 was established. Blind validation yielded correct classification of 6 out of 8 cases. CONCLUSIONS: The MALDI-TOF MS analysis of APL patients serum protein can be used as a promising dynamic method for MRD detection and the two peptides with m/z 4642 and 9289 may be better biomarkers.

Identification of a novel circularized transcript of the AML1 gene.

The AML1 gene is an essential transcription factor regulating the differentiation of hematopoietic stem cells into mature blood cells. Though at least 12 different alternatively spliced AML1 mRNAs are generated, three splice variants (AML1a, AML1b and AML1c) have been characterized. Here, using the reverse transcription-polymerase chain reaction with outward-facing primers, we identified a novel non-polyadenylated transcript from the AML1 gene, with exons 5 and 6 scrambled. The novel transcript resisted RNase R digestion, indicating it is a circular RNA structure that may originate from products of mRNA alternative splicing. The expression of the novel transcript in different cells or cell lines of human and a number of other species matched those of the canonical transcripts. The discovery provides additional evidence that circular RNA could stably exist in vivo in human, and may also help to understand the mechanism of the regulation of the AML1 gene transcription.

Prenatal diagnosis of anhidrotic ectodermal dysplasia with unconventional loci abnormalities: a case report.

Anhidrotic ectodermal dysplasia (EDA) is a relatively rare congenital hereditary disease. Because of a reduced number of sweat glands, patients are unable to perspire and consequently suffer from hyperthermia and infection. This is a potential cause of death in childhood. Domestic prenatal diagnosis methods focus on genetic diagnosis. But for some conditions, because of the uncertain molecular pathology, we need other methods to assist to in prenatal diagnosis. Here, we report one case of a new mutation locus which may be associated with EDA and the prenatal diagnosis of EDA by fetal skin biopsy under fetoscopy in mid pregnancy, combined with a review of the literature.

Malaria parasite colonisation of the mosquito midgut--placing the Plasmodium ookinete centre stage.

Vector-borne diseases constitute an enormous burden on public health across the world. However, despite the importance of interactions between infectious pathogens and their respective vector for disease transmission, the biology of the pathogen in the insect is often less well understood than the forms that cause human infections. Even with the global impact of Plasmodium parasites, the causative agents of malarial disease, no vaccine exists to prevent infection and resistance to all frontline drugs is emerging. Malaria parasite migration through the mosquito host constitutes a major population bottleneck of the lifecycle and therefore represents a powerful, although as yet relatively untapped, target for therapeutic intervention. The understanding of parasite-mosquito interactions has increased in recent years with developments in genome-wide approaches, genomics and proteomics. Each development has shed significant light on the biology of the malaria parasite during the mosquito phase of the lifecycle. Less well understood, however, is the process of midgut colonisation and oocyst formation, the precursor to parasite re-infection from the next mosquito bite. Here, we review the current understanding of cellular and molecular events underlying midgut colonisation centred on the role of the motile ookinete. Further insight into the major interactions between the parasite and the mosquito will help support the broader goal to identify targets for transmission-blocking therapies against malarial disease.