[Prognostic Value of IGF2BP3 Gene Expression Levels in Patients with Acute Myeloid Leukemia].

OBJECTIVE: To investigate the relationship between IGF2BP3 gene expression and prognosis in patients with acute myeloid leukemia (AML). METHODS: High throughput transcriptome sequencing was performed on bone marrow primary leukemia cells from 27 patients with AML in our center, the relationship between IGF2BP3 expression levels and clinical characteristics were analyzed and verify the samples from patients with newly treated AML and refractory AML. The expression level of IGF2BP3 gene were analyzed in 20 healthy subjects and 26 patients with AML. The expression of IGF2BP3 in two anthracycline-resistant cell lines (HL60/ADR, K562/ADR) was detected by RT-qPCR and Western blot, and the expression difference of IGF2BP3 was compared with that in sensitive cells (HL60, K562). The relationship between the expression level of IGF2BP3 in patients with AML and prognostic were analyzed through data analysis of 746 patients with AML, and the prognostic value of IGF2BP3 in AML was analyzed by multivariate Cox regression analysis. RESULTS: In the bone marrow primary leukemia cells of 27 AML patients in our center, the expression level of IGF2BP3 in patients with refractory AML was significantly higher than that in chemotherapy sensitive patients (P =0.0343). The expression of IGF2BP3 in leukemia patients with extramedullary infiltration (EMI) was significantly higher than that in AML patients without extramedullary infiltration (P =0.0049). Compared with healthy subjects (n=20), IGF2BP3 expression in AML patients (n=26) was higher (P =0.0009). The expression of IGF2BP3 mRNA in the anthracycline resistant cell lines (HL60/ADR, K562/ADR) was significantly higher than that in the sensitive cell lines (K562/ADR vs K562,P =0.0430; HL60/ADR vs HL60, P =0.7369). Western blot results showed that the expression of IGF2BP3 protein in mycin resistant cells was significantly higher than that in sensitive cells (P < 0.001). qPCR results showed that the expression level of IGF2BP3 mRNA in refractory AML patients was significantly higher than that in patients with chemotherapy sensitive (P =0.002). High expression of IGF2BP3 was associated with poor prognosis in AML (P < 0.05) in 3 large sample cohorts of AML patients. Univariate and multivariate prognostic analyses demonstrated that high expression of IGF2BP3 was significantly associated with shorter event-free survival (EFS, HR=1.887, P =0.024) and overall survival (OS, HR=1.619, P =0.016). CONCLUSION: The high expression of IGF2BP3 gene may be an important factor in the poor prognosis of AML, suggesting that IGF2BP3 gene may be a new molecular marker for the clinical prognosis evaluation and treatment strategy of AML.

[Venetoclax Combined with CACAG Regimen in the Treatment of Patients with Refractory/Relapse Acute Myeloid Leukemia: A Prospective Clinical Study].

OBJECTIVE: To investigate the efficacy and safety of Venetoclax combined with CACAG regimen in treatment of patients with refractory/relapse acute myeloid leukemia(R/R AML). METHODS: The study was a singlecenter prospective clinical trial. The enrolled patients met the criteria for R/R AML. Treatment included Azacidine(75 mg/m2，d 1-7), Ara-C (75-100 mg/m2, q12h, d 1-5), Aclacinomycin(20 mg d1,d3,d5), Chidamide(30 mg d1,d4), Venetoclax(100 mg d1, 200 mg d2, 400 mg d3-d14, in combination with Triazole Drug, reduced to 100 mg/d), and granulocyte colony-stimulating factor (300 µg /d until neutrophil recovery). The primary endpoint of observation was overall response rate after 1 course of treatment. RESULTS: A total of 19 patients were enrolled from January 2022 to April 2023. After 1 course of treatmen, the overall response rate was 81.3%(13/16), the CR rate was 68.8%(11/16), and the PR was 12.5%(2/16). Among the 11 patients who got CR/CRi, 8 cases achieved CRm (minimal residual disease negative CR) and 3 cases did not. As of March 27, 2023, the median follow-up time was 111(19-406) days. The six-month overall survival and progression-free survival rates were both 55.7%, the 1-year overall survival and progression-free survival rates were 46.4% and 47.7%, respectively. In addition, compared with the non-CRm group, CRm patients had a better PFS (377 days vs 111 days, P =0.046). Treatment-related adverse events were mainly 3-4 degrees of bone marrow suppression, complicated by various degrees of infection(n=12), hypokalemia(n=12) and hypocalcemia (n=10) and elevated liver enzymes (n=8), of which 3/4 degrees accounted for 47.4%(9/19). CONCLUSION: The Venetoclax combined with CACAG regimen is an effective salvage therapy for patients with R/R AML, with high remission rate and safety profile.

Structural insights into mechanisms of Argonaute protein-associated NADase activation in bacterial immunity.

Nicotinamide adenine dinucleotide (NAD+) is a central metabolite in cellular processes. Depletion of NAD+ has been demonstrated to be a prevalent theme in both prokaryotic and eukaryotic immune responses. Short prokaryotic Argonaute proteins (Agos) are associated with NADase domain-containing proteins (TIR-APAZ or SIR2-APAZ) encoded in the same operon. They confer immunity against mobile genetic elements, such as bacteriophages and plasmids, by inducing NAD+ depletion upon recognition of target nucleic acids. However, the molecular mechanisms underlying the activation of such prokaryotic NADase/Ago immune systems remain unknown. Here, we report multiple cryo-EM structures of NADase/Ago complexes from two distinct systems (TIR-APAZ/Ago and SIR2-APAZ/Ago). Target DNA binding triggers tetramerization of the TIR-APAZ/Ago complex by a cooperative self-assembly mechanism, while the heterodimeric SIR2-APAZ/Ago complex does not assemble into higher-order oligomers upon target DNA binding. However, the NADase activities of these two systems are unleashed via a similar closed-to-open transition of the catalytic pocket, albeit by different mechanisms. Furthermore, a functionally conserved sensor loop is employed to inspect the guide RNA-target DNA base pairing and facilitate the conformational rearrangement of Ago proteins required for the activation of these two systems. Overall, our study reveals the mechanistic diversity and similarity of Ago protein-associated NADase systems in prokaryotic immune response.

A novel scoring system for the quantitative prediction of prognosis in acute myeloid leukemia.

Background: Acute myeloid leukemia (AML) is a heterogeneous hematopoietic malignancy. Patient prognosis cannot be accurately assessed in National Comprehensive Cancer Network (NCCN) risk stratification subgroups based on the current criteria. This study aimed to develop a novel prognostic score model for the quantitative prediction of prognosis in AML. Results: We developed a prognostic risk scoring model of AML using differentially expressed genes to predict prognosis in patients with AML. Furthermore, we evaluated the effectiveness and clinical significance of this prognostic model in 4 AML cohorts and 905 patients with AML. A prognostic risk scoring model of AML containing eight prognosis-related genes was constructed using a multivariate Cox regression model. The model had a higher predictive value for the prognosis of AML in the training and validation sets. In addition, patients with lower scores had significantly better overall survival (OS) and even-free survival (EFS) than those with higher scores among patients with intermediate-risk AML according to the NCCN guidelines, indicating that the model could be used to further predict the prognosis of the intermediate-risk AML populations. Similarly, patients with high scores had remarkably poor OS and EFS in the normal-karyotype populations, indicating that the scoring model had an excellent predictive performance for patients with AML having normal karyotype. Conclusions: Our study provided an individualized prognostic risk score model that could predict the prognosis of patients with AML.

Characteristics of Dissolved Organic Matter Impacted by Different Land Use in Haihe River Watershed, China.

It is important to explore characteristics of dissolved organic matter (DOM) in the riverine system due to its critical role in the carbon cycle. This study investigated the distribution characteristics and sources of DOM based on excitation emission matrix three-dimensional fluorescence technology and parallel factor (EEM-PARAFAC) analysis at two rivers in northern China strongly impacted by human activities. The results show that the fluorescence intensity of terrestrial humic-like substances increased during summer in Haihe River. The intensity was significantly higher than in spring due to terrestrial detritus from runoff conveyance. The fluorescence intensity of protein-like substances in spring was the highest and decreased in summer. This feature of DOM in the Duliujian River was related to the increase in precipitation and surface runoff in the wet season and the rapid degradation of mixed DOM in the dry season. An analysis of HIX, BIX and FI showed a low degree of DOM humification and more endogenous contributions from microbial and phytoplankton degradation. Seasonal variations of dissolved organic carbon (DOC) and chromophoric DOM (CDOM, a335, thereinto C1) suggest that chromophores, particularly terrestrial substances, regulate the temporal patterns of DOM in the two rivers. Combined with the analysis of the proportion of land use types in riparian buffers, tillage had a great impact on DOM content and hydrophobicity in Haihe River watershed. Domestic wastewater and industrial sewage discharge contribute more DOM to Duliujian River watershed, which was indicated by more abundant protein-like components (212.17 ± 94.63 QSU in Duliujian River;186.59 ± 238.72 QSU in Haihe River). This study highlights that different land use types resulted in distinctive sources and seasonal dynamics of DOM in rivers. Meanwhile, it should be considered that the estimation of carbon cycling should involve monitoring and evaluating anthropogenic inputs into rivers.

Target RNA-guided protease activity in type III-E CRISPR-Cas system.

The type III-E CRISPR-Cas systems are newly identified adaptive immune systems in prokaryotes that use a single Cas7-11 protein to specifically cleave target RNA. Cas7-11 could associate with Csx29, a putative caspase-like protein encoded by the gene frequently found in the type III-E loci, suggesting a functional linkage between the RNase and protease activities in type III-E systems. Here, we demonstrated that target RNA recognition would stimulate the proteolytic activity of Csx29, and protein Csx30 is the endogenous substrate. More interestingly, while the cognate target RNA recognition would activate Csx29, non-cognate target RNA with the complementary 3' anti-tag sequence inhibits the enzymatic activity. Csx30 could bind to the sigma factor RpoE, which may initiate the stress response after proteolytic cleavage. Combined with biochemical and structural studies, we have elucidated the mechanisms underlying the target RNA-guided proteolytic activity of Csx29. Our work will guide further developments leveraging this simple RNA targeting system for RNA and protein-related applications.

Reduced Risk of Chronic Graft-Versus-Host Disease (cGVHD) by Rabbit Anti-Thymocyte Globulin (ATG) in Patients Undergoing Matched Sibling Donor Transplantation in Hematological Malignancies.

BACKGROUND With the addition of anti-thymocyte globulin (ATG) to GVHD prophylaxis in patients undergoing transplantation of peripheral blood stem cells (PBSCT), the incidence of cGVHD decreases. However, the optimal dose and timing of ATG remain undetermined. MATERIAL AND METHODS In this historical controlled trial, data from 85 patients who had hematological malignancies and underwent matched sibling donor (MSD)-PBSCT were used to analyze the effectiveness of rabbit ATG (rATG) for prophylaxis of GVHD. Forty patients received 5 mg/kg rATG used for days -5 to -2, and 45 patients did not receive ATG. RESULTS All patients had successful engraftment except for 2 in the non-ATG group, who had platelet engraftment failure. The 2-year cumulative incidence of chronic GVHD (cGVHD) in the ATG group versus non-ATG group was 19.3% (95% CI, 8.4-33.6%) versus 61.4% (95% CI, 45.4-73.9%) (P<0.001), and in those with moderate to severe cGVHD it was 11.0% (95% CI, 3.4-23.6%) versus 31.8% (95% CI, 18.8-45.6%) (P=0.029), respectively. The 2-year cumulative incidence of non-relapse mortality and relapse (CIR) were 0% versus 15.5% (95% CI, 6.8-27.5%) (P=0.018), and 53.3% (95% CI, 35.6-68.1%) versus 26.7% (95% CI, 14.9-40.0%) (P=0.019), respectively. No differences were found in other survival outcomes. In the multivariate analysis, ATG was an independent protective factor for moderate to severe cGVHD (HR=0.314, 95% CI, 0.103-0.958, P=0.042), and was an independent poor risk factor for CIR (HR=2.337, 95% CI, 1.133-4.822, P=0.022). CONCLUSIONS ATG in our strategy was effective for prophylaxis of cGVHD, whereas the relapse rate was increased in patients with rATG.

Structure and function of a bacterial type III-E CRISPR-Cas7-11 complex.

The type III-E CRISPR-Cas system uses a single multidomain effector called Cas7-11 (also named gRAMP) to cleave RNA and associate with a caspase-like protease Csx29, showing promising potential for RNA-targeting applications. The structural and molecular mechanisms of the type III-E CRISPR-Cas system remain poorly understood. Here we report four cryo-electron microscopy structures of Cas7-11 at different functional states. Cas7-11 has four Cas7-like domains, which assemble into a helical filament to accommodate CRISPR RNA (crRNA), and a Cas11-like domain facilitating crRNA-target RNA duplex formation. The Cas7.1 domain is critical for crRNA maturation, whereas Cas7.2 and Cas7.3 are responsible for target RNA cleavage. Target RNA binding induces the structural arrangements of Csx29, potentially exposing the catalytic site of Csx29. These results delineate the molecular mechanisms underlying pre-crRNA processing, target RNA recognition and cleavage for Cas7-11, and provide a structural framework to understand the role of Csx29 in type III-E CRISPR system.

A C-terminal glutamine recognition mechanism revealed by E3 ligase TRIM7 structures.

The E3 ligase TRIM7 has emerged as a critical player in viral infection and pathogenesis. However, the mechanism governing the TRIM7-substrate association remains to be defined. Here we report the crystal structures of TRIM7 in complex with 2C peptides of human enterovirus. Structure-guided studies reveal the C-terminal glutamine residue of 2C as the primary determinant for TRIM7 binding. Leveraged by this finding, we identify norovirus and SARS-CoV-2 proteins, and physiological proteins, as new TRIM7 substrates. Crystal structures of TRIM7 in complex with multiple peptides derived from SARS-CoV-2 proteins display the same glutamine-end recognition mode. Furthermore, TRIM7 could trigger the ubiquitination and degradation of these substrates, possibly representing a new Gln/C-degron pathway. Together, these findings unveil a common recognition mode by TRIM7, providing the foundation for further mechanistic characterization of antiviral and cellular functions of TRIM7.

Transcriptome-Wide Analysis of RNA N6-Methyladenosine Modification in Adriamycin-Resistant Acute Myeloid Leukemia Cells.

Acute myeloid leukemia (AML) is one of the most aggressive hematopoietic malignancies. Patients still suffer from refractory/relapsed disease after anthracycline-based therapy, which leads to a poor prognosis. N6-Methyladenosine (m6A) is the most abundant post-transcriptional modification in eukaryotes, the imbalance of which is reported to be associated with various pathological processes, including drug resistance. However, the relationship between m6A modification and drug resistance has not been well defined in AML. In this study, we analyzed the sequencing data of HL60 and its Adriamycin-resistant cell line HL60/ADR. We found a total of 40,550 m6A-methylated peaks, representing 15,640 genes in HL60, and 38,834 m6A-methylated peaks, representing 15,285 genes in HL60/ADR. KEGG pathway analysis showed that pathways were enriched in the FoxO signaling pathway, p53 signaling pathway, and Notch signaling pathway. MeRIP-seq results showed that the fold enrichment of the global m6A level in HL60/ADR was higher than that in HL60, and dot blot assay results indicated that the global m6A level was elevated in HL60/ADR cells compared with that in HL60 cells. Further analysis revealed that the expression level of METTL3 was elevated in HL60/ADR cells compared with that in HL60 cells. After a combined treatment of STM2457 (an inhibitor of METTL3) and Adriamycin, the proliferation of HL60/ADR was inhibited. Thus, we hypothesized that the abnormality of m6A modification played an important role in Adriamycin-resistant AML.

Study on the Influence of Low-Price Bid Winning and General Subcontracting Management on the Unsafe Behavior Intention of Construction Workers.

In recent years, there are many reasons for the frequent safety accidents in the construction field. The most controversial and typical one that firmly correlated with China's national condition is the low-price bid winning and the general subcontracting management, which probably have a great impact on the unsafe behavior intention of workers on the construction site. In order to figure out their internal relation, a quantitative statistical analysis of the unsafe behavior intentions of construction workers in the Beijing area was conducted through the on-site questionnaire considering three main variables, namely, general subcontract management, reasonable low-cost bid winning, and construction experiences. Meanwhile, the correlation, regression, and mediating effects of different influencing factors were analyzed through a regressive model to quantify the impact of each variable on the unsafe behavior intention of construction workers. The results showed that the influence of low-price bid winning on the unsafe behavior intention of on-site workers is faint. This is mainly because, in the case of labor buyer's market, the actual salary of workers is not relevant to whether the project is awarded at a low price. However, the general subcontracting management has a great impact on the unsafe behavior intention of on-site workers. At the same time, low-price bid winning also indirectly affects the strength of general subcontracting safety management, which has an indirect impact on the unsafe behavior intention of on-site workers. Generally, it is of greater significance to enhance the strength of the general subcontracting management and to formulate relevant regulations to guarantee the safety of construction workers.

A global record of annual terrestrial Human Footprint dataset from 2000 to 2018.

Human Footprint, the pressure imposed on the eco-environment by changing ecological processes and natural landscapes, is raising worldwide concerns on biodiversity and ecological conservation. Due to the lack of spatiotemporally consistent datasets of Human Footprint over a long temporal span, many relevant studies on this topic have been limited. Here, we mapped the annual dynamics of the global Human Footprint from 2000 to 2018 using eight variables that reflect different aspects of human pressures. The accuracy assessment revealed a good agreement between our mapped results and the previously developed datasets in different years. We found more than two million km2 of wilderness (i.e., regions with Human Footprint values below one) were lost over the past two decades. The biome dominated by mangroves experienced the most significant loss (i.e., above 5%) of wilderness, likely attributed to intensified human activities in coastal areas. The derived annual and spatiotemporally consistent global Human Footprint can be a fundamental dataset for many relevant studies about human activities and natural resources.

Optimization of cationic polymer-mediated transfection for RNA interference.

Transfection efficiency was estimated to optimize the conditions for RNA interference (RNAi), including transfection time, validity, and nucleic acid concentration and type, using the EZ Trans Cell Reagent, a cationic polymer. An shRNA against GFP was designed and transfected into cells using the EZ transfection reagent. The shRNA significantly decreased the expression of GFP. In addition, pre-diluted transfection reagent at room temperature and small nucleic acids increased the transfection efficiency, which peaked at 24 h. Compared with circular nucleic acids, linear nucleic acids showed higher transfection efficiency and a higher genome integration rate. We optimized cationic polymer-mediated RNAi conditions, and our data will be useful for future RNAi studies.

[Kinetics of MDSC in Patients Treated Steroids-Ruxolitinib as the First Line Therapy for aGVHD].

OBJECTIVE: To analyze the kinetic characteristics of lymphocyte subsets and myeloid-derived suppressor cell (MDSC) in patients who newly diagnosed intermediate- to high-risk aGVHD and treated with steroids-ruxolitinib as the first line therapy from a single-arm, open clinical trial (NCT04061876). METHODS: We prospectively observed the efficacy of 23 patients having intermediate- to high-risk aGVHD and treated with steroids-ruxolitinib as the first line therapy. The kinetic characteristics of lymphocyte subsets and MDSC were monitored, and then we compared them in steroids-ruxolitinib group (n=23), free-aGVHD group (n=20) and steroids group (n=23). RESULTS: Of the 23 patients, the CR rate was 78.26% (18/23) on day 28 after first-line treatment with steroids-ruxolitinib. On day 28 after treatment, patients had lower level of CD4+CD29+ T cells (P=0.08) than that of pre-treatment, whereas levels of other lymphocyte subsets in this study were higher than that of pre-treatment; CD4+CD29+ T cells in CR patients decreased, compared with refractory aGVHD patients. On day 28 of treatment, CD8+CD28- T cells (P=0.03) significantly increased in patients with aGVHD than that in patients without aGVHD, so did CD8+CD28- T / CD8+CD28+ T cell ratio (P=0.03). Compared with patients without aGVHD, patients with aGVHD had lower level of G-MDSC, especially on day 14 after allo-HSCT (P=0.04). Compared with pre-treatment, M-MDSC was higher in CR patients on day 3 and 7 post-treatment (P3=0.01, P7=0.03), e-MDSC was higher on day 28 post-treatment (P=0.01). Moreover, compared with CR patients, M-MDSC was lower in refractory aGVHD patients on day 3 post-treatment (P=0.01) and e-MDSC was lower on day 28 post-treatment (P=0.01). Compared with steroids group, MDSC in steroids-ruxolitinib group was higher, with the most significant difference in M-MDSC (P3=0.0351; P7=0.0142; P14=0.0369). CONCLUSION: We found that patients newly diagnosed intermediate- to high-risk aGVHD receiving first-line therapy with steroids-ruxolitinib achieved high response rate. Moreover, the novel first-line therapy has a small impact on the immune reconstitution of patients after allo-HSCT. Elevated MDSC might predict a better response in aGVHD patients receiving this novel first-line therapy. M-MDSC responded earlier to steroids-ruxolitinib than e-MDSC, G-MDSC.

The Optical Characterization and Distribution of Dissolved Organic Matter in Water Regimes of Qilian Mountains Watershed.

The constituents and content of dissolved organic matter (DOM) in the Qilian Mountain watershed were characterized with a spectroscopic technique, especially 3-DEEM fluorescence assisted by parallel factor (PARAFAC) analysis. The level of DOM in the surrounding area of Qinghai lake (thereafter the lake in this article specifically refers to Qinghai Lake)was highest at 9.45 mg C·L-1 and about 3 times less (3.09 mg C·L-1) in a cropland aquatic regime (the lowest value). In general, DOM was freshly autochthonously generated by plankton and plant debris, microorganisms and diagenetic effects in the aquatic environment (FI > 1.8). Component 1 (humic acid-like) and 3 (fulvic acid-like) determined the humification degree of chromophoric dissolved organic matter (CDOM). The spatial variation of sulfate and nitrate in the surrounding water regime of the lake revealed that organic molecules were mainly influenced by bacterial mediation. Mineral disintegration was an important and necessary process for fluorescent fraction formation in the cropland water regime. Exceptionally, organic moiety in the unused land area was affected by anespecially aridclimate in addition to microbial metabolic experience. Salinity became the critical factor determining the distribution of DOM, and the total normalized fluorescent intensity and CDOM level were lower in low-salinity circumstances (0.2-0.5 g·L-1) with 32.06 QSU and 1.38 m-1 in the grassland area, and higher salinity (0.6~0.8 g·L-1) resulted in abnormally high fluorescence of 150.62 QSU and absorption of 7.83 m-1 in the cropland water regime. Climatic conditions and microbial reactivity controlled by salinity were found to induce the above results. Our findings demonstrated that autochthonous inputs regulated DOM dynamics in the Qilian Mountains watershed of high altitude.

Toxic effects of the Emamectin Benzoate exposure on cultured human bronchial epithelial (16HBE) cells.

Pesticides pollution has caused serious environmental problems in recent years, and mounting evidence has shown that more and more insecticides have serious risk in human health. Emamectin Benzoate formally regarded as a highly safety insecticide based on its exclusive targets, but the cytotoxicity to human lung was ignored for a long time. In the present study, bioassay experiments were used to assess the toxicity of the Emamectin Benzoatein on human non-target cells including cell viability assay, DNA damage assay, flow cytometer assay and western blotting assay. The results indicated that Emamectin Benzoatecan cause the inhibition of the proliferation, cytochrome c release, activation of caspase-3/9 and increase Bax/Bcl-2 ratio, which means it induced the cytotoxicity on 16HBE cells associated with the mitochondrial apoptosis. Besides, the DNA damge caused by the Emamectin Benzoate suggest it has a potential genotoxic effect on human lung cells.

High-sensitivity quantification of glycosphingolipid glycans by ESI-MS utilizing ozonolysis-based release and isotopic Girard's reagent labeling.

Quantitative analysis of glycosphingolipids (GSLs) has been hindered by the lack of chromogenic groups for spectral detection or active functional groups for specific derivatization. In this study, a highly sensitive method based on ozonolysis-induced release and isotopic Girard's reagent P labeling of GSL glycans coupled with mass spectrometric detection for the quantification of animal tissue GSLs is developed. First, different approaches for the release of glycans from GSLs were compared with each other and the ozonolysis-based method was found to have the highest glycan yield under relative mild reaction conditions. Then a relative quantification method of ozonolysis-released GSL glycans based on stable isotope labeling using nondeuterated (d0-) and 2,3,4,5,6-pentadeuterated (d5-) Girard's reagent P (GP) was established, and its good linearity, accuracy and reproducibility were statistically verified. Finally, the new method was successfully applied to revealing the difference between porcine brain and liver as well as between the brain of normal and aging rats in GSL glycome by online hydrophilic interaction liquid chromatography coupling with ultraviolet detection and tandem mass spectrometry (HILIC-UV-MS/MS). This novel method is versatile and sensitive, enabling accurate quantitative analysis of tissue GSLs and showing great significance for glycomic studies.

Sensitive and robust MALDI-TOF-MS glycomics analysis enabled by Girard's reagent T on-target derivatization (GTOD) of reducing glycans.

Sensitive glycomics analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is of great importance but significantly hampered by their low ionization efficiency and labile sialic acid moieties. Chemical derivatization offers a viable way to improve both the ionization efficiency and analytical sensitivity of the glycans in MS analysis by altering their hydrophobicity or charge property. Here we employed Girard's reagent T (GT) for on-target derivatization (GTOD) of reducing glycan under mild acid condition to form stable hydrazones at room temperature, allowing rapid and sensitive identification of neutral and sialylated glycans in positive-ion mode as only permanently positive charged molecular ions without multiple ion adducts by MALDI-TOF-MS. The MS signal intensities of lactose, sialylated N-glycans derived from bovine fetuin and neutral N-glycans derived from RNaseB and ovalbumin were boosted by 7.44, 9.13, 12.96 and 13.47 folds on average (n = 3), respectively. More importantly, after GTOD strategy, unwanted desialylation of sialylated glycans during MS was suppressed. The detection limit of the assay is desirable since the nanogram of N-glycans derived from 0.16 µg ovalbumin could be detected. The assay demonstrated good stability (RSD≤2.95%, within 10 days), reliable reproducibility (RSD = 2.96%, n = 7) and a desirable linear dynamic range from 78 nmol/mL to 10 µmol/mL. The strategy has been successfully applied to MS analysis of reducing glycans from human milks, neutral and sialylated O-, N-glycans from glycoproteins, and reducing glycans derived from glycosphingolipids, presenting neater [M]+ signals which allow detection of more low-abundance glycans and assignation of Neu5Ac vs. Neu5Gc or fucose vs. hexose in glycans due to the absence of the ambiguous interpretation from multiple peaks (ion adducts [M+Na]+ and [M+K]+). Moreover, the GTOD assay prevents desialylation during MALDI-TOF-MS profiling and enables distinct linkage-specific characterization of terminal sialic acids of N-glycans derived from human serum protein when combines with an esterification.

3-Amino-1-phenyl-2-pyrazoline-5-ketone as a heterobifunctional chromogenic reagent to derivatize reducing glycans for subsequent online LC/MS analysis.

Sensitive analysis of glycans by liquid chromatography/mass spectrometry is significantly hampered by the lack of chromogenic or fluorescent groups on the glycan structures, as well as, their poor ionization properties. In the present, a heterobifunctional chromogenic reagent 3-amino-1-phenyl-2-pyrazoline-5-ketone (PAP) bearing amino and active methylene groups, which readily reacts with reducing glycans, was used for detection of the pre-column-labeled glycans via high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS). The PAP derivatives with active methylene and amino groups were obtained via reductive amination in acidic medium and condensation of an active PAP methylene group with the reducing end of glycans in alkaline medium, respectively, and the PAP derivatives could be further functionalized, e.g., via glycan microarray preparation. The conditions for the two reaction modes were optimized, the HPLC separation method of PAP derivatives was investigated, and the PAP derivatives of some glycans derived from biological samples were obtained and analyzed by ESI-MS and LC-MS. Using this new reagent, reducing glycans can be selectively derivatized by different reaction mechanisms, having great importance for functional glycomics studies.

The invasive MED/Q Bemisia tabaci genome: a tale of gene loss and gene gain.

BACKGROUND: Sweetpotato whitefly, Bemisia tabaci MED/Q and MEAM1/B, are two economically important invasive species that cause considerable damages to agriculture crops through direct feeding and indirect vectoring of plant pathogens. Recently, a draft genome of B. tabaci MED/Q has been assembled. In this study, we focus on the genomic comparison between MED/Q and MEAM1/B, with a special interest in MED/Q's genomic signatures that may contribute to the highly invasive nature of this emerging insect pest. RESULTS: The genomes of both species share similarity in syntenic blocks, but have significant divergence in the gene coding sequence. Expansion of cytochrome P450 monooxygenases and UDP glycosyltransferases in MED/Q and MEAM1/B genome is functionally validated for mediating insecticide resistance in MED/Q using in vivo RNAi. The amino acid biosynthesis pathways in MED/Q genome are partitioned among the host and endosymbiont genomes in a manner distinct from other hemipterans. Evidence of horizontal gene transfer to the host genome may explain their obligate relationship. Putative loss-of-function in the immune deficiency-signaling pathway due to the gene loss is a shared ancestral trait among hemipteran insects. CONCLUSIONS: The expansion of detoxification genes families, such as P450s, may contribute to the development of insecticide resistance traits and a broad host range in MED/Q and MEAM1/B, and facilitate species' invasions into intensively managed cropping systems. Numerical and compositional changes in multiple gene families (gene loss and gene gain) in the MED/Q genome sets a foundation for future hypothesis testing that will advance our understanding of adaptation, viral transmission, symbiosis, and plant-insect-pathogen tritrophic interactions.