Phosphorylation of Orc6 During Mitosis Regulates DNA Replication and Ribosome Biogenesis.

The human Origin Recognition Complex (ORC) is required not only for the initiation of DNA replication, but is also implicated in diverse cellular functions, including chromatin organization, centrosome biology, and cytokinesis. The smallest subunit of ORC, Orc6, is poorly conserved amongst eukaryotes. Recent studies from our laboratory have suggested that human Orc6 is not required for replication licensing, but is needed for S-phase progression. Further, ATR-dependent phosphorylation of Orc6 at T229 is implicated in DNA damage response during S-phase. In this study, we demonstrate that the CDK-dependent phosphorylation of Orc6 at T195 occurs during mitosis. While the phosphorylation at T195 does not seem to be required to exit mitosis, cells expressing the phosphomimetic T195E mutant of Orc6 impede S-phase progression. Moreover, the phosphorylated form of Orc6 associates with ORC more robustly, and Orc6 shows enhanced association with the ORC outside of G1, supporting the view that Orc6 may prevent the role of Orc1-5 in licensing outside of G1. Finally, Orc6 and the phosphorylated Orc6 localize to the nucleolar organizing centers and regulate ribosome biogenesis. Our results suggest that phosphorylated Orc6 at T195 prevents replication.

Monoallelically expressed noncoding RNAs form nucleolar territories on NOR-containing chromosomes and regulate rRNA expression.

Out of the several hundred copies of rRNA genes arranged in the nucleolar organizing regions (NOR) of the five human acrocentric chromosomes, ~50% remain transcriptionally inactive. NOR-associated sequences and epigenetic modifications contribute to the differential expression of rRNAs. However, the mechanism(s) controlling the dosage of active versus inactive rRNA genes within each NOR in mammals is yet to be determined. We have discovered a family of ncRNAs, SNULs (Single NUcleolus Localized RNA), which form constrained sub-nucleolar territories on individual NORs and influence rRNA expression. Individual members of the SNULs monoallelically associate with specific NOR-containing chromosomes. SNULs share sequence similarity to pre-rRNA and localize in the sub-nucleolar compartment with pre-rRNA. Finally, SNULs control rRNA expression by influencing pre-rRNA sorting to the DFC compartment and pre-rRNA processing. Our study discovered a novel class of ncRNAs influencing rRNA expression by forming constrained nucleolar territories on individual NORs.

MALDI-TOF MS-Based Clustering and Antifungal Susceptibility Tests of Talaromyces marneffei Isolates from Fujian and Guangxi (China).

Introduction: Talaromyces marneffei is a life-threatening pathogen that causes systemic talaromycosis in immunocompromised and acquired immunodeficiency syndrome (AIDS) patients. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) as a tool to cluster T. marneffei isolates is rarely reported and the data on antifungal susceptibility of T. marneffei isolated in the southern region of China, especially in Fujian, is hardly found. Methods: MALDI-TOF MS was used to cluster 135 T. marneffei isolates, and the minimum inhibitory concentration (MIC) values of amphotericin B, itraconazole, posaconazole, voriconazole, fluconazole, anidulafungin, micafungin, caspofungin and 5-fluorocytosine with Sensititre YeastOne™ YO10 assay were measured during January 2017 to October 2020 in Fujian and Guangxi. Results: MALDI-TOF MS correctly identified 100% of the T. marneffei isolates. Hierarchical clustering of MALDI-TOF peak profiles identified four different clusters. MICs for itraconazole, posaconazole, voriconazole and amphotericin B were as follows: ≤0.015-0.03 µg/mL, ≤0.008-0.03 µg/mL, ≤0.008-0.06 µg/mL, ≤0.12-1 µg/mL, respectively. MICs for echinocandins and fluconazole were comparatively high. Conclusion: Since only simple sample preparation is required and since results are available in a short period of time, MALDI-TOF MS can be considered as a method for identification and clustering of T. marneffei. Itraconazole, posaconazole, voriconazole and amphotericin B can be used to treat T. marneffei infected patients due to the low MICs.

Oxidation of 1-propanol to propionic acid with hydrogen peroxide catalysed by heteropolyoxometalates.

BACKGROUND: Propionic acid as a very valuable chemical is in high demand, and it is industrially produced via the oxo-synthesis of ethylene or ethyl alcohol and via the oxidation of propionaldehyde with oxygen. It is urgent to discover a new preparation method for propionic acid via a green route. Recyclable amino-acid-based organic-inorganic heteropolyoxometalates were first used to high-efficiently catalyse the selective oxidation of 1-propanol to propionic acid with H2O2 as an oxidant. RESULT: A series of amino-acid-based heteropoly catalysts using different types of amino acids and heteropoly acids were synthesized, and the experimental results showed proline-based heteropolyphosphatotungstate (ProH)3[PW12O40] exhibited excellent catalytic activity for the selective catalytic oxidation of 1-propanol to propionic acid owing to its high capacity as an oxygen transfer agent and suitable acidity. Under optimized reaction conditions, the conversion of 1-propanol and the selectivity of propionic acid reached 88% and 75%, respectively. Over four cycles, the conversion remained at >80%, and the selectivity was >60%. (ProH)3[PW12O40] was also used to catalyse the oxidations of 1-butanol, 1-pentanol, 1-hexanol, and benzyl alcohol. All the reactions had high conversions, with the corresponding acids being the primary oxidation product. CONCLUSIONS: Proline-based heteropolyoxometalate (ProH)3[PW12O40] has been successfully used to catalyse the selective oxidation of primary alcohols to the corresponding carboxylic acids with H2O2 as the oxidant. The new developed catalytic oxidation system is mild, high-efficient, and reliable. This study provides a potential green route for the preparation propionic acid.

The p53-induced RNA-binding protein ZMAT3 is a splicing regulator that inhibits the splicing of oncogenic CD44 variants in colorectal carcinoma.

p53 is an intensely studied tumor-suppressive transcription factor. Recent studies suggest that the RNA-binding protein (RBP) ZMAT3 is important in mediating the tumor-suppressive effects of p53. Here, we globally identify ZMAT3-regulated RNAs and their binding sites at nucleotide resolution in intact colorectal cancer (CRC) cells. ZMAT3 binds to thousands of mRNA precursors, mainly at intronic uridine-rich sequences and affects their splicing. The strongest alternatively spliced ZMAT3 target was CD44, a cell adhesion gene and stem cell marker that controls tumorigenesis. Silencing ZMAT3 increased inclusion of CD44 variant exons, resulting in significant up-regulation of oncogenic CD44 isoforms (CD44v) and increased CRC cell growth that was rescued by concurrent knockdown of CD44v Silencing p53 phenocopied the loss of ZMAT3 with respect to CD44 alternative splicing, suggesting that ZMAT3-mediated regulation of CD44 splicing is vital for p53 function. Collectively, our findings uncover a p53-ZMAT3-CD44 axis in growth suppression in CRC cells.

[Relationship Between the VNTR Loci Polymorphism and MALDI-TOF MS Protein Profile Diversity in Mycobacterium tuberculosis Complex].

OBJECTIVE: To study the relationship between the copy numbers of repetitive units at variable number tandem repeat (VNTR) loci of Mycobacterium tuberculosis complex (MTBC) with its diversity of protein profiles. METHODS: The MTBC strains were subjected to genotyping using multiple locus variable number tandem repeat analysis (MLVA). Also, the principal component analysis (PCA) was performed for bacterial protein profiles of MTBC using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The relationship between the polymorphism of VNTR loci and PCA clustering was analyzed. RESULTS: A total of 157 MTBC strains were collected. 146 MTBC strains (MS identification score values ≥1.700) were performed PCA and three clusters, clusterⅠ(61 strains), clusterⅡ(26 strains) and cluster Ⅲ(59 strains), were generated. Polymorphic diversities were observed in 24 VNTR loci, among them, 7 were highly various, 7 were moderately, and 10 were low various. The polymorphism of Mtub39, QUB26 and QUB4156 loci were correlated with the results of MALDI-TOF MS clustering (P=0.000, P=0.035, P=0.017). CONCLUSION: The polymorphism of Mtub39, QUB26 and QUB4156 loci in MTBC was correlated with the difference of MALDI-TOF MS protein profiles, suggesting that these loci may play a role in regulating the composition of protein profiles of MTBC strains.

[Triton X-100 Enhanced the Detection of Hodge Test and Carba NP Test for Carbapenemase-producing Acinetobacter baumannii Complex].

OBJECTIVE: To find out highly effective phenotypic methods to detect carbapenemase-producing Acinetobacter baumannii (A. baumannii complex) so as to support the epidemiological investigation and clinical application. METHODS: We included 113 A. baumannii complex and compared the detection performance of modified Hodge test, Carba NP test, Triton Hodge test, and the simplified Carba NP-direct test with Tritont X-100. RESULTS: We tested 83 carbapenemase-producing A. baumannii complex and 30 non-carbapenemase producers. The sensitivity and specificity of Hodge test were significantly higher than those of Carba NP test (71.1% versus 35.0%, 100% versus 86.7%, P<0.05, respectively). The sensitivity of Triton Hodge test and Carba NP-direct test was respectively significantly higher than Hodge test and Carba NP test (98.8% versus 71.1%, 85.5% versus 35.0%, P<0.001, respectively ). However, the specificities were comparable (P>0.05). The positive additive effects of the two methods with Triton X-100 were more obvious than those of the methods without Triton X-100 (P<0.001). CONCLUSION: Triton X-100 could increase the sensitivity and positive additive effect on phenotypic detection of A. baumannii complex. Triton Hodge test and Carba NP-direct test were more applicable for clinical routine procedure.

Rapid antimicrobial susceptibility testing by matrix-assisted laser desorption ionization-time of flight mass spectrometry using a qualitative method in Acinetobacter baumannii complex.

The transmission and infections of multidrug-resistant bacteria can be prevented by rapid identification and antibiotic susceptibility testing (AST) for pathogenic bacteria in a clinical microbiology laboratory. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been routinely used as a tool for the identification of pathogens; however, a simple and accurate method for a rapid determination of the antimicrobial susceptibility profile is an urgent requisite. The present study established a method based on mass spectrometry to determine the drug resistance. Acinetobacter baumannii complex isolates were tested as an example. After short-term culture, the isolates were incubated with meropenem of different concentrations to determine the growth or the inhibition of the growth by MALDI-TOF MS. The agreement of minimum inhibitory concentration (MIC) values between MALDI-TOF MS-based rapid AST and broth microdilution method in susceptible and resistant strains was 77.1% and 70.1%, respectively. The susceptibility-breakpoint concentration (2 µg/mL) achieved a 98.9% sensitivity and 100% specificity with respect to resistance detection. Similarly, 96.9% sensitivity and 100% specificity were obtained for resistance detection with meropenem concentration at 8 µg/mL. MALDI-TOF MS-based rapid AST was applied to determine the drug resistance at breakpoint concentration, although MS-MICs might shift to a low dilution. Thus, it is critical for patients to accelerate the AST result from two days to several hours.

Extended-spectrum ß-lactamase-producing E. coli septicemia among rectal carriers in the ICU.

The aim of this study was to identify risk factors for extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli (E coli) bloodstream infection (BSI) among carriers hospitalized between March 2011 and June 2016 at the ICU of the West China Hospital.The cases were patients with at least 1 episode of ESBL-producing E coli BSI within 1 week after a positive rectal swab. Controls were selected randomly 1:2 among ESBL-producing E coli rectal carriers who did not develop BSI.Among 19,429 ICU patients, 9015 (46.4%) had a positive rectal swab for ESBL-producing E coli. Of them, 42 (0.5%) were diagnosed with ESBL-producing E coli BSI. The in-hospital mortality was higher for the BSI patients compared with controls (19.1% vs. 6.0%, P = .031). In the past 72 hours, patients in case group were more likely to use penicillin (odds ratio [OR] = 12.076; 95% confidence interval [CI]: 1.397-104.251, P = .02), cephalosporin (OR = 6.900; 95% CI: 1.493-31.852, P = .01), and carbapenem (OR = 5.422; 95% CI: 1.228-23.907, P = .03) as compared to patients in control group. Also, when compared to patients in control group, patients in case group were likely to stay for a longer time in ICU before positive rectal swab test (OR = 1.041, 95% CI: 1.009-1.075, P = .01) and have higher maximum body temperature before positive rectal swab (OR = 8.014; 95% CI: 2.408-26.620, P = .001).Bacteremia owing to ESBL-producing E coli was associated with high antimicrobial exposure, hospital stay, and maximum body temperature.