Paclitaxel, a natural antitumor compound, is produced by yew trees at very low concentrations, causing a worldwide shortage of this important anticancer medicine. These plants also produce significant amounts of 7-␤-xylosyl-10-deacetyltaxol, which can be bio-converted into 10-deacetyltaxol for the semi-synthesis of paclitaxel. Some microorganisms can convert 7-␤-xylosyl-10-deacetyltaxol into 10-deacetyltaxol, but the bioconversion yield needs to be drastically improved for industrial applications. In addition, the related ␤-xylosidases of these organisms have not yet been defined. We set out to discover an efficient enzyme for 10-deacetyltaxol production. By combining the de novo sequencing of ␤-xylosidase isolated from Lentinula edodes with RT-PCR and the rapid amplification of cDNA ends, we cloned two cDNA variants, Lxyl-p1-1 and Lxyl-p1-2, which were previously unknown at the gene and protein levels. Both variants encode a specific bifunctional ␤-D-xylosidase/␤-D-glucosidase with an identical ORF length of 2412 bp (97% identity). The enzymes were characterized, and their 3.6-kb genomic DNAs (G-Lxyl-p1-1, G-Lxyl-p1-2), each harboring 18 introns, were also obtained. Putative substrate binding motifs, the catalytic nucleophile, the catalytic acid/base, and potential N-glycosylation sites of the enzymes were predicted. Kinetic analysis of both enzymes showed kcat/K m of up to 1.07 s ؊1 mM ؊1 against 7-␤-xylosyl-10-deacetyltaxol. Importantly, at substrate concentrations of up to 10 mg/ml (oversaturated), the engineered yeast could still robustly convert 7-␤-xylosyl-10-deacetyltaxol into 10-deacetyltaxol with a conversion rate of over 85% and a highest yield of 8.42 mg/ml within 24 h, which is much higher than those reported previously. Therefore, our discovery might lead to significant progress in the development of new 7-␤-xylosyl-10-deacetyltaxol-converting enzymes for more efficient use of 7-␤-xylosyltaxanes to semi-synthesize paclitaxel and its analogues. This work also might lead to further studies on how these enzymes act on 7-␤-xylosyltaxanes and contribute to the growing database of glycoside hydrolases. Molecular &

Abstract-For more than 50 years, it has been assumed that ventricular fibrillation (VF) is maintained solely by reentry in the working myocardium. This hypothesis has never been tested by recording VF with electrodes spaced sufficiently close to map activation sequences in 3D. We recorded the first 10 minutes of electrically induced VF from the anterior left ventricular (LV) free wall near the insertion of the anterior papillary muscle in 6 pigs. A 3D transmural unipolar electrode array consisting of a 9ϫ9 array of needles with 2-mm spacing and 6 electrodes 2 mm apart on each needle was used for recordings. Automatic analyses were performed to recognize 3D reentry and foci. Our results showed that intramural reentry is present early but not late during VF in the mapped region. The incidence of reentry in working myocardium decreases almost to 0 after 3 minutes of VF. In contrast, intramural foci are present during early VF and, as VF continues, increase in incidence, so that by 10 minutes of VF, 27% of wavefronts arise from intramural foci. These results suggest that, particularly after the first 3 minutes of VF, mechanisms other than local reentry in the working myocardium maintain VF in the anterior LV free wall near the root of the anterior papillary muscle.

Background Since December 2019, a cluster of coronavirus disease 2019 (COVID-19) occurred in Wuhan, Hubei Province, China and spread rapidly from China to other countries. In-hospital mortality are high in severe cases and cardiac injury characterized by elevated cardiac troponin are common among them. The mechanism of cardiac injury and the relationship between cardiac injury and in-hospital mortality remained unclear. Studies focused on cardiac injury in COVID-19 patients are scarce.Objectives To investigate the association between cardiac injury and in-hospital mortality of patients with confirmed or suspected COVID-19.Methods Demographic, clinical, treatment, and laboratory data of consecutive confirmed or suspected COVID-19 patients admitted in Wuhan No.1 Hospital from 25 th December, 2019 to 15 th February, 2020 were extracted from electronic medical records and were retrospectively reviewed and analyzed. Univariate and multivariate Cox regression analysis were used to explore the risk factors associated with in-hospital death.Results A total of 110 patients with confirmed (n=80) or suspected (n=30) COVID-19 were screened and 48 patients (female 31.3%, mean age 70.58±13.38 year old) among them with high-sensitivity cardiac troponin I (hs-cTnI) test within 48 hours after admission were included, of whom 17 (17/48, 35.4%) died in hospital while 31 (31/48, 64.6%) were discharged or transferred to other hospital. High-sensitivity cardiac troponin I was elevated in 13 (13/48, 27.1%) patents. Multivariate Cox regression analysis showed pulse oximetry of oxygen saturation (SpO2) on admission (HR 0.704, 95% CI 0.546-0.909, per 1% decrease, p=0.007), elevated hs-cTnI (HR 10.902, 95% 1.279-92.927, p=0.029) and elevated d-dimer (HR 1.103, 95%CI 1.034-1.176, per 1mg/L increase, p=0.003) on admission were independently associated with in-hospital mortality.

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The diversity and biological activities of fungi associated with the two sponges, Haliclona simulans and Gelliodes carnosa, were investigated using a culturedependent method followed by analysis of the fungal rDNA-ITS sequences. The two sponges were collected from the coastal waters of Lingshui Bay of Hainan Island in the South China Sea. A total of 37 independent fungal isolates corresponding to 30 different species were obtained from the two sponges. Nearly two thirds of the strains (n=24, 64.9%) had close affiliations (identity (ID) or similarity≥98%) with their best matches in GenBank. Another one third of the isolates (n=13, 35.1%) were distantly related to their closest relatives (ID<98%), implying that these species are possibly different from those previously reported. The two sponges possessed similar fungal diversities. Haliclona simulans harbored a mainly different fungal consortium as compared with that of the same sponge species collected from Irish coastal waters, suggesting that the fungal diversity associated with the sponges is more dependent on the surrounding environment than on the sponge species. Biological activities of the fungal culture extracts were tested against the human tumor cell lines, mainly, a human lung carcinoma cell line (A-549), a human liver carcinoma cell line (Bel-7402), and a human colon carcinoma cell line (HCT-8), and against the Gram positive bacterium Bacillus subtilis. A relatively high proportion of positive results were obtained in this study, demonstrating that fungi isolated from sponges could be a rich source of new biologically active natural products.

Objective: To clarify whether hyperthyroidism (HT) itself confers an additional effect on the hypercoagulable state and the risk of ischemic stroke among patients with hyperthyroid atrial fibrillation (AF). Methods: We prospectively evaluated plasma D-dimer levels and thromboembolic events among three groups of patients (hyperthyroid AF, n = 62; nonthyroid AF, n = 107, and HT without AF, n = 100). Plasma D-dimer levels were used to evaluate the hypercoagulable state. Results: The D-dimer level was significantly higher in patients with hyperthyroid AF than in nonthyroid AF (0.66 ± 0.06 vs. 0.34 ± 0.02 mg/l, p < 0.001) and HT without AF (0.66 ± 0.06 vs. 0.27 ± 0.02 mg/l, p < 0.001). During a 3-year follow-up, patients with hyperthyroid AF had a significantly higher incidence of ischemic stroke compared with patients with nonthyroid AF (hazard ratio, HR: 3.2, 95% confidence interval, CI: 1.01-5.59, p = 0.04). Cox regression analysis revealed that age (HR: 2.5, 95% CI: 1.01-1.21, p = 0.05), CHADS₂-VAS score (HR: 5.5, 95% CI: 1.51-7.43, p = 0.01) and anticoagulation (HR: 0.45, 95% CI: 0.07-0.54, p = 0.01) were independent predictors of risk for the occurrence of ischemic stroke. Conclusions: The present study suggests that HT may enhance the hypercoagulable state and the risk of ischemic stroke in patients with AF.

The low amplitudes of F wave in leads aVF and V1 could predict the recurrence of patients with persistent AF who underwent catheter ablation.

The gene encoding squalene synthase (GfSQS) was cloned from Fusarium fujikuroi (Gibberella fujikuroi MP-C) and characterized. The cloned genomic DNA is 3,267 bp in length, including the 5'-untranslated region (UTR), 3'-UTR, four exons, and three introns. A noncanonical splice-site (CA-GG, or GC-AG) was found at the first intron. The open reading frame of the gene is 1,389 bp in length, corresponding to a predicted polypeptide of 462 amino acid residues with a MW 53.4 kDa. The predicted GfSQS shares at least four conserved regions involved in the enzymatic activity with the SQSs of varied species. The recombinant protein was expressed in E. coli and detected by SDS-PAGE and western blot. GC-MS analysis showed that the wild-type GfSQS could catalyze the reaction from farnesyl diphosphate (FPP) to squalene, while the mutant mGfSQS (D82G) lost total activity, supporting the prediction that the aspartate-rich motif (DTXED) in the region I of SQS is essential for binding of the diphosphate substrate.