Isolation and characterization of the 1,4-beta-D-glucan glucanohydrolases of Talaromyces emersonii.

Culture filtrates of Talaromyces emersonii were found to contain four endocellulases termed I, II, III and IV, the last having the greatest electrophoretic mobility towards the anode in homogeneous 5%-(w/v)-polyacrylamide gels at pH 4.5. All four are glycoproteins, the carbohydrate contents being: I, 27.7%; II, 29.0%; III, 44.7%; IV, 50.8. Each form is eluted as a single peak corresponding to an Mr value of 68000 on gel filtration at pH 3.5 and as a single band corresponding to an Mr value of 35000 on reductive sodium dodecyl sulphate/polyacrylamide-gradient-gel electrophoresis. However, we believe that the latter represents the native Mr value. The pI values for each lie between pH 2.8 and 3.2. Activity in each case is optimal at pH 5.5-5.8 and at 75-80 degrees C. Half-life values at pH5 and 75 degrees C were from 2 to 4h. The specific activity with any individual substrate was much the same for each enzyme, as was the ratio of activity from one substrate to the next. Possible reasons for the observation that plots of velocity versus substrate concentration are sigmoidal are discussed. We believe that the finding of four endocellulases reflects differential glycosylation of a single enzyme form rather than genetically determined differences in primary structure.     

Lack of a circadian rhythm in the ability of the gametocytes of Plasmodium falciparum to infect Anopheles gambiae

Batches of Anopheles gambiae were fed once during the day or once or twice during the night on 12 carriers naturally infected with gametocytes of P. falciparum. No overall difference was noted in the oocyst numbers in batches of mosquitoes fed during the night as compared to the daytime.     

Lipocalin‐2 released in response to cerebral ischaemia mediates reperfusion injury in mice

Thrombolysis remains the only effective therapy to reverse acute ischaemic stroke. However, delayed treatment may cause serious complications including hemorrhagic transformation and reperfusion injury. The level of lipocalin‐2 (LCN2) is elevated in the plasma of ischaemic stroke patients, but its role in stroke is unknown. Here, we show that LCN2 was acutely induced in mice after ischaemic stroke and is an important mediator of reperfusion injury. Increased levels of LCN2 were observed in mouse serum as early as 1 hr after transient middle cerebral artery occlusion (tMCAO), reaching peak levels at 23 hrs. LCN2 was also detected in neutrophils infiltrating into the ipsilateral hemisphere, as well as a subset of astrocytes after tMCAO, but not in neurons and microglia. Stroke injury, neurological deficits and infiltration of immune cells were markedly diminished in LCN2 null mice after tMCAO, but not after permanent MCAO (pMCAO). In vitro, recombinant LCN2 protein induced apoptosis in primary cultured neurons in a dose‐dependent manner. Our results demonstrate that LCN2 is a neurotoxic factor secreted rapidly in response to cerebral ischaemia, suggesting its potential usage as an early stroke biomarker and a novel therapeutic target to reduce stroke‐reperfusion injury.     

Molecular biology of rotaviruses. III. Isolation and characterization of temperature-sensitive mutants of bovine rotavirus

Twenty-six temperature-sensitive (ts) mutants of United Kingdom tissue culture-adapted bovine rotavirus were isolated and characterized. Fourteen of these mutants were determined to be ts both by efficiency of plating and by virus yield at the nonpermissive temperature of 39.5 degrees C as compared with that at the permissive temperature of 32 degrees C. The remaining mutants were only ts by the criterion of efficiency of plating. High-frequency recombination (gene reassortment) was observed when some pairs of mutants were crossed, and this allowed the classification of the mutants into five separate recombination groups. Groups III and V have prototype ts mutants (ts34 and ts115, respectively) that do not synthesize RNA or polypeptides at 39.5 degrees C. The other groups, I, II, and IV, have prototype mutants (ts17, ts7, and ts6, respectively) that synthesize both RNA and polypeptides at 39.5 degrees C, although ts17 does so only at a reduced level.     

Comparative molecular docking and simulation analysis of molnupiravir and remdesivir with SARS-CoV-2 RNA dependent RNA polymerase (RdRp)

Treatment of SARS-CoV-2 targeting its RNA dependent RNA polymerase (RdRp) is of current interest. Remdesivir has been approved for the treatment of COVID-19 around the world. However, the drug has been linked with pharmacological limitations like adverse effects and reduced efficiency. Nevertheless, recent advancements have depicted molnupiravir as an effective therapeutic agent to target the SARS-CoV-2 RdRp. The drug has cleared both in vitro and in vivo screening. It is in phase-III clinical trial. Nonetheless, there are no data on themolecular binding interaction of molnupiravir with RdRp. Therefore, it is of interest to report the binding interaction of molnupiravir using molecular docking. It is also of interest to show its stability during interaction using molecular dynamics and binding free energy calculations along with drug likeliness and pharmacokinetic properties in comparison with remdesivir.     

The isolation, purification and properties of the cellobiohydrolase component of Penicillium funiculosum cellulase.

1. A cellobiohydrolase component was isolated from a Penicillium funiculosum cellulase preparation by chromatography on DEAE-Sephadex, and purified by isoelectric focusing. 2. Purified in this way, the enzyme was homogeneous as judged by electrophoresis on sodium dodecyl sulphate/polyacrylamide gels and isoelectric focusing in polyacrylamide gels. 3. Acting in isolation, the enzyme had little hydrolytic activity to highly ordered celluloses such as cotton fibre, but, when recombined in the original proportions with the other components [endo-(1 leads to 4)-beta-D-glucanase and beta-D-glucosidase] of the complex, 98% of the original activity was recovered. 4. Synergistic effects were also observed when the enzyme was acting in concert with endo-(1 leads to 4)-beta-D-glucanase from other fungal sources. 5. Less-well-ordered celluloses, such as that swollen in H3PO4, were extensively hydrolysed, the principal product being cellobiose. 6. Attack on carboxymethyl-cellulose (CM-cellulose), which is the substrate normally used to assay for endo-(1 leads to 4)-beta-D-glucanase activity, was minimal. 7. The enzyme was associated with 9% of neutral sugar, 88% of which was mannose. It was isoelectric at pH 4.36 (4 degrees C) and had a mol.wt. of 46 300 (determined by gel chromatography on a calibrated column of Ultrogel). 8. The enzyme was specific for the beta-(1 leads to 4)-linkage.     

Two-chain high molecular weight kininogen induces endothelial cell apoptosis and inhibits angiogenesis: partial activity within domain 5.

We previously reported that the binding of two-chain high molecular weight kininogen (HKa) to endothelial cells may occur through interactions with endothelial urokinase receptors. Since the binding of urokinase to urokinase receptors activates signaling responses and may stimulate mitogenesis, we assessed the effect of HKa binding on endothelial cell proliferation. Unexpectedly, HKa inhibited proliferation in response to several growth factors, with 50% inhibition caused by approximately 10 nM HKa. This activity was Zn(2+) dependent and not shared by either single-chain high molecular weight kininogen (HK) or low molecular weight kininogen. HKa selectively inhibited the proliferation of human umbilical vein and dermal microvascular endothelial cells, but did not affect that of umbilical vein or human aortic smooth muscle cells, trophoblasts, fibroblasts, or carcinoma cells. Inhibition of endothelial proliferation by HKa was associated with endothelial cell apoptosis and unaffected by antibodies that block the binding of HK or HKa to any of their known endothelial receptors. Recombinant HK domain 5 displayed activity similar to that of HKa. In vivo, HKa inhibited neovascularization of subcutaneously implanted Matrigel plugs, as well as rat corneal angiogenesis. These results demonstrate that HKa is a novel inhibitor of angiogenesis, whose activity is dependent on the unique conformation of the two-chain molecule.     

Re-evaluation of the Carcinogenic Significance of Hepatitis B Virus Integration in Hepatocarcinogenesis

To examine the role of hepatitis B virus (HBV) integration in hepatocarcinogenesis, a systematic comparative study of both tumor and their corresponding non-tumor derived tissue has been conducted in a cohort of 60 HBV associated hepatocellular carcinoma (HCC) patients. By using Alu-polymerase chain reaction (PCR) and ligation-mediated PCR, 233 viral-host junctions mapped across all human chromosomes at random, no difference between tumor and non-tumor tissue was observed, with the exception of fragile sites (P = 0.0070). HBV insertions in close proximity to cancer related genes such as hTERT were found in this study, however overall they were rare events. No direct correlation between chromosome aberrations and the number of HBV integration events was found using a sensitive array-based comparative genomic hybridization (aCGH) assay. However, a positive correlation was observed between the status of several tumor suppressor genes (TP53, RB1, CDNK2A and TP73) and the number of chromosome aberrations (r = 0.6625, P = 0.0003). Examination of the viral genome revealed that 43% of inserts were in the preC/C region and 57% were in the HBV X gene. Strikingly, approximately 24% of the integrations examined had a breakpoint in a short 15 nt viral genome region (1820–1834 nt). As a consequence, all of the confirmed X gene insertions were C-terminal truncated, losing their growth-suppressive domain. However, the same pattern of X gene C-terminal truncation was found in both tumor and non-tumor derived samples. Furthermore, the integrated viral sequences in both groups had a similar low frequency of C1653T, T1753V and A1762T/G1764A mutations. The frequency and patterns of HBV insertions were similar between tumor and their adjacent non-tumor samples indicating that the majority of HBV DNA integration events are not associated with hepatocarcinogenesis.     

Dendrite Growth—Microstructure—Stress—Interrelations in Garnet Solid-State Electrolyte

This study illustrates how the microstructure of garnet solid-state electrolytes (SSE) affects the stress-state and dendrite growth. Tantalum-doped lithium lanthanum zirconium oxide (LLZTO, Li_6.4La₃Zr_1.4Ta_0.6O₁₂) is synthesized by powder processing and sintering (AS), or with the incorporation of intermediate-stage high-energy milling (M). The M compact displays higher density (91.5% vs 82.5% of theoretical), and per quantitative stereology, lower average grain size (5.4 ± 2.6 vs 21.3 ± 11.1 µm) and lower AFM-derived RMS surface roughness contacting the Li metal (45 vs 161 nm). These differences enable symmetric M cells to electrochemically cycle at constant capacity (0.1 mAh cm⁻²) with enhanced critical current density (CCD) of 1.4 versus 0.3 mA cm⁻². It is demonstrated that LLZTO grain size distribution and internal porosity critically affect electrical short-circuit failure, indicating the importance of electronic properties. Lithium dendrites propagate intergranularly through regions where LLZTO grains are smaller than the bulk average (7.4 ± 3.8 µm for AS in a symmetric cell, 3.1 ± 1.4 µm for M in a half-cell). Metal also accumulates in the otherwise empty pores of the sintered compact present along the dendrite path. Mechanistic modeling indicates that reaction and stress heterogeneities are interrelated, leading to current focusing and preferential plating at grain boundaries.