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The outbreak of COVID-19 has now become a global pandemic that has severely impacted lives and economic stability. There is, however, no effective antiviral drug that can be used to treat COVID-19 to date. Built on the fact that SARS-CoV-2 initiates its entry into human cells by the receptor binding domain (RBD) of its spike protein binding to the angiotensin-converting enzyme 2 (hACE2), we extended a recently developed approach, EvoDesign, to design multiple peptide sequences that can competitively bind to the SARS-CoV-2 RBD to inhibit the virus from entering human cells. The protocol starts with the construction of a hybrid peptidic scaffold by linking two fragments grafted from the interface of the hACE2 protein (a.a. 22-44 and 351-357) with a linker glycine, which is followed by the redesign and refinement simulations of the peptide sequence to optimize its binding affinity to the interface of the SARS-CoV-2 RBD. The binding experiment analyses showed that the designed peptides exhibited a significantly stronger binding potency to hACE2 than the wild-type hACE2 receptor (with -53.35 vs. -46.46 EvoEF2 energy unit scores for the top designed and wild-type peptides, respectively). This study demonstrates a new avenue to utilize computationally designed peptide motifs to treat the COVID-19 disease by blocking the critical spike-RBD and hACE2 interactions.

In this study, the major secretome components of Penicillium oxalicum 16 and Trichoderma reesei RUT-C30 under wheat bran (WB) and rice straw (RS) solid-state fermentation were systematically analyzed. The activities of the major components, e.g., cellulase, hemicellulase, and amylase, were consistent with their abundance in the secretomes. P. oxalicum 16 secreted more abundant glycoside hydrolases than T. reesei RUT-C30. The main up-regulated proteins from the induction of WB, compared with that from RS, were amylase, pectinase, and protease, whereas the main down-regulated enzymes were cellulase, hemicellulase, swollenin, and lytic polysaccharide monooxygenase (LPMO). Specifically, WB induced more β-1,4-glucosidases, namely, S8B0F3 (UniProt ID), and A0A024RWA5 than RS, but RS induced more β-1,4-exoglucanases and β-1,4-endoglucanases, namely, A0A024RXP8, A024SH76, S7B6D6, S7ZP52, A024SH20, A024S2H5, S8BGM3, S7ZX22, and S8AIJ2. The P. oxalicum 16 xylanases S8AH74 and S7ZA57 were the major components responsible for degrading soluble xylan, and S8BDN2 probably acted on solid-state hemicellulose instead of soluble xylan. The main hemicellulase component of T. reesei RUT-C30 in RS was the xyloglucanase A0A024S9Z6 with an abundance of 16%, but T. reesei RUT-C30 lacked the hemicellulase mannanase and had a small amount of the hemicellulase xylanase. P. oxalicum 16 produced more amylase than T. reesei RUT-C30, and the results suggest amylase S7Z6T2 may degrade soluble starch. The percentage of the glucoamylase S8B6D7 did not significantly change, and reached an average abundance of 5.5%. The major auxiliary degradation enzymes of P. oxalicum 16 were LPMOs S7Z716 and S7ZPW1, whereas those of T. reesei RUT-C30 were swollenin and LPMOs A0A024SM10, A0A024SFJ2, and A0A024RZP7.

Positively charged groups that mimic arginine or lysine in a natural substrate of trypsin are necessary for drugs to inhibit the trypsin-like serine protease TMPRSS2 that is involved in the viral entry and spread of coronaviruses, including SARS-CoV-2. Based on this assumption, we identified a set of 13 approved or clinically investigational drugs with positively charged guanidinobenzoyl and/or aminidinobenzoyl groups, including the experimentally verified TMPRSS2 inhibitors Camostat and Nafamostat. Molecular docking using the C-I-TASSER-predicted TMPRSS2 catalytic domain model suggested that the guanidinobenzoyl or aminidinobenzoyl group in all the drugs could form putative salt bridge interactions with the side-chain carboxyl group of Asp435 located in the S1 pocket of TMPRSS2. Molecular dynamics simulations further revealed the high stability of the putative salt bridge interactions over long-time (100 ns) simulations. The molecular mechanics/generalized Born surface area-binding free energy assessment and per-residue energy decomposition analysis also supported the strong binding interactions between TMPRSS2 and the proposed drugs. These results suggest that the proposed compounds, in addition to Camostat and Nafamostat, could be effective TMPRSS2 inhibitors for COVID-19 treatment by occupying the S1 pocket with the hallmark positively charged groups.

Cerenkov Emission (CE) during external beam radiation therapy (EBRT) from a linear accelerator (Linac) has been demonstrated as a useful tool for radiotherapy quality assurance and potentially other applications for online tracking of tumors during treatment delivery. However, some of the current challenges that are impacting the potential of CE are related to the limited detection sensitivity and the lack of flexible tools to fit into an already complex treatment delivery environment. Silicon photomultiplier (SiPM) solid-state devices are new promising tools for low light detection due to their extreme sensitivity that mirrors photomultiplier tubes and yet have a form factor that is similar to silicon photodiodes, allowing for improved flexibility in device design that may help in the process of wider clinical applicability. In this paper, we assess the feasibility of using SiPMs to detect CE during EBRT from a Linac and contrast their performance with commercially available silicon photodiodes (PDs). We demonstrate the feasibility of the SiPM-based probes for standard dosimetry measurements. We also demonstrate that CE optical signals can be detected from tissue depths about five times greater than that for standard probes based on PDs, making our SiPM probe an enabling technology of CE measurements, particularly for deep tissue applications.

The outbreak of COVID-19 has now become a global pandemic and it continues to spread rapidly worldwide, severely threatening lives and economic stability. Making the problem worse, there is no specific antiviral drug that can be used to treat COVID-19 to date. SARS-CoV-2 initiates its entry into human cells by binding to angiotensin-converting enzyme 2 (hACE2) via the receptor binding domain (RBD) of its spike protein. Therefore, molecules that can block SARS-CoV-2 from binding to hACE2 may potentially prevent the virus from entering human cells and serve as an effective antiviral drug. Based on this idea, we designed a series of peptides that can strongly bind to SARS-CoV-2 RBD in computational experiments. Specifically, we first constructed a 31-mer peptidic scaffold by linking two fragments grafted from hACE2 (a.a. 22-44 and 351-357) with a linker glycine, and then redesigned the peptide sequence to enhance its binding affinity to SARS-CoV-2 RBD. Compared with several computational studies that failed to identify that SARS-CoV-2 shows higher binding affinity for hACE2 than SARS-CoV, our protein design scoring function, EvoEF2, makes a correct identification, which is consistent with the recently reported experimental data, implying its high accuracy. The top designed peptide binders exhibited much stronger binding potency to hACE2 than the wild-type (−53.35 vs. −46.46 EvoEF2 energy unit for design and wild-type, respectively). The extensive and detailed computational analyses support the high reasonability of the designed binders, which not only recapitulated the critical native binding interactions but also introduced new favorable interactions to enhance binding. Due to the urgent situation created by COVID-19, we share these computational data to the community, which should be helpful to develop potential antiviral peptide drugs to combat this pandemic.

Pore structure is known to play a role in the strength of coke. However, across cokes with similar porosities, strength can be variable, indicating that certain features of the pore structure are playing a role. An understanding of what those features are and how they form can pave the way for better coke strength prediction models and reveal new ways to manipulate the coking process to prevent or enable certain features forming. In this paper, rheometry has been used to characterise the viscoelastic properties and follow the pore structure development through normal force measurements as coal is heated at 3 °C/min. Coal samples were quenched in the rheometer at various temperatures corresponding to key events of bubble growth, bubble coalescence, minimum viscosity and resolidification and these quenched samples underwent micro-CT imaging, using the Imaging and Medical Beamline of the Australian Synchrotron to obtain 3D representations of the structure with approximately 10 μm resolution. The number of isolated pores, the pore size distributions of the isolated and connected pores, and the overall porosity were determined. Pore connectivity was found to be established very early on in the softening phase and coincides with the bubble coalescence phenomenon. For the high fluidity coals, virtually all of the pore space in the semi-coke is connected. It is proposed that bubble coalescence and applied forces play key roles in the contraction process and pore structure properties, in particular the size of the pore openings. In addition, during resolidification, the porosity reached a limit, however, the size of the pore openings continued to change, with a reduction in smaller openings and corresponding increase in larger openings. This trend suggests that the pore space becomes more channel-like, which could be due to the gas flow behaviour or could be due to surface tension effects. Semi-fusinite appears to reduce expansion. The precise mechanism by which it reduces expansion is not known. It may act as a conduit for the release of volatiles. This research is part of a broad set of activities which include understanding behaviour in the sole heated oven (SHO) and studying the mechanisms of coke breakage.

Micro-CT imaging of samples of metallurgical coke was achieved at high resolution (~ 10 μm), using the Imaging and Medical Beamline of the Australian Synchrotron. The coke samples were then stressed to breaking point and imaged again. The result is that we can examine, in fine detail in 3D, the locations of the fracture surfaces in the coke, and their relationship to the different aspects of the microstructure. In addition, we have performed 3D finite element linear elasticity analysis, using the microstructure of the cokes as input to the calculations. The result is a 3D map of the von Mises stress in the structure, which provides information about the likely locations of fracture. Finally, we have performed a fractographic analysis of the fracture surfaces, in order to characterise the nature of the fracture mechanisms. The imaging and analysis has been performed for both compressive and mixed-mode loading of the coke samples. The work provides the opportunity to identify the key mechanisms for fracturing of coke due to the aspects of their microstructure, and has the potential to aid in the production of stronger metallurgical coke, by means of blending coals to produce coke with improved strength properties.

We report the wet-state glycation of Bovine Serum Albumin (BSA) with xylose through the Maillard reaction and its effect on protein aggregations during heat treatment. Using low doses of xylose, glycation stabilized the conformations of BSA and reduced the formation of large aggregates during heat treatment. However, using high doses of xylose, glycation promoted the conversion of α-helices to unfolded structures and β-sheets, and induced the formation of short amyloid-like aggregates after heat treatment. Using high-resolution mass spectrometry, three possible glycation sites were determined on BSA treated with low dose of xylose, while six glycations sites were detected when high doses of xylose were used instead. Considering the importance of glycation to the food industry, this work not only provides new insights into the nonlinear effects of glycation on protein stability, but also provides a strategy to tune the nutritional value and functionality of proteins during food processing.

Metal ions perform a wide variety of functions associated with life processes. In this work, we investigated the influence of Mg2+ on the denaturation and aggregation of bovine serum albumin (BSA) during heat treatment. The unfolding and denaturation of proteins during food processing, especially heat treatment, has a major influence on their functional properties. Our results show that Mg2+ significantly stabilized the secondary structure of BSA when the protein was heated at 80 °C, preventing the transformation of α-helices into unfolded structures. Mg2+ also inhibited the aggregation of BSA into larger, heterogeneous structures. Moreover, after adding Mg2+, the melting temperature of BSA increased from 70.20 °C to 73.74 °C, further indicating that Mg2+ has a stabilizing effect. Finally, molecular dynamics (MD) simulations revealed that Mg2+ preferentially binds multivalently to the carboxylate groups of Asp and Glu BSA residues, and the observed stabilizing effect could be attributed to the crosslinking of secondary structure elements within BSA induced by Mg2+ binding. Considering the ubiquity of Mg2+, our work provides a facile strategy to control BSA unfolding and aggregation, which is helpful for enhancing the processing and storage properties of BSA or other proteins during food processing.

Tamoxifen is the major therapeutic agent for the treatment of hormone-dependent breast cancer. Tamoxifen treatment appears to be associated with an increased incidence of endometrial carcinoma in humans and hepatocellular carcinoma in rats. These carcinogenic effects of tamoxifen might be induced by the formation of a tamoxifen reactive intermediate that binds covalently to macromolecules. Liver microsomal cytochrome P450s (CYPs) catalyze the metabolism of tamoxifen, forming a reactive intermediate that binds irreversibly to microsomal proteins, primarily to a 54 kDa protein (Mani, C. and Kupfer, D., Cancer Res., 51, 6052-6058, 1991). The current study identifies the P450 enzymes that catalyze the activation of tamoxifen to a reactive intermediate in rats and humans. Among the species examined, rats, chickens and humans demonstrate low tamoxifen binding activity, ranging from 0.1 to 0.4 nmol bound/mg protein/h. In contrast, hamsters and mice exhibit high binding, 1.2 and 1.6 nmol/mg protein/h respectively. Treatment of male rats with phenobarbital or pregnenolone-16 alpha-carbonitrile (PCN) markedly elevated the binding of tamoxifen to liver microsomal proteins. Methylcholanthrene treatment had no effect on binding. These findings suggested the involvement of CYP3A in catalysis of the covalent binding. Alternate substrates of CYP3A, cortisol and erythromycin, inhibited tamoxifen binding in liver microsomes from PCN- and phenobarbital-treated rats. Treatment of rats with troleandomycin (TAO), an inducer of CYP3A, followed by the dissociation of the TAO-CYP3A complex, elevated the covalent binding to liver microsomes approximately 3-fold. Antibodies against rat CYP3A1 strongly inhibited tamoxifen binding to liver microsomes from PCN- and phenobarbital-treated rats, whereas the antibodies anti-CYP2B1/2B2 did not inhibit binding. In humans, tamoxifen binding was inhibited by the anti-rat CYP3A1 IgG and also by alternate substrates of CYP3A. These results indicate that the activation of tamoxifen to a reactive intermediate by rat and human liver microsomes is principally catalyzed by CYP3A enzymes.

Antibody against purified CYP2A1 recognizes two rat liver microsomal P450 enzymes, CYP2A1 and CYP2A2, that catalyze the 7 alpha- and 15 alpha-hydroxylation of testosterone, respectively. In human liver microsomes, this antibody recognizes a single protein, namely CYP2A6, which catalyzes the 7-hydroxylation of coumarin. To examine species differences in CYP2A function, liver microsomes from nine mammalian species (rat, mouse, hamster, rabbit, guinea pig, cat, dog, cynomolgus monkey, and human) were tested for their ability to catalyze the 7 alpha- and 15 alpha-hydroxylation of testosterone and the 7-hydroxylation of coumarin. Antibody against rat CYP2A1 recognized one or more proteins in liver microsomes from all mammalian species examined. However, liver microsomes from cat, dog, cynomolgus monkey, and human catalyzed negligible rates of testosterone 7 alpha- and/or 15 alpha-hydroxylation, whereas rat and cat liver microsomes catalyzed negligible rates of coumarin 7-hydroxylation. Formation of 7-hydroxycoumarin accounted for a different proportion of the coumarin metabolites formed by liver microsomes from each of the various species examined. 7-Hydroxycoumarin was the major metabolite (greater than 70%) in human and monkey, but only a minor metabolite (less than 1%) in rat. The 7-hydroxylation of coumarin by human liver microsomes was catalyzed by a single, high-affinity enzyme (Km 0.2-0.6 microM), which was markedly inhibited (greater than 95%) by antibody against rat CYP2A1. The rate of coumarin 7-hydroxylation varied approximately 17-fold among liver microsomes from 22 human subjects. This variation was highly correlated (r2 = 0.956) with interindividual differences in the levels of CYP2A6, as determined by immunoblotting. These results indicate that CYP2A6 is largely or entirely responsible for catalyzing the 7-hydroxylation of coumarin in human liver microsomes. Treatment of monkeys with phenobarbital or dexamethasone increased coumarin 7-hydroxylase activity, whereas treatment with beta-naphthoflavone caused a slight decrease. These results suggest that environmental factors can increase or decrease CYP2A expression in cynomolgus monkeys, which implies that environmental factors may be responsible for the large variation in CYP2A6 levels in humans, although genetic factors may also be important. In contrast to rats and mice, the expression of CYP2A enzymes in cynomolgus monkeys and humans was not sexually differentiated. Despite their structural similarity to coumarin, the anticoagulants dicumarol and warfarin do not appear to be substrates for CYP2A6. The overall rate of dicumarol metabolism varied approximately 5-fold among the human liver microsomal samples, but this variation correlated poorly (r2 = 0.126) with the variation observed in CYP2A6 levels and coumarin 7-hydroxylase activity.(ABSTRACT TRUNCATED AT 400 WORDS)

The purpose of the present work was to characterize the effect of musk xylene (MX) and musk ketone (MK) treatment on rat hepatic cytochrome P450 enzymes. Male F344 rats were dosed orally with MX (10, 50 or 200 mg/kg) or MK (20, 100 or 200 mg/kg) for 7 days, after which CYP1A, 2B and 3A enzyme activities and protein levels were determined. MX treatment resulted in a two- to four-fold increase in the activity of CYP1A, 2B and 3A enzymes. For CYP1A and 3A, these changes were consistent with small increases in immunoreactive proteins. However, for CYP2B, despite only a three-fold increase in enzyme activity, protein levels were increased nearly 50-fold relative to control. This induction occurred by transcriptional activation of the CYP2B1 gene as evidenced by increased steady state CYP2B1 mRNA levels. In contrast to MX, MK treatment increased CYP2B activity, protein and mRNA levels. However MK treatment also increased CYP1A enzyme activity nearly 30-fold higher than control rats, a profile that was markedly different from MX, and very different from its effects in mice (Stuard, S.B., Caudill, D., Lehman-Mc-Keeman, L.D., 1997. Characterization of the effects of musk ketone on mouse cytochrome P450 enzymes. Fund. Appl. Toxicol. 40, 264-271). These results indicate that in rats, MX is an inducer of CYP2B enzymes, but these enzymes are not functionally active. In contrast, MK also induces CYP2B enzymes, with no concurrent inactivation. MK also exhibits a unique pattern of cytochrome P450 induction by increasing both CYP1A and CYP2B in rats.

The stability of cytochrome P450 enzymes, cytochrome b5, and NADPH-cytochrome c reductase was examined in (A) human liver samples frozen in liquid nitrogen and stored at -80 degrees C, (B) human liver microsomes suspended in 250 mM sucrose and stored at -80 degrees C, and (C) human liver microsomes subjected to as many as 10 cycles of thawing and freezing. In study A, microsomes from five human livers were prepared from fresh (unfrozen) tissue and from tissue that was stored frozen at -80 degrees C for 1, 2, 4, or 6 months. The apparent concentration of cytochromes P450 and b5 and the activity of NADPH-cytochrome c reductase decreased 20-40% as a result of freezing the liver, regardless of whether the liver was stored for 1 or 6 months. Similar decreases were observed in the activities of cytochrome P450 enzymes belonging to several gene families, namely CYP1A2 (7-ethoxyresorufin O-dealkylation and caffeine N3-demethylation), CYP2A6 (coumarin 7-hydroxylation), CYP2C9 (tolbutamide methylhydroxylation), CYP2C19 (S-mephenytoin 4'- hydroxylation), CYP2D6 (dextromethorphan O-de-methylation), CYP2E1 (chlorzoxazone 6-hydroxylation), CYP3A4solidus5 (testosterone 6beta-hydroxylation), and CYP4A9solidus11 (lauric acid 12-hydroxylation). Freezing human liver did not convert cytochrome P450 to its inactive form, cytochrome P420, but it increased the contamination of liver microsomes with hemoglobin or other heme-containing proteins, which resulted in a uniform decrease in the specific activity of cytochromes P450 and b5 and in the specific activity of all P450 enzymes. In study B, the concentration of cytochromes P450 and b5, the activity of NADPH-cytochrome c reductase, and the activity of individual cytochrome P450 enzymes were determined in 10 samples of human liver microsomes stored at -80 degrees C for approximately 0, 1, or 2 years. The sample-to-sample variation in the concentration and activity of cytochrome P450, cytochrome b5, and NADPH-cytochrome c reductase was nominally affected by long-term storage of human liver microsomes at -80 degrees C, indicating there was no differential loss of cytochrome P450 activity, cytochrome b5 concentration, or NADPH-cytochrome c reductase activity. In study C, microsomes from a pool of human livers were subjected to 1, 2, 3, 5, 7, or 10 cycles of freezing at -80 degrees C followed by thawing at room temperature. Freezing/thawing liver microsomes for up to 10 cycles did not convert cytochrome P450 to P420, nor did it cause significant loss of CYP1A2, CYP2A6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4/5, or CYP4A9/11 activity. Overall, these results suggest that our current methods for storing and processing human liver are well suited to preserving microsomal P450 enzyme activity.