Mutational analysis of Aspergillus awamori glucoamylase selectivity to improve glucose yield

Three single mutations, Lys108→Arg, Lys108→Met and Tyr312→Trp, and one insertional mutation, 322-314 Loop, in Aspergillus awamori glucoamylase were constructed and tested along with four previously constructed thermostable mutations: Asn20→Cys/Ala27→Cys (SS), Ser30→Pro, Gly137→Ala, and Ser436→Pro, and one previously made thermosensitive mutation, Ala27→Pro, for their selectivity by high-concentration glucose condensation and maltodextrin hydrolysis reactions. Lys108 is important in substrate binding during maltooligosaccharide hydrolysis. Both SS and 311-314 Loop mutant GAs successfully decrease isomaltose formation rate and thus increase peak glucose yield. Tyr312→Trp GA helps to form a tighter hydrogen bond network between Tyr48, Tyr311 and Glu400, and therefore increases peak glucose yield up to 2% at 55°C;The following multiple mutations were made to improve selectivity, thermostability, and specific activity according to the principle of cumulative mutational effects of proteins: SS/311-314 Loop, SS/Ser411→Ala, Ser30→Pro/311-314 Loop, Ser30→Pro/Ser411 →Ala, Gly137→Ala/311-314 Loop, Gly137→Ala/Ser411 →Ala, 311-314 Loop/Ser411→Ala, Ser30→Pro/Gly137→Ala/311-314 Loop, and Ser30→Pro/Gly137→ Ala/Ser411→Ala. Some previously constructed multiple thermostable mutations, SS/Ser30→Pro, SS/Gly137→Ala, SS/Ser436→Pro, Ser30→Pro/Gly137→ Ala, and Gly137→Ala/Ser436→Pro, were also tested for their selectivity. Only SS/Ser436→Pro and Glyl37→ Ala/Ser43 6→Pro GAs have generally lower peak glucose yields than does wild-type GA, while SS/Ser411→Ala GA has the lowest initial rates of glucose and isomaltose formation from 30% (w/v) maltodextrin hydrolysis and 30% (w/v) glucose condensation reactions, respectively, among all double mutations. 311-314 Loop/Ser411→Ala GA is the most thermosensitive mutated GA with the lowest specific activity. Both Ser30→ Pro/Gly137→Ala/311-314 Loop and Ser30→Pro/Glyl 37→Ala/Ser411→Ala GAs are the best mutant GAs so far regarding to their thermostability and selectivity;In general, there is an inverse linear correlation between the peak glucose yield and the initial rate ratio if isomaltose formation over glucose formation. The peak glucose yield is slightly higher for DE 25 maltodextrin than for DE 10 or DE 18 maltodextrins. Peak glucose yields decrease with increasing temperatures. Temperature plays a very important in determining GA selectivity. There is no strong correlation between substrate selectivity and thermostability of GA

Immunogenic cell death: The cornerstone of oncolytic viro-immunotherapy

According to the World Health Organization, cancer is one of the leading global health concerns, causing nearly 10 million deaths in 2020. While classical chemotherapeutics produce strong cytotoxicity on cancer cells, they carry limitations of drug resistance and off-target effects and sometimes fail to elicit adequate antitumor protection against tumor relapse. Additionally, most cancer cells have developed various ways to escape immune surveillance. Nevertheless, novel anticancer strategies such as oncolytic viro-immunotherapy can trigger immunogenic cell death (ICD), which can quickly grasp the attention of the host defense machinery, resulting in an ensuing antitumor immune response. Specifically, oncolytic viruses (OVs) can infect and destroy targeted cancer cells and stimulate the immune system by exposing pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) to promote inflammatory reactions, and concomitantly prime and induce antitumor immunity by the release of neoantigens from the damaged cancer cells. Thus, OVs can serve as a novel system to sensitize tumor cells for promising immunotherapies. This review discusses the concept of ICD in cancer, centralizing ICD-associated danger signals and their consequence in antitumor responses and ICD induced by OVs. We also shed light on the potential strategies to enhance the immunogenicity of OVs, including the use of genetically modified OVs and their combination with ICD-enhancing agents, which are helpful as forthcoming anticancer regimens

Strategies to Preclude Hepatitis C Virus Entry

Without a preventive vaccine, hepatitis C virus (HCV) remains an important pathogen worldwide with millions of carriers at risk of end-stage liver diseases. Despite the introduction of novel direct-acting antivirals (DAAs), resistance problems, challenges with the difficult-to-treat populations and high costs limit the widespread application of these drugs. Antivirals with alternative mechanism(s) of action, such as by restricting viral entry or cell-to-cell spread, could help expand the scope of antiviral strategies for the management of hepatitis C. Transfusion-associated HCV infection remains another issue in endemic and resource-limited areas around the world. This chapter describes some of the latest developments in antiviral strategies to preclude HCV entry, such as through monoclonal antibodies and small molecules, as well as measures to enhance the safety of therapeutic plasma products in blood transfusion

Replacement and deletion mutations in the catalytic domain and belt region of Aspergillus awamori glucoamylase to enhance thermostability

Three single-residue mutations, Asp71→Asn, Gln409→Pro and Gly447→Ser, two long-to-short loop replacement mutations, Gly23-Ala24-Asp25-Gly26-Ala27-Trp28-Val29-Ser30→Asn-Pro-Pro (23–30 replacement) and Asp297-Ser298-Glu299-Ala300-Val301→Ala-Gly-Ala (297–301 replacement) and one deletion mutation removing Glu439, Thr440 and Ser441 (Δ439–441), all based on amino acid sequence alignments, were made to improve Aspergillus awamori glucoamylase thermostability. The first and second single-residue mutations were designed to introduce a potential N-glycosylation site and to restrict backbone bond rotation, respectively, and therefore to decrease entropy during protein unfolding. The third single-residue mutation was made to decrease flexibility and increase O-glycosylation in the already highly O-glycosylated belt region that extends around the globular catalytic domain. The 23–30 replacement mutation was designed to eliminate a very thermolabile extended loop on the catalytic domain surface and to bring the remainder of this region closer to the rest of the catalytic domain, therefore preventing it from unfolding. The 297–301 replacement mutant GA was made to understand the function of the random coil region between α-helices 9 and 10. Δ439–441 was constructed to decrease belt flexibility. All six mutations increased glucoamylase thermostability without significantly changing enzyme kinetic properties, with the 23–30 replacement mutation increasing the activation free energy for thermoinactivation by about 4 kJ/mol, which leads to a 4°C increase in operating temperature at constant thermostability

The Methanolic Extract of Perilla frutescens Robustly Restricts Ebola Virus Glycoprotein-Mediated Entry

Ebola virus (EBOV), one of the most infectious human viruses and a leading cause of viral hemorrhagic fever, imposes a potential public health threat with several recent outbreaks. Despite the difficulties associated with working with this pathogen in biosafety level-4 containment, a protective vaccine and antiviral therapeutic were recently approved. However, the high mortality rate of EBOV infection underscores the necessity to continuously identify novel antiviral strategies to help expand the scope of prophylaxis/therapeutic management against future outbreaks. This includes identifying antiviral agents that target EBOV entry, which could improve the management of EBOV infection. Herein, using EBOV glycoprotein (GP)-pseudotyped particles, we screened a panel of natural medicinal extracts, and identified the methanolic extract of Perilla frutescens (PFME) as a robust inhibitor of EBOV entry. We show that PFME dose-dependently impeded EBOV GP-mediated infection at non-cytotoxic concentrations, and exerted the most significant antiviral activity when both the extract and the pseudoparticles are concurrently present on the host cells. Specifically, we demonstrate that PFME could block viral attachment and neutralize the cell-free viral particles. Our results, therefore, identified PFME as a potent inhibitor of EBOV entry, which merits further evaluation for development as a therapeutic strategy against EBO

Age, but not short-term intensive swimming, affects chondrocyte turnover in zebrafish vertebral cartilage

Both age and intensive exercise are generally considered critical risk factors for osteoarthritis. In this work, we intend to establish zebrafish models to assess the role of these two factors on cartilage homeostasis. We designed a swimming device for zebrafish intensive exercise. The body measurements, bone mineral density (BMD) and the histology of spinal cartilages of 4- and 12-month-old zebrafish, as well the 12-month-old zebrafish before and after a 2-week exercise were compared. Our results indicate that both age and exercise affect the body length and body weight, and the micro-computed tomography reveals that both age and exercise affect the spinal BMD. However, quantitative analysis of immunohistochemistry and histochemistry indicate that short-term intensive exercise does not affect the extracellular matrix (ECM) of spinal cartilage. On the other hand, the cartilage ECM significantly grew from 4 to 12 months of age with an increase in total chondrocytes. dUTP nick end labeling staining shows that the percentages of apoptotic cells significantly increase as the zebrafish grows, whereas the BrdU labeling shows that proliferative cells dramatically decrease from 4 to 12 months of age. A 30-day chase of BrdU labeling shows some retention of labeling in cells in 4-month-old spinal cartilage but not in cartilage from 12-month-old zebrafish. Taken together, our results suggest that zebrafish chondrocytes are actively turned over, and indicate that aging is a critical factor that alters cartilage homeostasis. Zebrafish vertebral cartilage may serve as a good model to study the maturation and homeostasis of articular cartilage

The discovery of potential acetylcholinesterase inhibitors: A combination of pharmacophore modeling, virtual screening, and molecular docking studies

<p>Abstract</p> <p>Background</p> <p>Alzheimer's disease (AD) is the most common cause of dementia characterized by progressive cognitive impairment in the elderly people. The most dramatic abnormalities are those of the cholinergic system. Acetylcholinesterase (AChE) plays a key role in the regulation of the cholinergic system, and hence, inhibition of AChE has emerged as one of the most promising strategies for the treatment of AD.</p> <p>Methods</p> <p>In this study, we suggest a workflow for the identification and prioritization of potential compounds targeted against AChE. In order to elucidate the essential structural features for AChE, three-dimensional pharmacophore models were constructed using Discovery Studio 2.5.5 (DS 2.5.5) program based on a set of known AChE inhibitors.</p> <p>Results</p> <p>The best five-features pharmacophore model, which includes one hydrogen bond donor and four hydrophobic features, was generated from a training set of 62 compounds that yielded a correlation coefficient of R = 0.851 and a high prediction of fit values for a set of 26 test molecules with a correlation of R²= 0.830. Our pharmacophore model also has a high Güner-Henry score and enrichment factor. Virtual screening performed on the NCI database obtained new inhibitors which have the potential to inhibit AChE and to protect neurons from Aβ toxicity. The hit compounds were subsequently subjected to molecular docking and evaluated by consensus scoring function, which resulted in 9 compounds with high pharmacophore fit values and predicted biological activity scores. These compounds showed interactions with important residues at the active site.</p> <p>Conclusions</p> <p>The information gained from this study may assist in the discovery of potential AChE inhibitors that are highly selective for its dual binding sites.</p