Intrinsic factors behind long COVID: III. Persistence of SARS-CoV-2 and its components.

Considerable research has been done in investigating SARS-CoV-2 infection, its characteristics, and host immune response. However, debate is still ongoing over the emergence of post-acute sequelae of SARS-CoV-2 infection (PASC). A multitude of long-lasting symptoms have been reported several weeks after the primary acute SARS-CoV-2 infection that resemble several other viral infections. Thousands of research articles have described various post-COVID-19 conditions. Yet, the evidence around these ongoing health problems, the reasons behind them, and their molecular underpinnings are scarce. These persistent symptoms are also known as long COVID-19. The persistence of SARS-CoV-2 and/or its components in host tissues can lead to long COVID. For example, the presence of viral nucleocapsid protein and RNA was detected in the skin, appendix, and breast tissues of some long COVID patients. The persistence of viral RNA was reported in multiple anatomic sites, including non-respiratory tissues such as the adrenal gland, ocular tissue, small intestine, lymph nodes, myocardium, and sciatic nerve. Distinctive viral spike sequence variants were also found in non-respiratory tissues. Interestingly, prolonged detection of viral subgenomic RNA was observed across all tissues, sometimes in multiple tissues of the same patient, which likely reflects recent but defective viral replication. Moreover, the persistence of SARS-CoV-2 RNA was noticed throughout the brain at autopsy, as late as 230 days following symptom onset among unvaccinated patients who died of severe infection. Here, we review the persistence of SARS-CoV-2 and its components as an intrinsic factor behind long COVID. We also highlight the immunological consequences of this viral persistence.

HLA-I and HLA-II Peptidomes of SARS-CoV-2: A Review.

The adaptive (T-cell-mediated) immune response is a key player in determining the clinical outcome, in addition to neutralizing antibodies, after SARS-CoV-2 infection, as well as supporting the efficacy of vaccines. T cells recognize viral-derived peptides bound to major histocompatibility complexes (MHCs) so that they initiate cell-mediated immunity against SARS-CoV-2 infection or can support developing a high-affinity antibody response. SARS-CoV-2-derived peptides bound to MHCs are characterized via bioinformatics or mass spectrometry on the whole proteome scale, named immunopeptidomics. They can identify potential vaccine targets or therapeutic approaches for SARS-CoV-2 or else may reveal the heterogeneity of clinical outcomes. SARS-CoV-2 epitopes that are naturally processed and presented on the human leukocyte antigen class I (HLA-I) and class II (HLA-II) were identified for immunopeptidomics. Most of the identified SARS-CoV-2 epitopes were canonical and out-of-frame peptides derived from spike and nucleocapsid proteins, followed by membrane proteins, whereby many of which are not caught by existing vaccines and could elicit effective responses of T cells in vivo. This review addresses the detection of SARS-CoV-2 viral epitopes on HLA-I and HLA-II using bioinformatics prediction and mass spectrometry (HLA peptidomics). Profiling the HLA-I and HLA-II peptidomes of SARS-CoV-2 is also detailed.

Volatile Compounds, Fatty Acids Constituents, and Antimicrobial Activity of Cultured Spirulina (Arthrospira fusiformis) Isolated from Lake Mariout in Egypt.

In this study, Arthrospira fusiformis previously isolated from Lake Mariout (Alexandria, Egypt) was cultivated in the laboratory using a medium for pharmaceutical grade Arthrospira, named as Amara and Steinbüchel medium. Hot water extract of the Egyptian Spirulina was prepared by autoclaving dried biomass in distilled water at 121°C for 15 min. This algal water extract was analyzed by GC-MS to evaluate its volatile compounds and fatty acids composition. The antimicrobial activity of phycobiliprotein extract from Arthrospira fusiformis using phosphate buffer was evaluated against thirteen microbial strains (two Gram-positive bacteria, eight Gram-negative bacteria, one yeast, and two filamentous fungi). The major components of fatty acids in the hot extract of Egyptian A. fusiformis were hexadecanoic acid (palmitic acid, 55.19%) and octadecanoic acid (stearic acid, 27.14%). The main constituents of its volatile compounds were acetic acid (43.33%) and oxalic acid (47.98%). The most potent antimicrobial effect of phycobiliprotein extract was obtained against two Gram-negative bacteria Salmonella typhi and Proteus vulgaris, filamentous fungus Aspergillus niger, and the pathogenic yeast Candida albicans (all of which showed MIC values of 58.1 µg/ml). Escherichia coli and Salmonella typhimurium come second in their susceptibility to the phycobiliprotein extract from Arthrospira fusiformis and Serratia marcescens and Aspergillus flavus are the least in susceptibility, with MIC values of 116.2 and 232.5 µg/ml, respectively, while phycobiliprotein extract has no antibacterial effect on methicillin-resistant as well as susceptible Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Shigella sonnei. These findings confirmed the nutritional value of Egyptian A. fusiformis isolated from Lake Mariout and suggest the potential use of this strain as an ingredient in the cooking of some foods to increase the level of stearic acid and palmitic acid. Moreover, its effective antibacterial activities against some important and highly resistant to antibiotics bacterial pathogens in addition to its antifungal effects recommend the therapeutic use of its biomass.

Fungi as a Source of Edible Proteins and Animal Feed.

It is expected that the world population will reach 9 billion by 2050. Thus, meat, dairy or plant-based protein sources will fail to meet global demand. New solutions must be offered to find innovative and alternative protein sources. As a natural gift, edible wild mushrooms growing in the wet and shadow places and can be picked by hand have been used as a food. From searching mushrooms in the forests and producing single cell proteins (SCP) in small scales to mega production, academia, United Nations Organizations, industries, political makers and others, play significant roles. Fermented traditional foods have also been reinvestigated. For example, kefir, miso, and tempeh, are an excellent source for fungal isolates for protein production. Fungi have unique criteria of consuming various inexpensive wastes as sources of carbon and energy for producing biomass, protein concentrate or amino acids with a minimal requirement of other environmental resources (e.g., light and water). Fungal fermented foods and SCP are consumed either intentionally or unintentionally in our daily meals and have many applications in food and feed industries. This review addresses fungi as an alternative source of edible proteins and animal feed, focusing mainly on SCP, edible mushrooms, fungal fermented foods, and the safety of their consumption.

Novel Approach Using shRNA of IQGAP1 for Colon Cancer Therapy: HCT116 as a Surrogate Model Colorectal Carcinoma

BACKGROUND: Colorectal carcinoma (CRC) represents life-threatening problems worldwide. IQ motif containing GTPase activating protein 1 (IQGAP1) is acting as oncogenesis regulators. RNAi is proposed as promising cancer therapeutics. OBJECTIVE: The objective of this work to explore the consequences of the IQGAP1 silence as a goal for treating CRC using the HCT166 cells as a model for human colon cancer. METHODS: RNAi technology was used to design a short specific sequence of RNA (shRNA) to silence the IQGAP1 oncogene. The impact of IQGAP1 silencing on IQGAPs, Ras, IL-8, and TRAIL was investigated. Furthermore, the effect of IQGAP1 silencing on cell viability, proliferation, apoptosis, and invasive capacity was investigated. RESULTS: The present results revealed that IQGAP1 shRNA-treated HCT166 cells showed no invasive capacity compared to the control cells. The silencing of IQGAP1 induced remarkable downregulation of IQGAP1, RAS (H&K), IL-8, CXCR1, CXCR2, NF-kB, BCL-2, and apoptosis of HCT166 cells. On the contrary, IQGAP2, IQGAP3, DR4, DR5, CASP-3, and BAX genes were significantly up-regulated. CONCLUSION: The IQGAP1 regulates the expression of IQGAPs, Ras, IL-8 receptors, and the apoptotic network. Therefore, the silence of IQGAP1 is a promising strategy for colon cancer therapy.

Bacterial Ghosts of Pseudomonas aeruginosa as a Promising Candidate Vaccine and Its Application in Diabetic Rats.

Infections with Pseudomonas aeruginosa (PA) pose a major clinical threat worldwide especially to immunocompromised patients. As a novel vaccine network for many kinds of bacteria, bacterial ghosts (BGs) have recently been introduced. In the present research, using Sponge-Like Reduced Protocol, P. aeruginosa ghosts (PAGs) were prepared to maintain surface antigens and immunogenicity. This is the first study, to our knowledge, on the production of chemically induced well-structured bacterial ghosts for PA using concentrations of different chemicals. The research was carried out using diabetic rats who were orally immunized at two-week intervals with three doses of PAGs. Rats were subsequently challenged either by the oral route or by the model of ulcer infection with PA. In challenged rats, in addition to other immunological parameters, organ bioburden and wound healing were determined, respectively. Examination of the scanning and transmission electron microscope (EM) proved that PAGs with a proper three-dimensional structure were obtained. In contrast to control groups, oral PAGs promoted the generation of agglutinating antibodies, the development of IFN-γ, and the increase in phagocytic activity in vaccinated groups. Antibodies of the elicited PAGs were reactive to PA proteins and lipopolysaccharides. The defense against the PA challenge was observed in PAGs-immunized diabetic rats. The resulting PAGs in orally vaccinated diabetic rats were able to evoke unique humoral and cell-mediated immune responses and to defend them from the threat of skin wound infection. These results have positive implications for future studies on the PA vaccine.

Itraconazole-hydroxypropyl-ß-cyclodextrin loaded deformable liposomes: in vitro skin penetration studies and antifungal efficacy using Candida albicans as model.

The study aimed to develop novel ITZ-loaded deformable liposomes (DL) in presence of hydroxypropyl-ß-cyclodextrin (HPßCD) (DL-CD) to enhance antifungal activity. These formulations have been reported as conceivable vesicles to deliver drug molecules to the skin layers. The efficiency of the prepared systems was compared with conventional liposomes (CL) and ITZ solution. The developed liposomes were characterized for particle size, entrapment efficiency (EE %), deformability, stability, and morphology of the vesicles. In addition, ex vivo penetration and antifungal activity were evaluated. It was found that the presence of HPßCD played a significant role in reducing the vesicle size to nano range. The deformability study and TEM images revealed that membrane deformability of DL and DL-CD was much higher than that of CL. Moreover, DL-CD enhanced the amount of ITZ in SC and deeper skin layers compared to DL and CL. The antifungal activity of ITZ-loaded deformable liposomes remained intact compared to ITZ solution. It can be concluded that deformable liposomes in the presence of HPßCD may be a promising carrier for effective cutaneous delivery of ITZ.

Production and characterization of keratinolytic protease from new wool-degrading Bacillus species isolated from Egyptian ecosystem.

Novel keratin-degrading bacteria were isolated from sand soil samples collected from Minia Governorate, Egypt. In this study, the isolates were identified as Bacillus amyloliquefaciens MA20 and Bacillus subtilis MA21 based on morphological and biochemical characteristics as well as 16S rRNA gene sequencing. B. amyloliquefaciens MA20 and B. subtilis MA21 produced alkaline keratinolytic serine protease when cultivated in mineral medium containing 1% of wool straight off sheep as sole carbon and nitrogen source. The two strains were observed to degrade wool completely to powder at pH 7 and 37°C within 5 days. Under these conditions the maximum activity of proteases produced by B. amyloliquefaciens MA20 and B. subtilis MA21 was 922 and 814 U/ml, respectively. The proteases exhibited optimum temperature and pH at 60°C and 9, respectively. However, the keratinolytic proteases were stable in broad range of temperature and pH values towards casein Hammerstein. Furthermore the protease inhibitor studies indicated that the produced proteases belong to serine protease because of their sensitivity to PMSF while they were inhibited partially in presence of EDTA. The two proteases are stable in most of the used organic solvents and enhanced by metals suggesting their potential use in biotechnological applications such as wool industry.

Sponge-like: a new protocol for preparing bacterial ghosts.

Bacterial Ghosts (BGs) received an increasing interest in the recent years for their promising medicinal and pharmaceutical applications. In this study, for the first time we introduce a new protocol for BGs production. E. coli BL21 (DE3) pLysS (Promega) was used as a model to establish a general protocol for BGs preparation. The protocol is based on using active chemical compounds in concentrations less than the Minimum Inhibition Concentration (MIC). Those chemical compounds are SDS, NaOH, and H2O2. Plackett-Burman experimental design was used to map the best conditions for BGs production. Normal and electronic microscopes were used to evaluate the BGs quality (BGQ). Spectrophotometer was used to evaluate the amount of the released protein and DNA. Agarose gel electrophoresis was used to determine the existence of any residue of DNA after each BGs preparation. Viable cells, which existed after running this protocol, were subjected to lysis by inducing the lysozyme gene carried on pLysS plasmid. This protocol is able to produce BGs that can be used in different biotechnological applications.

New Medium for Pharmaceutical Grade Arthrospira.

The aim of this study is to produce a pharmaceutical grade single cell product of Arthrospira from a mixed culture. We have designed a medium derived from a combination between George's and Zarrouk's media. Our new medium has the ability to inhibit different forms of cyanobacterium and microalgae except the Chlorella. The medium and the cultivation conditions have been investigated to map the points where only Arthrospira could survive. For that, a mixed culture of pure Chlorella and Arthrospira (~90 : 10) has been used to develop the best medium composition that can lead to the enrichment of the Arthrospira growth and the inhibition of the Chlorella growth. To enable better control and to study its growth, an 80 l photobioreactor has been used. We have used high saline (2xA-St) medium which has been followed by in fermentor reducing its concentration to 1.5x. The investigation proves that Chlorella has completely disappeared. A method and a new saline medium have been established using a photobioreactor for in fermentor production of single cell Arthrospira. Such method enables the production of pure pharmaceutical grade Arthrospira for medicinal and pharmaceutical applications or as a single cell protein.

Back to natural fiber: wool color influences its sensitivity to enzymatic treatment.

There are many missed biotechnological opportunities in the developmental countries. Wool quality improvement is one of them. This study is concerning with improving the wool quality using technical enzymes. White wool proves to be more susceptible to the enzymatic treatment than blackish brown wool. This proves that the enzymatic reaction is sensitive to the natural color differences between wool fibers. A simple enzymatic method has been used to improve the wool quality as well as to investigate the changes happened in the wool fibers. Geobacillus stearothermophilus has been used under mesophilic and static cultivation conditions using wool as the main carbon source. These conditions prove to be more suitable for maintaining the fiber structure, less expensive, and reliable as an in-house biotechnological process that can be adapted everywhere. The enzyme activity in case of white wool was 4 Units/ml and for blackish brown wool was 1.5 Units/ml. Electron microscope has been used to evaluate the end result. By following the process included in this paper using probable microbial strain(s), the wool quality improvement can be applied globally and can add another value to the economy of the developmental countries.

Leucocytes show improvement growth on PHA polymer surface.

Polyhydroxyalkanoate (PHA) from one fermentation process shows diverse physical properties when extracted using different methods. Pseudomonas aeruginosa strain has been previously isolated from the Egyptian ecosystem was cultivated on olive oil as a carbon source under PHA accumulation conditions. PHA was extracted using four different extraction methods and the polymer give different biological properties. Leucocytes grown in different rate on each preparation. RBCs haemolysis test was used to determine the polymers toxicity. PHA isolated directly with chloroform give the highest leucocytes number (19.4 10(4) cells/48 hr) and the lowest Haemolytic index (2.28). Bioassays used in this study are recommended for evaluating the in vitro polymer biocompatibility aiming to in vivo application or as a cell line-supporting matrix.

Case-by-case study using antibiotic-EDTA combination to control pseudomonas aeruginosa.

Ps. aeruginosa a well-known opportunistic bacterium infects various tissues and organs causing sever problems. In tropical and semitropical countries and because of environmental and sanitizations reasons Ps. aeruginosa acquiring more virulent factors, gained multi-resistant genes, adapt resistant mechanisms for antibiotics, drugs, disinfectants and in general for any toxic compound. Six multidrugs resistant strains out of 260 different bacteria isolated from patients at Tanta University hospital, Tanta, Egypt, used in this study. To evaluate the role of divalent cations in antibiotic resistance we used a medium containing Ca(2+) and Mg(2+). Various antibiotics tested with or without the LC(10) and LC(50) equivalent amount of EDTA against each strain. We found that after adding EDTA, 70% of the strains turned from resistant to sensitive, especially considering those antibiotics, which inhibit protein synthesis, such, as tetracycline. Simple experiment for testing the effect of Antibiotic-EDTA combination on Rabbits skin artificial ulcer(s) infected by Ps. aeruginosa significantly improve the efficiency of using such combination in superficial treatment. We propose using media other than Muller-Hinton agar in antibiotic sensitivity test to select the best antibiotics could be used in vivo or in superficial treatment. A combination between a proper amount of EDTA and antibiotics especially protein inhibitors will improve the control of Ps. aeruginosa.

Replacement of the catalytic nucleophile cysteine-296 by serine in class II polyhydroxyalkanoate synthase from Pseudomonas aeruginosa-mediated synthesis of a new polyester: identification of catalytic residues.

The class II PHA (polyhydroxyalkanoate) synthases [PHA(MCL) synthases (medium-chain-length PHA synthases)] are mainly found in pseudomonads and catalyse synthesis of PHA(MCL)s using CoA thioesters of medium-chain-length 3-hydroxy fatty acids (C6-C14) as a substrate. Only recently PHA(MCL) synthases from Pseudomonas oleovorans and Pseudomonas aeruginosa were purified and in vitro activity was achieved. A threading model of the P. aeruginosa PHA(MCL) synthase PhaC1 was developed based on the homology to the epoxide hydrolase (1ek1) from mouse which belongs to the alpha/beta-hydrolase superfamily. The putative catalytic residues Cys-296, Asp-452, His-453 and His-480 were replaced by site-specific mutagenesis. In contrast to class I and III PHA synthases, the replacement of His-480, which aligns with the conserved base catalyst of the alpha/beta-hydrolases, with Gln did not affect in vivo enzyme activity and only slightly in vitro enzyme activity. The second conserved histidine His-453 was then replaced by Gln, and the modified enzyme showed only 24% of wild-type in vivo activity, which indicated that His-453 might functionally replace His-480 in class II PHA synthases. Replacement of the postulated catalytic nucleophile Cys-296 by Ser only reduced in vivo enzyme activity to 30% of wild-type enzyme activity and drastically changed substrate specificity. Moreover, the C296S mutation turned the enzyme sensitive towards PMSF inhibition. The replacement of Asp-452 by Asn, which is supposed to be required as general base catalyst for elongation reaction, did abolish enzyme activity as was found for the respective amino acid residue of class I and III enzymes. In the threading model residues Cys-296, Asp-452, His-453 and His-480 reside in the core structure with the putative catalytic nucleophile Cys-296 localized at the highly conserved gamma-turns of the alpha/beta-hydrolases. Inhibitor studies indicated that catalytic histidines reside in the active site. The conserved residue Trp-398 was replaced by Phe and Ala, respectively, which caused inactivation of the enzyme indicating an essential role of this residue. In the threading model this residue was found to be surface-exposed. No evidence for post-translational modification by 4-phosphopantetheine was obtained. Overall, these data suggested that in class II PHA synthases the conserved histidine which was found as general base catalyst in the catalytic triad of enzymes related to the alpha/beta-hydrolase superfamily, was functionally replaced by His-453 which is conserved among all PHA synthases.

Biochemical characterization of the Pseudomonas putida 3-hydroxyacyl ACP:CoA transacylase, which diverts intermediates of fatty acid de novo biosynthesis.

The 3-hydroxyacyl ACP:CoA transacylase (PhaG) was recently identified in various Pseudomonas species and catalyzes the diversion of ACP thioester intermediates of fatty acid de novo biosynthesis toward the respective CoA thioesters, which serve as precursors for polyester and rhamnolipid biosynthesis. PhaG from Pseudomonas putida was overproduced in Escherichia coli as a C-terminal hexahistidine-tagged (His(6)) fusion protein in high yield. The His(6)-PhaG was purified to homogeneity by refolding of PhaG obtained from inclusion bodies, and a new enzyme assay was established. Kinetic analysis of the 3-hydroxyacyl transfer to ACP, catalyzed by His(6)-PhaG, gave K(0.5) values of 28 microm (ACP) and 65 microm (3-hydroxyacyl-CoA) considering V(max) values of 11.7 milliunits/mg and 12.4 milliunits/mg, respectively. A Hill coefficient of 1.38 (ACP) and 1.32 (3-hydroxyacyl-CoA) indicated a positive substrate cooperativity. Subcellular localization studies showed that PhaG is not attached to polyester granules and resides in the cytosol. Gel filtration chromatography analysis in combination with light scattering analysis indicated substrate-induced dimerization of the transacylase. A threading model of PhaG was developed based on the homology to an epoxide hydrolase (1cqz). In addition, the alignment with the alpha/beta-hydrolase fold region indicated that PhaG belongs to alpha/beta-hydrolase superfamily. Accordingly, CD analysis suggested a secondary structure composition of 29% alpha-helix, 22% beta-sheet, 18% beta-turn, and 31% random coil. Site-specific mutagenesis of seven highly conserved amino acid residues (Asp-60, Ser-102, His-177, Asp-182, His-192, Asp-223, His-251) was used to validate the protein model and to investigate organization of the transacylase active site. Only the D182(A/E) mutation was permissive with about 30% specific activity of the wild type enzyme. Furthermore, this mutation caused a change in substrate specificity, indicating a functional role in substrate binding. The serine-specific agent phenylmethylsulfonyl fluoride (PMSF) or the histidine-specific agent diethylpyrocarbonate (DEPC) caused inhibition of 3-hydroxyacyl transfer to holo-ACP, and the S102(A/T) or H251(A/R) PhaG mutant was incapable of catalyzing 3-hydroxyacyl transfer, suggesting that these residues are part of a catalytic triad.

Molecular characterization of the poly(3-hydroxybutyrate) (PHB) synthase from Ralstonia eutropha: in vitro evolution, site-specific mutagenesis and development of a PHB synthase protein model.

A threading model of the Ralstonia eutropha polyhydroxyalkanoate (PHA) synthase was developed based on the homology to the Burkholderia glumae lipase, whose structure has been resolved by X-ray analysis. The lid-like structure in the model was discussed. In this study, various R. eutropha PHA synthase mutants were generated employing random as well as site-specific mutagenesis. Four permissive mutants (double and triple mutations) were obtained from single gene shuffling, which showed reduced activity and whose mutation sites mapped at variable surface-exposed positions. Six site-specific mutations were generated in order to identify amino acid residues which might be involved in substrate specificity. Replacement of residues T323 (I/S) and C438 (G), respectively, which are located in the core structure of the PHA synthase model, abolished PHA synthase activity. Replacement of the two amino acid residues Y445 (F) and L446 (K), respectively, which are located at the surface of the protein model and adjacent to W425, resulted in reduced activity without changing substrate specificity and indicating a functional role of these residues. The E267K mutant exhibited only slightly reduced activity with a surface-exposed mutation site. Four site-specific deletions were generated to evaluate the role of the C-terminus and variant amino acid sequence regions, which link highly conserved regions. Deleted regions were D281-D290, A372-C382, E578-A589 and V585-A589 and the respective PHA synthases showed no detectable activity, indicating an essential role of the variable C-terminus and the linking regions between conserved blocks 2 and 3 as well as 3 and 4. Moreover, the N-terminal part of the class II PHA synthase (PhaC(Pa)) from Pseudomonas aeruginosa and the C-terminal part of the class I PHA synthase (PhaC(Re)) from R. eutropha were fused, respectively, resulting in three fusion proteins with no detectable in vivo activity. However, the fusion protein F1 (PhaC(Pa)-1-265-PhaC(Re)-289-589) showed 13% of wild type in vitro activity with the fusion point located at a surface-exposed loop region.