Genome sequencing of Pseudomonas aeruginosa phages; UF_RH7 and UF_RH9.

We have sequenced the genomes of two lytic phages, UF_RH7 and UF_RH9, which infect Pseudomonas aeruginosa. UF_RH7 belongs to Casjensviridae family and has a genome length of 58,217 bp and encodes 82 proteins. UF_RH9 belongs to Caudoviricetes class and has a genome length of 42,609 bp and encodes 55 proteins.

Complete Genome Sequencing of a Novel Pseudomonas aeruginosa Phage, UF_RH5.

Here, we introduce UF_RH5, a novel lytic phage targeting clinically isolated Pseudomonas aeruginosa. It belongs to the Siphovirus morphology family, Septimatrevirus genus, with a 42,566-bp genome with a GC content of 53.60%, encoding 58 proteins. Under electron microscopy, UF_RH5 exhibits a length of 121 nm and a capsid size of 45 nm.

Complete Genome Sequence of Pseudomonas aeruginosa Phage UF_RH6, Isolated from Human Lung.

We report the genome sequence of a lytic phage named UF_RH6, which infects Pseudomonas aeruginosa. This phage was isolated from a respiratory secretion sample from a patient with pulmonary P. aeruginosa. UF_RH6 belongs to the family Caudoviricetes and the genus Samunavirus. Its genome is 94,715 bp in length and encodes 130 proteins.

Complete Genome Sequencing of the Novel Pseudomonas aeruginosa Phage UF_RH1.

Here, we present the genome sequence of a novel Pseudomonas aeruginosa bacteriophage called UF_RH1. This lytic phage has a genome size of 42,567 bp and is classified as a member of the Siphoviridae family and the Septimatrevirus genus. UF_RH1 shares genetic similarities with Stenotrophomonas phage vB_SmaS-DLP_2.

T-Ridership: A web tool for reprogramming public transportation fleets to minimize COVID-19 transmission.

As the outbreak of novel coronavirus disease (COVID-19) continues to spread throughout the world, steps are being taken to limit the impact on public health. In the realm of infectious diseases like COVID-19, social distancing is one of the effective measures to avoid exposure to the virus and reduce its spread. Traveling on public transport can meaningfully facilitate the propagation of the transmission of infectious diseases. Accordingly, responsive actions taken by public transit agencies against risk factors can effectively limit the risk and make transit systems safe. Among the multitude of risk factors that can affect infection spread on public transport, the likelihood of exposure is a major factor that depends on the number of people riding the public transport and can be reduced by socially distanced settings. Considering that many individuals may not act in the socially optimal manner, the necessity of public transit agencies to implement measures and restrictions is vital. In this study, we present a novel web-based application, T-Ridership, based on a hybrid optimized dynamic programming inspired by neural networks algorithm to optimize public transit for safety with respect to COVID-19. Two main steps are taken in the analysis through Metropolitan Transportation Authority (MTA): detecting high-density stations by input data normalization, and then, using these results, the T-Ridership tool automatically determines optimal station order to avoid overcrowded transit vehicles. Effectively our proposed web tool helps public transit to be safe to ride under risk of infections by reducing the density of riders on public transit vehicles as well as trip duration. These results can be used in expanding on and improving policy in public transit, to better plan the scheduled time of trains and buses in a way that prevents high-volume human contact, increases social distance, and reduces the possibility of disease transmission (available at:http://t-ridership.com and GitHub at: https://github.com/Imani-Saba/TRidership).

BML: a versatile web server for bipartite motif discovery.

Motif discovery and characterization are important for gene regulation analysis. The lack of intuitive and integrative web servers impedes the effective use of motifs. Most motif discovery web tools are either not designed for non-expert users or lacking optimization steps when using default settings. Here we describe bipartite motifs learning (BML), a parameter-free web server that provides a user-friendly portal for online discovery and analysis of sequence motifs, using high-throughput sequencing data as the input. BML utilizes both position weight matrix and dinucleotide weight matrix, the latter of which enables the expression of the interdependencies of neighboring bases. With input parameters concerning the motifs are given, the BML achieves significantly higher accuracy than other available tools for motif finding. When no parameters are given by non-expert users, unlike other tools, BML employs a learning method to identify motifs automatically and achieve accuracy comparable to the scenario where the parameters are set. The BML web server is freely available at http://motif.t-ridership.com/ (https://github.com/Mohammad-Vahed/BML).

Optimization of expression, purification and secretion of functional recombinant human growth hormone in Escherichia coli using modified staphylococcal protein a signal peptide.

BACKGROUND: Human Growth Hormone (hGH) is a glycoprotein released from the pituitary gland. Due to the wide range of effects in humans, any disruption in hGH secretion could have serious consequences. This highlights the clinical importance of hGH production in the treatment of different diseases associated with a deficiency of this hormone. The production of recombinant mature hormone in suitable hosts and secretion of this therapeutic protein into the extracellular space can be considered as one of the best cost-effective approaches not only to obtain the active form of the protein but also endotoxin-free preparation. Since the natural growth hormone signal peptide is of eukaryotic origin and is not detectable by any of the Escherichia coli secretory systems, including Sec and Tat, and is therefore unable to secrete hGH in the prokaryotic systems, designing a new and efficient signal peptide is essential to direct hGh to the extracellular space. RESULTS: In this study, using a combination of the bioinformatics design and molecular genetics, the protein A signal peptide from Staphylococcus aureus was modified, redesigned and then fused to the mature hGH coding region. The recombinant hGH was then expressed in E. coli and successfully secreted to the medium through the Sec pathway. Secretion of the hGH into the medium was verified using SDS-PAGE and western blot analysis. Recombinant hGH was then expressed in E. coli and successfully secreted into cell culture medium via the Sec pathway. The secretion of hGH into the extracellular medium was confirmed by SDS-PAGE and Western blot analysis. Furthermore, the addition of glycine was shown to improve hGH secretion onto the culture medium. Equations for determining the optimal conditions were also determined. Functional hGH analysis using an ELISA-based method confirmed that the ratio of the active form of secreted hGH to the inactive form in the periplasm is higher than this ratio in the cytoplasm. CONCLUSIONS: Since the native signal protein peptide of S. aureus protein A was not able to deliver hGH to the extracellular space, it was modified using bioinformatics tools and fused to the n-terminal region of hGh to show that the redesigned signal peptide was functional.

Structural Analysis of the Novel Variants of SARS-CoV-2 and Forecasting in North America.

BACKGROUND: little is known about the forecasting of new variants of SARS-COV-2 in North America and the interaction of variants with vaccine-derived neutralizing antibodies. METHODS: the affinity scores of the spike receptor-binding domain (S-RBD) of B.1.1.7, B. 1.351, B.1.617, and P.1 variants in interaction with the neutralizing antibody (CV30 isolated from a patient), and human angiotensin-converting enzyme 2 (hACE2) receptor were predicted using the template-based computational modeling. From the Nextstrain global database, we identified prevalent mutations of S-RBD of SARS-CoV-2 from December 2019 to April 2021. Pre- and post-vaccination time series forecasting models were developed based on the prediction of neutralizing antibody affinity scores for S-RBD of the variants. RESULTS: the proportion of the B.1.1.7 variant in North America is growing rapidly, but the rate will reduce due to high affinity (~90%) to the neutralizing antibody once herd immunity is reached. Currently, the rates of isolation of B. 1.351, B.1.617, and P.1 variants are slowly increasing in North America. Herd immunity is able to relatively control these variants due to their low affinity (~70%) to the neutralizing antibody. The S-RBD of B.1.617 has a 110% increased affinity score to the human angiotensin-converting enzyme 2 (hACE2) in comparison to the wild-type structure, making it highly infectious. CONCLUSION: The newly emerged B.1.351, B.1.617, and P.1 variants escape from vaccine-induced neutralizing immunity and continue circulating in North America in post- herd immunity era. Our study strongly suggests that a third dose of vaccine is urgently needed to cover novel variants with affinity scores (equal or less than 70%) to eliminate developing viral mutations and reduce transmission rates.

CCL113, a novel sulfonamide, induces selective mitotic arrest and apoptosis in HeLa and HepG2 cells.

Targeting cell­cycle regulation to hinder cancer cell proliferation is a promising anticancer strategy. The present study investigated the effects of a novel sulfonamide, CCL113, on cell cycle progression in cancer cell lines (HeLa and HepG2), a noncancerous cell line (Vero) and a normal human fibroblast cell line (TIG­1­20). The present results showed that treatment with CCL113 significantly decreased the viability of the cancer cells. FACS analyses showed that CCL113 treatment increased the proportion of cancerous and noncancerous cells in the G2/M phase. Analyses of cell cycle regulatory proteins showed that CCL113 treatment inhibited the activity of CDK1 in HeLa cells, possibly due to the decrease in the level of Cdc25B/C proteins and arrest in the M phase. Using time­lapse imaging­assisted analyses of HeLa and Vero cells expressing fluorescent ubiquitination­based cell cycle indicator (FUCCI), it was observed that CCL113 treatment led to a prolonged G2 phase at the G2/M checkpoint and arrest in the M phase in both cell lines. This possibly activated the DNA damage response in noncancerous cells, while inducing mitotic arrest leading to apoptosis in the cancer cells. The results of molecular docking studies suggested that CCL113 might have the potential to bind to the taxol­binding site on ß­tubulin. In conclusion, CCL113 holds potential as a reliable anticancer drug due to its ability to induce mitotic arrest followed by apoptosis of cancer cells and to activate the DNA damage response in noncancerous cells, thereby facilitating exit from the cell cycle.

The initial stage of structural transformation of Aß42 peptides from the human and mole rat in the presence of Fe2+ and Fe3+: Related to Alzheimer's disease.

The early stage of secondary structural conversion of amyloid beta (Aß) to misfolded aggregations is a key feature of Alzheimer's disease (AD). Under normal physiological conditions, Aß peptides can protect neurons from the toxicity of highly concentrated metals. However, they become toxic under certain conditions. Under conditions of excess iron, amyloid precursor proteins (APP) become overexpressed. This subsequently increases Aß production. Experimental studies suggest that Aß fibrillation (main-pathway) and amorphous (off-pathway) aggregate formations are two competitive pathways driven by factors such as metal binding, pH and temperature. In this study, we performed molecular dynamic (MD) simulations to examine the initial stage of conformational transformations of human Aß (hAß) and rat Aß (rAß) peptides in the presence of Fe2+ and Fe3+ ions. Our results demonstrated that Fe2+ and Fe3+ play key roles in Aßs folding and aggregation. Fe3+ had a greater effect than Fe2+on Aßs' folding during intermolecular interactions and subsequently, had a greater effect in decreasing structural diversity. Fe2+ was observed to be more likely than Fe3+ to interact with nitrogen atoms from the residues of imidazole rings of His. rAß peptides are more energetically favorable than hAß for intermolecular interactions and amorphous aggregations. We concluded that most hAß structures were energetically unfavorable. However, hAßs with intermolecular ß-sheet formations in the C-terminal were energetically favorable. It is notable that Fe2+ can change the surface charge of hAß. Furthermore, Fe3+ can promote C-terminal folding by binding to Glu22 and Ala42, and by forming stable ß-sheet formations on the C-terminal. Fe3+ can also pause the main-pathway by inducing random aggregations.

G-rich VEGF aptamer as a potential inhibitor of chitin trafficking signal in emerging opportunistic yeast infection.

The alarm is rang for friendly fire; Saccharomyces cerevisiae (S. cerevisiae) newfound as a fungal pathogen with an individual feature. S. cerevisiae has food safety and is not capable of producing infection but, when the host defenses are weakened, there is room for opportunistic S. cerevisiae strains to cause a health issues. Fungal diseases are challenging to treat because, unlike bacteria, the fungal are eukaryotes. Antibiotics only target prokaryotic cells, whereas compounds that kill fungi also harm the mammalian host. Small differences between mammalian and fungal cells regarding genes and proteins sequence and function make finding a drug target more challenging. Recently, Chitin synthase has been considered as a promising target for antifungal drug development as it is absent in mammals. In S. cerevisiae, CHS3, a class IV chitin synthase, produces 90% of the chitin and essential for cell growth. CHS3 from the trans-Golgi network to the plasma membrane requires assembly of the exomer complex (including proteins cargo such as CHS5, CHS6, Bach1, and Arf1). In this work, we performed SELEX (Systematic Evolution of Ligands by EXponential enrichment) as high throughput virtual screening of the RCSB data bank to find an aptamer as potential inhibit of the class IV chitin synthase of S. cerevisiae. Among all the candidates, G-rich VEGF (GVEGF) aptamer (PDB code: 2M53) containing locked sugar parts was observed as potential inhibitor of the assembly of CHS5-CHS6 exomer complex a subsequently block the chitin biosynthesis pathway as an effective anti-fungal. It was suggested from the simulation that an assembly of exomer core should begin CHS5-CHS6, not from CHS5-Bach1. It is notable that secondary structures of CHS6 and Bach1 was observed very similar, but they have only 25% identity at the amino acid sequence that exhibited different features in exomer assembly.

Analysis of Physicochemical Interaction of Aß40 with a GM1 Ganglioside-Containing Lipid Membrane.

The interaction of amyloid beta (Aß) peptides with the cell membrane is one of the factors enhancing Aß aggregation, which is closely related to neurodegenerative disease. In this work, we performed molecular dynamics (MD) simulation to investigate the initial stage of adhesion of Aß40 to a GM1 ganglioside-containing membrane. Conformational change of Aß40 due to interaction with the membrane was monitored and compared with that of Aß42 observed in the previous study. Multiple computational trials were executed to analyze the probability of Aß binding using a calculation model consisting of a GM1-containing mixed lipid membrane, a water layer, ions, and Aß40. A single long-time MD simulation was also carried out. It was suggested from the simulation that a cluster of sialic acids of GM1 head groups often caught the side chain of His13 or His14 of Aß40 in the early stage of the MD simulations. Afterward, the main chain of Leu34 formed many hydrogen bonds with gangliosides. These residues cooperatively work for Aß40 to be held on the lipid membrane. It is notable that Aß40 was observed to be deeply inserted into the head group region of the lipid membrane in some computational trials. In the insertion, Aß40 occasionally formed a hydrogen bond with sphingomyelin. The difference in the secondary structure between Aß40 and Aß42 was an important factor for Aß40 to be deeply inserted into the membrane.

Simulation Study on Complex Conformations of Aß42 Peptides on a GM1 Ganglioside-Containing Lipid Membrane.

Aggregation and complex formation of amyloid beta (Aß) peptides on a neuronal cell membrane is a hallmark of neuro-disturbance diseases. In this work, we performed molecular dynamics (MD) simulations to investigate the initial stage of interactions of multiple Aß42 peptides on a GM1 ganglioside-containing membrane that mimics a micro-domain on the neuronal cell surface. Conformational changes of Aßs due to adhesion on the membrane and subsequent molecular interactions among the Aßs were monitored. It was suggested from results of the two 1.0 µs simulation trials that stable complexes of Aß peptides were not rapidly generated but that a steady binding of two Aßs was gradually formed. Observation of two Aßs that will be a complex with steady binding revealed that one Aß was bound to the membrane surface, while the other was attached to the first one without strong contact with the membrane. The motion of the first one was restricted and its conformational change was limited, with the basic side-chains of Arg5 and Lys28 working as anchors to hold the Aß helix region on the membrane. In contrast, the second one had high flexibility and showed diversity in its conformation. The second Aß can search for an energetically favorable binding position on the first one. A parallel ß-sheet structure was formed between the C-terminal sides of the two Aßs. Ala30 was critically important to lead the stable ß-sheet conformation at the C-terminal hydrophobic domains of Aßs. In the N-terminal sides, helix structures were kept in both Aßs.

Improving the chitinolytic activity of Bacillus pumilus SG2 by random mutagenesis.

Bacillus pumilus SG2, a halotolerant strain, expresses two major chitinases designated ChiS and ChiL that were induced by chitin and secreted into the supernatant. The present work aimed to obtain a mutant with higher chitinolytic activity through mutagenesis of Bacillus pumilus SG2 using a combination of UV irradiation and nitrous acid treatment. Following mutagenesis and screening on chitin agar and subsequent formation of halos, the mutated strains were examined for degradation of chitin under different conditions. A mutant designated AV2-9 was selected owing to its higher chitinase activity. To search for possible mutations in the whole operon including ChiS and ChiL, the entire chitinase operon, including the intergenic region, promoter, and two areas corresponding to the ChiS and ChiL ORF, was suquenced. Nucleotide sequence analysis of the complete chitinase operon from the SG2 and AV2-9 strains showed the presence of a mutation in the catalytic domain (GH18) of chitinase (ChiL). The results demonstrated that a single base change had occurred in the ChiL sequence in AV2- 9. The wild-type chitinase, ChiL, and the mutant (designated ChiLm) were cloned, expressed, and purified in E. coli. Both enzymes showed similar profiles of activity at different ranges of pH, NaCl concentration, and temperature, but the mutant enzyme showed approximately 30% higher catalytic activity under all the conditions tested. The results obtained in this study showed that the thermal stability of chitinase increased in the mutant strain. Bioinformatics analysis was performed to predict changes in the stability of proteins caused by mutation.