Application of functional proteomics in understanding RNA virus-mediated infection.

Together with the expansion of genome sequencing research, the number of protein sequences whose function is yet unknown is increasing dramatically. The primary goals of functional proteomics, a developing area of study in the realm of proteomic science, are the elucidation of the biological function of unidentified proteins and the molecular description of cellular systems at the molecular level. RNA viruses have emerged as the cause of several human infectious diseases with large morbidity and fatality rates. The introduction of high-throughput sequencing tools and genetic-based screening approaches over the last few decades has enabled researchers to find previously unknown and perplexing elements of RNA virus replication and pathogenesis on a scale never feasible before. Viruses, on the other hand, frequently disrupt cellular proteostasis, macromolecular complex architecture or stoichiometry, and post-translational changes to take over essential host activities. Because of these consequences, structural and global protein and proteoform monitoring is highly necessiated. Mass spectrometry (MS) has the potential to elucidate key details of virus-host interactions and speed up the identification of antiviral targets, giving precise data on the stoichiometry of cellular and viral protein complexes as well as mechanistic insights, has lately emerged as a key part of the RNA virus biology toolbox as a functional proteomics approach. Affinity-based techniques are primarily employed to identify interacting proteins in stable complexes in living organisms. A protein's biological role is strongly suggested by its relationship with other members of a certain protein complex that is involved in a particular process. With a particular emphasis on the most recent advancements in defining host responses and their translational implications to uncover novel tractable antiviral targets, this chapter provides insight on several functional proteomics techniques in RNA virus biology.

The interplay between circadian clock and viral infections: A molecular perspective.

The circadian clock influences almost every aspect of mammalian behavioral, physiological and metabolic processes. Being a hierarchical network, the circadian clock is driven by the central clock in the brain and is composed of several peripheral tissue-specific clocks. It orchestrates and synchronizes the daily oscillations of biological processes to the environment. Several pathological events are influenced by time and seasonal variations and as such implicate the clock in pathogenesis mechanisms. In context with viral infections, circadian rhythmicity is closely associated with host susceptibility, disease severity, and pharmacokinetics and efficacies of antivirals and vaccines. Leveraging the circadian molecular mechanism insights has increased our understanding of clock infection biology and proposes new avenues for viral diagnostics and therapeutics. In this chapter, we address the molecular interplay between the circadian clock and viral infections and discuss the importance of chronotherapy as a complementary approach to conventional medicines, emphasizing the significance of virus-clock studies.

Mechanism of cell cycle regulation and cell proliferation during human viral infection.

Over the history of the coevolution of Host viral interaction, viruses have customized the host cellular machinery into their use for viral genome replication, causing effective infection and ultimately aiming for survival. They do so by inducing subversions to the host cellular pathways like cell cycle via dysregulation of important cell cycle checkpoints by viral encoded proteins, arresting the cell cycle machinery, blocking cytokinesis as well as targeting subnuclear bodies, thus ultimately disorienting the cell proliferation. Both DNA and RNA viruses have been active participants in such manipulation resulting in serious outcomes of cancer. They achieve this by employing different mechanisms-Protein-protein interaction, protein-phosphorylation, degradation, redistribution, viral homolog, and viral regulation of APC at different stages of cell cycle events. Several DNA viruses cause the quiescent staged cells to undergo cell cycle which increases nucleotide pools logistically significantly persuading viral replication whereas few other viruses arrest a particular stage of cell cycle. This allows the latter group to sustain the infection which allows them to escape host immune response and support viral multiplication. Mechanical study of signaling such viral mediated pathways could give insight into understanding the etiology of tumorigenesis and progression. Overall this chapter highlights the possible strategies employed by DNA/RNA viral families which impact the normal cell cycle but facilitate viral infected cell replication. Such information could contribute to comprehending viral infection-associated disorders to further depth.

Exploring the role of secretory proteins in the human infectious diseases diagnosis and therapeutics.

Secretory proteins are playing important role during the host-pathogen interaction to develop the infection or protection into the cell. Pathogens developing infectious disease to human being are taken up by host macrophages or number of immune cells, play an important role in physiological, developmental and immunological function. At the same time, infectious agents are also secreting various proteins to neutralize the resistance caused by host cells and also helping the pathogens to develop the infection. Secretory proteins (secretome) are only developed at the time of host-pathogen interaction, therefore they become very important to develop the targeted and potential therapeutic strategies. Pathogen specific secretory proteins released during interaction with host cell provide opportunity to develop point of care and rapid diagnostic kits. Proteins secreted by pathogens at the time of interaction with host cell have also been found as immunogenic in nature and numbers of vaccines have been developed to control the spread of human infectious diseases. This chapter highlights the importance of secretory proteins in the development of diagnostic and therapeutic strategies to fight against human infectious diseases.

Nanobody-peptide-conjugate (NPC) for passive immunotherapy against SARS-CoV-2 variants of concern (VoC): a prospective pan-coronavirus therapeutics.

The COVID-19 crisis, incited by the zoonotic SARS-CoV-2 virus, has quickly escalated into a catastrophic public health issue and a grave threat to humankind owing to the advent of mutant viruses. Multiple pharmaceutical therapies or biologics envision stopping the virus from spreading further; however, WHO has voiced concerns about the variants of concern (VoCs) inability to respond. Nanobodies are a new class of antibody mimics with binding affinity and specificity similar to classical mAbs, as well as the privileges of a small molecular weight, ease of entry into solid tissues, and binding cryptic epitopes of the antigen. Herein, we investigated multiple putative anti-SARS-CoV-2 nanobodies targeting the Receptor binding domain of the WHO-listed SARS-CoV-2 variants of concern using a comprehensive computational immunoinformatics methodology. With affinity maturation via alanine scanning mutagenesis, we remodeled an ultrapotent nanobody with substantial breadth and potency, exhibiting pico-molar binding affinities against all the VoCs. An antiviral peptide with specificity for ACE-2 receptors was affixed to make it multispecific and discourage viral entry. Collectively, we constructed a broad-spectrum therapeutic biparatopic nanobody-peptide conjugate (NPC) extending coverage to SARS-CoV-2 VoCs RBDs. We PEGylated the biparatopic construct with 20kD maleimide-terminated PEG (MAL-(PEG)n-OMe) to improve its clinical efficacy limiting rapid renal clearance, and performed in silico cloning to facilitate future experimental studies. Our findings suggest that combining biparatopic nanobody conjugate with standard treatment may be a promising bivariate tool for combating viral entry during COVID-19 illness.

High-throughput virtual screening of small-molecule inhibitors targeting immune cell checkpoints to discover new immunotherapeutics for human diseases.

Immunotherapy is widely used to treat various cancers, and the drugs used are called immune checkpoint (ICP) inhibitors. Overexpression of immune cell checkpoints is reported for other human diseases such as acute infections (malaria), chronic viral infection (HIV, hepatitis B virus, TB infections), allergy, asthma, neurodegeneration, and autoimmune diseases. Some mAbs (monoclonal antibodies) are available against ICPs, but they have side effects. Small molecule seems to be safer in comparison with mAbs. Three independent small-molecule inhibitor libraries consisting of 9466 compounds were screened against seven immune cell checkpoints by applying high-throughput virtual screening approach. A total of 13 ICP inhibitors were finalized based on docking, MM-GBSA scores, and ADME properties. Six compounds were selected for MD simulation, and then, rutin hydrate (targeting all seven immune cell checkpoints), amikacin hydrate (targeting six), and 6-hydroxyluteolin (targeting three) were found to be the best immune cell checkpoint inhibitors. These three potential inhibitors have shown the potential to activate human immune cells and thus may control the spread of human lifestyle or infectious diseases. Proposed inhibitors warrant the in vitro and in vivo validation to develop it as an immunotherapeutic.

Clinical Profile and Outcomes of Multisystem Inflammatory Syndrome in Children: A Multicentric Observational Study.

Background and objective Multisystem inflammatory syndrome in children (MIS-C) is a postinfectious, generalized, hyperimmune state and is potentially lethal. There is scarce data on the clinical presentation and epidemiology of MIS-C in India. In light of this, we conducted this study to describe clinical presentations and outcomes in children diagnosed with MIS-C. Methodology This was a 15-month hospital-based prospective observational study conducted in the Departments of Pediatrics at Jagannath Hospital and Hitech Medical College, Bhubaneswar. The study included all patients diagnosed with MIS-C and treated at these hospitals between May 1, 2020, and August 31, 2021. The inclusion criteria were as follows: patients who were reverse transcription-polymerase chain reaction (RT-PCR)-positive, antibody-positive, or had known contact with those infected with coronavirus disease 2019 (COVID-19). We reviewed patient medical records to collect demographic data such as age, sex, body mass index (BMI), duration of illness, clinical symptomatology, findings of initial echocardiography, and outcomes. We followed each case for three months. We analyzed descriptive statistics using percentages and means and conducted the statistical analysis using SPSS Statistics for Windows, Version 25.0. (IBM Corp., Armonk, NY). Results A total of 30 cases were included in the study, consisting of 16 boys (53.3%) and 14 girls (46.7%). The mean age of the study population was 6.7 years, and 43% had a BMI in the overweight range. All patients (100%) had a fever, 66.7% had lethargy (n=20), and 64.3% (n=19) had abdominal symptoms in the form of vomiting, diarrhea, and abdominal pain. Respiratory distress at admission was found in 16 cases (53.3%), while hypotension at admission was found in 18 (60%) cases. Our population's average duration of pediatric ICU stay was 3.7 ± 1.2 days, and the average duration of inotropy was 2.2 ± 0.5 days. Fifteen cases (50%) required only oxygen support; 10 (33%) required noninvasive ventilation, and only one patient required invasive ventilation. Twenty-two patients (74%) needed fluid boluses. Outcomes of coronary artery dilatations were favorable, regressing to normal (Z-score <2.5) in affected patients within 90 days of follow-up. Conclusions MIS-C has myriad presenting signs, symptoms, and severity. It is often associated with circulatory failure or shock. However, most patients demonstrated good early outcomes, improved left ventricle (LV) function, normalization of coronary abnormalities, and no mortality. This study provides additional data on the clinical presentation of MIS-C and highlights the importance of close, long-term follow-up monitoring of this patient population.

MiRNA-SARS-CoV-2 dialogue and prospective anti-COVID-19 therapies.

COVID-19 is a highly transmissible disease caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), affects 226 countries and continents, and has resulted in >6.2 million deaths worldwide. Despite the efforts of all scientific institutions worldwide to identify potential therapeutics, no specific drug has been approved by the FDA to treat the COVID-19 patient. SARS-CoV-2 variants of concerns make the potential of publicly known therapeutics to respond to and detect disease onset highly improbable. The quest for universal therapeutics pointed to the ability of RNA-based molecules to shield and detect the adverse effects of the COVID-19 illness. One such candidate, miRNA (microRNA), works on regulating the differential expression of the target gene post-transcriptionally. The prime focus of this review is to report the critical miRNA molecule and their regular expression in patients with COVID-19 infection and associated comorbidities. Viral and host miRNAs control the etiology of COVID-19 infection throughout the life cycle and host inflammatory response, where host miRNAs are identified as a double-edged showing as a proviral and antiviral response. The review also covered the role of viral miRNAs in mediating host cell signaling expression during disease pathology. Studying molecular interactions between the host and the SARS-CoV-2 virus during COVID-19 pathogenesis offers the chance to use miRNA-based therapeutics to reduce the severity of the illness. By utilizing an appropriate delivery vehicle, these small non-coding RNA could be envisioned as a promising biomarker in designing a practical RNAi-based treatment approach of clinical significance.