Psychological morbidities and fatigue in patients with confirmed COVID-19 during disease outbreak: prevalence and associated biopsychosocial risk factors.

Objective: The coronavirus disease 2019 (COVID-19) - a novel and highly infectious pneumonia - has now spread across China and beyond for over four months. However, its psychological impact on patients is unclear. We aim to examine the prevalence and associated risk factors for psychological morbidities and fatigue in patients with confirmed COVID-19 infection. Methods: Amidst the disease outbreak, 41 out of 105 COVID-19 patients in a local designated hospital in China were successfully assessed using a constellation of psychometric questionnaires to determine their psychological morbidities and fatigue. Several potential biopsychosocial risk factors (including pre-existing disabilities, CT severity score of pneumonia, social support, coping strategies) were assessed through multivariable logistic regression analyses to clarify their association with mental health in patients. Results: 43.9% of 41 patients presented with impaired general mental health, 12.2% had post-traumatic stress disorder (PTSD) symptoms, 26.8% had anxiety and/or depression symptoms, and 53.6% had fatigue. We did not find any association between pneumonia severity and psychological morbidities or fatigue in COVID-19 patients. However, high perceived stigmatization was associated with an increased risk of impaired general mental health and high perceived social support was associated with decreased risk. Besides, negative coping inclination was associated with an increased risk of PTSD symptoms; high perceived social support was associated with a decreased risk of anxiety and/or depression symptoms. Conclusions: Psychological morbidities and chronic fatigue are common among COVID-19 patients. Negative coping inclination and being stigmatized are primary risk factors while perceived social support is the main protective factor.

A Novel Phosphopeptide Microarray Based Interactome Map in Breast Cancer Cells Reveals Phosphoprotein-GRB2 Cell Signaling Networks.

The architecture of cellular proteins connected to form signaling pathways in response to internal and external cues is much more complex than a group of simple protein-protein interactions. Post translational modifications on proteins (e.g., phosphorylation of serine, threonine and tyrosine residues on proteins) initiate many downstream signaling events leading to protein-protein interactions and subsequent activation of signaling cascades leading to cell proliferation, cell differentiation and cell death. As evidenced by a rapidly expanding mass spectrometry database demonstrating protein phosphorylation at specific motifs, there is currently a large gap in understanding the functional significance of phosphoproteins with respect to their specific protein connections in the signaling cascades. A comprehensive map that interconnects phospho-motifs in pathways will enable identification of nodal protein interactions that are sensitive signatures indicating a disease phenotype from the physiological hemostasis and provide clues into control of disease. Using a novel phosphopeptide microarray technology, we have mapped endogenous tyrosine-phosphoproteome interaction networks in breast cancer cells mediated by signaling adaptor protein GRB2, which transduces cellular responses downstream of several RTKs through the Ras-ERK signaling cascade. We have identified several previously reported motif specific interactions and novel interactions. The peptide microarray data indicate that various phospho-motifs on a single protein are differentially regulated in various cell types and shows global downregulation of phosphoprotein interactions specifically in cells with metastatic potential. The study has revealed novel phosphoprotein mediated signaling networks, which warrants further detailed analysis of the nodes of protein-protein interaction to uncover their biomarker or therapeutic potential.

Web-based design of peptide microarrays using microPepArray Pro.

Peptide microarrays (peptide arrays) have increasingly become an important research tool for studying protein detection, profiling, and protein-protein interactions, and they have the potential to foster high-throughput protein analysis as DNA arrays did for genomics research a decade ago. Recently, technologies have emerged that allow flexible synthesis of high-density peptide arrays based on specific application needs (e.g., phosphopeptide microarrays). To fully unleash the power of this promising research tool, significant efforts are required to develop computational and informatics resources that facilitate the experimental design and data analysis for a wide range of peptide array-based applications. The design of peptide arrays is inherently more complex than that of DNA arrays. We herein introduce microPepArray Pro, a Web-based general-purpose peptide array design program. microPepArray Pro features strong content design capabilities and maximized user control. The program suits the needs of a diversity of design tasks, works with a variety of peptide array configurations, and is highly expandable: new functionalities can be developed and added to microPepArray Pro with relative ease.

Qualitative and quantitative analysis of peptide microarray binding experiments using SVM-PEPARRAY.

A main objective of analyzing peptide array-based binding experiments is to uncover the relationship between a peptide sequence and the binding outcome. Limited by the peptide array technologies available for applications, few attempts have been made to construct qualitative or quantitative models that depict the peptide sequence:binding strength relationships in peptide microarray-based binding studies. There has been a long history of similar modeling efforts based on low-throughput binding data in the areas of T-cell epitope screening and kinase substrate mapping, however. The keen needs in peptide array applications and the success of the modeling efforts in related fields have prompted us to develop SVM-PEPARRAY, a Web-based program capable of constructing qualitative and quantitative models based on peptide microarray binding datasets using support vector machine (SVM) modeling methods. We expect that such modeling analysis will allow researchers to quickly extract sequence-based biological information from improved peptide array binding results and provide more precise and accurate information about the biological systems investigated.

Microfluidic Reactor Array Device for Massively Parallel In-situ Synthesis of Oligonucleotides.

We have designed and fabricated a microfluidic reactor array device for massively parallel in-situ synthesis of oligonucleotides (oDNA). The device is made of glass anodically bonded to silicon consisting of three level features: microreactors, microchannels and through inlet/outlet holes. Main challenges in the design of this device include preventing diffusion of photogenerated reagents upon activation and achieving uniform reagent flow through thousands of parallel reactors. The device embodies a simple and effective dynamic isolation mechanism which prevents the intermixing of active reagents between discrete microreactors. Depending on the design parameters, it is possible to achieve uniform flow and synthesis reaction in all of the reactors by proper design of the microreactors and the microchannels. We demonstrated the use of this device on a solution-based, light-directed parallel in-situ oDNA synthesis. We were able to synthesize long oDNA, up to 120 mers at stepwise yield of 98 %. The quality of our microfluidic oDNA microarray including sensitivity, signal noise, specificity, spot variation and accuracy was characterized. Our microfluidic reactor array devices show a great potential for genomics and proteomics researches.

PepCyber:P~PEP: a database of human protein protein interactions mediated by phosphoprotein-binding domains.

Phosphoprotein-binding domains (PPBDs) mediate many important cellular and molecular processes. Ten PPBDs have been known to exist in the human proteome, namely, 14-3-3, BRCT, C2, FHA, MH2, PBD, PTB, SH2, WD-40 and WW. PepCyber:P approximately PEP is a newly constructed database specialized in documenting human PPBD-containing proteins and PPBD-mediated interactions. Our motivation is to provide the research community with a rich information source emphasizing the reported, experimentally validated data for specific PPBD-PPEP interactions. This information is not only useful for designing, comparing and validating the relevant experiments, but it also serves as a knowledge-base for computationally constructing systems signaling pathways and networks. PepCyber:P approximately PEP is accessible through the URL, http://www.pepcyber.org/PPEP/. The current release of the database contains 7044 PPBD-mediated interactions involving 337 PPBD-containing proteins and 1123 substrate proteins.

microParaflo biochip for nucleic acid and protein analysis.

We describe in this chapter the use of oligonucleotide or peptide microarrays (arrays) based on microfluidic chips. Specifically, three major applications are presented: (1) microRNA/small RNA detection using a microRNA detection chip, (2) protein binding and function analysis using epitope, kinase substrate, or phosphopeptide chips, and (3) protein-binding analysis using oligonucleotide chips. These diverse categories of customizable arrays are based on the same biochip platform featuring a significant amount of flexibility in the sequence design to suit a wide range of research needs. The protocols of the array applications play a critical role in obtaining high quality and reliable results. Given the comprehensive and complex nature of the array experiments, the details presented in this chapter is intended merely as a useful information source of reference or a starting point for many researchers who are interested in genome- or proteome-scale studies of proteins and nucleic acids and their interactions.

Microfluidic PicoArray synthesis of oligodeoxynucleotides and simultaneous assembling of multiple DNA sequences.

Large DNA constructs of arbitrary sequences can currently be assembled with relative ease by joining short synthetic oligodeoxynucleotides (oligonucleotides). The ability to mass produce these synthetic genes readily will have a significant impact on research in biology and medicine. Presently, high-throughput gene synthesis is unlikely, due to the limits of oligonucleotide synthesis. We describe a microfluidic PicoArray method for the simultaneous synthesis and purification of oligonucleotides that are designed for multiplex gene synthesis. Given the demand for highly pure oligonucleotides in gene synthesis processes, we used a model to improve key reaction steps in DNA synthesis. The oligonucleotides obtained were successfully used in ligation under thermal cycling conditions to generate DNA constructs of several hundreds of base pairs. Protein expression using the gene thus synthesized was demonstrated. We used a DNA assembly strategy, i.e. ligation followed by fusion PCR, and achieved effective assembling of up to 10 kb DNA constructs. These results illustrate the potential of microfluidics-based ultra-fast oligonucleotide parallel synthesis as an enabling tool for modern synthetic biology applications, such as the construction of genome-scale molecular clones and cell-free large scale protein expression.

Orientation of d-tubocurarine in the muscle nicotinic acetylcholine receptor-binding site.

Ligand modification and receptor site-directed mutagenesis were used to examine binding of the competitive antagonist, d-tubocurarine (dTC), to the muscle-type nicotinic acetylcholine receptor (AChR). By using various dTC analogs, we measured the interactions of specific dTC functional groups with amino acid positions in the AChR gamma-subunit. Because data for mutations at residue gammaTyr(117) were the most consistent with direct interaction with dTC, we focused on that residue. Double mutant thermodynamic cycle analysis showed apparent interactions of gammaTyr(117) with both the 2-N and the 13'-positions of dTC. Examination of a dTC analog with a negative charge at the 13'-position failed to reveal electrostatic interaction with charged side-chain substitutions at gamma117, but the effects of side-chain substitutions remained consistent with proximity of Tyr(117) to the cationic 2-N of dTC. The apparent interaction of gammaTyr(117) with the 13'-position of dTC was likely mediated by allosteric changes in either dTC or the receptor. The data also show that cation-pi electron stabilization of the 2-N position is not required for high affinity binding. Molecular modeling of dTC within the binding pocket of the acetylcholine-binding protein places the 2-N in proximity to the residue homologous to gammaTyr(117). This model provides a plausible structural basis for binding of dTC within the acetylcholine-binding site of the AChR family that appears consistent with findings from photoaffinity labeling studies and with site-directed mutagenesis studies of the AChR.