Longitudinal Plasma Proteomics Analysis Reveals Novel Candidate Biomarkers in Acute COVID-19.

The host response to COVID-19 pathophysiology over the first few days of infection remains largely unclear, especially the mechanisms in the blood compartment. We report on a longitudinal proteomic analysis of acute-phase COVID-19 patients, for which we used blood plasma, multiple reaction monitoring with internal standards, and data-independent acquisition. We measured samples on admission for 49 patients, of which 21 had additional samples on days 2, 4, 7, and 14 after admission. We also measured 30 externally obtained samples from healthy individuals for comparison at baseline. The 31 proteins differentiated in abundance between acute COVID-19 patients and healthy controls belonged to acute inflammatory response, complement activation, regulation of inflammatory response, and regulation of protein activation cascade. The longitudinal analysis showed distinct profiles revealing increased levels of multiple lipid-associated functions, a rapid decrease followed by recovery for complement activation, humoral immune response, and acute inflammatory response-related proteins, and level fluctuation in the regulation of smooth muscle cell proliferation, secretory mechanisms, and platelet degranulation. Three proteins were differentiated between survivors and nonsurvivors. Finally, increased levels of fructose-bisphosphate aldolase B were determined in patients with exposure to angiotensin receptor blockers versus decreased levels in those exposed to angiotensin-converting enzyme inhibitors. Data are available via ProteomeXchange PXD029437.

Second messenger c-di-AMP regulates multiple antibiotic sensitivity pathways in Mycobacterium smegmatis by discrete mechanisms.

One of the debilitating causes of high mortality in the case of tuberculosis and other bacterial infections is the resistance development against standard drugs. There are limited studies so far to describe how a bacterial second messenger molecule can directly participate in distinctive antibiotic tolerance characteristics of a cell in a mechanism-dependent manner. Here we show that intracellular cyclic di-AMP (c-di-AMP) concentration can modulate drug sensitivity of Mycobacterium smegmatis by interacting with an effector protein or interfering with the 5'-UTR regions in mRNA of the genes and thus causing transcriptional downregulation of important genes in the pathways. We studied four antibiotics with different mechanisms of action: rifampicin, ciprofloxacin, erythromycin, and tobramycin and subsequently found that the level of drug sensitivity of the bacteria is directly proportional to the c-di-AMP concentration inside the cell. Further, we unraveled the underlying molecular mechanisms to delineate the specific genes and pathways regulated by c-di-AMP and hence result in differential drug sensitivity in M. smegmatis.

Elucidating the role of c-di-AMP in Mycobacterium smegmatis: Phenotypic characterization and functional analysis.

Cyclic-di-AMP (c-di-AMP) is an important secondary messenger molecule that plays a critical role in monitoring several important cellular processes, especially in several Gram-positive bacteria. In this study, we seek to unravel the physiological significance of the molecule c-di-AMP in Mycobacterium smegmatis under different conditions, using strains with altered c-di-AMP levels: c-di-AMP null mutant (ΔdisA) and a c-di-AMP over-expression mutant (Δpde). Our thorough analysis of the mutants revealed that the intracellular concentration of c-di-AMP could determine many basic phenotypes such as colony architecture, cell shape, cell size, membrane permeability etc. Additionally, it was shown to play a significant role in multiple stress adaptation pathways in the case of different DNA and membrane stresses. Our study also revealed how the biofilm phenotypes of M. smegmatis cells are altered with high intracellular c-di-AMP concentration. Next, we checked how c-di-AMP contributes to antibiotic resistance or susceptibility characteristics of M. smegmatis, which was followed by a detailed transcriptome profile analysis to reveal key genes and pathways such as translation, arginine biosynthesis, cell wall and plasma membrane are regulated by c-di-AMP in mycobacteria.

Sub-operon promoter arrangement of disA facilitates c-di-AMP homeostasis and selective stress responses in Mycobacterium smegmatis.

Bacterial second messenger signaling often plays an important role in cellular physiology. In this study, we have attempted to understand how c-di-AMP synthesis and degradation are transcriptionally regulated in Mycobacterium smegmatis. We found that although a c-di-AMP synthesis gene, disA, exists in a multi-gene operon, a sub-operon promoter arrangement facilitates disA gene expression under normal conditions to maintain intracellular c-di-AMP concentration and is induced further during certain stress adaptations. Individual gene-specific promoters also play a key role under various genotoxic stress conditions to shut down c-di-AMP synthesis, which could otherwise be detrimental for cells. Further, we learned that a high c-di-AMP concentration plays a role in the autoregulation of the disA promoter to limit intracellular c-di-AMP concentration. This study was helpful to understand how c-di-AMP synthesis is regulated under normal and stress conditions linked to its physiological relevance in M. smegmatis.

C-4-Modified Isotetrones Prevent Biofilm Growth and Persister Cell Resuscitation in Mycobacterium smegmatis.

Hyperphosphorylated nucleotide (p)ppGpp, synthesized by Rel protein, regulates the stringent response pathway responsible for biofilm and persister cell growth in mycobacteria. The discovery of vitamin C as an inhibitor of Rel protein activities raises the prospect of tetrone lactones to prevent such pathways. The closely related isotetrone lactone derivatives are identified herein as inhibitors of the above processes in a mycobacterium. Synthesis and biochemical evaluations show that an isotetrone possessing phenyl substituent at C-4 inhibit the biofilm formation at 400 µg mL-1, 84 h post-exposure, followed by moderate inhibition by the isotetrone possessing the p-hydroxyphenyl substituent. The latter isotetrone inhibits the growth of persister cells at 400 µg mL-1 f.c. when monitored for 2 weeks, under PBS starvation. Isotetrones also potentiate the inhibition of antibiotic-tolerant regrowth of cells by ciprofloxacin (0.75 µg mL-1) and thus act as bioenhancers. Molecular dynamics studies show that isotetrone derivatives bind to the RelMsm protein more efficiently than vitamin C at a binding site possessing serine, threonine, lysine, and arginine.

c-di-AMP signaling plays important role in determining antibiotic tolerance phenotypes of Mycobacterium smegmatis.

In this study, we probe the role of secondary messenger c-di-AMP in drug tolerance, which includes both persister and resistant mutant characterization of Mycobacterium smegmatis. Specifically, with the use of c-di-AMP null and overproducing mutants, we showed how c-di-AMP plays a significant role in resistance mutagenesis against antibiotics with different mechanisms of action. We elucidated the specific molecular mechanism linking the elevated intracellular c-di-AMP level and high mutant generation and highlighted the significance of non-homology-based DNA repair. Further investigation enabled us to identify the unique mutational landscape of target and non-target mutation categories linked to intracellular c-di-AMP levels. Overall fitness cost of unique target mutations was estimated in different strain backgrounds, and then we showed the critical role of c-di-AMP in driving epistatic interactions between resistance genes, resulting in the evolution of multi-drug tolerance. Finally, we identified the role of c-di-AMP in persister cells regrowth and mutant enrichment upon cessation of antibiotic treatment.

Intra-protein interactions of SARS-CoV-2 and SARS: a bioinformatic analysis for plausible explanation regarding stability, divergency, and severity.

The current nightmare for the whole world is COVID-19. The occurrence of concentrated pneumonia cases in Wuhan city, Hubei province of China, was first reported on December 30, 2019. SARS-CoV first disclosed in 2002 but had not outspread worldwide. After 18 years, in 2020, it reemerged and outspread worldwide as SARS-CoV-2 (COVID-19), as the most dangerous virus-creating disease in the world. Is it possible to create a favorable evolution within the short time (18 years)? If possible, then what are those properties or factors that are changed in SARS-CoV-2 to make it undefeated? What are the fundamental differences between SARS-CoV-2 and SARS? The study is one of the initiatives to find out all those queries. Here, four types of protein sequences from SARS-CoV-2 and SARS were retrieved from the database to study their physicochemical and structural properties. Results showed that charged residues are playing a pivotal role in SARS-CoV-2 evolution and contribute to the helix stabilization. The formation of the cyclic salt bridge and other intra-protein interactions specially network aromatic-aromatic interaction also play the crucial role in SAS-CoV-2. This comparative study will help to understand the evolution from SARS to SARS-CoV-2 and helpful in protein engineering.

Understanding the Meaning of Recovery to Patients Undergoing Abdominal Surgery.

Importance: Postoperative recovery is difficult to define or measure. Research addressing interventions aimed to improve recovery after abdominal surgery often focuses on measures such as duration of hospital stay and complication rates. Although these clinical parameters are relevant, understanding patients' perspectives regarding postoperative recovery is fundamental to guiding patient-centered care. Objective: To elucidate the meaning of recovery from the perspective of patients undergoing abdominal surgery. Design, Setting, and Participants: This international qualitative study involved semistructured interviews with patients recovering from abdominal surgery from October 2016 to November 2018 in tertiary hospitals in 4 countries (Canada, Italy, Brazil, and Japan). A purposive maximal variation sampling method was used to ensure the recruitment of patients with varying demographic, clinical, and surgical characteristics. Data on race were not collected. Each interview lasted between 1 and 2 hours. Interviews were recorded and then transcribed verbatim. Transcripts were then analyzed using an inductive thematic analysis approach. Data analysis was conducted from July 2019 to September 2019. Main Outcomes and Measures: The qualitative analysis revealed themes reflecting the meaning of recovery from the perspective of patients undergoing abdominal surgery. Results: Thirty patients recovering from abdominal surgery were interviewed (15 [50%] female; mean [SD] age, 57 [18] years; 10 [33%] underwent major surgery; 16 [53%] underwent laparoscopic surgery). The interviews revealed that for patients undergoing abdominal surgery, the meaning of recovery embodied 5 overarching themes: (1) returning to habits and routines, (2) resolution of symptoms, (3) overcoming mental strains, (4) regaining independence, and (5) enjoying life. Themes associating the meaning of recovery to traditional parameters, such as earlier hospital discharge or absence of complications, were not identified in the interviews. Conclusions and Relevance: This qualitative study suggests that the meaning of recovery from the perspective of patients undergoing abdominal surgery goes beyond traditional clinical parameters. The elements of recovery identified in this study should be taken into account in patient-surgeon discussions about recovery and when developing patient-centered strategies to improve postoperative outcomes.