Hypophosphatemia in Chronic Obstructive Pulmonary Disease Patients Requiring Mechanical Ventilation and Its Impact on Weaning in an Intensive Care Unit of a Tertiary Care Hospital in Eastern India.

Background Hypophosphatemia, defined as a serum phosphate level less than 2.5 mg/dL, is a frequent finding in patients with chronic obstructive pulmonary disease (COPD) and has been speculated to negatively affect weaning outcomes. This study aimed to determine the incidence of hypophosphatemia in COPD patients requiring mechanical ventilation and evaluate the predictive role of hypophosphatemia as an indicator of successful weaning from mechanical ventilation in such patients admitted to the intensive care unit (ICU) in a tertiary care hospital in eastern India. Methodology This prospective observational study included 60 adult patients aged 18 to 75 years with acute exacerbations of COPD on mechanical ventilation in the ICU who were planned to undergo a weaning trial. Serum phosphate levels were assessed at the time of admission and before each weaning attempt. Weaning outcomes at each attempt, length of ventilator and ICU stay, and mortality were recorded. Data collection was initiated after approval of the Institutional Ethics Committee. Receiver operating curve (ROC) analysis was done to identify the cut-off value of serum phosphate which predicted successful weaning. Results Of 60 participants, hypophosphatemia on admission was present in 15 (25%) patients. Despite the correction, 13 (21.7%) patients had hypophosphatemia before the first weaning attempt. Only 22 patients out of 60 were successfully weaned off from mechanical ventilation in the first trial, accounting for a success rate of 36.7%, of whom 20 were normophosphatemic (90.9%). In the second and third weaning trials, hypophosphatemia was significantly associated with weaning failure. Overall differences in mean serum phosphate levels among those who failed to wean in each weaning trial and the successful attempt were statistically significant (p < 0.001). On ROC analysis of serum phosphate level before the first weaning trial, a cut-off value of ≥3.0 mg/dL was identified to have 86.4% sensitivity, 55.3% specificity, 52.8% positive predictive value, 87.5% negative predictive value, and 66.7% diagnostic accuracy in predicting weaning success. Five patients died, accounting for a mortality rate of 8.3%. Lower mean serum phosphate levels before the first weaning trial, higher mean age, and longer ventilator and ICU days were significantly associated with mortality among our study participants (p < 0.05). Conclusions Our findings suggest that maintaining normal serum phosphate levels is critical to successfully weaning off patients with COPD from ventilator support.

Saccharomyces cerevisiae DJ-1 paralogs maintain genome integrity through glycation repair of nucleic acids and proteins.

Reactive carbonyl species (RCS) such as methylglyoxal and glyoxal are potent glycolytic intermediates that extensively damage cellular biomolecules leading to genetic aberration and protein misfolding. Hence, RCS levels are crucial indicators in the progression of various pathological diseases. Besides the glyoxalase system, emerging studies report highly conserved DJ-1 superfamily proteins as critical regulators of RCS. DJ-1 superfamily proteins, including the human DJ-1, a genetic determinant of Parkinson's disease, possess diverse physiological functions paramount for combating multiple stressors. Although S. cerevisiae retains four DJ-1 orthologs (Hsp31, Hsp32, Hsp33, and Hsp34), their physiological relevance and collective requirement remain obscure. Here, we report for the first time that the yeast DJ-1 orthologs function as novel enzymes involved in the preferential scavenge of glyoxal and methylglyoxal, toxic metabolites, and genotoxic agents. Their collective loss stimulates chronic glycation of the proteome, and nucleic acids, inducing spectrum of genetic mutations and reduced mRNA translational efficiency. Furthermore, the Hsp31 paralogs efficiently repair severely glycated macromolecules derived from carbonyl modifications. Also, their absence elevates DNA damage response, making cells vulnerable to various genotoxins. Interestingly, yeast DJ-1 orthologs preserve functional mitochondrial content, maintain ATP levels, and redistribute into mitochondria to alleviate the glycation damage of macromolecules. Together, our study uncovers a novel glycation repair pathway in S. cerevisiae and a possible neuroprotective mechanism of how hDJ-1 confers mitochondrial health during glycation toxicity.

Nanoparticle Induced Conformational Switch Between α-Helix and ß-Sheet Attenuates Immunogenic Response of MPT63.

Although significant efforts have been devoted to develop nanoparticle-based biopharmaceuticals, it is not understood how protein conformation and nanoparticle surface modulate each other in optimizing the activity and/or toxicity of the biological molecules. This is particularly important for a protein, which can adopt different conformational states separated by a relatively small energy barrier. In this paper, we have studied nanoparticle binding-induced conformational switch from ß-sheet to α-helix of MPT63, a small major secreted protein from Mycobacterium tuberculosis and a drug target against Tuberculosis. The binding of magnetite nanoparticles to MPT63 results in a ß-sheet to α-helix switch near the sequence stretch between the 19th and 30th amino acids. As a consequence, the immunogenic response of the protein becomes compromised, which could be restored by protein engineering. This study emphasizes that conformational stability toward NP surface binding may require optimization involving genetic engineering for development of a nanoparticle conjugated pharmaceutical.

Small Molecules Attenuate the Interplay between Conformational Fluctuations, Early Oligomerization and Amyloidosis of Alpha Synuclein.

Aggregation of alpha synuclein has strong implications in Parkinson's disease. The heterogeneity of folding/aggregation landscape and transient nature of the early intermediates result in difficulty in developing a successful therapeutic intervention. Here we used fluorescence measurements at ensemble and single molecule resolution to study how the late and early events of alpha synuclein aggregation modulate each other. In-vitro aggregation data was complemented using measurements inside live neuroblastoma cells by employing a small molecule labeling technique. An inhibitor molecule (arginine), which delayed the late event of amyloidosis, was found to bind to the protein, shifting the early conformational fluctuations towards a compact state. In contrast, a facilitator of late aggregation (glutamate), was found to be excluded from the protein surface. The presence of glutamate was found to speed up the oligomer formation at the early stage. We found that the effects of the inhibitor and facilitator were additive and as a result they maintained a ratio at which they cancelled each other's influence on different stages of alpha synuclein aggregation.

Understanding the Effect of Single Cysteine Mutations on Gold Nanoclusters as Studied by Spectroscopy and Density Functional Theory Modeling.

Fluorescent metal nanoclusters have generated considerable excitement in nanobiotechnology, particularly in the applications of biolabeling, targeted delivery, and biological sensing. The present work is an experimental and computational study that aims to understand the effects of protein environment on the synthesis and electronic properties of gold nanoclusters. MPT63, a drug target of Mycobacterium tuberculosis, was used as the template protein to synthesize, for the first time, gold nanoclusters at a low micromolar concentration of the protein. Two single cysteine mutants of MPT63, namely, MPT63Gly20Cys (mutant I) and MPT63Gly40Cys (mutant II) were employed for this study. The experimental results show that cysteine residues positioned in two different regions of the protein induce varying electronic states of the nanoclusters depending on the surrounding amino acids. A mixture of five-atom and eight-atom clusters was generated for each mutant, and the former was found to be predominant in both cases. Computational studies, including density functional theory (DFT), frontier molecular orbital (FMO), and natural bond orbital (NBO) calculations, validated the experimental observations. The as-prepared protein-stabilized nanoclusters were found to have applications in the imaging of live cells.

The presence of non-native helical structure in the unfolding of a beta-sheet protein MPT63.

MPT63, a major secreted protein from Mycobacterium tuberculosis, has been shown to have immunogenic properties and has been implicated in virulence. MPT63 is a ß-sandwich protein containing 11 ß strands and a very short stretch of 310 helix. The detailed experimental and computational study reported here investigates the equilibrium unfolding transition of MPT63. It is shown that in spite of being a complete ß-sheet protein, MPT63 has a strong propensity toward helix structures in its early intermediates. Far UV-CD and FTIR spectra clearly suggest that the low-pH intermediate of MTP63 has enhanced helical content, while fluorescence correlation spectroscopy suggests a significant contraction. Molecular dynamics simulation complements the experimental results indicating that the unfolded state of MPT63 traverses through intermediate forms with increased helical characteristics. It is found that this early intermediate contains exposed hydrophobic surface, and is aggregation prone. Although MPT63 is a complete ß-sheet protein in its native form, the present findings suggest that the secondary structure preferences of the local interactions in early folding pathway may not always follow the native conformation. Furthermore, the Gly25Ala mutant supports the proposed hypothesis by increasing the non-native helical propensity of the protein structure.