The Cardiac Comeback-Beating Stronger: Exploring the Remarkable Resilience of the Heart in COVID-19 Recovery through Cardiac Autonomic Analysis.

Background and Objectives: Analyzing the cardiac autonomic function in COVID-19 patients can provide insights into the impact of the virus on the heart's regulatory mechanisms and its recovery. The autonomic nervous system plays a crucial role in regulating the heart's functions, such as heart rate, blood pressure, and cardiac output. This study aimed to investigate the impact of COVID-19 on heart rate variability (HRV) during a 6-min walk test (6MWT). Materials and Methods: The study included 74 participants, consisting of 37 individuals who had recovered from mild to moderate COVID-19 and 37 healthy controls. The study assessed heart rate variability (HRV) and blood pressure both before and after a 6-min walk test (6MWT). Results: The study found significant differences in a few time domains (SDNN and pNN50) and all frequency domain measures, whereas there were no significant differences in demographic characteristics or blood pressure between COVID-19-recovered individuals and healthy controls at rest. There were significant 6MWT effects on average HR, time-domain (SDNN and pNN50) measures of HRV, and all frequency domain measures of HRV. A significant group × 6MWT interaction was found for SDNN, pNN50, total power, Ln total power, LF, HF, Ln LF, Ln HF, and LF nu. Conclusions: Cardiac Autonomic analysis through HRV is essential to ensure the continued health and well-being of COVID-19 survivors and to minimize the potential long-term impacts of the disease on their cardiovascular system. This suggests that HRV analysis during the recovery phase following exercise could serve as a valuable tool for evaluating the physiological effects of COVID-19 and monitoring the recovery process.

eIF5A promotes translation of polyproline motifs.

Translation factor eIF5A, containing the unique amino acid hypusine, was originally shown to stimulate Met-puromycin synthesis, a model assay for peptide bond formation. More recently, eIF5A was shown to promote translation elongation; however, its precise requirement in protein synthesis remains elusive. We use in vivo assays in yeast and in vitro reconstituted translation assays to reveal a specific requirement for eIF5A to promote peptide bond formation between consecutive Pro residues. Addition of eIF5A relieves ribosomal stalling during translation of three consecutive Pro residues in vitro, and loss of eIF5A function impairs translation of polyproline-containing proteins in vivo. Hydroxyl radical probing experiments localized eIF5A near the E site of the ribosome with its hypusine residue adjacent to the acceptor stem of the P site tRNA. Thus, eIF5A, like its bacterial ortholog EFP, is proposed to stimulate the peptidyl transferase activity of the ribosome and facilitate the reactivity of poor substrates like Pro.

Hypusine-containing protein eIF5A promotes translation elongation.

Translation elongation factors facilitate protein synthesis by the ribosome. Previous studies identified two universally conserved translation elongation factors, EF-Tu in bacteria (known as eEF1A in eukaryotes) and EF-G (eEF2), which deliver aminoacyl-tRNAs to the ribosome and promote ribosomal translocation, respectively. The factor eIF5A (encoded by HYP2 and ANB1 in Saccharomyces cerevisiae), the sole protein in eukaryotes and archaea to contain the unusual amino acid hypusine (N(epsilon)-(4-amino-2-hydroxybutyl)lysine), was originally identified based on its ability to stimulate the yield (endpoint) of methionyl-puromycin synthesis-a model assay for first peptide bond synthesis thought to report on certain aspects of translation initiation. Hypusine is required for eIF5A to associate with ribosomes and to stimulate methionyl-puromycin synthesis. Because eIF5A did not stimulate earlier steps of translation initiation, and depletion of eIF5A in yeast only modestly impaired protein synthesis, it was proposed that eIF5A function was limited to stimulating synthesis of the first peptide bond or that eIF5A functioned on only a subset of cellular messenger RNAs. However, the precise cellular role of eIF5A is unknown, and the protein has also been linked to mRNA decay, including the nonsense-mediated mRNA decay pathway, and to nucleocytoplasmic transport. Here we use molecular genetic and biochemical studies to show that eIF5A promotes translation elongation. Depletion or inactivation of eIF5A in the yeast S. cerevisiae resulted in the accumulation of polysomes and an increase in ribosomal transit times. Addition of recombinant eIF5A from yeast, but not a derivative lacking hypusine, enhanced the rate of tripeptide synthesis in vitro. Moreover, inactivation of eIF5A mimicked the effects of the eEF2 inhibitor sordarin, indicating that eIF5A might function together with eEF2 to promote ribosomal translocation. Because eIF5A is a structural homologue of the bacterial protein EF-P, we propose that eIF5A/EF-P is a universally conserved translation elongation factor.

Controlled hypotension for FESS: A randomised double-blinded comparison of magnesium sulphate and dexmedetomidine.

BACKGROUND AND AIMS: Intense bleeding during general anaesthesia (GA) is the major limitation during functional endoscopic sinus surgery (FESS). This study was aimed to compare the efficacy of dexmedetomidine and magnesium sulphate (MgSO4) for controlled hypotension in FESS. METHODS: Sixty eight patients undergoing FESS were randomised to receive either dexmedetomidine 1 µg/kg over 10 min followed by infusion at 0.2 to 0.7 µg/kg/h (Group D) or MgSO4 40 mg/kg over 10 min followed by an infusion at 10 to 15 mg/kg/h (Group M). Anaesthesia and infusion rates for study drugs were maintained with sevoflurane to keep MAP between 60-70 mmHg throughout the surgery. The time to reach the target MAP, the number of patients requiring a minimum and maximum infusion doses of study drugs were noted. RESULTS: The mean time to achieve target mean arterial pressure (MAP) was less in group D (10.59 ± 2.04) as compared with (21.32 ± 4.65 min) group M (P < 0.001). The target MAP was achieved between 5-15 min in 73.52% patients (Group D) with an infusion dose of 0.2-0.4 µg/kg/h of dexmedetomidine without the use of sevoflurane, while 82.35% patients in group M required 4% sevoflurane along with >12-15 mg/kg/hr infusion of MgSO4 to achieve target MAP in 10-20 min. CONCLUSION: Dexmedetomidine is superior to MgSO4 in achieving target MAP in lesser time with minimum infusion dose.

Candida drug resistance protein 1, a major multidrug ATP binding cassette transporter of Candida albicans, translocates fluorescent phospholipids in a reconstituted system.

Candida albicans drug resistance protein 1 (Cdr1p), an ATP-dependent drug efflux pump, contributes to multidrug resistance in Candida-infected immunocompromised patients. Previous cell-based assays suggested that Cdr1p also acts as a phospholipid translocator. To investigate this, we reconstituted purified Cdr1p into sealed membrane vesicles. Comparison of the ATPase activities of sealed and permeabilized proteoliposomes indicated that Cdr1p was asymmetrically reconstituted such that approximately 70% of the molecules had their ATP binding sites accessible to the extravesicular space. Fluorescent glycerophospholipids were incorporated into the outer leaflet of the proteoliposomes, and their transport into the inner leaflet was tracked with a quenching assay using membrane-impermeant dithionite. We observed ATP-dependent transport of the fluorescent lipids into the inner leaflet of the vesicles. With approximately 6 molecules of Cdr1p per vesicle on average, the half-time to reach the maximal extent of transport was approximately 15 min. Transport was reduced in vesicles reconstituted with Cdr1p variants with impaired ATPase activity and could be competed out to different levels by a molar excess of drugs such as fluconazole and miconazole that are known to be effluxed by Cdr1p. Transport was not affected by ampicillin, a compound that is not effluxed by Cdr1p. Our results suggest a direct link between the ability of Cdr1p to translocate fluorescent phospholipids and efflux drugs. We note that only a few members of the ABC superfamily of Candida have a well-defined role as drug exporters; thus, lipid translocation mediated by Cdr1p could reflect its cellular function.

Chimeras of the ABC drug transporter Cdr1p reveal functional indispensability of transmembrane domains and nucleotide-binding domains, but transmembrane segment 12 is replaceable with the corresponding homologous region of the non-drug transporter Cdr3p.

The molecular basis of the broad substrate recognition and the transport of substrates by Cdr1p, a major drug efflux protein of Candida albicans, is not well understood. To investigate the role of transmembrane domains and nucleotide-binding domains (NBDs) of Cdr1p in drug transport, two sets of protein chimeras were constructed: one set between homologous regions of Cdr1p and the non-drug transporter Cdr3p, and another set consisting of Cdr1p variants comprising either two N- or two C-terminal NBDs of Cdr1p. The replacement of either the N- or the C-terminal half of Cdr1p by the homologous segments of Cdr3p resulted in non-functional recombinant strains expressing chimeric proteins. The results suggest that the chimeric protein could not reach the plasma membrane, probably because of misfolding and subsequent cellular trafficking problems, or the rapid degradation of the chimeras. As an exception, the replacement of transmembrane segment 12 (TMS12) of Cdr1p by the corresponding region of Cdr3p resulted in a functional chimera which displayed unaltered affinity for all the tested substrates. The variant protein comprising either two N-terminal or two C-terminal NBDs of Cdr1p also resulted in non-functional recombinant strains. However, the N-terminal NBD variant, which also showed poor cell surface localization, could be rescued to cell surface, if cells were grown in the presence of drug substrates. The rescued chimera remained non-functional, as was evident from impaired ATPase and efflux activities. Taken together, the results suggest that the two NBDs of Cdr1p are asymmetric and non-exchangeable and that the drug efflux by Cdr1p involves complex interactions between the two halves of the protein.

Functional analysis of CaIPT1, a sphingolipid biosynthetic gene involved in multidrug resistance and morphogenesis of Candida albicans.

In the present study we describe the isolation and functional analysis of a sphingolipid biosynthetic gene, IPT1, of Candida albicans. The functional consequence of the disruption of both alleles of IPT1 was confirmed by mass analysis of its sphingolipid composition. The disruption of both alleles or a single allele of IPT1 did not lead to any change in growth phenotype or total sphingolipid, ergosterol, or phospholipid content of the mutant cells. The loss of mannosyl diinositol diphosphoceramide [M(IP)(2)C] in the ipt1 disruptant, however, resulted in increased sensitivity to drugs like 4-nitroquinoline oxide, terbinafine, o-phenanthroline, fluconazole, itraconazole, and ketoconazole. The increase in drug susceptibilities of ipt1 cells was linked to an altered sphingolipid composition, which appeared to be due to the impaired functionality of Cdr1p, a major drug efflux pump of C. albicans that belongs to the ATP binding cassette superfamily. Our confocal and Western blotting results demonstrated that surface localization of green fluorescent protein-tagged Cdr1p was affected in ipt1 disruptant cells. Poor surface localization of Cdr1p resulted in an impaired ability to efflux fluconazole and rhodamine 6G. The effect of mannosyl inositol phosphoceramide accumulation in the ipt1 mutant and the absence of M(IP)(2)C from the ipt1 mutant on the efflux of drug substrates was very selective. The efflux of methotrexate, a specific substrate of CaMdr1p, another major efflux pump of major facilitator superfamily, remained unaffected in ipt1 mutant cells. Interestingly, changes in sphingolipid composition affected the ability of mutant cells to form proper hyphae in various media. Taken together, our results demonstrate that an altered composition of sphingolipid, which is among the major constituents of membrane rafts, affects the drug susceptibilities and morphogenesis of C. albicans.

Expression of the CDR1 efflux pump in clinical Candida albicans isolates is controlled by a negative regulatory element.

Resistance to azole antifungal drugs in clinical isolates of the human fungal pathogen Candida albicans is often caused by constitutive overexpression of the CDR1 gene, which encodes a multidrug efflux pump of the ABC transporter superfamily. To understand the relevance of a recently identified negative regulatory element (NRE) in the CDR1 promoter for the control of CDR1 expression in the clinical scenario, we investigated the effect of mutation or deletion of the NRE on CDR1 expression in two matched pairs of azole-sensitive and resistant clinical isolates of C. albicans. Expression of GFP or lacZ reporter genes from the wild type CDR1 promoter was much higher in the azole-resistant C. albicans isolates than in the azole-susceptible isolates, reflecting the known differences in CDR1 expression in these strains. Deletion or mutation of the NRE resulted in enhanced reporter gene expression in azole-sensitive strains, but did not further increase the already high CDR1 promoter activity in the azole-resistant strains. In agreement with these findings, electrophoretic mobility shift assays showed a reduced binding to the NRE of nuclear extracts from the resistant C. albicans isolates as compared with extracts from the sensitive isolates. These results demonstrate that the NRE is involved in maintaining CDR1 expression at basal levels and that this repression is overcome in azole-resistant clinical C. albicans isolates, resulting in constitutive CDR1 overexpression and concomitant drug resistance.

Alanine scanning of transmembrane helix 11 of Cdr1p ABC antifungal efflux pump of Candida albicans: identification of amino acid residues critical for drug efflux.

OBJECTIVES: To investigate the role of transmembrane segment 11 (TMS11) of Candida albicans drug resistance protein (Cdr1p) in drug extrusion. METHODS: We replaced each of the 21 putative residues of TMS11 with alanine by site-directed mutagenesis. The Saccharomyces cerevisiae AD1-8u(-) strain was used to overexpress the green fluorescent protein tagged wild-type and mutant variants of TMS11 of Cdr1p. The cells expressing mutant variants were functionally characterized. RESULTS: Out of 21 residues of TMS11, substitution of seven residues, i.e. A1346G, A1347G, T1351A, T1355A, L1358A, F1360A and G1362A, affected differentially the substrate specificity of Cdr1p, while 14 mutants had no significant effect on Cdr1p function. TMS11 projection in an alpha-helical configuration revealed with few exceptions (A1346 and F1360), a distinct segregation of mutation-sensitive residues (A1347, T1351, T1355, L1358 and G1362) towards the more hydrophilic face. Interestingly, mutation-insensitive residues seem to cluster towards the hydrophobic side of the helix. Competition of rhodamine 6G efflux, in the presence of excess of various substrates in the cells expressing native Cdr1p, revealed for the first time the overlapping binding site between azoles (such as ketoconazole, miconazole and itraconazole) and rhodamine 6G. The ability of these azoles to compete with rhodamine 6G was completely lost in mutants F1360A and G1362A, while it was selectively lost in other variants of Cdr1p. We further confirmed that fungicidal synergism of calcineurin inhibitor FK520 with azoles is mediated by Cdr1p; wherein in addition to conserved T1351, substitution of T1355, L1358 and G1362 of TMS11 also resulted in abrogation of synergism. CONCLUSIONS: Our study for the first time provides an insight into the possible role of TMS11 of Cdr1p in drug efflux.

ABC multidrug transporter Cdr1p of Candida albicans has divergent nucleotide-binding domains which display functional asymmetry.

In order to ascertain the molecular basis of ATP-mediated drug extrusion by Cdr1p, a multidrug transporter of Candida albicans, we recently have reported that the Walker A motif of the N-terminal nucleotide biding domain (NBD) of this protein contains an uncommon cysteine residue (C193; GXXGXGCS/T) which is indispensable for ATP hydrolysis. This residue is exceptionally conserved in N-terminal NBDs of fungal ABC transporters and hence makes these transporters an evolutionarily divergent group. However, the presence of a conventional lysine residue at a similar position in the Walker A motif of the C-terminal NBD warrants the individual contribution of both the NBDs in the ATP-driven efflux function of such transporters. In this study we have investigated the contribution of this divergent Walker A motif in the context of the full Cdr1p protein under in vivo conditions by swapping these two crucial amino acids (C193K in Walker A motif of N-terminal NBD and K901C in Walker A motif of C-terminal NBD) between the two NBDs. Both the native and the mutant variants of Cdr1p were integrated at the PDR5 locus as GFP-tagged fusion proteins and were hyper-expressed. Our study shows that both C193K- and K901C-expressing cells elicit a severe impairment of Cdr1p's ATPase function. However, both these mutations have distinct phenotypes with respect to other functional parameters such as substrate efflux and drug resistance profiles. In contrast to C193K, K901C mutant cells were substantially hypersensitive to the tested drugs (fluconazole, ansiomycin, miconazole and cycloheximide) and were unable to expel rhodamine 6G. Our results for the first time show that both NBDs influence the Cdr1p function asymmetrically, and that the positioning of the cysteine and lysine residues within the respective Walker A motifs is functionally not interchangeable.

Membrane sphingolipid-ergosterol interactions are important determinants of multidrug resistance in Candida albicans.

In this study, we examined the importance of membrane ergosterol and sphingolipids in the drug susceptibilities of Candida albicans. We used three independent methods to test the drug susceptibilities of erg mutant cells, which were defective in ergosterol biosynthesis. While spot and filter disk assays revealed that erg2 and erg16 mutant cells of C. albicans became hypersensitive to almost all of the drugs tested (i.e., 4-nitroquinoline oxide, terbinafine, o-phenanthroline, itraconazole, and ketoconazole), determination of the MIC at which 80% of the cells were inhibited revealed more than fourfold increase in susceptibility to ketoconazole and terbinafine. Treatment of wild-type C. albicans cells with fumonisin B1 resulted in 45% inhibition of sphingolipid biosynthesis and caused cells to become hypersensitive to the above drugs. Although erg mutants displayed enhanced membrane fluidity and passive diffusion, these changes alone were not sufficient to elicit the observed hypersusceptibility phenotype of erg mutants. For example, the induction in vitro of a 12% change in the membrane fluidity of C. albicans cells by a membrane fluidizer, benzyl alcohol, did not affect the drug susceptibilities of Candida cells. Additionally, the surface localization of green fluorescent protein-tagged Cdr1p, a major drug efflux pump protein of C. albicans, revealed that any disruption in ergosterol and sphingolipid interactions also interfered with its proper surface localization and functioning. A 50% reduction in the efflux of the Cdr1p substrate, rhodamine 6G, in erg mutant cells or in cells with a reduced sphingolipid content suggested a strong correlation between these membrane lipid components and this major efflux pump protein. Taken together, the results of our study demonstrate for the first time that there is an interaction between membrane ergosterol and sphingolipids, that a reduction in the content of either of these two components results in a disruption of this interaction, and that this disruption has deleterious effects on the drug susceptibilities of C. albicans cells.

Functional characterization of Candida albicans ABC transporter Cdr1p.

In view of the importance of Candida drug resistance protein (Cdr1p) in azole resistance, we have characterized it by overexpressing it as a green fluorescent protein (GFP)-tagged fusion protein (Cdr1p-GFP). The overexpressed Cdr1p-GFP in Saccharomyces cerevisiae is shown to be specifically labeled with the photoaffinity analogs iodoarylazidoprazosin (IAAP) and azidopine, which have been used to characterize the drug-binding sites on mammalian drug-transporting P-glycoproteins. While nystatin could compete for the binding of IAAP, miconazole specifically competed for azidopine binding, suggesting that IAAP and azidopine bind to separate sites on Cdr1p. Cdr1p was subjected to site-directed mutational analysis. Among many mutant variants of Cdr1p, the phenotypes of F774A and DeltaF774 were particularly interesting. The analysis of GFP-tagged mutant variants of Cdr1p revealed that a conserved F774, in predicted transmembrane segment 6, when changed to alanine showed increased binding of both photoaffinity analogues, while its deletion (DeltaF774), as revealed by confocal microscopic analyses, led to mislocalization of the protein. The mislocalized DeltaF774 mutant Cdr1p could be rescued to the plasma membrane as a functional transporter by growth in the presence of a Cdr1p substrate, cycloheximide. Our data for the first time show that the drug substrate-binding sites of Cdr1p exhibit striking similarities with those of mammalian drug-transporting P-glycoproteins and despite differences in topological organization, the transmembrane segment 6 in Cdr1p is also a major contributor to drug substrate-binding site(s).

Purification and characterization of the N-terminal nucleotide binding domain of an ABC drug transporter of Candida albicans: uncommon cysteine 193 of Walker A is critical for ATP hydrolysis.

The Candida drug resistance protein Cdr1p (approximately 170 kDa) is a member of ATP binding cassette (ABC) superfamily of drug transporters, characterized by the presence of 2 nucleotide binding domains (NBD) and 12 transmembrane segments (TMS). NBDs of these transporters are the hub of ATP hydrolysis activity, and their sequence contains a conserved Walker A motif (GxxGxGKS/T). Mutations of the lysine residue within this motif abrogate the ability of NBDs to hydrolyze ATP. Interestingly, the sequence alignments of Cdr1p NBDs with other bacterial and eukaryotic transporters reveal that its N-terminal NBD contains an unusual Walker A sequence (GRPGAGCST), as the invariant lysine is replaced by a cysteine. In an attempt to understand the significance of this uncommon positioning of cysteine within the Walker A motif, we for the first time have purified and characterized the N-terminal NBD (encompassing first N-terminal 512 amino acids) of Cdr1p as well as its C193A mutant protein. The purified NBD-512 protein could exist as an independent functional general ribonucleoside triphosphatase with strong divalent cation dependence. It exhibited ATPase activity with an apparent K(m) in the 0.8-1.0 mM range and V(max) in the range of 147-160 nmol min(-)(1) (mg of protein)(-)(1). NBD-512-associated ATPase activity was also sensitive to inhibitors such as vanadate, azide, and NEM. The Mut-NBD-512 protein (C193A) showed a severe impairment in its ability to hydrolyze ATP (95%); however, no significant effect on ATP (TNP-ATP) binding was observed. Our results show that C193 is critical for N-terminal NBD-mediated ATP hydrolysis and represents a unique feature distinguishing the ATP-dependent functionality of the ABC transporters of fungi from those found in bacteria and other eukaryotes.