Baseline findings of a multicentric ambispective cohort study (2021-2022) among hospitalised mucormycosis patients in India.

In India, the incidence of mucormycosis reached high levels during 2021-2022, coinciding with the COVID-19 pandemic. In response to this, we established a multicentric ambispective cohort of patients hospitalised with mucormycosis across India. In this paper, we report their baseline profile, clinical characteristics and outcomes at discharge. Patients hospitalized for mucormycosis during March-July 2021 were included. Mucormycosis was diagnosed based on mycological confirmation on direct microscopy (KOH/Calcofluor white stain), culture, histopathology, or supportive evidence from endoscopy or imaging. After consent, trained data collectors used medical records and telephonic interviews to capture data in a pre-tested structured questionnaire. At baseline, we recruited 686 patients from 26 study hospitals, of whom 72.3% were males, 78% had a prior history of diabetes, 53.2% had a history of corticosteroid treatment, and 80% were associated with COVID-19. Pain, numbness or swelling of the face were the commonest symptoms (73.3%). Liposomal Amphotericin B was the commonest drug formulation used (67.1%), and endoscopic sinus surgery was the most common surgical procedure (73.6%). At discharge, the disease was stable in 43.3%, in regression for 29.9% but 9.6% died during hospitalization. Among survivors, commonly reported disabilities included facial disfigurement (18.4%) and difficulties in chewing/swallowing (17.8%). Though the risk of mortality was only 1 in 10, the disability due to the disease was very high. This cohort study could enhance our understanding of the disease's clinical progression and help frame standard treatment guidelines.

Constraining Cosmological Phase Transitions with the Parkes Pulsar Timing Array.

A cosmological first-order phase transition is expected to produce a stochastic gravitational wave background. If the phase transition temperature is on the MeV scale, the power spectrum of the induced stochastic gravitational waves peaks around nanohertz frequencies, and can thus be probed with high-precision pulsar timing observations. We search for such a stochastic gravitational wave background with the latest data set of the Parkes Pulsar Timing Array. We find no evidence for a Hellings-Downs spatial correlation as expected for a stochastic gravitational wave background. Therefore, we present constraints on first-order phase transition model parameters. Our analysis shows that pulsar timing is particularly sensitive to the low-temperature (T∼1-100 MeV) phase transition with a duration (ß/H_{*})^{-1}∼10^{-2}-10^{-1} and therefore can be used to constrain the dark and QCD phase transitions.

Mechanisms of Colorectal Cancer Prevention by Aspirin-A Literature Review and Perspective on the Role of COX-Dependent and -Independent Pathways.

Aspirin, synthesized and marketed in 1897 by Bayer, is one of the most widely used drugs in the world. It has a well-recognized role in decreasing inflammation, pain and fever, and in the prevention of thrombotic cardiovascular diseases. Its anti-inflammatory and cardio-protective actions have been well studied and occur through inhibition of cyclooxygenases (COX). Interestingly, a vast amount of epidemiological, preclinical and clinical studies have revealed aspirin as a promising chemopreventive agent, particularly against colorectal cancers (CRC); however, the primary mechanism by which it decreases the occurrences of CRC has still not been established. Numerous mechanisms have been proposed for aspirin's chemopreventive properties among which the inhibition of COX enzymes has been widely discussed. Despite the wide attention COX-inhibition has received as the most probable mechanism of cancer prevention by aspirin, it is clear that aspirin targets many other proteins and pathways, suggesting that these extra-COX targets may also be equally important in preventing CRC. In this review, we discuss the COX-dependent and -independent pathways described in literature for aspirin's anti-cancer effects and highlight the strengths and limitations of the proposed mechanisms. Additionally, we emphasize the potential role of the metabolites of aspirin and salicylic acid (generated in the gut through microbial biotransformation) in contributing to aspirin's chemopreventive actions. We suggest that the preferential chemopreventive effect of aspirin against CRC may be related to direct exposure of aspirin/salicylic acid or its metabolites to the colorectal tissues. Future investigations should shed light on the role of aspirin, its metabolites and the role of the gut microbiota in cancer prevention against CRC.

Do Aspirin and Flavonoids Prevent Cancer through a Common Mechanism Involving Hydroxybenzoic Acids?-The Metabolite Hypothesis.

Despite decades of research to elucidate the cancer preventive mechanisms of aspirin and flavonoids, a consensus has not been reached on their specific modes of action. This inability to accurately pinpoint the mechanism involved is due to the failure to differentiate the primary targets from its associated downstream responses. This review is written in the context of the recent findings on the potential pathways involved in the prevention of colorectal cancers (CRC) by aspirin and flavonoids. Recent reports have demonstrated that the aspirin metabolites 2,3-dihydroxybenzoic acid (2,3-DHBA), 2,5-dihydroxybenzoic acid (2,5-DHBA) and the flavonoid metabolites 2,4,6-trihydroxybenzoic acid (2,4,6-THBA), 3,4-dihydroxybenzoic acid (3,4-DHBA) and 3,4,5-trihydroxybenzoic acid (3,4,5-THBA) were effective in inhibiting cancer cell growth in vitro. Limited in vivo studies also provide evidence that some of these hydroxybenzoic acids (HBAs) inhibit tumor growth in animal models. This raises the possibility that a common pathway involving HBAs may be responsible for the observed cancer preventive actions of aspirin and flavonoids. Since substantial amounts of aspirin and flavonoids are left unabsorbed in the intestinal lumen upon oral consumption, they may be subjected to degradation by the host and bacterial enzymes, generating simpler phenolic acids contributing to the prevention of CRC. Interestingly, these HBAs are also abundantly present in fruits and vegetables. Therefore, we suggest that the HBAs produced through microbial degradation of aspirin and flavonoids or those consumed through the diet may be common mediators of CRC prevention.

Aspirin metabolites 2,3­DHBA and 2,5­DHBA inhibit cancer cell growth: Implications in colorectal cancer prevention.

Although compelling evidence exists on the ability of aspirin to treat colorectal cancer (CRC), and numerous theories and targets have been proposed, a consensus has not been reached regarding its mechanism of action. In this regard, a relatively unexplored area is the role played by aspirin metabolites 2,3­dihydroxybenzoic acid (2,3­DHBA) and 2,5­dihydroxybenzoic acid (2,5­DHBA) in its chemopreventive actions. In a previous study, we demonstrated that 2,3­DHBA and 2,5­DHBA inhibited CDK1 enzyme activity in vitro. The aim of the present study was to understand the effect of these metabolites on the enzyme activity of all CDKs involved in cell cycle regulation (CDKs 1, 2, 4 and 6) as well as their effect on clonal formation in three different cancer cell lines. Additionally, in silico studies were performed to determine the potential sites of interactions of 2,3­DHBA and 2,5­DHBA with CDKs. We demonstrated that 2,3­DHBA and 2,5­DHBA inhibits CDK­1 enzyme activity beginning at 500 µM, while CDK2 and CDK4 activity was inhibited only at higher concentrations (>750 µM). 2,3­DHBA inhibited CDK6 enzyme activity from 250 µM, while 2,5­DHBA inhibited its activity >750 µM. Colony formation assays showed that 2,5­DHBA was highly effective in inhibiting clonal formation in HCT­116 and HT­29 CRC cell lines (250­500 µM), and in the MDA­MB­231 breast cancer cell line (~100 µM). In contrast 2,3­DHBA was effective only in MDA­MB­231 cells (~500 µM). Both aspirin and salicylic acid failed to inhibit all four CDKs and colony formation. Based on the present results, it is suggested that 2,3­DHBA and 2,5­DHBA may contribute to the chemopreventive properties of aspirin, possibly through the inhibition of CDKs. The present data and the proposed mechanisms should open new areas for future investigations.

The Flavonoid Metabolite 2,4,6-Trihydroxybenzoic Acid Is a CDK Inhibitor and an Anti-Proliferative Agent: A Potential Role in Cancer Prevention.

Flavonoids have emerged as promising compounds capable of preventing colorectal cancer (CRC) due to their anti-oxidant and anti-inflammatory properties. It is hypothesized that the metabolites of flavonoids are primarily responsible for the observed anti-cancer effects owing to the unstable nature of the parent compounds and their degradation by colonic microflora. In this study, we investigated the ability of one metabolite, 2,4,6-trihydroxybenzoic acid (2,4,6-THBA) to inhibit Cyclin Dependent Kinase (CDK) activity and cancer cell proliferation. Using in vitro kinase assays, we demonstrated that 2,4,6-THBA dose-dependently inhibited CDKs 1, 2 and 4 and in silico studies identified key amino acids involved in these interactions. Interestingly, no significant CDK inhibition was observed with the structurally related compounds 3,4,5-trihydroxybenzoic acid (3,4,5-THBA) and phloroglucinol, suggesting that orientation of the functional groups and specific amino acid interactions may play a role in inhibition. We showed that cellular uptake of 2,4,6-THBA required the expression of functional SLC5A8, a monocarboxylic acid transporter. Consistent with this, in cells expressing functional SLC5A8, 2,4,6-THBA induced CDK inhibitory proteins p21Cip1 and p27Kip1 and inhibited cell proliferation. These findings, for the first time, suggest that the flavonoid metabolite 2,4,6-THBA may mediate its effects through a CDK- and SLC5A8-dependent pathway contributing to the prevention of CRC.

Salicylic acid metabolites and derivatives inhibit CDK activity: Novel insights into aspirin's chemopreventive effects against colorectal cancer.

Aspirin's potential as a drug continues to be evaluated for the prevention of colorectal cancer (CRC). Although multiple targets for aspirin and its metabolite, salicylic acid, have been identified, no unifying mechanism has been proposed to clearly explain its chemopreventive effects. Our goal here was to investigate the ability of salicylic acid metabolites, known to be generated through cytochrome P450 (CYP450) enzymes, and its derivatives as cyclin dependent kinase (CDK) inhibitors to gain new insights into aspirin's chemopreventive actions. Using in vitro kinase assays, for the first time, we demonstrate that salicylic acid metabolites, 2,3-dihydroxybenzoic acid (2,3-DHBA) and 2,5-dihydroxybenzoic acid (2,5-DHBA), as well as derivatives 2,4-dihydroxybenzoic acid (2,4-DHBA), 2,6-dihydroxybenzoic acid (2,6-DHBA), inhibited CDK1 enzyme activity. 2,3-DHBA and 2,6-DHBA did not inhibit CDK2 and 4; however, both inhibited CDK-6 activity. Interestingly, another derivative, 2,4,6-trihydroxybenzoic acid (2,4,6-THBA) was highly effective in inhibiting CDK1, 2, 4 and 6 activity. Molecular docking studies showed that these compounds potentially interact with CDK1. Immunoblotting experiments showed that aspirin acetylated CDK1, and pre-incubation with salicylic acid and its derivatives prevented aspirin-mediated CDK1 acetylation, which supported the data obtained from molecular docking studies. We suggest that intracellularly generated salicylic acid metabolites through CYP450 enzymes within the colonic epithelial cells, or the salicylic acid metabolites generated by gut microflora may significantly contribute to the preferential chemopreventive effect of aspirin against CRC through inhibition of CDKs. This novel hypothesis and mechanism of action in aspirin's chemopreventive effects opens a new area for future research. In addition, structural modification to salicylic acid derivatives may prove useful in the development of novel CDK inhibitors in cancer prevention and treatment.

Aspirin inhibits glucose­6­phosphate dehydrogenase activity in HCT 116 cells through acetylation: Identification of aspirin-acetylated sites.

Glucose-6-phosphate dehydrogenase (G6PD) catalyzes the first reaction in the pentose phosphate pathway, and generates ribose sugars, which are required for nucleic acid synthesis, and nicotinamide adenine dinucleotide phosphate (NADPH), which is important for neutralization of oxidative stress. The expression of G6PD is elevated in several types of tumor, including colon, breast and lung cancer, and has been implicated in cancer cell growth. Our previous study demonstrated that exposure of HCT 116 human colorectal cancer cells to aspirin caused acetylation of G6PD, and this was associated with a decrease in its enzyme activity. In the present study, this observation was expanded to HT­29 colorectal cancer cells, in order to compare aspirin­mediated acetylation of G6PD and its activity between HCT 116 and HT­29 cells. In addition, the present study aimed to determine the acetylation targets of aspirin on recombinant G6PD to provide an insight into the mechanisms of inhibition. The results demonstrated that the extent of G6PD acetylation was significantly higher in HCT 116 cells compared with in HT­29 cells; accordingly, a greater reduction in G6PD enzyme activity was observed in the HCT 116 cells. Mass spectrometry analysis of aspirin­acetylated G6PD (isoform a) revealed that aspirin acetylated a total of 14 lysine residues, which were dispersed throughout the length of the G6PD protein. One of the important amino acid targets of aspirin included lysine 235 (K235, in isoform a) and this corresponds to K205 in isoform b, which has previously been identified as being important for catalysis. Acetylation of G6PD at several sites, including K235 (K205 in isoform b), may mediate inhibition of G6PD activity, which may contribute to the ability of aspirin to exert anticancer effects through decreased synthesis of ribose sugars and NADPH.

Cyclin A2 and CDK2 as Novel Targets of Aspirin and Salicylic Acid: A Potential Role in Cancer Prevention.

UNLABELLED: Data emerging from the past 10 years have consolidated the rationale for investigating the use of aspirin as a chemopreventive agent; however, the mechanisms leading to its anticancer effects are still being elucidated. We hypothesized that aspirin's chemopreventive actions may involve cell-cycle regulation through modulation of the levels or activity of cyclin A2/cyclin-dependent kinase-2 (CDK2). In this study, HT-29 and other diverse panel of cancer cells were used to demonstrate that both aspirin and its primary metabolite, salicylic acid, decreased cyclin A2 (CCNA2) and CDK2 protein and mRNA levels. The downregulatory effect of either drugs on cyclin A2 levels was prevented by pretreatment with lactacystin, an inhibitor of proteasomes, suggesting the involvement of 26S proteasomes. In-vitro kinase assays showed that lysates from cells treated with salicylic acid had lower levels of CDK2 activity. Importantly, three independent experiments revealed that salicylic acid directly binds to CDK2. First, inclusion of salicylic acid in naïve cell lysates, or in recombinant CDK2 preparations, increased the ability of the anti-CDK2 antibody to immunoprecipitate CDK2, suggesting that salicylic acid may directly bind and alter its conformation. Second, in 8-anilino-1-naphthalene-sulfonate (ANS)-CDK2 fluorescence assays, preincubation of CDK2 with salicylic acid dose-dependently quenched the fluorescence due to ANS. Third, computational analysis using molecular docking studies identified Asp145 and Lys33 as the potential sites of salicylic acid interactions with CDK2. These results demonstrate that aspirin and salicylic acid downregulate cyclin A2/CDK2 proteins in multiple cancer cell lines, suggesting a novel target and mechanism of action in chemoprevention. IMPLICATIONS: Biochemical and structural studies indicate that the antiproliferative actions of aspirin are mediated through cyclin A2/CDK2.

Aspirin acetylates wild type and mutant p53 in colon cancer cells: identification of aspirin acetylated sites on recombinant p53.

Aspirin's ability to inhibit cell proliferation and induce apoptosis in cancer cell lines is considered to be an important mechanism for its anti-cancer effects. We previously demonstrated that aspirin acetylated the tumor suppressor protein p53 at lysine 382 in MDA-MB-231 human breast cancer cells. Here, we extended these observations to human colon cancer cells, HCT 116 harboring wild type p53, and HT-29 containing mutant p53. We demonstrate that aspirin induced acetylation of p53 in both cell lines in a concentration-dependent manner. Aspirin-acetylated p53 was localized to the nucleus. In both cell lines, aspirin induced p21(CIP1). Aspirin also acetylated recombinant p53 (rp53) in vitro suggesting that it occurs through a non-enzymatic chemical reaction. Mass spectrometry analysis and immunoblotting identified 10 acetylated lysines on rp53, and molecular modeling showed that all lysines targeted by aspirin are surface exposed. Five of these lysines are localized to the DNA-binding domain, four to the nuclear localization signal domain, and one to the C-terminal regulatory domain. Our results suggest that aspirin's anti-cancer effect may involve acetylation and activation of wild type and mutant p53 and induction of target gene expression. This is the first report attempting to characterize p53 acetylation sites targeted by aspirin.

An eccentric binary millisecond pulsar in the galactic plane.

Binary pulsar systems are superb probes of stellar and binary evolution and the physics of extreme environments. In a survey with the Arecibo telescope, we have found PSR J1903+0327, a radio pulsar with a rotational period of 2.15 milliseconds in a highly eccentric (e = 0.44) 95-day orbit around a solar mass (M(middle dot in circle)) companion. Infrared observations identify a possible main-sequence companion star. Conventional binary stellar evolution models predict neither large orbital eccentricities nor main-sequence companions around millisecond pulsars. Alternative formation scenarios involve recycling a neutron star in a globular cluster, then ejecting it into the Galactic disk, or membership in a hierarchical triple system. A relativistic analysis of timing observations of the pulsar finds its mass to be 1.74 +/- 0.04 M solar symbol, an unusually high value.