A study on the impact of CYP2C19 genotype and platelet reactivity assay on patients undergoing PCI

Background

A thorough understanding of the patient''s genotype and their functional response to a medication is necessary for improving event free survival. Several outcome studies support this view particularly if the patient is to be started on clopidogrel due to the prevalence of clopidogrel resistance. Such guided therapy has reduced the incidence of Major Adverse Cardiac Events (MACE) after stent implantation.

Methods

Between August 2013 and August 2014, 200 patients with coronary artery disease undergoing percutaneous coronary intervention (PCI) were prescribed any one of the anti-platelet medications such as clopidogrel, prasugrel or ticagrelor and offered testing to detect CYP2C19 gene mutations along with a platelet reactivity assay (PRA). Intended outcome was modification of anti-platelet therapy defined as either dose escalation of clopidogrel or replacement of clopidogrel with prasugrel or ticagrelor for the patients in clopidogrel arm, and replacement of ticagrelor or prasugrel with clopidogrel if those patients were non-carrier of mutant genes and also if they demonstrated bleeding tendencies in the ticagrelor and prasugrel arms.

Conclusion

Clopidogrel resistance was observed to be 16.5% in our study population. PRA was useful in monitoring the efficacy of thienopyridines. By having this test, one can be safely maintained on clopidogrel in non-carriers, or with increased dose of clopidogrel in intermediate metabolizers or with newer drugs such as ticagrelor or prasugrel in poor metabolizers. Patients on ticagrelor and prasugrel identified as non-carriers of gene mutations for clopidogrel metabolism could be offered clopidogrel resulting in economic benefits to the patients. Patients at high risk of bleeding were also identified by the PRA.     

Protective immunity in human Bancroftian filariasis: inverse relationship between antibodies to microfilarial sheath and circulating filarial antigens

The existence and the nature of protective immunity in human filariasis continues to be a subject of intense debate. While there is no broad consensus on functional immunity against larval and adult stage parasites, anti-microfilarial immunity has been demonstrated to be mediated by antibodies to the microfilarial sheath. In the present study, circulating filarial antigens (CFA), a marker of active filarial infection in human Bancroftian filariasis, was found to be inversely associated with antibodies to microfilarial sheath in a cohort of 411 subjects representing all categories of filariasis across the clinical spectrum of the disease. Approximately 80% of humans of all age groups (5-65 years) were found to have either CFA or anti-sheath antibodies. The inverse relationship observed between these two parameters was found to be independent of the clinical manifestation; both symptomatic and asymptomatic cases were found to display similar inverse association between CFA and anti-sheath antibodies. The prevalence of anti-sheath antibodies in the paediatric group was found to be very high as compared to adults; 78% of children below the age of 10 years tested positive for anti-sheath antibodies although the mf rate and CFA rate were only 4.5% and 22.7%, respectively, in this age group, indicating that developing larvae or juvenile adult stage parasites could have been the source of antigenic stimulus for induction of antibodies to the microfilarial sheath.     

An unusual case of a pleural effusion with an abdominal mass

A 38-year-old man presented to us with a left sided pleural effusion. Pleural fluid was aspirated and analysis revealed it to be an exudate with predominant lymphocytes and an elevated ADA level. He was discharged on antituberculous treatment. Patient returned with re-accumulation of pleural fluid. Computed tomography done in our institute picked up not only parenchymal disease in the lung which was not evident on chest radiographs but also picked up an abdominal mass in the left renal fossa. Pathological examination of excised mass revealed its tuberculous nature. The repeated recollection of pleural fluid was attributed to a "paradoxical response"; the patient was reassured and his anti-tuberculous treatment continued. Recognition of the fact that evidence of tuberculosis at distant sites may occasionally be needed to substantiate the diagnosis of tuberculous pleural effusion in a difficult and bacteriologically "negative" case prompted us to report this case.     

Heme impairs the ball-and-chain inactivation of potassium channels

Fine-tuned regulation of K⁺ channel inactivation enables excitable cells to adjust action potential firing. Fast inactivation present in some K⁺ channels is mediated by the distal N-terminal structure (ball) occluding the ion permeation pathway. Here we show that Kv1.4 K⁺ channels are potently regulated by intracellular free heme; heme binds to the N-terminal inactivation domain and thereby impairs the inactivation process, thus enhancing the K⁺ current with an apparent EC₅₀ value of ∼20 nM. Functional studies on channel mutants and structural investigations on recombinant inactivation ball domain peptides encompassing the first 61 residues of Kv1.4 revealed a heme-responsive binding motif involving Cys13:His16 and a secondary histidine at position 35. Heme binding to the N-terminal inactivation domain induces a conformational constraint that prevents it from reaching its receptor site at the vestibule of the channel pore.A-type K⁺ channels, a family of voltage-gated K⁺ (Kv) channels, play a vital role in the control of neuronal excitability, regulation of presynaptic spike duration, Ca²⁺ entry, and neurotransmitter release (1). One of the prominent features of A-type K⁺ channels is their inactivation, which is mediated by two structurally distinct processes (2, 3). The fast inactivation is initiated by the N-terminal protein structure, thereby termed N-type inactivation, whereas the slow C-type inactivation is related to the pore structure (2, 3). N-type inactivation proceeds according to a “ball-and-chain” mechanism; the positive charges of the N-terminal ball domains bring the structures to the pore domain of the channel and the distal segment of one of the four intrinsically disordered N-terminal ball domains enters the hydrophobic central cavity/vestibule of the inner pore of the channel thus obstructing the flow of K⁺ (2–5).Acute enzymatic or mutational removal of the distal N terminus eliminates N-type inactivation, and in such inactivation-removed channels, intracellular application of peptides corresponding to the N-terminal sequence restores inactivation (4, 6, 7). Structural analysis suggests that the N-terminal inactivation structure needs to be flexible or even intrinsically disordered to reach the receptor in the channel’s cavity (8, 9).“Tuning” of rapid N-type inactivation is an effective way of adapting cells to specific needs. For example, molecular processes affecting the speed and degree of N-type inactivation in Kv1.4 (KCNA4) channels include redox regulation of a cysteine residue in the N-terminal ball structure (C13) (10), protonation of histidine at position 16 (11), interaction with membrane lipids (12), and Ca²⁺-dependent phosphorylation (13). Furthermore, low-molecular-weight compounds affecting N-type inactivation (N-type disinactivators) have been discussed as potential drugs regulating cellular excitability (14).Heme [Fe(II) protoporphyrin-IX] is well known as a protein cofactor, often conferring gas sensitivity as exemplified in hemoglobin, cytochromes, myoglobin, and soluble guanylyl cyclase. In many heme proteins including soluble guanylyl cyclase, heme is bound or coordinated in part by an amino acid sequence typically containing a histidine or cysteine residue, which acts as an axial fifth ligand (in addition to the four bonds provided by the nitrogen atoms of the protoporphyrin-IX ring to the iron center) to the redox-sensitive iron center, and water or a bound gas molecule acts as the sixth ligand (15). However, recent advances revealed a novel role of heme as a nongenomic modulator of ion channel functions, first exemplified for the large-conductance voltage- and Ca²⁺-dependent K⁺ channel (Slo1 BK) (16) and later for the epithelial Na⁺ channel (17). Detailed analysis of the biophysical action of heme [ferrous iron (Fe²⁺)] or hemin [ferric iron (Fe³⁺)] on the Slo1 BK channel demonstrated that hemin is a potent modulator of the allosteric gating mechanism of the channel (18), and mutagenesis studies have indicated the sequence CKACH located in the cytoplasmic C terminus of the channel plays a critical role (16, 19). However, neither for Slo1 BK channels nor for epithelial Na⁺ channels, the interaction of heme with the ion channel protein is structurally resolved. In this study, we found that the fast N-type inactivation of Kv1.4 A-type K⁺ channels is potently modulated by heme/hemin. Furthermore, we provide structural insight into heme interaction with a channel explaining how heme prevents A-type channels from entering an inactivated state.     

Botryomycosis: A surprising revelation in the lacrimal sac

A middle aged woman presented to us with a localised well defined swelling of 3 months duration. It was located just below the lower eyelid punctum and was constantly discharging whitish granules. We suspected it to be arising from the lacrimal apparatus and posted the patient for Dacryocystectomy. On the operating table we found a swelling in the region of the lacrimal sac which was later excised. Histopathology revealed Botryomycosis and Chronic Dacryocystitis. Botryomycosis is a rare condition and requires a high index of suspicion to diagnose it. It is confirmed by histopathology and culture. Surgical debridement is the treatment of choice in such cases with an assessment of the immune status. Long term antibiotic treatment is required in all conditions as recurrence is common.     

Effectiveness and future implications of COVID-19-related risk stratification for managing retinopathy of prematurity: The Indian twin cities retinopathy of prematurity study report number 10

Purpose:To evaluate the effectiveness and future implications of COVID-related risk stratification for managing retinopathy of prematurity (ROP).Methods:A prospective study was conducted at a tertiary eye care center from the beginning of the lockdown in India from 23 March 2020 till the end of the first phase of lockdown on 29 May 2020. We evaluated 200 prematurely born infants (< 34 weeks of gestational age) using the new safety guideline protocols for low-risk babies developed in conjunction with the Indian ROP Society for care during the COVID-19 pandemic. Low risk included babies born at more than 30 weeks of gestational age, post menstrual age 34 weeks or above at presentation, more than 1000 grams of birth weight, and stable systemically with good weight gain.Results:New guidelines were implemented in 106 (53%) infants who were low risk while 94 (47%) infants with high risk were followed up as per the old guidelines. Out of the 106 infants (212 eyes) managed by the new guidelines, good outcome (group 1) was seen in 102 (96.2%) infants. Twenty-seven of the 102 infants had some form of ROP and 5 of these infants needed treatment. None of the low-risk babies with no detachment at presentation managed by new guidelines required surgery later (group 2). Two (1.9%) infants came with retinal detachment at presentation and underwent successful surgery (group 3) and two infants (1.9%) were lost to follow up.Conclusion:New risk stratification during the COVID-19 pandemic was an efficient and safe strategy in managing low-risk ROP babies.     

Fundamental understanding of the size and surface modification effects on r1, the relaxivity of Prussian blue nanocube@m-SiO2: a novel targeted chemo-photodynamic theranostic agent to treat colon cancer

A targeted multimodal strategy on a single nanoplatform is attractive in the field of nanotheranostics for the complete ablation of cancer. Herein, we have designed mesoporous silica (m-SiO₂)-coated Prussian blue nanocubes (PBNCs), functionalized with hyaluronic acid (HA) to construct a multifunctional PBNC@m-SiO₂@HA nanoplatform that exhibited good biocompatibility, excellent photodynamic activity, and in vitro T₁-weighted magnetic resonance imaging ability (r₁ ∼ 3.91 mM⁻¹ s⁻¹). After loading doxorubicin into the as-prepared PBNC@m-SiO₂@HA, the developed PBNC@m-SiO₂@HA@DOX displayed excellent pH-responsive drug release characteristics. Upon irradiation with 808 nm (1.0 W cm⁻²) laser light, PBNC@m-SiO₂@HA@DOX exhibited synergistic photodynamic and chemotherapeutic efficacy (∼78% in 20 minutes) for human colorectal carcinoma (HCT 116) cell line compared to solo photodynamic or chemotherapy. Herein, the chemo-photodynamic therapeutic process was found to follow the apoptotic pathway via ROS-mediated mitochondrion-dependent DNA damage with a very low cellular uptake of PBNC@m-SiO₂@HA@DOX for the human embryonic kidney (HEK 293) cell line, illustrating its safety. Hence, it may be stated that the developed nanoplatform can be a potential theranostic agent for future applications. Most interestingly, we have noted variation in r₁ at each step of the functionalization along with size variation that has been the first time modelled on the basis of the Solomon–Bloembergen–Morgan theory considering changes in the defect crystal structure, correlation time, water diffusion rate, etc., due to varied interactions between PBNC and water molecules.

A targeted multimodal strategy on a single nanoplatform is attractive in the field of nanotheranostics for the complete ablation of cancer.     

2′-Deoxy-5-(hydroxymethyl)cytidine: estimation in human cancer cells with a simple chemosensor

A pyrrole-based rhodamine conjugate (CS-1) has been developed and characterized for the selective detection and quantification of 2′-deoxy-5-(hydroxymethyl)cytidine (5hmC) in human cancer cells with a simple chemosensing method.

A new chemosensor, CS-1, has been developed and characterized for the selective detection and quantification of 2′-deoxy-5-(hydroxymethyl)cytidine (5hmC) in human cancer cells.

2′-Deoxy-5-(hydroxymethyl)cytidine (5hmC) is found in both neuronal cells and embryonic stem cells. It is a modified pyrimidine and used to quantify DNA hydroxymethylation levels in biological samples^1–3 as it is capable of producing interstrand cross-links in double-stranded DNA. It is produced through an enzymatic pathway carried out by the Ten-Eleven Translocation (TET1, TET2, TET3) enzymes, iron and 2-oxoglutarate dependent dioxygenase.^4–7 In the DNA demethylation process, methylcytosine is converted to cytosine and generates 5hmC as an intermediate in the first step of this process which is then further oxidized to 5-formylcytosine (fC) and 5-carboxycytosine (caC) of very low levels compared to the cytosine level.⁸ Though the biological function of 5hmC in the mammalian genome is still not revealed, the presence of a hydroxymethyl group can regulate gene expression (switch ON & OFF). Reports say that in artificial DNA 5hmC is converted to unmodified cytosine when introduced into mammalian cells.^9,10Levels of 5hmC substantially vary in different tissues and cells. It is found to be highest in the brain, particularly in nervous system and in moderate percentage in liver, colon, rectum and kidney tissues, whereas it is relatively low in lung and very low in breast and placenta.^11,12 The percentage of 5hmC content is much less in cancer and tumor tissues compared to the healthy ones. The reason behind this loss is the absence of TET1, TET2, TET3, IDH1, or IDH2 mutations in most of the human cancer cells which means decrease of methylcytosine oxidation.^13–15 This loss of 5hmC in cancer cells is being used as a diagnostic tool for the detection of early-stage of malignant disease. Few analytical methods^16–19 such as glucosyltransferase assays, tungsten-based oxidation systems, and TET-assisted bisulfite sequencing (TAB-Seq) or oxidative bisulfite sequencing (oxBS-Seq) protocols are now developed to differentiate 5hmC from other nucleotide which are naturally occurred. There are also few methods such as liquid chromatography/tandem mass spectroscopy (LC/MS-MS), which determine the level of 5hmC in mammalian cancer cell.^20–22 However, these procedures are highly toxic and expensive due to requirement of catalyzation through enzymes or heavy metal ion and these techniques require expertise, facilities, much time and costs even beyond standard DNA sequencing. As a result, these detection techniques are currently inappropriate for the high-throughput screening of genome-wide 5hmC levels (performance comparison is shown in Table S1, ESI^†).Among all reputed methods fluorescence detection method using chemosensors is significantly important due to its indispensable role in medicinal and biological applications.^23–27 Chemosensors have been effectively explored to monitor biochemical processes and assays through in situ analysis in living systems and abiotic samples with much less time and cost.In this contribution we prepared and characterize (Scheme S1 and Fig. S1–S3, ESI^†) a pyrrole–rhodamine based chemosensor (CS-1) which shows efficient and selective fluorescence signal for 5hmC in aqueous medium (Scheme 1). A transparent single crystal of CS-1 (Fig. 1) was obtained by slow evaporation of the solvent from a solution of CS-1 in CH₃CN. It crystallizes as monoclinic with space group P2₁/n (Fig. S4 and Table S2, ESI^†).Open in a separate windowScheme 15hmC-induced FRET OFF–ON mechanism of the chemosensor CS-1.Open in a separate windowFig. 1ORTEP diagram of CS-1 (ellipsoids are drawn at 40% probability level).Spectrophotometric and spectrofluorimetric titrations were carried out to understand the CS-1–5hmC interaction with 1 : 1 binding stoichiometry (Fig. S5, ESI^†) upon adding varying concentrations of 5hmC to a fixed concentration of CS-1 (1 μM) in aqueous medium at neutral pH. Upon the addition of increasing concentrations of the 5hmC, a clear absorption band (K_a = 4.47 × 10⁵ M⁻¹, Fig. S6, ESI^†) appeared to be centered at 556 nm with increasing intensity (Fig. 2a). On the other hand, for the fluorescence emission spectra of CS-1 (Fig. 2b), upon irradiation at 325 nm, an emission maxima at 390 nm was observed, which was attributed to the fluorescence emission from the donor unit i.e. the pyrrole moiety of CS-1. When 5hmC were added, due to rhodamine moiety CS-1 showed a 95-fold increase in fluorescence at 565 nm (K_a = 4.61 × 10⁵ M⁻¹, Fig. S7, ESI^†) with the detection limit of 8 nM (Fig. S8, ESI^†). The binding of 5hmC induces opening of the spirolactam ring in CS-1, inducing a shift of the emission spectrum. Subsequently, increased overlap between the emission of the energy-donor (pyrrole) and the absorption of the energy-acceptor (rhodamine) greatly enhances the intramolecular FRET process,^28,29 producing an emission from the energy acceptor unit in CS-1.Open in a separate windowFig. 2(a) UV-vis absorption spectra of CS-1 (1 μM) upon gradual addition of 5hmC up to 1.2 equiv. in H₂O–CH₃CN (15 : 1, v/v) at neutral pH. (b) Fluorescence emission spectra of CS-1 (1 μM) upon addition of 1.2 equiv. of 5hmC in H₂O–CH₃CN (15 : 1, v/v) at neutral pH (λ_ex = 325 nm).In order to establish the sensing selectivity of the chemosensor CS-1, parallel experimentations were carried out with other pyrimidine/purine derivatives such as 5-methylcytosine, cytosine, cytidine, thymine, uracil, 5-hydroxymethyluracil, adenine and guanine. Comparing with other pyrimidine/purine derivatives the abrupt fluorescence enhancement was found upon addition of 5hmC to CS-1 while others do not make any fluorescence changes under UV lamp (Fig. 3, lower panel). Furthermore, the prominent color change from colorless to deep pink allows 5hmC to be detected by naked eye (Fig. 3, upper panel). The above observation shows consistency with the fluorescence titration experiments where no such binding of CS-1 with other pyrimidine/purine derivatives was found (Fig. S9, ESI^†).Open in a separate windowFig. 3Visible color (top) and fluorescence changes (bottom) of CS-1 (1 μM) in aqueous medium upon addition of 1.2 equiv. of various pyrimidine/purine derivatives (λ_ex = 325 nm) in H₂O–CH₃CN (15 : 1, v/v) at neutral pH.pH titration reveals that CS-1 becomes fluorescent below pH 5 due to the spirolactam ring opening of rhodamine. However, it is non-fluorescent at pH range of 5–13. Upon addition of 5hmC to CS-1 shows deep red fluorescence in the pH range of 5–8 (Fig. S10, ESI^†). Considering the biological application and the practical applicability of the chemosensor pH 7.4 has been preferred to accomplish all experiments successfully.In ¹H NMR titration (Fig. S11, ESI^†), the most interesting feature is the continuous downfield shift of aromatic protons on the pyrrole moiety of CS-1 upon gradual addition of 5hmC. This may be explained as the decrease in electron density of the pyrrole moiety upon binding with 5hmC through hydrogen bonding. Xanthene protons to be shifted downfield upon spirolactam ring opening indicates the probe to coordinate with 5hmC and electrons are accumulated around 5hmC. In ¹³C NMR titration the spiro cycle carbon peak at 65 ppm was shifted to 138 ppm along with a little downfield shift of the aromatic region of CS-1 (Fig. S12, ESI^†). This coordination led to the spiro cycle opening and changes to the absorption and emission spectra, further evident by mass spectrometry (Fig. S13, ESI^†), which corroborates the stronger interaction of CS-1 with 5hmC.The experimental findings were validated by density functional theory (DFT) calculations using the 6-31G+(d,p) method basis set implemented at Gaussian 09 program. Energy optimization calculations presented the conformational changes at the spirolactam position of CS-1 while 5hmC takes part to accommodate a probe molecule. After CS-1–5hmC complexation the energy is minimized by 19.45 kcal from the chemosensor CS-1, indicating a stable complex structure (Fig. 4 and Table S3, ESI^†). This theoretical study strongly correlates the experimental findings.Open in a separate windowFig. 4Energy diagram showing the energy differences between CS-1 and CS-1–5hmC complex.The desirable features of CS-1 such as high sensitivity and high selectivity at physiological pH encouraged us to further evaluate the potential of the chemosensor for imaging 5hmC in live cells (Fig. 5). A549 cells (Human cancer cell A549, ATCC no. CCL-185) treated with CS-1 (1 μM) exhibited weak fluorescence, whereas a deep red fluorescence signal was observed in the cells stained with CS-1 (1 μM) and 5hmC (10 μM), which is in good agreement with the FRET OFF–ON profile of the chemosensor CS-1 in presence of 5hmC, thus corroborating the in-solution observation (Fig. S14, ESI^†). Cytotoxicity assay measurement shows that the chemosensor CS-1 does not have any toxicity on the tested cells and CS-1–5hmC complex does not exert any significant adverse effect on cell viability at tested concentrations (Fig. S15, ESI^†). As far as we are aware, this is the first report where we are executing the possible use of the pyrrole–rhodamine based chemosensor for selective recognition of 5hmC in living cells. These findings open an avenue for future biomedical applications of the chemosensor to recognize 5hmC.Open in a separate windowFig. 5Confocal microscopic images of A549 cells treated with CS-1 and 5hmC. (a) Cells treated with only CS-1 at 1 μM concentration. (b) Bright field image of (a). (c) Cells treated with CS-1 and 5hmC at concentration 10 μM. (d) Bright field image of (c). All images were acquired with a 60× objective lens with the applied wavelengths: For (a) and (b), E_ex = 341 nm, E_em = 414 nm, filter used: DIDS; for (c) and (d) E_ex = 550 nm, E_em = 571 nm, filter used: Rhod-2.The concentration of 5hmC was also quantified from A549 human cancer cells. Lysate of 10⁷ A549 cells was added to 1 μM of CS-1 and the fluorescence signal was recorded. Presence of 5hmC in these cancer cells was detected with the help of CS-1–5hmC standard fluorescence curve (Fig. 6) using the selective detection ability of the chemosensor CS-1.Open in a separate windowFig. 6(a) Calibration curve obtained for the estimation of 5hmC. (b) Estimation of the concentration of 5hmC (red point) from the calibration curve.From the standard curve it was found that the concentration of 5hmC in the tested sample was 0.034 μM present in 16.7 mm³ A549 cell volume (†). Assay of 5hmC was further validated from multiple samples of A549 human cancer cells using CS-1. Increasing fold of fluorescence signals was also statistically validated after calculating the Z′ value (Table S5, ESI^†). All tested samples shows the Z′ score value more than 0.9, indicating an optimized and validated assay of 5hmC.Quantification of 5hmC in human cancer cell A549