Metal oxide QD based ultrasensitive microsphere fluorescent sensor for copper,chromium and iron ions in water

Herein we developed a rapid, cheap, and water-soluble ultra-sensitive ZnO quantum dot (QD) based metal sensor for detecting different hazardous metal ions up to the picomolar range in water. Various spectroscopic and microscopic techniques confirmed the formation of 2.15 ± 0.46 μm of ZnO QD conjugated CMC microspheres (ZCM microspheres) which contain 5.5 ± 0.5 nm fluorescent zinc oxide (ZnO) QDs. Our system, as a promising sensor, exhibited excellent photostability and affinity towards various heavy metal ions. The detection limits were calculated to be 16 pM for Cu²⁺ and 0.18 nM for Cr⁶⁺ ions which are better than previously reported values. The simple fluorescence ‘turn off’ property of our ZCM microsphere sensor system can serve a two-in-one purpose by not only detecting the heavy metals but also quantifying them. Nonetheless, pattern recognition for different heavy metals helped us to detect and identify multiple heavy metal ions. Finally, their practical applications on real samples also demonstrated that the ZCM sensor can be effectively utilized for detection of Cr⁶⁺, Fe³⁺, Cu²⁺ present in the real water samples. This study may inspire future research and design of target fluorescent metal oxide QDs with specific functions.

Herein we developed a rapid, cheap, and water-soluble ultra-sensitive ZnO quantum dot (QD) based metal sensor for detecting different hazardous metal ions up to the picomolar range in water.     

Human APOBEC3G-mediated hypermutation is associated with antiretroviral therapy failure in HIV-1 subtype C-infected individuals

Introduction

Human APOBEC3G/F (hA3G/F) restricts retroviral replication through G-to-A hypermutations, which can generate drug-resistant progenies in vitro. The clinical relevance is still inconclusive. To bridge this gap, we aim to study the role of these hypermutations in evolution of drug resistance; we characterised hA3G/F-mediated hypermutations in the RT region of the pol gene of patients with or without antiretroviral therapy (ART).

Methods

In 88 HIV-1-positive individuals, drug resistance genotyping was carried out in plasma virus and provirus by population sequencing. Hypermutations were determined by three different approaches using Hypermut 2.0 software, cluster analysis and APOBEC3G-mediated defectives indices. Clinical and demographic characteristics of these individuals were studied in relation to these hypermutations.

Results

hA3G/F-mediated hypermutated sequences in proviral DNA, but not in plasma virus, were identified in 11.4% (10/88) subjects. Proviral hypermutations were observed more frequently in patients with ART failure than in ART-naïve individuals (p=0.03). In therapy failure patients, proviral hypermutation were associated with greater intra-compartmental genetic diversity (p<0.001). In therapy-naïve individuals, hypermutated proviral DNA with M184I and M230I mutations due to the editing of hA3G, had stop codons in the open reading frames and the same mutations were absent in the plasma virus. Only a limited concordance was found between the drug resistance mutations in plasma RNA and proviral DNA.

Conclusions

hA3G lethal hypermutation was significantly associated with ART failure in Indian HIV-1 subtype C patients. It is unlikely that viral variants, which exhibit hypermutated sequences and M184I and/or M230I, will mature and expand in vivo.     

Traumatic abdominal wall hernia: Delayed repair: Advantageous or taxing

INTRODUCTIONTraumatic abdominal wall hernia (TAWH) is a rare entity. Most cases occur in children, following an injury from the bicycle handle bar. In adults, it usually results from road traffic accidents (RTA). We present one of the largest reported cases of TAWH following RTA managed by delayed mesh repair.PRESENTATION OF CASEA 35 yr old obese male with RTA was diagnosed with TAWH with 19 cm × 15 cm defect in left flank. As there were no intra abdominal injuries and overlying skin was abraded, he was planned for elective repair after 6 months. On exploration a defect of 30 cm × 45 cm was found extending from midline anteriorly to 8 cm short of midline posteriorly in transverse axis and costal margin to iliac crest in craniocaudal axis. After restoration of bowel into abdominal cavity, primary closure or even approximation of muscular defect was not possible thus a mesh closure using 60 cm × 60 cm prolene mesh in subcutaneous plane was done. After 4 months follow up, patient is healthy and has no recurrence.DISCUSSIONEmergent surgical management of TAWH is usually favoured due to high incidence of associated intra abdominal injuries. Delayed repair may be undertaken in selected cases.CONCLUSIONTAWH, although rare, should be suspected in cases of RTA with abdominal wall swellings. With time, the hernia defect may enlarge and muscles may undergo atrophy making delayed repair difficult.     

Synthesis and antimicrobial screening of some novel chalcones and flavanones substituted with higher alkyl chains

As a part of our program to generate some novel flavonoid frameworks substituted with higher alkyl groups as possible antimicrobial agents, we have in total synthesized twelve novel chalcones (11–16) and their corresponding flavanones (17–22) substituted with either nonyl or dodecyl chains in ring B in very good to excellent yields. The synthesized compounds have been screened for their antimicrobial potential against six bacterial and four fungal strains. The tested compounds, in general, showed significant antibacterial and comparable antifungal activities. While the chalcone (16) with a dodecyl chain showed highly promising antibacterial activity against almost all the organisms tested, the chalcone (13) with nonyl chain showed promising antifungal activity against Candida rugosa and Aspergillus niger strains.     

Chemopreventive potential of Apium leptophyllum (Pers.) against DMBA induced skin carcinogenesis model by modulatory influence on biochemical and antioxidant biomarkers in Swiss mice

Aim:

The study was designed to investigate the chemopreventive potential of flavonoidal fractions of Apium leptophyllum fruits (FFALF) on Swiss mice.

Materials and Methods:

Skin tumor or papilloma was developed by topical application of DMBA (25 μg in 0.1 ml acetone) on intrascapular region of mice, twice weekly for 8 weeks. The animals were divided into six groups: Group I (vehicle control); group II (FFALF control, 5 mg/kg); group III (carcinogenic control, DMBA treated initially for 8 weeks); and group IV, V and VI as pre-treated group (FFALF 5, 10 and 20 mg/kg respectively for 16 weeks along with DMBA treatment). After the 16^th week of treatment; the tumor morphology, skin histopathology, and biochemical and antioxidant biomarkers were measured and compared with carcinogenic control as well as vehicle control.

Results:

The co-administration of FFALF with DMBA-treated groups showed significant (P ≤ 0.001) prevention against skin papilloma and normalized the status of lipid peroxidation with antioxidant biomarkers in a dose-dependent manner as compared to carcinogenic control.

Conclusions:

Thus, the present study suggests that the FFALF is non-carcinogenic and has chemopreventive potential on DMBA-induced carcinogenesis in mouse, which may be due to the modulation of cutaneous lipid peroxidation or enhancement of total antioxidant capacity.KEY WORDS: Apium leptophyllum, antioxidant biomarker, chemoprevention, DMBA, skin papilloma     

Microwave irradiation: a green approach for the synthesis of functionalized N-methyl-1,4-dihydropyridines

An eco-friendly and cost-effective, microwave-assisted green approach has been developed for the synthesis of diverse functionalized N-methyl-1,4-dihydropyridines (1,4-DHPs). This pseudo three-component reaction was carried out between two equivalents of (E)-N-methyl-1-(methylthio)-2-nitroethenamine (NMSM) and one equivalent of aromatic aldehydes under microwave irradiation at 100 °C without catalyst and solvent. Short reaction times, avoidance of toxic solvents or expensive, metallic and corrosive catalysts and no need for column chromatographic purification are among the valuable features of the presented method. Moreover, the “greenness” of the method was evaluated within the ambits of the defined green metrics such as atom economy, carbon efficiency, E-factor, reaction mass efficiency, overall efficiency, process mass intensity and solvent intensity and the method exhibited a good to excellent score.

Microwave-assisted green synthesis of N-methyl-1,4-dihydropyridines under eco-friendly conditions.     

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