Molecular characterization and differential expression of an aromatic heptaketide producing type III plant polyketide synthase from Himalayan rhubarb

Pandith Shahzad A.,Dhar Niha,Bhosale Sumedha,Barvkar Vitthal T.,Razdan Sumeer,Shah Manzoor A.,Lattoo Surrinder K. 한국식물생명공학회 2023 Plant biotechnology reports Vol.17 No.2

Rheum australe (Himalayan Rhubarb, Polygonaceae), an endangered medicinal and vegetable herb owes its age-old remedying properties to the bio-active phyto-constituents viz. anthraquinones, stilbenoids, chromones and dietary flavonoids. Polyketide pathway primarily involving the intricate Type III polyketide synthases (PKSs) contributes to the biosynthesis of these phyto-constituents. In the present study, we perform a homology-based approach to isolate an 1176 bp full-length cds sequence of the RaALS gene showing an equitable level of sequence similarity to related Type III PKSs at both nucleic acid and amino acid levels. In silico characterization revealed the presence of highly conserved amino acid residues found in nearly all Type III PKSs including the conserved active-site residues, signature motif and cyclization pocket residues with an exception of Ile256 and Gly258. Docking studies established major interactions between the starter acetyl-CoA and RaALS. Copy number analysis suggested slender evolution in Type III PKS in R. australe having a single copy of RaALS gene. qRT-PCR analyses revealed corroboration between the higher expression of RaALS in leaves followed by stem and root with that of the metabolite concentration. Expression studies further showed a direct increase of RaALS transcripts with the growing metabolite accretion in relation to altitude suggesting a probable involvement of specific Type III PKS in biosynthesis of the major phyto-constituents. Furthermore, abiotic stressors viz. methyl jasmonate, salicylic acid and UV light enhanced RaALS transcription hinting towards its role in defense mechanism in R. australe and highlighting the significance of RaALS as a prospective target for metabolic engineering.

GSTP1 Gene Ile105Val Polymorphism Causes an Elevated Risk for Bladder Carcinogenesis in Smokers

Pandith, Arshad Ahmad,Lateef, Adil,Shahnawaz, Sheikh,Hussain, Aashaq,Malla, Tahir Mohiuddin,Azad, Niyaz,Shehjar, Fahim,Salim, Mosin,Shah, Zafar Amin Asian Pacific Journal of Cancer Prevention 2013 Asian Pacific journal of cancer prevention Vol.14 No.11

Background: The glutathione S transferase (GST) family of enzymes plays a vital role in the phase II biotransformation of environmental carcinogens, pollutants, drugs and other xenobiotics. GSTs are polymorphic and polymorphisms in GST genes have been associated with cancer susceptibility and prognosis. GSTP1 is associated with risk of various cancers including bladder cancer. A case control study was conducted to determine the genotype distribution of GSTP1 A>G SNP, to elucidate the possible role of this SNP as a risk factor in urinary bladder cancer (UBC) development and to examine its correlation with clinico-pathologic variables inUBC cases. Materials and Methods: Using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) approach, we tested the genotype distribution of 180 bladder cancer patients in comparison with 210 cancer-free controls from the same geographical region with matched frequency in age and gender. Results: We did not observe significant genotype differences between the control and bladder cancer patients overall with an odds ratio (OR)=1.23 (p>0.05). The rare allele (AG+GG) was found to be present more in cases (28.3%) than in controls (24%), though the association was not significant (p<0.05). However, a significant risk of more than 2-fold was found for the variant allele (AG+GG) with smokers in cases as compared to controls (p>0.05). Conclusions: Thus, it is evident from our study that GSTP1 SNP is not implicated overall in bladder cancer, but that the rare, valine-related allele is connected with higher susceptibility to bladder cancer in smokers and also males.

Novel fluorescent C₂-symmetric sequential <i>on-off-on</i> switch for Cu²⁺ and pyrophosphate and its application in monitoring of endogenous alkaline phosphatase activity

Pandith, Anup,Bhattarai, Kashi Raj,Guralamatta Siddappa, Ravi Kumara,Chae, Han-Jung,Seo, Young Jun Elsevier 2019 Sensors and actuators. B Chemical Vol.282 No.-

<P><B>Abstract</B></P> <P>A doubly armed hydrazone-based <B>FLRHYDDFP</B> probe selectively detects Cu<SUP>2+</SUP> and pyrophosphate (PPi) ions through an colorimetric response-<I>“colorless → yellow → colorless</I>”- as well as “<I>on-off-on</I>” photonic switching response under physiological conditions in a sequential manner. The binding stoichiometries of the analytes Cu<SUP>2+</SUP> and PPi were 1:2 and 2:4 for <B>FLRHYDDFP</B>-Cu<SUP>2+</SUP> and Cu<SUP>2+</SUP>/PPi, respectively. The sequential sensing ability of <B>FLRHYDDFP</B> toward Cu<SUP>2+</SUP> and PPi, attributed to effective complexation-aided d→π* electron transfer (ET) from Cu<SUP>2+</SUP> to <B>FLRHYDDFP</B> and intramolecular charge/electron transfer from <B>FLRHYDDFP</B> to <B>FLRHYDDFP<SUP>+</SUP> </B>, resulted in the formation of a non-symmetric Cu<SUP>2+</SUP> chelate that provided a yellow-colored solution with a significant bathochromic shift from 376 to 446 nm in the UV–vis spectrum and quenching in the emission spectrum. Upon addition of PPi, Cu<SUP>2+</SUP> was extruded from the complex, resulting in a revival of the fluorescence centered at 572 nm. Thus, sequential addition of Cu<SUP>2+</SUP> and PPi yielded a colorless–yellow–colorless transition under visible light and <I>on-off-on</I> switching under 365-nm light (fluorescence). The lowest detection limits for Cu<SUP>2+</SUP> and PPi, when using colorimetric and fluorimetric methods, were in the sub-micromolar and nanomolar levels, respectively. By exploiting this PPi sensing strategy, invitro as well as endogenous alkaline phosphatase activity could be monitored effectively, as demonstrated by exploiting the intracellular production or residual PPi in human salivary glands (normal) and cancer cell lines.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Novel and highly sensitive colorimetric and fluorimetric probe <B>FLRHYDDFP</B> designed and synthesized to identify the Cu<SUP>2+</SUP> and PPi ions in invitro studies with revsersibility. </LI> <LI> By the aid of PPi sensing strategy, alkaline phosphatase activities (ALP) were monitored with lowest detection limit of 0.012 U/mL. </LI> <LI> For the first time endogenous PPi productions aided ALP recognition demonstrated using fluorescence confocal image guided technique in the presence of ALP inhibitor. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

Highly selective imidazole-appended 9,10-<i>N,N′</i>-diaminomethylanthracene fluorescent probe for switch-on Zn²⁺ detection and switch-off H₂PO₄ ⁻ and CN⁻ detection in 80% aqueous DMSO

Pandith, Anup,Uddin, Nizam,Choi, Cheol Ho,Kim, Hong-Seok Elsevier 2017 Sensors and actuators. B Chemical Vol.247 No.-

<P><B>Abstract</B></P> <P>A new imidazole-appended anthracene-based photoinduced electron transfer (PET) probe (<B>P</B>) was designed and synthesized. Probe <B>P</B> detected Zn<SUP>2+</SUP> selectively in an 80% aqueous solution of dimethyl sulfoxide (DMSO) through a switch-on response at pH 7.5 without any interference from other metal ions. Probe <B>P</B> exhibited a moderately strong association constant (<I>K<SUB>a</SUB> </I>) of 1.41×10<SUP>5</SUP> M<SUP>−1</SUP> and associated with Zn<SUP>2+</SUP> ions with 1:1 stoichiometry. The in situ-generated <B>P</B>•Zn<SUP>2+</SUP> (1:1) ensemble detected H<SUB>2</SUB>PO<SUB>4</SUB> <SUP>−</SUP> and CN<SUP>−</SUP> ions sequentially through switch-off responses. The selective discrimination of H<SUB>2</SUB>PO<SUB>4</SUB> <SUP>−</SUP> and CN<SUP>−</SUP> ions occurred both in the absence and presence of Ca<SUP>2+</SUP> ions at pH 7.5 in a <I>N</I>-2-hydroxyethylpiperazine-<I>N</I>-2-ethanesulfonic acid (HEPES) buffer. The switch-on response was driven by the selective complexation of <B>P</B> with Zn<SUP>2+</SUP> ions, mediated by the combined effects of PET and chelation-induced enhanced fluorescence, resulting in a more structurally rigid complex. Applications of <B>P</B> to real water sample analysis and sequential logic gate operations were demonstrated. The lowest detection limits for Zn<SUP>2+</SUP>, H<SUB>2</SUB>PO<SUB>4</SUB> <SUP>−</SUP>, and CN<SUP>−</SUP> ions in 80% aqueous DMSO were determined to be 1.0×10<SUP>−9</SUP>, 1.0×10<SUP>−9</SUP>, and 8.0×10<SUP>−9</SUP> M, respectively.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Imidazole-appended anthracene probe P for sensing Zn<SUP>2+</SUP> was synthesized. </LI> <LI> Zn<SUP>2+</SUP> ions “<I>switched on</I>” the emission of P in 80% aqueous DMSO. </LI> <LI> In situ generated P-Zn<SUP>2+</SUP> (1:1) ensemble detected H<SUB>2</SUB>PO<SUB>4</SUB> <SUP>−</SUP> and CN<SUP>−</SUP> ions sequentially through “<I>switch off</I>” response in the absence and presence of Ca<SUP>2+</SUP> ions respectively. </LI> <LI> Sensing ability of P towards Zn<SUP>2+</SUP> is attributed to PET and CHEF upon complexation. </LI> <LI> Application of P to real water samples and sequential logic gate operations were demonstrated. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

A new fluorogenic sensing platform for salicylic acid derivatives based on π-π and NH-π interactions between electron-deficient and electron-rich aromatics

Pandith, Anup,Hazra, Giridhari,Kim, Hong-Seok Elsevier 2017 Spectrochimica acta. Part A, Molecular and biomole Vol.178 No.-

<P><B>Abstract</B></P> <P>A novel simple fluorescent probe was designed for the recognition of electron-rich salicylic acid derivatives (SAs). The imidazole-appended aminomethyl perylene probe <B>1</B> selectively differentiated between electron-rich amino-SAs and electron-deficient nitro-SAs in EtOH, exhibiting the highest selectivity and sensitivity toward 5-aminosalicylic acid (5-ASA) and showing strong 1:1 binding (<I>K</I> <SUB>a</SUB> =1.37×10<SUP>7</SUP> M<SUP>−1</SUP>). This high selectivity and sensitivity resulted from the synergistic multiple hydrogen bonding interactions of secondary amine and imidazole units and π-π interactions between electron-rich and electron-deficient rings, along with the unusual NH-π interactions between 5-ASA and the perylene moiety of <B>1</B>. The limit of detection (LOD) for 5-ASA in EtOH was 0.012ppb.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Imidazole-appended aminomethyl perylene probe <B>1</B> for sensing 5-aminosalycylic acid (5-ASA) was synthesized. </LI> <LI> 5-ASA “<I>switched on</I>” the emission of probe <B>1</B>. </LI> <LI> Fluorescence mechanism explained by multiple hydrogen bonding, π-π, and unusual NH-π interactions between 5-ASA and <B>1</B>. </LI> <LI> The limit of detection for 5-ASA in EtOH was 0.012ppb. </LI> </UL> </P> <P><B>Graphical Abstract</B></P> <P>[DISPLAY OMISSION]</P>

Daphnetin: A novel blue-green photonic switch for disodium phosphates that allows monitoring of polymerase chain reactions

Pandith, Anup,Koo, Jachoon,Seo, Young Jun Elsevier 2018 Spectrochimica acta. Part A, Molecular and biomole Vol.204 No.-

<P><B>Abstract</B></P> <P>This paper describes the very simple and robust ratiometric photonic switching properties of daphnetin (<B>DP</B>) toward HPO<SUB>4</SUB> <SUP>2−</SUP> ions selectively in complex biological fluids, without any interference from other relevant anions under physiological conditions. The sensing ability of <B>DP</B> toward HPO<SUB>4</SUB> <SUP>2−</SUP> ions was first demonstrated using UV–Vis and fluorescence spectroscopy, dynamic light scattering (DLS), and one- and two-dimensional NMR spectroscopy. <B>DP</B> can detect HPO<SUB>4</SUB> <SUP>2−</SUP> ions at concentrations up to the sub-micromolar/nanomolar level very effectively, with a ratiometric response resulting from intramolecular charge transfer aided by aggregated-induced emission. The interactions between <B>DP</B> and HPO<SUB>4</SUB> <SUP>2−</SUP> ions resulted in new bands appearing in the UV–Vis (at 385 nm) and emission (at 535 nm) spectra. The noncovalently held HPO<SUB>4</SUB> <SUP>2−</SUP> ions induced pronounced specific aggregation of <B>DP</B> molecules, resulting in the new excimer band at 535 nm while retaining the monomer band centered at 445 nm. In contrast, reciprocal absorptivity changes were observed at 320 and 385 nm, with exponential decrements and increments, respectively. This probe could effectively monitor the consumption of dNTPs during various cycles of the polymerase chain reaction performed with relatively short oligonucleotides as well as genomic DNA from <I>Agrobacterium tumefaciens</I> (AcH5α strain).</P> <P><B>Highlights</B></P> <P> <UL> <LI> A novel, simple and robust blue-green fluorescent probe <B>DP</B>, used to detect the HPO<SUB>4</SUB> <SUP>2−</SUP> ions using colorimetric and fluorimetric methods. </LI> <LI> <B>DP</B> able to sense the HPO<SUB>4</SUB> <SUP>2−</SUP> ions very selectively, through the ratiometric response, without any interferences from other biologically relevant anions at physiological conditions. </LI> <LI> For the first time HPO<SUB>4</SUB> <SUP>2−</SUP> ions sensing aided application were effectively demonstrated in polymerase chain reactions (PCR) monitoring process. </LI> </UL> </P> <P><B>Graphical Abstract</B></P> <P>[DISPLAY OMISSION]</P>

A simple and robust PET-based anthracene-appended O-<i>N</i>-O chelate for sequential recognition of Fe³⁺/CN^– ions in aqueous media and its multimodal applications

Pandith, Anup,Choi, Jun-Hyeak,Jung, Ok-Sang,Kim, Hong-Seok Elsevier Sequoia [etc.] 2018 Inorganica chimica acta Vol.482 No.-

<P><B>Abstract</B></P> <P>A very simple, robust, and pico/nanomolar-sensitive 9,10-diethanolamine-substituted fluorescent Fe<SUP>3+</SUP>/CN<SUP>–</SUP> probe (<B>PD</B>) was synthesised, and its sensing abilities towards various ions were studied in mixed aqueous media. <B>PD</B> selectively recognised Fe<SUP>3+</SUP> ions through a ‘<I>turn on</I>’ response with an excellent binding constant (<I>K</I> <SUB>a</SUB>, 9.29 × 10<SUP>6</SUP> M<SUP>−1</SUP>) in 1:2 binding stoichiometry at pH 7.0 in phosphate-buffered saline (PBS). The <I>in situ</I> generated Fe<SUP>3+</SUP>·<B>PD</B> ensemble sequentially recognised CN<SUP>–</SUP> ions with an excellent binding constant (<I>K</I> <SUB>a</SUB> 1.72 × 10<SUP>8</SUP> M<SUP>−1</SUP>) via a ‘<I>turn off</I>’ mode by extruding Fe<SUP>3+</SUP> ions from the ensemble. The highly selective sequential ‘<I>on-off</I>’ responses towards Fe<SUP>3+</SUP> and CN<SUP>–</SUP> ions were attributed to inhibition and restoration of photoinduced electron transfer (PET) and chelation-induced enhanced fluorescence (CHEF) effects from the chelating N and O heteroatoms. <B>PD</B> was able to detect Fe<SUP>3+</SUP> and CN<SUP>–</SUP> ions in real water samples satisfactorily at picomolar to sub-nanomolar levels. A colorimetric assay based on pyrocatechol violet (<B>PCV</B>) was also able to detect Fe<SUP>3+</SUP>/CN<SUP>–</SUP> in a sequential manner (up to sub-micromolar level) by a change in colour from colourless to yellow/pale green without any interferences from other ions. Based on the complexation and decomplexation mechanism, bio-imaging photonic INHIBIT logic circuit strips were prepared for use under physiological conditions. In addition, solid-phase recognition of Fe<SUP>3+</SUP>/CN<SUP>–</SUP> ions was demonstrated using cost-effective paper-based strips.</P> <P><B>Highlights</B></P> <P> <UL> <LI> 9,10-Diethanolamine-appended anthracene probe PD was synthesized and sensing abilities were studied in aqueous medium. </LI> <LI> Probe PD detected Fe<SUP>3+</SUP> and CN<SUP>–</SUP> sequentially through ‘<I>switch-on</I>’ and ‘<I>switch-off</I>’ responses. </LI> <LI> Pyrocatechol violet based colorimetric assay also is able to detect Fe<SUP>3+</SUP>/CN<SUP>–</SUP> in sequential manner by changing the color from colorless to yellow/pale green. </LI> <LI> Bio-imaging, photonic INHIBIT logic circuit strips were demonstrated at physiological conditions based on complexations and decomplexation mechanism. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

Oncogenic Activation of Fibroblast Growth Factor Receptor-3 and RAS Genes as Non-Overlapping Mutual Exclusive Events in Urinary Bladder Cancer

Pandith, Arshad A,Hussain, Aashaq,Khan, Mosin S,Shah, Zafar A,Wani, M Saleem,Siddiqi, Mushtaq A Asian Pacific Journal of Cancer Prevention 2016 Asian Pacific journal of cancer prevention Vol.17 No.6

Background: Urinary bladder cancer is a common malignancy in the West and ranks as the $7^{th}$ most common cancer in our region of Kashmir, India. FGFR3 mutations are frequent in superficial urothelial carcinoma (UC) differing from the RAS gene mutational pattern. The aim of this study was to analyze the frequency and association of FGFR3 and RAS gene mutations in UC cases. Materials and Methods: Paired tumor and adjacent normal tissue specimens of 65 consecutive UC patients were examined. DNA preparations were evaluated for the occurrence of FGFR3 and RAS gene mutations by PCR-SCCP and DNA sequencing. Results: Somatic point mutations of FGFR3 were identified in 32.3% (21 of 65). The pattern and distribution were significantly associated with low grade/stage (p<0.05). The overall mutations in exon 1 and 2 in all the forms of RAS genes aggregated to 21.5% and showed no association with any clinic-pathological parameters. In total, 53.8% (35 of 65) of the tumors studied had mutations in either a RAS or FGFR3 gene, but these were totally mutually exclusive in and none of the samples showed both the mutational events in mutually exclusive RAS and FGFR3. Conclusions: We conclude that RAS and FGFR3 mutations in UC are mutually exclusive and non-overlapping events which reflect activation of oncogenic pathways through different elements.

Selective Detection of 2,4,6-Trinitrophenol Based on In Situ-generated Fluorescent Zn2+–Anthracene Ensembles in 80% Aqueous Dimethyl Sulfoxide

Anup Pandith,김홍석 대한화학회 2018 Bulletin of the Korean Chemical Society Vol.39 No.1

Novel in situ-generated highly fluorescent chelated Zn2+–anthracene (Zn2+•P: 1:1) complex detected 2,4,6-trinitrophenol (TNP) more selectively by a “switch-off” response with a very high Stern–Volmer quenching constant (Ksv = 3.75 × 106 M−1) at a 1:2 stoichiometry in 80% aqueous DMSO. The selective quenching response that occurred with TNP is due to the effective destruction of the chelated complex by efficient protonation at secondary amines, which created an effective resonance energy transfer (RET) from the anthracene unit to the TNP. Formation of a stable P2+•(TNP)−2 ionic charge-transfer complex showed that the anthracene monomer band quenching resulted from efficient RET, accompanied by synergistic multiple hydrogen bonding, electron-rich–electron-deficient πAn–πTNP interactions, and columbic interactions. An application was demonstrated using the biocompatible ensemble-coated hydrogel for ultraviolet-assisted naked eye sensing, and its sensing mechanism was postulated. The lowest detection limit for TNP was 3.01 × 10−10 M in 80% aqueous DMSO.

Pyrene-appended imidazolium probe for 2,4,6-trinitrophenol in water

Kumar, A.,Pandith, A.,Kim, H.S. Elsevier Sequoia 2016 Sensors and actuators. B, Chemical Vol.231 No.-

Pyrene-appended imidazolium salts (1-3) were synthesized for sensing 2,4,6-trinitrophenol (TNP); the salts selectively and sensitively detected TNP in a pH 7.4 PBS-DMSO (99.5:0.5v/v) solution. The binding of TNP to probe 3 completely quenched its green excimer emission because of the loss of π-π interactions between the pyrene rings and energy transfer from electron-rich pyrene to electron-deficient TNP. Probe 3 showed the highest association constant with TNP (K<SUB>a</SUB>=8.34x10<SUP>6</SUP>M<SUP>-1</SUP>) in a 1:2 stoichiometry; the limit of detection (LOD) of TNP in a pH 7.4 PBS-DMSO (99.5:0.5v/v) solution was 10nM. Probe 3 also detected TNP in tap water and river water with an LOD of 10nM and a good association constant; the calculated LOD was 0.067 ppb. Filter paper strips coated with 3 detected TNP in an aqueous solution. The interactions of TNP with probes 1-3 were investigated by UV-vis, fluorescence, and <SUP>1</SUP>H NMR spectroscopies as well as density functional theory calculations.