First report of Athelia rolfsii (=Sclerotium rolfsii) associated with foot rot disease of Chrysanthemum morifolium in India.

Chrysanthemum morifolium L. is an important flower crop grown in different parts of Karnataka for its striking cut flowers and international market value. During a field survey (Mysore district, Karnataka, February, 2022), chrysanthemum fields were found infected with foot rot disease. The presence of white mycelial structures with sclerotia were recorded near the stem-soil interface. The disease incidence ranged 10-12% measured in an area of approximately 10 hectares. The infected plants showed quick wilt, yellowing and toppling of the entire plant. Infected plants from Doddamaragowdanahally and Rayanahally (n=15) were collected and associated fungal pathogen isolated after surface sterilization with NaOCl (1%) on potato dextrose agar (PDA) amended with chloramphenicol (50 mg/L). Fungal mycelia developed from the infected tissues were inoculated on to fresh PDA plates to obtained pure cultures for further identification. Fungal colonies with dense, aerial whitish-cottony mycelia with uniformly globoid sclerotia (0.284.2 mm) were observed after 15 days of incubation (28 ± 2°C). Sclerotia were white in the beginning and turned brown at maturity. The average number of sclerotia produced per plate ranged from 240 to >480 (n = 10). To further to confirm the identity of the isolates, two representative isolates (CmSr1 and CmSr2) was subjected to molecular identification based on ITS-rDNA sequences. Briefly, genomic DNA was isolated from 12 day old cultures using the CTAB method and ITS-rDNA was amplified using ITS1-ITS4 primers (White et al., 1990). An expected amplicon of >650 bp (ITS) was obtained and later sequenced from both the directions. The consensus sequences were analysed through nBLAST search which revealed that 100% sequence similarity with reference sequences of Athelia rolfsii (S. rolfsii) from GenBank database (MT127465, MN974137, KC292637; identity 656/656; 0 gaps). A phylogenetic tree obtained by the neighbor-joining method using MEGAX shared a common clade with the reference sequences retrieved and computed, thus confirming the identification based on sequence analysis and molecular phylogeny. The representative sequence of A. rolfsii isolates CmSr1 and CmSr2 isolates deposited in GenBank with Accession nos. ON456153 and ON456154, respectively. Based on etiology, morphological, cultural and molecular data the pathogen was identified as Athelia rolfsii (Curzi) Tu & Kimbrough (Syn: Sclerotium rolfsii Sacc.) (Mordue, 1974; Mahadevakumar et al., 2016, 2018). Plants (n=60) were inoculated with sclerotial bodies (2 sclerotia/plant) near stem soil interface under green house and covered with polythene bags (at 27 ± 2°C and 80% RH). Non-inoculated plants (n=20) served as controls. The development of foot rot disease was observed eight days after inoculation. A total of 48 plants showed the foot rot symptoms and 12 inoculated plants and control plants remained healthy. The identity of the fungus was confirmed by morphological and cultural characters after re-isolation. C. morifolium is an important flower crop in Karnataka. S. rolfsii is known to be associated with blight and collar rot of Chrysanthemum spp. from Kerala (Beena et al., 2002) but no species (host) identity provided. Therefore, to the best of our knowledge, this is the first report of foot rot disease caused by Athelia rolfsii on C. morifolium in India. Early diagnosis of this disease will help the farmers to adopt suitable management practices to avoid loss.

A hybrid LIBS-Raman system combined with chemometrics: an efficient tool for plastic identification and sorting.

Classification of plastics is of great importance in the recycling industry as the littering of plastic wastes increases day by day as a result of its extensive use. In this paper, we demonstrate the efficacy of a combined laser-induced breakdown spectroscopy (LIBS)-Raman system for the rapid identification and classification of post-consumer plastics. The atomic information and molecular information of polyethylene terephthalate, polyethylene, polypropylene, and polystyrene were studied using plasma emission spectra and scattered signal obtained in the LIBS and Raman technique, respectively. The collected spectral features of the samples were analyzed using statistical tools (principal component analysis, Mahalanobis distance) to categorize the plastics. The analyses of the data clearly show that elemental information and molecular information obtained from these techniques are efficient for classification of plastics. In addition, the molecular information collected via Raman spectroscopy exhibits clearly distinct features for the transparent plastics (100% discrimination), whereas the LIBS technique shows better spectral feature differences for the colored samples. The study shows that the information obtained from these complementary techniques allows the complete classification of the plastic samples, irrespective of the color or additives. This work further throws some light on the fact that the potential limitations of any of these techniques for sample identification can be overcome by the complementarity of these two techniques. Graphical Abstract ᅟ.

Effect of Telmisartan on Arsenic-Induced (Sub-chronic) Perturbations in Redox Homeostasis, Pro-inflammatory Cascade and Aortic Dysfunction in Wistar Rats.

An experimental study was conducted in male Wistar rats to explore the antioxidant potential of telmisartan (an AT1 receptor blocker) to overcome arsenic ('As')-induced perturbations in redox homeostasis pro-inflammatory cytokines, prostaglandin-E2 levels and aortic dysfunction in Wistar rats. Wistar rats were randomly divided into four groups of six each. Group-I served as untreated control, while group-II received sodium (meta) arsenite (NaAsO2) (10 mg/kg b.wt. p.o) for a period of 60 days. Experimental rats in group-III received treatment similar to group-II, but in addition received telmisartan (with 1% aqueous solution of Tween 80) @ 10 mg/kg b.wt. (p.o) for a similar duration, while rats in group-IV received telmisartan alone. Arsenic exposure resulted in significant (p < 0.05) elevation in the levels of superoxide anion ([Formula: see text]) radicals (control: 768.20 ± 126.77 vs group-II: 1232.75 ± 97.85 pmol of NBT reduced/min/mg protein). Telmisartan administration showed significant (p < 0.05) reduction in [Formula: see text] generation (815.34 ± 43.41 pmol of NBT reduced/min/mg protein). Sub-chronic exposure to 'As' significantly (p < 0.05) decreased the activities of SOD, CAT, GPx and GR activity and GSH levels in the aorta, thus induced lipid peroxidation (LPO) measured as measured in terms of thiobarbituric acid reactive substances (TBARS) called as malondialdehyde (MDA). However, the administration of telmisartan effectively countered the LPO (24.03 ± 1.18 nmol of MDA/g) on account of restoring the levels of aforesaid antioxidant defense system. Telmisartan administration effectively attenuated the 'As'-induced surge in pro-inflammatory cytokines (viz., IL-1ß, IL-6 and TNF-α) levels, as well as countered the activity of cyclooxygenase (COX2) as indicated by a significant (p < 0.05) decrease in PGE2 level in the aorta. In addition to it, there was a significant (p < 0.05) decrease in plasma angiotensin II (Ang-II) levels in experimental rats receiving telmisartan. Quantitative RT-PCR studies revealed that sub-chronic exposure to 'As' upregulated the Nox2 mRNA expression, but there was a 1.2-fold reduction in expression level upon co-administration of telmisartan. Histopathological examination revealed marked recovery from 'As'-induced disruption of tunica adventitia and loss of connective tissue in experimental rats receiving telmisartan. The study concludes that telmisartan can overcome aortic dysfunction induced by sub-chronic exposure to arsenic through drinking water in experimental rats through restoration of redox balance, attenuation of pro-inflammatory cytokines and mediators and downregulation of Nox2 mRNA expression.

Echelle LIBS-Raman system: A versatile tool for mineralogical and archaeological applications.

There are a number of analytical techniques used to study material related problems. Most of them imply experimental determination of parameters and functions by means of which elemental, optical and other properties of materials can be described. Laser Induced Breakdown Spectroscopy (LIBS) and Raman spectroscopy are highly complementary spectroscopic techniques used for lab, in-situ, and remote analyses of materials. The LIBS-Raman system provides further information compared to other conventional techniques since it can detect the presence of low atomic number-elements, isotopic composition, hydration and structure of trace materials which may be present as surface layers etc., which are very difficult, if not impossible, by portable X-ray based systems. A setup for LIBS and Raman spectroscopy measurements in a single unit has been developed and reported recently by us using an echelle spectrograph system. The system utilizes a single nanosecond pulsed Nd:YAG laser (532 nm) and an ICCD coupled echelle spectrograph for both measurements. The unit has been successfully used for multipurpose applications such as identification of minerals, pigments etc and also for checking quality assurance. The combined atomic and molecular information from the same location on a sample, at several locations, can provide more comprehensive information regarding its properties than using either of these quantities taken singly. In many cases, it has been observed that the high resolution of the echelle spectrograph provides better quality Raman signals by virtue of the small degeneracy/crystal field splitting of many fundamental Raman bands. This can provide valuable information on inclusions in bulk samples, changes due to symmetry alteration or complex formation with surroundings, hydrogen bonding etc. The advantage of echelle system to identify natural and artificial pigments by identifying the minor and trace components by the spectra of the elements therein, is also helpful in many situations. In the present paper, the orthogonal use of LIBS and Raman spectroscopy is assessed and highlighted. The results clearly demonstrate the potential of echelle-based LIBS-Raman system in applications where more detailed information on complex samples like minerals, archaeological artifacts etc, is required with minimum sample damage or consumption.

Anticancer efficacy of 6-thioguanine loaded chitosan nanoparticles with or without curcumin.

6-Thioguanine encapsulated chitosan nanoparticles (6-TG-CNPs) has formulated by the ionic-gelation method. Morphologically, the 6-TG-CNPs were spherical and showed mean size, PDI, zeta potential, and entrapment efficiency of 261.63 ± 6.01 nm, 0.34 ± 0.10, +15.97 ± 0.46 mV and 44.27%, respectively. The IR spectra confirmed the 6-TG complex with chitosan. The in vitro drug release profile of 6-TG-CNPs revealed an increase in sustained-release (91.40 ± 1.08% at 48 h) at pH 4.8 compared to less sustained-release (73.96 ± 1.12% at 48 h) at pH 7.4. The MTT assay was conducted on MCF-7 and PA-1 cell lines at 48 h incubation to determine % cell viability. The IC50 values of 6-TG, 6-TG-CNPs, and curcumin for MCF-7 were 23.09, 17.82, and 15.73 µM, respectively. Likewise, IC50 values of 6-TG, 6-TG-CNPs, and curcumin for PA-1 were 5.81, 3.92, and 12.89 µM, respectively. A combination of 6-TG-CNPs (IC25) with curcumin (IC25) on PA-1 and MCF-7 showed % cell viability of 43.67 ± 0.02 and 49.77 ± 0.05, respectively. The in vitro cytotoxicity potential in terms of % cell viability, early apoptosis, G2/M phase arrest, and DNA demethylating activity of 6-TG-CNPs alone and combination with curcumin proved to be more effective than that of 6-TG on PA-1 cells.

A lentiviral vector with novel multiple cloning sites: stable transgene expression in vitro and in vivo.

Gene delivery has become an important tool for biological research and gene therapy trials. Lentiviral vector (LV) mediated gene transfer is a preferred approach for stable, sustained transgenic expression. We report here step-wise development of an Indian human immunodeficiency virus type 2 (HIV-2) isolate derived third generation lentiviral vector with a novel, versatile multiple cloning site (MCS) that can also facilitate single step sub-cloning of a PCR amplified transgene cassette by T/A cloning strategy apart from useful cohesive/blunt end cloning. Efficiency of the vector systems was functionally demonstrated by development of a transgenic enhanced green fluorescence protein (GFP) expressing cell line. Further, a GFP down regulated cell line was derived from the said cell line through LV mediated shRNA expression by cloning the GFP-shRNA cassette using the T/A cloning strategy. Subsequently long term expression of GFP transgene in nude mouse spleen/liver was also documented till 30 days. This LV platform with the enhanced user friendly cloning options will be an important advancement in gene transfer technology.

Role of cation-pi interactions in single chain 'all-alpha' proteins.

Cation-pi interactions are known to be important contributors to protein stability and ligand-protein interactions. In this study, we have analyzed the influence of cation-pi interactions in single chain 'all-alpha' proteins. We observed 135 cation-pi interactions in a data set of 75 proteins. No significant correlation was observed between the total number of amino acid residues and number of cation-pi interactions. These interactions are mainly formed by long-range contacts and there is preference of Arg over Lys in these interactions. Arg-Phe interactions are predominant among the various pairs analyzed. Despite the scarcity of interactions involving Trp, the average energy for Trp-cation interactions, was quite high. This information implies that the cation-pi interactions involving Trp, maybe of high relevance to the proteins. Secondary structure analysis reveals that cation-pi interactions are formed preferably between residues, in which at least one of them, is in the secondary structure of alpha-helical segments. Among the various types of folds of 'all-alpha' proteins considered for the analysis, proteins belonging to alpha-alpha superhelix fold have the highest number of cation-pi interaction forming residues.

A hyphenated echelle LIBS-Raman system for multi-purpose applications.

We have developed and standardized a novel hybrid laser-induced breakdown spectroscopy (LIBS)-Raman system using a single pulsed laser and a high-resolution intensified charge coupled device coupled echelle spectrograph. LIBS and Raman spectroscopy are highly complementary techniques which yield elemental and molecular information. Both techniques share an apparently similar instrumental configuration but need entirely different requirements like spectral range covered, resolution, and light-gathering efficiencies. There are thus many challenges to be faced in developing a combined system. In the present work, we show that an echelle spectrograph combined with a compact Q-switched Nd:YAG laser operating at 532 nm as an excitation source in a portable configuration can be efficiently used for such multi-purpose spectroscopy. Atomic and molecular emissions from the sample surface have been recorded in a gated mode using this setup. Compared to conventional spectrographs, echelle provides simultaneous broad bandpass (250-900 nm) and better spectral resolution at an extremely small fixed slit width of 10 × 50 µm without moving the dispersive elements. The echelle-based hyphenated system provides fast and reliable analysis of materials with combined atomic and molecular spectra of the same spot with better reliability. In this paper, we discuss the optimization of various instrumental parameters and optical components of this hyphenated system using a medium Raman cross section sample, CaCO3. The feasibility of single shot LIBS-Raman measurement capabilities of echelle has also been demonstrated using the developed system.

Laser-induced breakdown spectroscopy-Raman: An effective complementary approach to analyze renal-calculi.

Presence of renal-calculi (kidney stones) in human urethra is being increasingly diagnosed over the last decade and is considered as one of the most painful urological disorders. Accurate analysis of such stones plays a vital role in the evaluation of urolithiasis patients and in turn helps the clinicians toward exact etiologies. Two highly complementary laser-based analytical techniques; laser-induced breakdown spectroscopy (LIBS) and micro-Raman spectroscopy have been used to identify the chemical composition of different types of renal-calculi. LIBS explores elemental characteristics while Raman spectroscopy provides molecular details of the sample. This complete information on the sample composition might help clinicians to identify the key aspects of the formation of kidney stones, hence assist in therapeutic management and to prevent recurrence. The complementarity of both techniques has been emphasized and discussed. LIBS spectra of different types of stones suggest the probable composition of it by virtue of the major, minor and trace elements detected from the sample. However, it failed to differentiate the crystalline form of different hydrates of calcium oxalate stone. This lacuna was overcome by the use of Raman spectroscopy and these results are compared with conventional chemical analysis.

Studies on the luteolytic, oestrogenic and follicle-stimulating hormone like activity of plant Rhaphidophora pertusa (Roxb.).

The phytochemical, pharmacological and toxicological feature of plant Rhaphidophora pertusa (Roxb.) was done. Phytosteroids, flavonoids, tannins and glucosides were detected in the plant extracts. In cross-bred (Zebu X Holstein-Friesian or Jersey) dairy cows, subsequent to prostaglandin (PG) induced oestrus, to each group (n=4), cloprostenol (PG control) 100 microg i.m. on day 10, the rice gruel (vehicle) was fed on day 10 or the fresh stem (1 kg/animal/day) in rice gruel on day 9, or days 9 and 10, or days 9-11 of the oestrous cycle. Each group received subcutaneously either 5% gum acacia suspension or the plant ethyl acetate or methanol extract (1g in gum acacia) on days 8 (to bannur ewes) or 10 (to dairy cows) of the oestrous cycle. In PG control cows or ewes, there was induction of oestrus in 48 h and a fall in serum progesterone concentration. The feeding of fresh stem in the rice gruel or the s.c. administration of the plant extract did not induce oestrus or significantly (P>0.05) alter the serum progesterone, bilirubin, calcium, creatinine, phosphorus, magnesium and glucose concentrations or the total erythrocyte and leucocyte count, differential leucocyte count and haemoglobin concentration. The plant did not cause any toxicity in the cow or ewe. In immature rats, the aqueous or methanol (hot or cold) extract did not cause any follicle-stimulating hormone (FSH)-like activity. The methanol extract increased the uterine weight in ovariectomised rats. This suggested the presence of oestrogenic activity in the plant. In conclusion, the present study revealed the presence of oestrogenic activity in the plant and the absence of luteolytic or FSH-like or toxic activity.

Structural analysis of cation-pi interactions in DNA binding proteins.

Cation-pi interactions play an important role in the stability of protein structures. In this work, we have analyzed the influence of cation-pi interactions in DNA binding proteins. We observed cation-pi interactions in 45 out of 62 DNA binding proteins and there is no significant correlation between the number of amino acid residues and number of cation-pi interactions. These interactions are mainly formed by long-range contacts, and the role of short and medium-range contacts is minimal. The preference of Arg is higher than Lys to form cation-pi interactions. The pair-wise cation-pi interaction energy between aromatic and positively charged residues shows that Arg-Tyr energy is the strongest among the possible six pairs. The structural analysis of cation-pi interaction forming residues shows that Lys, Trp, and Tyr prefer to be in the binding site of protein-DNA complexes. Further, the accessible surface areas of cation-pi interaction forming cationic residues are significantly less than that of other residues. The preference of cation-pi interaction forming residues in different secondary structures shows that Lys prefers to be in strand and Phe prefers to be in turn regions. The results obtained in the present study will be useful in understanding the contribution of cation-pi interactions to the stability and specificity of protein-DNA complexes.

Spectroscopic evidence for structural oscillations in DNA caused by oxygen.

It has been shown that interaction of dissolved oxygen with DNA in an aqueous solution causes oscillations in the DNA structure and leads to the formation of a loose DNA-oxygen complex. These results have been compared with those obtained with solution of DNA in high salt concentration.

Molecular electrostatic potential mapping and structure-activity relationship for 3-methoxy flavones.

Molecular electrostatic potential (MEP) maps of certain 3-methoxy flavone derivatives having different anti-picornavirus activities have been studied. Geometries of the molecules were optimised and charge distributions computed using the AM1 molecular orbital method. Hybridization displacement charges (HDC) were combined with the Löwdin charge distributions to compute the MEP maps. Reliability of the method of computing MEP maps was tested by studying certain other molecules for which ab initio MEP results are available. The anti-picornavirus activities of the flavones have been shown to be related with negative MEP values in two regions, one near the 3-methoxy group and another in a diagonally opposite region near the substituent attached to the C7 atom of the molecules.

Primary splenic lymphoma: a case report.

Primary splenic lymphomas (PSL) constitute a rare variety of splenic neoplasms. As a secondary lymphoid organ, the spleen is usually involved by lymphomas as part of the systemic illness. However, rarely it can be the exclusive site of disease burden. An elderly lady presented with symptoms and signs of splenomegaly. After evaluation she was found to have a splenic tumor. Splenectomy was done which revealed primary splenic lymphoma. This case report highlights the evaluation and management of this illness.

Probing oxidative stress in single erythrocytes with Raman Tweezers.

Raman Tweezers have been successfully applied to characterize chemically-induced oxidative stress on optically-trapped live, single erythrocytes. There is significant enhancement in Raman peak intensities corresponding to SS and C-S stretching modes that are induced by oxidative stress. This is consistent with the formation of mixed disulphides between protein SH groups and low-molecular-mass thiols such as glutathione during oxidative damage to cells. Enhancement in glutathione level as a protective response against oxidative stress has been observed. Principal component analysis of the data yields good discrimination between spectra of normal and stress-induced red blood cells.

Classification of protein profiles using fuzzy clustering techniques: an application in early diagnosis of oral, cervical and ovarian cancer.

Present study has brought out a comparison of PCA and fuzzy clustering techniques in classifying protein profiles (chromatogram) of homogenates of different tissue origins: Ovarian, Cervix, Oral cancers, which were acquired using HPLC-LIF (High Performance Liquid Chromatography-Laser Induced Fluorescence) method developed in our laboratory. Study includes 11 chromatogram spectra each from oral, cervical, ovarian cancers as well as healthy volunteers. Generally multivariate analysis like PCA demands clear data that is devoid of day-to-day variation, artifacts due to experimental strategies, inherent uncertainty in pumping procedure which is very common activities during HPLC-LIF experiment. Under these circumstances we demonstrate how fuzzy clustering algorithm like Gath Geva followed by Sammon mapping outperform PCA mapping in classifying various cancers from healthy spectra with classification rate up to 95 % from 60%. Methods are validated using various clustering indexes and shows promising improvement in developing optical pathology like HPLC-LIF for early detection of various cancers in all uncertain conditions with high sensitivity and specificity.

Protein profile analysis of cellular samples from the cervix for the objective diagnosis of cervical cancer using HPLC-LIF.

Protein profiles of cytologic samples from the cervix were studied using High Performance Liquid Chromatographic (HPLC) separation combined with ultra-sensitive laser induced fluorescence (LIF) detection. HPLC-LIF protein profiles of samples from clinically normal subjects, individuals suffering from cervical cancer (different stages), and subjects who had other gynecological problems related to cervix, like erosion of cervix and Nabothian cyst, but no malignancy, were subjected to Principal Component Analysis (PCA). The application of HPLC-LIF protein profiling combined with PCA was found to be a highly efficient method for discrimination of different classes of samples with high sensitivity and specificity. Diagnostic accuracy and optimal threshold - decision criterion - for objective discrimination were estimated using sensitivity-specificity pairs and Youden's index (J) plots.

Full-length genome characterization of an HIV type 2 isolate from India.

An infectious Indian human immunodeficiency virus-2 (HIV-2) subtype A isolate was completely sequenced and analyzed and its phylogenetic relatedness was investigated. The unusual limited size of the long terminal repeat (LTR) from the isolate was caused due to a truncation within the nef open reading frame (ORF) located at the U3 region of the LTR. The genetic relatedness and lineage of this HIV-2 strain were investigated. The close relatedness of this isolate to West African HIV-2 isolates confirms a geographical entry route of HIV-2 to this part of the Indian subcontinent. This is the first report of an HIV-2 full genome analysis from the Indian subcontinent as well as from Asia.

Optical screening of oral cancer: technology for emerging markets.

Oral cancer is the sixth most common cancer in the world. It is one of the most prevalent cancers in the developing countries of South Asia accounting for one third of the world burden. Sixty percent of the cancers are advanced by the time they are detected. Two methods of optical spectroscopy for detection of oral cancer have been discussed here. These methods are simple, easy to handle and non-invasive. The evaluation of the data is done automatically using pattern recognition techniques, making the screening subjective.