Stimuli-directed selective detection of Cu2+ and Cr2O72- ions using a pH-responsive chitosan-poly(aminoamide) fluorescent microgel in aqueous media.

In this work, the preparation of a pH-responsive fluorescent microgel, (NANO-PAMAM-CHT), is presented for the selective detection of Cu2+ and Cr2O72- ions. The NANO-PAMAM-CHT (nanosized polyaminoamide-chitosan microgel) is synthesized via aza-Michael addition reactions in a controlled and stepwise manner in water, using easily affordable starting materials like 1,4-diaminobutane, N,N'-methylene-bis-acrylamide, NIPAM and chitosan. NANO-PAMAM-CHT shows pH-responsive fluorescent properties, whereas the fluorescence intensity shows a pH-responsive change. Due to the selective fluorescence quenching, the microgel can detect both Cu2+ ions and Cr2O72- ions selectively at ambient pH in aqueous medium. Moreover, it can selectively differentiate between Cu2+ ion and Cr2O72- ions at pH ∼3 in water. The limits of detection for Cu2+ ions and Cr2O72- ions are reported as 16.9 µM and 2.62 µM, respectively (lower than the minimum allowed level in drinking water) at pH ∼7. Mechanistic study further reveals the dynamic quenching phenomenon in the presence of Cu2+ ions and static quenching in the presence of Cr2O72- ions.

Stimuli-responsive fluorescent nanogel: a nonconventional donor for ratiometric temperature and pH sensing.

A reactive stimuli responsive fluorescent polyaminoamide nanogel (NANO-PAMAM) is synthesized via an aza-Michael polyaddition reaction in water. In the next stage, doxorubicin (a FRET acceptor) is covalently linked with the nanogel (NANO-PAMAM, a nonconventional FRET donor) to form a ratiometric nanosensor for temperature and pH sensing. MTT assay revealed comparable biocompatibility of the donor nanogels (NANO-PAMAM) and nanosensors (NANO-PAMAM-DOX).

Quantitative functionalization of chitosan using green and efficient azetidinium-amine reactions.

Despite significant advances in the field, quantitative post-polymer modification of chitosan, specifically attaching diverse hydrophobic moieties with experimentally predefined (via the ratio of reagents) degree of functionalization remain extremely challenging. In this context, we report our studies towards the use of green and efficient azetidinium-amine reactions to prepare a library of modified chitosan with a predefined degree of functionalization, with excellent conversion (>90%), atom economy and very low E-factor (<0.1). Initially six different azetidinium functionalized coupler molecules, which contains azetidinium functionality attached with other defined target functionality (ranging from hydrophobic to hydrophilic) are synthesized. In the next stage the azetidinium functionalized coupler molecules are reacted with chitosan (both low molecular weight and high molecular weight chitosans are used) in presence of 1,4-diazabicyclo[2.2.2]octane(DABCO, as base) using water as solvent and the other functional moiety are attached with the chitosan backbones. The degree of functionalization was calculated using 1H NMR and excellent match (80-100%) was noted between calculated degree of functionalization and feed ratio of the reagents used (experimentally defined ratio).

A regression-based three-phase approach to assess outdoor thermal comfort in informal micro-entrepreneurial settings in tropical Mumbai.

Urban heat poses a public health risk to the residents of megacities in developing countries because the population spends a significant amount of time outdoors to work and socialize with limited cooling resources. Understanding the drivers of outdoor comfort and heat stress in informal work settings is important to design climate-sensitive outdoor spaces and reduce heat vulnerability. We present outdoor thermal comfort perceptions (OTCPs) of people engaged in outdoor micro entrepreneurial activities in Mumbai using seasonal surveys and biometeorological observations. We propose a three-phase approach to analyze the relative importance of climatic and non-climatic variables for OTCPs. The first phase evaluates the seasonal and intra-neighborhood variation of thermal sensation votes (TSV) with respect to physiological equivalent temperature (PET) and air temperature. Second, we include physiological parameters to evaluate the seasonal and intra-neighborhood variation of overall sensation votes (OSV). Third, we consider aggregated survey responses and include behavioral and perceptual variables to determine their relative significance. We employ three linear modeling techniques to assess model performance in explaining the variability of OTCP using OSV as dependent variable. Results reveal that microclimatic parameters alone are unable to explain the variability of OTCP. Our results yield a neutral PET value (PETneutral) of 23.75 °C for Mumbai in the winter. PETneutral was higher for activities at the clothing market compared to other micro entrepreneurial activities. Acclimatization significantly improved comfort in the summer, while evaporative cooling was beneficial in the winter. Further, an ANCOVA and ordinal logistic regressions demonstrate the importance of behavioral attributes (presence in the location, expectation, beverage intake) in explaining the variance in OTCP. Our study also reveals that wind speed and humidity play an important role in shaping overall comfort in the Mumbai neighborhoods.

A BODIPY-O-glycoside based near-infrared fluorescent sensor for serum albumin.

Highly sensitive and selective near-infrared fluorescent bioprobes for serum albumin detection and quantification are in high demand for biomedical applications. Herein, we report a near-infrared emitting BODIPY-O-glycoside dye as a turn-on emission sensor for serum albumin. To the best of our knowledge, this is the first report of NIR-emitting BODIPY dyes for serum albumin sensing. Despite the various outstanding photophysical properties of the BODIPY dyes, their insolubility in water/biological media restricts their real biomedical applications. To overcome this issue, highly stable unadulterated BODIPY-O-glycoside nanoparticles (BDP-Glu-NPs) were prepared in aqueous solution by self-assembly of amphiphilic BODIPY-O-glycoside dyes. The BDP-Glu-NPs were characterized by spectroscopic, NMR, DLS and TEM studies. The ability of the BDP-Glu-NPs for the detection and quantification of serum albumin was demonstrated. It showed a 150-fold fluorescence enhancement in the presence of serum albumin, with excellent selectivity over other amino acids, porphyrin, proteins and various inorganic salts. Detection of human serum albumin (HSA) in urine samples showed that the bioprobe is applicable to a clinically significant range of the analytes with very low detection limit. These results suggested that the BDP-Glu-NPs can act as potential bioprobe to quantify albumin in biochemical and clinical samples.

Degradable and processable polymer monoliths with open-pore porosity for selective CO2 and iodine adsorption.

A task-specific design of biodegradable and processable porous polymers is one of the primary requisite for their efficient day-to-day use to minimize polymer waste. Herein, a surfactant (or additive)-free method is reported for the synthesis of a processable and degradable aliphatic open-pore porous polyelectrolyte monolith for the removal of gaseous pollutants such as iodine and CO2. This is achieved via a colloidal templating method. In the 1st stage, cationic colloidal nanoparticles containing reactive amines and acrylamide groups were formed via the phase separation of hyperbranched polyaminoamides in water (sol). These cationic nanoparticles (which acted as both templates and macromers) further reacted to form a gel, which upon freeze-drying leads to the formation of a polymer monolith with an open-pore porous morphology and hierarchical porosity throughout its structure. During gelation, the shape of the monolith can be controlled using suitable templates and a similar strategy was used to prepare porous thin films. The monolith has shown excellent iodine adsorption ability (5000 mg g-1 in the vapor phase and 2663 mg g-1 in the solution phase) with good reusability and CO2 adsorption ability (60 mg g-1), with CO2/CH4 and CO2/N2 selectivities of 18.5 and 6.7, respectively. The degradability of the materials was studied in detail at different pH, confirming their easy degradability in aqueous solutions and a higher degradability at basic pH.

Synthesis of Bioactive Diarylheptanoids from Alpinia officinarum and Their Mechanism of Action for Anticancer Properties in Breast Cancer Cells.

An efficient synthesis of the Alpinia officinarum-derived diarylheptanoids, viz., enantiomers of a ß-hydroxyketone (1) and an α,ß-unsaturated ketone (2) was developed starting from commercially available eugenol. Among these, compound 2 showed a superior antiproliferative effect against human breast adenocarcinoma MCF-7 cells. Besides reducing clonogenic cell survival, compound 2 dose-dependently increased the sub G1 cell population and arrested the G2-phase of the cell cycle, as revealed by flow cytometry. Mechanistically, compound 2 acts as an intracellular pro-oxidant by generating copious amounts of reactive oxygen species. Compound 2 also induced both loss of mitochondrial membrane potential (MMP) as well as lysosomal membrane permeabilization (LMP) in the MCF-7 cells. The impaired mitochondrial and lysosomal functions due to reactive oxygen species (ROS)-generation by compound 2 may contribute to its apoptotic property.

Pharmacological targeting of differential DNA repair, radio-sensitizes WRN-deficient cancer cells in vitro and in vivo.

Werner (WRN) expression is epigenetically downregulated in various tumors. It is imperative to understand differential repair process in WRN-proficient and WRN-deficient cancers to find pharmacological targets for radio-sensitization of WRN-deficient cancer. In the current investigation, we showed that pharmacological inhibition of CHK1 mediated homologous recombination repair (HRR), but not non-homologous end joining (NHEJ) repair, can causes hyper-radiosensitization of WRN-deficient cancers. This was confirmed in cancer cell lines of different tissue origin (osteosarcoma, colon adenocarcinoma and melanoma) with WRN silencing and overexpression. We established that WRN-depleted cells are dependent on a critical but compromised CHK1-mediated HRR-pathway for repairing ionizing radiation (IR) induced DSBs for their survival. Mechanistically, we unraveled a new finding that the MRE11, CTIP and WRN proteins are largely responsible for resections of late and persistent DSBs. In response to IR-treatment, MRE11 and CTIP-positively and WRN-negatively regulate p38-MAPK reactivation in a CHK1-dependent manner. A degradation resistant WRN protein, mutated at serine 1141, abrogates p38-MAPK activation. We also showed that CHK1-p38-MAPK axis plays important role in RAD51 mediated HRR in WRN-silenced cells. Like CHK1 inhibition, pharmacological-inhibition of p38-MAPK also hyper-radiosensitizes WRN-depleted cells by targeting HR-pathway. Combination treatment of CHK1-inhibitor (currently under various clinical trials) and IR exhibited a strong synergy against WRN-deficient melanoma tumor in vivo. Taken together, our findings suggest that pharmacological targeting of CHK1-p38-MAPK mediated HRR is an attractive strategy for enhancing therapeutic response of radiation treatment of cancer.

Iron modulatory property of a polysaccharide from Indian medicinal plant Ocimum sanctum.

Despite being an essential element for normal functioning of cells and organisms, iron, in excess, can induce oxidative stress by generating reactive oxygen species. A water-soluble, non-toxic iron chelator can reduce the iron-induced oxidative stress in the body as well as help in extricating excess iron. Herein, we report an Ocimum sanctum-derived antioxidant polysaccharide (OSP) that inhibits the deleterious effect of iron. Ocimum sanctum is a widely acknowledged medicinal plant contributing toward several biological benefits. Besides showing good hydroxyl radical scavenging activity, OSP could bind to ferric and ferrous ions to prevent their participation in redox reactions as revealed from modified 2-deoxyribose assays, carried out under various conditions. It also acted as an iron modulator to prevent site-specific damage and was effective in protecting mouse fibroblast L929 cells against iron induced death.

Monitoring the formation of insulin oligomers using a NIR emitting glucose-conjugated BODIPY dye.

Protein oligomers, which are formed due to the aggregation of protein molecules under physiological stress, are neurotoxic and responsible for several neurological diseases. Early detection of protein oligomers is essential for the timely intervention in the associated diseases. Although several probes have been developed for the detection of insoluble matured protein fibrils, fluorescent probes with emission in the near infrared (NIR) region for probing protein oligomers are very rare. In the present study we have designed and synthesized a glucose-conjugated BODIPY dye with emission in the NIR spectral range. Our detailed studies show that the new probe is not only capable of detecting matured fibrils but can also probe the formation of oligomers from the native protein. The new probe shows a large increase in its emission intensity upon association with oligomers and matured fibrils. Hence, the present probe has a great potential for the in vivo imaging of protein oligomers and matured fibrils. Detailed spectroscopic properties of the new probes in molecular solvents have been performed to understand its oligomers- and fibril- sensing mechanism.

Outdoor thermal comfort in various microentrepreneurial settings in hot humid tropical Kolkata: Human biometeorological assessment of objective and subjective parameters.

Extreme heat and associated health risks increasingly become threats to urban populations, especially in developing countries of the tropics. Although human thermal exposure in cities has been studied across the globe, current narratives insufficiently discuss mixed-used spaces, informal economic activity settings, and informal settlements. This study assessed outdoor human thermal comfort in the tropical city of Kolkata, India where uncomfortable hot and humid climatic conditions prevail year-round. Thermal Comfort Perception Surveys (TCPS) and biometeorological observations were conducted during summer and winter in three microentrepreneurial neighborhoods (Kumartuli, Boipara, and Mallickghat). A one-way ANOVA was performed to investigate the variance in Physiologically Equivalent Temperature (PET) values of 318 survey samples across neighborhoods. Through multiple linear regression and ANCOVA, significant relationships were established between various climatic and non-climatic parameters. No respondent reported a neutral thermal sensation during the summer. Annual neutral PET across neighborhoods was 23.6 °C with a neutral PET range of 19.5 °C to 27.6 °C. Annual neutral PET was 22.7 °C and 26.5 °C in Mallickghat and Boipara, respectively. Respondents in Boipara were more sensitive towards warmer sensation than in Mallickghat. Even in the winter, people reported warmer sensation votes. PET was a better predictor of the mean Thermal Sensation Vote (mTSV) compared to air temperature. In a few cases, acclimatization and expectations improved thermal comfort. Results can be useful in formulating strategies towards improving outdoor microclimate and heat health in tropical cities.

Tuning of electron tunneling: a case study using BODIPY molecular layers.

Redox active π-conjugated organic molecules have shown the potential to be used as electronic components such as diode and memory elements. Here, we demonstrate that using simple surface chemistry, rectification characteristics can be tuned to reproducible negative differential resistance (NDR) with a very high peak-to-valley ratio (PVR) up to 1000 in 2,6-diethyl-4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indecene (BODIPY) grafted on Si. The change in properties is related to oxidation and reduction of BODIPY, which results in the change in resonant to non-resonant tunneling of electrons under bias. This has been explained by the ab initio molecular-orbital theoretical calculations.

Thiol antioxidants sensitize malabaricone C induced cancer cell death via reprogramming redox sensitive p53 and NF-κB proteins in vitro and in vivo.

Specific focus on "redox cancer therapy" by targeting drugs to redox homeostasis of the cancer cells is growing rapidly. Recent clinical studies showed that N-acetyl cysteine (NAC) treatment significantly decreased the metabolic heterogeneity and reduced Ki67 (a proliferation marker) with simultaneous enhancement in apoptosis of tumor cells in patients. However, it is not yet precisely known how thiol antioxidants enhance killing of cancer cells in a context dependent manner. To this end, we showed that a dietary compound, malabaricone C (mal C) generated copious amounts of reactive oxygen species (ROS) and also reduced GSH level in lung cancer cells. Paradoxically, although antioxidants supplementation reduced mal C-induced ROS, thiol-antioxidants (NAC/GSH) restored intracellular GSH level but enhanced DNA DSBs and apoptotic cell death induced by mal C. Our results unraveled two tightly coupled biochemical mechanisms attributing this sensitization process by thiol antioxidants. Firstly, thiol antioxidants enable the "catechol-quinone redox cycle" of mal C and ameliorate ROS generation and bio-molecular damage (DNA and protein). Secondly, thiol antioxidants cause rapid glutathionylation of transcription factors [p53, p65 (NF-κB) etc.], oxidized by mal C, and abrogates their nuclear sequestration and transcription of the anti-apoptotic genes. Furthermore, analyses of the mitochondrial fractions of p53 expressing and silenced cells revealed that cytoplasmic accumulation of glutathionylated p53 (p53-SSG) triggers a robust mitochondrial death process. Interestingly, mutation of redox sensitive cysteine residues at 124, 141 and 182 position in p53 significantly reduces mal C plus NAC mediated sensitization of cancer cells. The preclinical results, in two different tumor models in mice, provides further support our conclusion that NAC is able to sensitize mal C induced suppression of tumor growth in vivo.

A bis-resorcinol resveratrol congener prevents indomethacin-induced gastric ulceration by inhibiting TNF-α as well as NF-κB and JNK pathways.

The cytoprotective action of the synthetic resveratrol (Resv) congener, E-3,3',5,5'-tetrahydroxystilbene (designated as HST-1) against indomethacin (IND)-induced stomach ulceration has been established using a mice model. HST-1 reversed the adverse effects of IND on several inflammatory (myeloperoxidase, cytokines, adhesion molecules etc.) and ulcer-healing (cyclooxygenases, prostaglandin, growth factors and their receptors etc.) parameters in mice. More importantly, HST-1 down-regulated TNF-α and the TNF-α-mediated activation of NF-κB and JNK/MAPK pathways that are the key determinants in the IND-gastropathy. The effect of HST-1 on all these factors was significantly better than that of Resv, misoprostol, and omeprazole. HST-1 also did not induce small intestinal mucosal injury, unlike some of the proton pump inhibitors. On the other hand, Resv reduced activation of the prosurvival ERK1/2 pathway that may explain its contraindicative property in the gastrointestinal tract.

Mechanism of coralyne-mediated DNA photo-nicking process.

Previously, we reported that coralyne and UVA combination sensitized a wide range of human carcinoma cells regardless of their p53 status. The coralyne induced photosensitization of cancer cells may be clinically attractive, as mutation in the p53 gene is prevalent in many types of tumors. Coralyne mediated photosensitization of cancer cells is attributable to its ability to cause extensive DNA single strand breaks (SSB). However, the precise mechanism of coralyne induced DNA photo-damage is not yet known. The present study was aimed to understand the hitherto unknown mechanism of the coralyne-induced DNA photo-cleavage process. To this end, we compared the DNA photo-nicking properties of berberine, jatrorrhizine and coralyne, and deciphered involvement of the photochemical processes in the photo-nuclease action of coralyne using absorption and electron spin resonance spectroscopy, high performance liquid chromatography and mass spectroscopy (MS) techniques in conjunction with relevant in vitro studies with plasmid DNA. In association with UVA, coralyne, but not berberine and jatrorrhizine induced significant nicking of plasmid DNA via an O2-independent photo-chemical process. The Job's plot of our spectrophotometric data suggested that one coralyne molecule remains intercalated with two DNA base pairs (i. e., 1:2) and starts forming aggregates beyond this molar ratio. The DNA photo-nicking by the combination of coralyne and UVA (designated as CUVA) was primarily caused by the coralyne aggregates without any significant contribution from the DNA-intercalated coralyne monomer.

A chemoenzymatic synthesis of ceramide trafficking inhibitor HPA-12.

A chemoenzymatic synthesis of the title compound has been developed using an efficient and highly enantioselective lipase-catalyzed acylation in a hydrophobic ionic liquid, [bmim][PF6], followed by a diastereoselective asymmetric dihydroxylation as the key steps for incorporating the stereogenic centers. The further conversion to the appropriate intermediates and subsequent acylation with lauric acid furnished the target compound.

Spice-derived phenolic, malabaricone B induces mitochondrial damage in lung cancer cells via a p53-independent pathway.

The spice-derived phenolic, malabaricone B (mal B) showed selective toxicity to human lung cancer (A549), malignant melanoma (A375) and T cell leukemia (Jurkat) cell lines, without showing toxicity to human normal intestinal (INT407), human kidney (HEK293) and lung fibroblast (WI-38) cells. Among the chosen cancer cell lines, mal B showed maximum cytotoxicity to the A549 cells (IC50 = 8.1 ± 1.0 µM), which was significantly better than that of curcumin (IC50 = 26.7 ± 3.1 µM). Further morphological studies by phase contrast microscopy and a clonogenic assay of the A549 cells revealed that mal B treatment increased the number of shrinking cells and also abolished the clonal proliferation of the cells. Mal B induced apoptotic cell death was confirmed by DNA laddering and quantified by cytoplasmic oligonucleosome formation and annexin V/PI assays. The mal B-induced apoptosis was mediated by an increase in the intracellular reactive oxygen species (ROS), because the cell-permeable antioxidants, N-acetylcysteine (NAC) and PEG-SOD, strongly inhibited its cytotoxicity to the A549 cells. Mal B increased the BAX level while simultaneously decreasing the BCL-2 and BCL-XL levels in the A549 cells, triggering the mitochondrial apoptotic pathway as revealed from the release of cytochrome c, and the activation of caspase-9 and caspase-3. Pre-treatment of cells with caspase-9, caspase-3 and pan-caspase inhibitors made them more resistant to mal B treatment. This effect of mal B was strongly associated with the concomitant decrease in anti-apoptotic (IAP1, IAP2 and survivin), angiogenic (growth factors) and cancer invasiveness (matrix metalloproteinase-9, COX-2) modulating proteins. Mal B induced cytotoxicity was unaffected by the shRNA-mediated depletion of p53 in A549 cells. Most importantly, mal B sensitized a wide range of human carcinoma cells regardless of their p53 status. Finally, mal B (100 mg kg-1) also inhibited lung tumor (xenograft) growth in SCID mice.

Bi-mediated allylation of aldehydes in [bmim][Br]: a mechanistic investigation.

The inexpensive room temperature ionic liquid (RTIL), [bmim][Br] has been found to be a superior medium for the Bi-mediated Barbier-type allylation of aldehydes compared to other conventional solvents. It plays the dual role of a solvent and a metal activator enabling higher yields of the products in a shorter reaction time using stoichiometric/near-stoichiometric amounts of reagents. Plausibly, [bmim][Br] activates Bi metal by a charge transfer mechanism. The 1H VT-NMR studies suggested that both the allylating species, allylbismuth dibromide and diallylbismuth bromide, are generated in situ.

Resveratrol analogue, trans-4,4'-dihydroxystilbene (DHS), inhibits melanoma tumor growth and suppresses its metastatic colonization in lungs.

The prevalence of melanoma and the lack of effective therapy for metastatic melanoma warrant extensive and systematic evaluations of small molecules in cellular and pre-clinical models. We investigated, herein, the antitumor and anti-metastatic effects of trans-4,4'-dihydroxystilbene (DHS), a natural product present in bark of Yucca periculosa, using in vitro and in vivo melanoma murine models. DHS showed potent melanoma cytotoxicity, as determined by MTT and clonogenic assay. Further, DHS induced cytotoxicity was mediated through apoptosis, which was assessed by annexin V-FITC/PI, sub-G1 and caspase activation assays. In addition, DHS inhibited cell proliferation by inducing robust cell cycle arrest in G1-phase. Imperatively, these inhibitory effects led to a significant reduction of melanoma tumor in pre-clinical murine model. DHS also inhibited cell migration and invasion of melanoma cells, which were examined using wound healing and Transwell migration/invasion assays. Mechanistically, DHS modulated the expressions of several key metastasis regulating proteins e.g., MMP-2/9, N-cadherin, E-cadherin and survivin. We also showed the anti-metastatic effect of DHS in a melanoma mediated lung metastasis model in vivo. DHS significantly reduced large melanoma nodule formation in the parenchyma of lungs. Therefore, DHS may represent a promising natural drug in the repertoire of treatment against melanoma tumor growth and metastasis.

Allylpyrocatechol attenuates methotrexate-induced hepatotoxicity in a collagen-induced model of arthritis.

The cornerstone of treatment for rheumatoid arthritis is low dose methotrexate (MTX), but its use is limited by concerns regarding its potential for hepatotoxicity. Allylpyrocatechol (APC), a phytoconstituent sourced from leaves of Piper betle demonstrated antioxidant, anti-inflammatory, and antiarthritic properties. The present study aimed to evaluate the combined effect of APC and MTX on limiting progression of lipopolysaccharide accelerated collagen-induced arthritis, along with reduction of MTX-induced hepatic damage. A collagen-induced arthritis (CIA) model was established by immunising Sprague-Dawley rats with bovine collagen type II (CII) and lipopolysaccharide, followed by a booster dose of CII on day 15. Rats from days 11-27 were administered APC (20 mg/kg), methotrexate (1.5 mg/kg), or a combination of MTX and APC. The combinatorial therapy of APC and MTX significantly improved the parameters of arthritis as evident from the reduction in paw oedema and arthritic score and was endorsed by radiological and histopathological changes. This combination prevented the rise in levels of proinflammatory cytokines, tumour necrosis factor (TNF-α), and interleukin 6 (IL-6). Furthermore, unlike MTX-monotherapy, the APC-MTX combination decreased the associated cachexia, splenomegaly, and oxidative stress. Importantly, the hepatic damage mediated by MTX monotherapy was effectively attenuated by the inclusion of APC. Taken together, antioxidants such as APC when combined with MTX not only potentiated the antiarthritic effect but importantly alleviated the MTX-induced hepatic damage, thus endorsing its effectiveness in preventing progression of articular diseases such as rheumatoid arthritis.