Stabilization of G-Quadruplex Structures of the SARS-CoV-2 Genome by TMPyP4, BRACO19, and PhenDC3.

The G-quadruplex is one of the non-canonical structures formed by nucleic acids, which can be formed by guanine-rich sequences. They became the focus of much research when they were found in several oncogene promoter regions and also in the telomeres. Later on, they were discovered in viruses as well. Various ligands have been developed in order to stabilize DNA G-quadruplexes, which were believed to have an anti-cancer or antiviral effect. We investigated three of these ligands, and whether they can also affect the stability of the G-quadruplex-forming sequences of the RNA genome of SARS-CoV-2. All three investigated oligonucleotides showed the G-quadruplex form. We characterized their stability and measured their thermodynamic parameters using the Förster resonance energy transfer method. The addition of the ligands caused an increase in the unfolding temperature, but this effect was smaller compared to that found earlier in the case of G-quadruplexes of the hepatitis B virus, which has a DNA genome.

Effect of macrocyclization and tetramethylrhodamine labeling on chemokine binding peptides.

Receptor-derived peptides have played an important role in elucidating chemokine-receptor interactions. For the inflammatory chemokine CXC-class chemokine ligand 8 (CXCL8), a site II-mimetic peptide has been derived from parts of extracellular loops 2 and 3 and adjacent transmembrane helices of its receptor CXC-class chemokine receptor 1 (Helmer et al., RSC Adv., 2015, 5, 25657). The peptide sequence with a C-terminal glutamine did not bind to CXCL8, whereas one with a C-terminal glutamate did but with low micromolar affinity. We sought to improve the affinity and protease stability of the latter peptide through cyclization while also cyclizing the former for control purposes. To identify a cyclization strategy that permits a receptor-like interaction, we conducted a molecular dynamics simulation of CXCL8 in complex with full-length CXC-class chemokine receptor 1. We introduced a linker to provide an appropriate spacing between the termini and used an on-resin side-chain-to-tail cyclization strategy. Upon chemokine binding, the fluorescence intensity of the tetramethylrhodamine (TAMRA)-labeled cyclic peptides increased whereas the fluorescence anisotropy decreased. Additional molecular dynamics simulations indicated that the fluorophore interacts with the peptide macrocycle so that chemokine binding leads to its displacement and observed changes in fluorescence. Macrocyclization of both 18-amino acid-long peptides led to the same low micromolar affinity for CXCL8. Likewise, both TAMRA-labeled linear peptides interacted with CXCL8 with similar affinities. Interestingly, the linear TAMRA-labeled peptides were more resistant to tryptic digestion than the unlabeled counterparts, whereas the cyclized peptides were not degraded at all. We conclude that the TAMRA fluorophore tends to interact with peptides altering their protease stability and behavior in fluorescence-based assays.

High-Resolution Confocal Fluorescence Imaging of Serine Hydrolase Activity in Cryosections - Application to Glioma Brain Unveils Activity Hotspots Originating from Tumor-Associated Neutrophils.

BACKGROUND: Serine hydrolases (SHs) are a functionally diverse family of enzymes playing pivotal roles in health and disease and have emerged as important therapeutic targets in many clinical conditions. Activity-based protein profiling (ABPP) using fluorophosphonate (FP) probes has been a powerful chemoproteomic approach in studies unveiling roles of SHs in various biological systems. ABPP utilizes cell/tissue proteomes and features the FP-warhead, linked to a fluorescent reporter for in-gel fluorescence imaging or a biotin tag for streptavidin enrichment and LC-MS/MS-based target identification. Existing ABPP approaches characterize global SH activity based on mobility in gel or MS-based target identification and cannot reveal the identity of the cell-type responsible for an individual SH activity originating from complex proteomes. RESULTS: Here, by using an activity probe with broad reactivity towards the SH family, we advance the ABPP methodology to glioma brain cryosections, enabling for the first time high-resolution confocal fluorescence imaging of global SH activity in the tumor microenvironment. Tumor-associated cell types were identified by extensive immunohistochemistry on activity probe-labeled sections. Tissue-ABPP indicated heightened SH activity in glioma vs. normal brain and unveiled activity hotspots originating from tumor-associated neutrophils (TANs), rather than tumor-associated macrophages (TAMs). Thorough optimization and validation was provided by parallel gel-based ABPP combined with LC-MS/MS-based target verification. CONCLUSIONS: Our study advances the ABPP methodology to tissue sections, enabling high-resolution confocal fluorescence imaging of global SH activity in anatomically preserved complex native cellular environment. To achieve global portrait of SH activity throughout the section, a probe with broad reactivity towards the SH family members was employed. As ABPP requires no a priori knowledge of the identity of the target, we envisage no imaginable reason why the presently described approach would not work for sections regardless of species and tissue source.

Identification of the xenograft and its ascendant sphere-forming cell line as belonging to EBV-induced lymphoma, and characterization of the status of sphere-forming cells.

BACKGROUND: We have characterized the human cell line arised from the Epstein-Barr virus (EBV) positive multiple myeloma aspirate subjected to the long-term cultivation. This cell line has acquired the ability to form free-floating spheres and to produce a xenograft upon transplantation into NOD/SCID mice. METHODS: Cells from both in vitro culture and developed xenografts were investigated with a number of analytical approaches, including pathomorphological analysis, FISH analysis, and analysis of the surface antigens and of the VDJ locus rearrangement. RESULTS: The obtained results, as well as the confirmed presence of EBV, testify that both biological systems are derived from B-cells, which, in turn, is a progeny of the EBV-transformed B-cellular clone that supplanted the primordial multiple myeloma cells. Next we assessed whether cells that (i) were constantly present in vitro in the investigated cell line, (ii) were among the sphere-forming cells, and (iii) were capable of internalizing a fluorescent TAMRA-labeled DNA probe (TAMRA+ cells) belonged to one of the three types of undifferentiated bone marrow cells of a multiple myeloma patient: CD34+ hematopoietic stem cells, CD90+ mesenchymal stem cells, and clonotypic multiple myeloma cell. CONCLUSION: TAMRA+ cells were shown to constitute the fourth independent subpopulation of undifferentiated bone marrow cells of the multiple myeloma patient. We have demonstrated the formation of ectopic contacts between TAMRA+ cells and cells of other types in culture, in particular with CD90+ mesenchymal stem cells, followed by the transfer of some TAMRA+ cell material into the contacted cell.

Tetramethylrhodamine is an essential scaffold of azide probe in detecting cellular acrolein.

Tetramethylrhodamine (TAMRA)-phenyl azide is a chemical probe used to detect intracellular acrolein directly in live cells. Herein, we demonstrated that TAMRA is the optimum fluorophore for the probe. TAMRA-phenyl azide was used to reveal that high levels of acrolein are generated in a variety of breast cancer cells, regardless of the tumor subtype. These findings corroborate the analysis presented in our previous report, in which TAMRA-phenyl azide was used to label breast cancer tissues resected from breast cancer patients. Because high levels of acrolein were generated in all cancer cell types, we believe that acrolein detection may be useful as a general method for labeling cancerous tissues.

Immunoassay for rapid on-site detection of glyphosate herbicide.

Glyphosate is the most widespread herbicide and its global use is steadily increasing. Although glyphosate is considered to have low toxicity, its wide application has raised concerns about its effects on human health. The extensive use of glyphosate has risen a need of its continuous monitoring in drinking and surface waters to assure in accordance with the set standards. Within the present study, we have developed a novel assay for the on-site detection of glyphosate by combining flow-through technology with the high specificity of immunorecognition. The proposed biosensing system was based on the detection of fluorescence signal generated by the quantitative replacement of glyphosate in antigen-antibody complex with IgY-type anti-glyphosate antibodies on microbeads by synthetic 5-carboxytetramethylrhodamine (5-TAMRA) conjugated glyphosate. The working range of this assay was in low millimolar range and the time required for glyphosate detection around 0.5 h. The applicability of the immunoassay for glyphosate detection in surface water was tested and the biosensor results were validated with high-performance liquid chromatography.

A novel Tc-99m and fluorescence-labeled arginine-arginine-leucine-containing peptide as a multimodal tumor imaging agent in a murine tumor model.

We developed a Tc-99m and TAMRA-labeled peptide, Tc-99m arginine-arginine-leucine (RRL) peptide (TAMRA-GHEG-ECG-RRL), to target tumor cells and evaluated the diagnostic performance of Tc-99m TAMRA-GHEG-ECG-RRL as a dual-modality imaging agent for tumor in a murine model. TAMRA-GHEG-ECG-RRL was synthesized using Fmoc solid-phase peptide synthesis. Binding affinity and in vitro cellular uptake studies were performed. Gamma camera imaging, biodistribution, and ex vivo imaging studies were performed in murine models with PC-3 tumors. Tumor tissue slides were prepared and analyzed with immunohistochemistry using confocal microscopy. After radiolabeling procedures with Tc-99m, Tc-99m TAMRA-GHEG-ECG-RRL complexes were prepared in high yield (>96%). The Kd of Tc-99m TAMRA-GHEG-ECG-RRL determined by saturation binding was 41.7 ± 7.8 nM. Confocal microscopy images of PC-3 cells incubated with TAMRA-GHEG-ECG-RRL showed strong fluorescence in the cytoplasm. Gamma camera imaging revealed substantial uptake of Tc-99m TAMRA-GHEG-ECG-RRL in tumors. Tumor uptake was effectively blocked by the coinjection of an excess concentration of RRL. Specific uptake of Tc-99m TAMRA-GHEG-ECG-RRL was confirmed by biodistribution, ex vivo imaging, and immunohistochemistry stain studies. In conclusion, in vivo and in vitro studies revealed substantial uptake of Tc-99m TAMRA-GHEG-ECG-RRL in tumors. Tc-99m TAMRA-GHEG-ECG-RRL has potential as a dual-modality tumor imaging agent.

Dissection of the angle of single fluorophore attached to the nucleotide in corkscrewing microtubules.

Direct dissection of the angles of single fluorophores under an optical microscope has been a challenging approach to study the dynamics of proteins in an aqueous solution. For angle quantifications of single substrates, however, there was only one report (Nishizaka et al., 2014) because of difficulties of construction of experimental systems with active proteins working at the single-molecule level. We here show precise estimation of orientation of single fluorescent nucleotides bound to single tubulins that comprise microtubule. When single-headed kinesins immobilized on a glass surface drive the sliding of microtubules, microtubules show corkscrewing with regular pitches (Yajima et al., 2005 & 2008). We found, by using a three-dimensional tracking microscope, that S8A mutant kinesin also showed precise corkscrewing with a 330-nm pitch, which is 13% longer than that of the wild type. The assay with the mutant was combined with a defocused imaging technique to visualize the rotational behavior of fluorescent nucleotide bound to corkscrewing microtubule. Notably, the defocused pattern of single TAMRA-GTP periodically changed, precisely correlating to its precession movement. The time course of the change in the fluorophore angle projected to the xy-plane enabled to estimate both the fluorophore orientation against microtubule axis and the precision of angle-determination of analyses system. The orientation showed main distribution with peaks at∼40°, 50° and 60°. To identify their molecular conformations, the rigorous docking simulations were performed using an atomic-level structure modeled by fitting x-ray crystal structures to the cryo-electron microscopy map. Among isomers, 2'-O-EDA-GDP labeled with 5- or 6-TAMRA were mainly specified as possible candidates as a substrate, which suggested the hydrolysis of TAMRA-GTP by tubulins.

Synthesis and evaluation of Tc-99m and fluorescence-labeled elastin-derived peptide, VAPG for multimodal tumor imaging in murine tumor model.

We developed a Tc-99m and fluorescence-labeled peptide, Tc-99m TAMRA-GHEG-ECG-VAPG to target tumor cells and evaluated the diagnostic performance as a dual-modality imaging agent for tumor in a murine model. TAMRA-GHEG-ECG-VAPG was synthesized by using Fmoc solid-phase peptide synthesis. Radiolabeling of TAMRA-GHEG-ECG-VAPG with Tc-99m was done by using ligand exchange via tartrate. Binding affinity and in vitro cellular uptake studies were performed. Gamma camera imaging, biodistribution, and ex vivo imaging studies were performed in murine models with SW620 tumors. Tumor tissue slides were prepared and analyzed with immunohistochemistry by using confocal microscopy. After radiolabeling procedures with Tc-99m, Tc-99m TAMRA-GHEG-ECG-VAPG complexes were prepared in high yield (>96%). The Kd of Tc-99m TAMRA-GHEG-ECG-VAPG determined by saturation binding was 16.8 ± 3.6 nM. Confocal microscopy images of SW620 cells incubated with TAMRA-GHEG-ECG-VAPG showed strong fluorescence in the cytoplasm. Gamma camera imaging revealed substantial uptake of Tc-99m TAMRA-GHEG-ECG-VAPG in tumors. Tumor uptake was effectively blocked by the coinjection of an excess concentration of VAPG. Specific uptake of Tc-99m TAMRA-GHEG-ECG-VAPG was confirmed by biodistribution, ex vivo imaging, and immunohistochemistry stain studies. In vivo and in vitro studies revealed substantial uptake of Tc-99m TAMRA-GHEG-ECG-VAPG in tumor cells. Tc-99m TAMRA-GHEG-ECG-VAPG has potential as a dual-modality tumor imaging agent.

Tetramethyl-6-carboxyrhodamine quenching-based aptasensing platform for aflatoxin B1: Analytical performance comparison of two aptamers.

In this study, a simple TAMRA (tetramethyl-6-carboxyrhodamine) quenching-based aptasensing platform was designed for the detection of aflatoxin B1 (AFB1). Here, we compared the analytical performance of two aptamer sequences: seqA and seqB. The AFB1 detection was based on the interactions of FAM (carboxyfluorescein)-labeled aptamer with TAMRA-labeled DNA complementary strand in the presence and absence of target analyte. Under optimized experimental conditions, TAMRA-labeled strand quenched the fluorescence response of FAM-labeled aptamer due to the noncovalent interaction between the two DNA strands. The binding of AFB1 induced the complex formation and weakened the interaction between FAM-labeled aptamer and TAMRA-labeled complementary strand, resulting in the fluorescence recovery. By using this principle concept, an assay was constructed for the detection of AFB1. The method exhibited good sensitivity, good selectivity with a limit of detection of 0.2 ng ml(-1), and a wide linear range from 0.25 to 32 ng ml(-1). For real sample application, the aptasensors were tested in beer and wine samples, with good recovery rates obtained for AFB1 detection.

Mechanistic studies for the role of cellular nucleic-acid-binding protein (CNBP) in regulation of c-myc transcription.

BACKGROUND: Guanine-rich sequence of c-myc nuclease hypersensitive element (NHE) III1 is known to fold in G-quadruplex and subsequently serves as a transcriptional silencer. Cellular nucleic-acid-binding protein (CNBP), a highly conserved zinc-finger protein with multiple biological functions, could bind to c-myc NHE III1 region, specifically to the single strand G-rich sequence. METHODS: In the present study, a variety of methods, including cloning, expression and purification of protein, EMSA, CD, FRET, Ch-IP, RNA interference, luciferase reporter assay, SPR, co-immunoprecipitation, and co-transfection, were applied to investigate the mechanism for the role of CNBP in regulating c-myc transcription. RESULTS: We found that human CNBP specifically bound to the G-rich sequence of c-myc NHE III1 region both in vitro and in cellulo, and subsequently promoted the formation of G-quadruplex. CNBP could induce a transient decrease followed by an increase in c-myc transcription in vivo. The interaction of CNBP with NM23-H2 was responsible for the increase of c-myc transcription. CONCLUSIONS: Based on above experimental results, a new mechanism, involving G-quadruplex related CNBP/NM23-H2 interaction, for the regulation of c-myc transcription was proposed. GENERAL SIGNIFICANCE: These findings indicated that the regulation of c-myc transcription through NHE III1 region might be governed by mechanisms involving complex protein-protein interactions, and suggested a new possibility of CNBP as a potential anti-cancer target based on CNBP's biological function in c-myc transcription.

Effect of tamra bhasma (calcined copper) on ponderal and biochemical parameters.

INTRODUCTION: Tamra Bhasma (TB) and its forms like Somnathi Tamra Bhasma (STB), etc., are in vogue since centuries in Ayurveda. The present study is carried out to evaluate the effect of TB and STB in different dose levels on ponderal and biochemical parameters in wistar strain albino rats to provide scientific basis for its safety profile. MATERIALS AND METHODS: TB and STB were prepared as per the classical guidelines and administered to wistar strain albino rats for 45 consecutive days. Blood was collected and rats were sacrificed on the 46(th) day. Ponderal and biochemical parameters were studied. RESULTS: Results showed significant decrease in serum cholesterol, High Density Lipoprotein (HDL) cholesterol, triglycerides, total protein, and serum alkaline phosphatase levels. Comparatively, all the differences in between the groups are insignificant and no pathological changes at ponderal and biochemical levels were observed. CONCLUSION: Based on these observations, it can be said that these formulations can be safely used in cases of hyperlipidemia.

Assessment of genotoxic potential of hridayarnava rasa (a herbo-mineralo-metallic ayurvedic formulation) using chromosomal aberration and sperm abnormality assays.

OBJECTIVES: Herbo-mineral formulations are being successfully used in therapeutics since centuries. But recently, they came under the scanner for their metallic contents especially the presence of heavy metals. Hence it is the need of the hour to assess and establish the safety of these formulations through toxicity studies. In line with the various toxicity studies that are being carried out, Government of India expressed the need for conducting genotoxicity studies of different metal- or mineral-based drugs. Till date very few Ayurvedic herbo-mineral formulations have been studied for their genotoxic potential. The present study is aimed to evaluate the genotoxic potential of Hridayarnava Rasa. MATERIALS AND METHODS: It was prepared as per classical guidelines and administered to Swiss albino mice for 14 consecutive days. Chromosomal aberration and sperm abnormality assay were done to evaluate the genotoxic potential of the test drugs. Cyclophosphamide (CP) was taken as positive group and results were compared. RESULTS: All treated groups exhibited significant body weight gain in comparison to CP group. Results revealed no structural deformity in the above parameters in comparison to the CP-treated group. CONCLUSION: Reported data showed that both tested samples of Hridayarnava Rasa does not possess genotoxic potential under the experimental conditions and can be safely used.

Product development and characterization of the Ayurvedic herbo-mineral-metallic compound- Hridayarnava Rasa.

BACKGROUND: Herbo-mineral-metallic formulations are an inseparable part of the Ayurveda system of traditional medicine. Hridayarnava Rasa (HR) is a preparation containing metals like copper, sulphur, and mercury in processed forms and other herbs that do not produce toxic effects and adverse drug reactions when taken in appropriate dosage. Ayurveda practitioners use it in treating cardiac diseases like hypertension, cardiotoxicity and many more. The rasa-aushadhis possess characteristics such as rapid efficacy, little dosage required, and extensive therapeutic applicability. Hridayarnava Rasa [AFI Part-1, 20:55] has been employed for the treatment of various diseases from ancient times. A systematic study of these formulations manufacturing is required to maintain their quality, safety, and efficacy is a need of time to protect the immense faith of patients in Ayurveda. OBJECTIVES: The present study aimed to prepare HR as per standard operating procedures mentioned in the classical text and to characterize it physio-chemically using advanced analytical techniques. MATERIALS AND METHODS: HR was prepared and physicochemical analyses and assay of elements by ICP-AES were carried out as per Ayurvedic Pharmacopoeia of India (API). Powder X-ray diffraction (XRD), Field emission gun scanning electron microscopy with energy dispersive spectroscopy (FEG SEM, EDAX), CHNS-O analysis, Fourier Transform Infrared Spectroscopy (FTIR), Thermo-gravimetric analysis (TGA), Particle size distribution analysis (PSD) was carried out. RESULTS: The XRD analysis of HR showed the presence of unreacted sulphur and sulfides of copper and mercury. FEG SEM revealed the particles in the form of aggregates as nanocrystallites in the range of 100-1000 nm. Elemental analysis showed the presence of copper, sulphur, and mercury in major, along with traces of iron, calcium, sodium, potassium, and magnesium. In FTIR analysis, 18 peaks were observed, which strongly suggests the presence of various organic groups. In the TGA, four peaks were seen, which can be attributed to sulphur volatilization and oxidative changes in mercury. In PSD analysis, 50% of the material was found below 16.40 µm. CONCLUSION: To establish a piece of fundamental knowledge and ensure uniformity of these rasa-aushadhis, it is imperative to conduct an analysis of their characteristics as per classical texts and modern analytical techniques. Additionally, it is crucial to investigate the significance of each procedural step included in the preparation process. The inferences drawn are helpful as an essential aid for quality assurance and standardization of this herbo-mineral-metallic formulation.

Steady-state and time-resolved fluorescence of tetramethylrhodamine attached to DNA: correlation with DNA sequences.

Time-resolved fluorescence as well as steady-state absorption and fluorescence were detected in order to study the interactions between tetramethylrhodamine (TAMRA) and DNA when TAMRA was covalently labeled on single- and double-stranded oligonucleotides. Fluorescence intensity quenching and lifetime changes were characterized and correlated with different DNA sequences. The results demonstrated that the photoinduced electron transfer interaction between guanosine residues and TAMRA introduced a short lifetime fluorescence component when guanosine residues were at the TAMRA-attached terminal of the DNA sequences. The discrepancy of two-state and three-state models in previous studies was due to the DNA sequence selection and sensitivity of techniques used to detect the short lifetime component. The results will help the design of fluorescence-based experiments related to a dye labeled probe.

A case series sharing novel experience of treating viral pandemic cases of morbid, mid aged, mild, moderate & severe grade with only Ayurvedic Medicines.

In ongoing viral pandemic named as COVID-19 also Severe Acute Respiratory illness (SARI) or Flue Like illness (FLI) reported surging in many cities of India and many of the patients opted for traditional medicine, in spite of they have been given a option of contemporary line of treatment instructed by health authorities, they opted to take traditional indian medicine that is Ayurvedic medicine. Present case series is a same novel experience of early diagnosing and treating mid aged, morbid individuals who took only Ayurvedic treatment and could get out of the disease without any complications. This case series had 10 mid aged, morbid patients with maximum symptoms of COVID-19 disease and their hemogram and CRP was suggestive of moderate to severe type COVID-19/FLI/SARI. They were diagnosed by contemporary methods of pathology and treated with Ayurvedic classical medicines Tamra Sinduradi Yoga and Bhunimbadi Kwath for 20 days along with continuing the medicines for their ongoing morbidities. All 10 patients showed recoveries without any complications, they reduced their all symptoms, drastic reduction in their CRP and corrections in their hemograms were observed and also they showed any complications neither physically nor in their pathological tests. Hence it can be concluded that early diagnosis and treating it with Ayurvedic medicine can manage viral pandemic issue in a very successful way.

Efficacy of the new therapeutic approach in curing malignant neoplasms on the model of human glioblastoma.

OBJECTIVE: Glioma is a highly invasive tumor, frequently disposed in essential areas of the brain, which makes its surgical excision extremely difficult; meanwhile adjuvant therapy remains quite ineffective. METHODS: In the current report, a new therapeutic approach in curing malignant neoplasms has been performed on the U87 human glioblastoma model. This approach, termed "Karanahan", is aimed at the eradication of cancer stem cells (CSCs), which were recently shown to be capable of internalizing fragments of extracellular double-stranded DNA. After being internalized, these fragments interfere in the process of repairing interstrand cross-links caused by exposure to appropriate cytostatics, and such an interference results either in elimination of CSCs or in the loss of their tumorigenic potency. Implementation of the approach requires a scheduled administration of cytostatic and complex composite double-stranded DNA preparation. RESULTS: U87 cells treated in vitro in accordance with the Karanahan approach completely lost their tumorigenicity and produced no grafts upon intracerebral transplantation into immunodeficient mice. In SCID mice with developed subcutaneous grafts, the treatment resulted in reliable slowing down of tumor growth rate (P < 0.05). In the experiment with intracerebral transplantation of U87 cells followed by surgical excision of the developed graft and subsequent therapeutic treatment, the Karanahan approach was shown to reliably slow down the tumor growth rate and increase the median survival of the mice twofold relative to the control. CONCLUSIONS: The effectiveness of the Karanahan approach has been demonstrated both in vitro and in vivo in treating developed subcutaneous grafts as well as orthotopic grafts after surgical excision of the tumor.

Design and Synthesis of Fluorescent Methylphenidate Analogues for a FRET-Based Assay of Synapsin III Binding.

We previously described synapsin III (Syn III) as a synaptic phosphoprotein that controls dopamine release in cooperation with α-synuclein (aSyn). Moreover, we found that in Parkinson's disease (PD), Syn III also participates in aSyn aggregation and toxicity. Our recent observations point to threo-methylphenidate (MPH), a monoamine re-uptake inhibitor that efficiently counteracts the freezing-gait characteristic of advanced PD, as a ligand for Syn III. We have designed and synthesised two different fluorescently labelled MPH derivatives, one with Rhodamine Red (RHOD) and one with 5-carboxytetramethylrhodamine (TAMRA), to be used for assessing MPH binding to Syn III by FRET. TAMRA-MPH exhibited the ideal characteristics to be used as a FRET acceptor, as it was able to enter into the SK-N-SH cells and could interact specifically with human green fluorescent protein (GFP)-tagged Syn III but not with GFP alone. Moreover, the uptake of TAMRA-MPH and co-localization with Syn III was also observed in primary mesencephalic neurons. These findings support that MPH is a Syn III ligand and that TAMRA-conjugated drug molecules might be valuable tools to study drug-ligand interactions by FRET or to detect Syn III in cytological and histological samples.

Study on physical properties of Ayurvedic nanocrystalline Tamra Bhasma by employing modern scientific tools.

BACKGROUND: Tamra Bhasma is derived from metallic copper that is recommended for different ailments of liver and spleen, dropsy, abdominal pain, heart disease, colitis, tumors, anemia, loss of appetite, tuberculosis, as well as eye problems. OBJECTIVES: The knowledge of crystallite size and active ingredients in Bhasma materials is limited restricting its use as nanomedicine in the modern era. Also, the 2015 Nobel prize in medicine has motivated many researchers towards traditional medicines. Therefore, the different chemical and physical properties of prepared Tamra Bhasma has been studied by modern experimental tools (XRD, VSM, SEM, FTIR and PL spectrometer) and the preliminary testing of Tamra Bhasma nanoparticles was examined on bacteria. MATERIALS AND METHODS: Bhasma is prepared by metals and minerals using three step procedures e.g. Shodhana, Bhavana and Marana. In the present work, for the preparation of Tamra Bhasma, pulverized copper wire was used and prepared by the principle of Puta (incineration) in an Electrical Muffle Furnace (EMF). RESULTS: X-ray diffraction analysis and scanning electron microscopy results revealed that the crystallite size of Bhasma powder was less than 100 nm and nanocrystallites of aglomerated size in micrometer. Magnetometer measurement supports its medicinal value. Photoluminescence (PL) properties of nanocrystalline Bhasma powder was investigated in UV-NIR region and shows luminescence in visible region. The antimicrobial study of Tamra Bhasma shows effectiveness on bacteria and, may be useful to control the bacterial infection disease. CONCLUSION: Scientific data obtained using modern scientific tools and evidence would support in utilizing the ancient Indian wisdom of Ayurveda for the development of newer drugs as a modern nanomedicine and in other possible technological applications.

Features of monocyte-derived dendritic cells encompassing a rare subpopulation of cells that are capable of natural internalization of extracellular dsDNA.

The present study demonstrates that monocyte-derived dendritic cells (moDCs) produced in vitro using a GM-CSF and IFN-α differentiation protocol encompass a rare (∼5%) subpopulation of cells showing classical dendritic cell morphology and capable of natural internalization of extracellular self-DNA. We established that DEFB, HMGB1, LL-37 and RAGE antigens, which mediate the process of DNA internalization, are expressed on the surface of moDCs similar to plasmacytoid dendritic cells. However, in constrast to the latter subpopulation, these cells do not produce interleukin (IL)-37. Nonetheless, the process of DNA internalization was not in direct relation to the presence of the above antigens on the surface of these cells. Dendritic cells were sorted into total and non-DNA-internalizing populations and cytokine production was analyzed at 24-48 hours post-DNA treatment. We show that massive secretion of cytokines by dendritic cells is associated with the dsDNA-internalizing subpopulation. A total pool of IFN-moDCs secrete pro-inflammatory "first-wave" cytokines (IL-2, IL-6, IL-8, TNF-α) at both 24 and 48 hours time points. The anti-inflammatory cytokines IL-4 and IL-10 were found to be modestly induced, whereas GM-CSF, G-CSF, and IFN-γ production was strongly induced. Treatment of moDCs with dsDNA results in the up-regulated transcription of IFN-α, IFN-ß, IFN-γ, IL-8, IL-10, and VEGF by 6 hours. Combined dsDNA + chloroquine treatment has a synergistic effect on transcription of only one of the genes tested, with the pro-inflammatory cytokine IFN-ß displaying the strongest fold induction by 24 hours.