LncRNA HULC augments high glucose-associated pancreatic cancer progression and drug resistance by enhancing YAP activity and autophagy.

BACKGROUND INFORMATION: One of the confounding factors in pancreatic cancer (PC) pathogenesis is hyperglycemia. The molecular mechanism by which high glucose (HG) influences PC severity is poorly understood. Our investigation delved into the impact of lncRNA highly upregulated in liver cancer (HULC) and its interaction with yes-associated protein (YAP) in regulating the fate of pancreatic ductal adenocarcinoma cells (PDAC) under HG-induced conditions. PDAC cells were cultured under normal or HG conditions. We thereafter measured the effect of HG on the viability of PDAC cells, their migration potential and drug resistance properties. The lncRNAs putatively dysregulated in PC and diabetes were shortlisted by bioinformatics analysis followed by wet lab validation of function. RESULTS: HG led to enhanced proliferation and drug refractoriness in PDAC cells. HULC was identified as one of the major deregulated lncRNAs following bioinformatics analysis. HULC was found to regulate the expression of the potent transcriptional regulator - YAP through selective histone modifications at the YAP promoter. siRNA-mediated ablation of HULC resulted in a concurrent decrease in YAP transcriptional activity. Importantly, HULC and YAP were found to co-operatively regulate the cellular homeostatic process autophagy, thus inculcating drug resistance and proliferative potential in PDAC cells. Moreover, inhibition of autophagy or YAP led to a decrease in HULC levels, suggesting the existence of an inter-regulatory feedback loop. CONCLUSIONS: We observed that HG triggers aggressive properties in PDAC cells. Mechanistically, up-regulation of lncRNA HULC resulted in activation of YAP and differential regulation of autophagy coupled to increased proliferation of PDAC cells. SIGNIFICANCE: Inhibition of HULC and YAP may represent a novel therapeutic strategy for PDAC. Furthermore, this study portrays the intricate molecular interplay between HULC, YAP and autophagy in PDAC pathogenesis.

Photodynamic Therapy Induced Mitochondrial Targeting Strategies for Cancer Treatment: Emerging Trends and Insights.

The perpetuity of cancer prevalence at a global level calls for development of novel therapeutic approaches with improved targetability and reduced adverse effects. Conventional cancer treatments have a multitude of limitations such as nonselectivity, invasive nature, and severe adverse effects. Chemotherapy is also losing its efficacy because of the development of multidrug resistance in the majority of cancers. To address these issues, selective targeting-based approaches are being explored for an effective cancer treatment. Mitochondria, being the moderator of a majority of crucial cellular pathways like metabolism, apoptosis, and reactive oxygen species (ROS) homeostasis, are an effective targeting site. Mitochondria-targeted photodynamic therapy (PDT) has arisen as a potential approach in this endeavor. By designing photosensitizers (PSs) that preferentially accumulate in the mitochondria, PDT offers a localized technique to induce cytotoxicity in cancer cells. In this review, we intend to explore the crucial principles and challenges associated with mitochondria-targeted PDT, including variability in mitochondrial function, mitochondria-specific PSs, targeted nanocarrier-based monotherapy, and combination therapies. The hurdles faced by this emerging strategy with respect to safety, optimization, clinical translation, and scalability are also discussed. Nonetheless, mitochondria-targeted PDT exhibits a significant capacity in cancer treatment, especially in combination with other therapeutic modalities. With perpetual research and technological advancements, this treatment strategy is a great addition to the arsenal of cancer treatment options, providing better tumor targetability while reducing the damage to surrounding healthy tissues. This review emphasizes the current status of mitochondria-targeted PDT, limitations, and future prospects in its pursuit of safe and efficacious cancer therapy.

One-Pot Synthesis of Vinylogous Cyano Aminoaryls (VinCAs) as Benzenic Fluorophores: Tailoring Diverse Emission Colors for White Light Emitting Materials and Cell Imaging.

Donor-acceptor-based organic small molecules with an electronic push-pull effect can demonstrate intramolecular charge transfer to show interesting photoluminescence properties. This is an essential criterion for designing fluorogenic probes for cell imaging studies and the development of organic light-emitting diodes. Now, to design such optical materials sometimes it is necessary to tune the band gap by controlling the energies of the highest occupied molecular orbital and lowest unoccupied molecular orbital. Typically, the band gaps could be modulated by installing unsaturated handles between electron-rich donors and electron-deficient acceptors. However, these methods are often synthetically and economically challenging due to the involvement of expensive catalysts and difficult reaction setups. In our present study, we show a straightforward, cost-effective method for obtaining a series of donor-acceptor-type Vinylogous Cyano Aminoaryls (VinCAs) with diverse emission colors. Further studies reveal that these VinCAs can serve as effective cell imaging agents, showcasing potential use in chemical biology. Additionally, these molecules could be further used to generate white light emission (WLE), showing their potential utility in advanced lighting technologies.

Chloroquine induces transitory attenuation of proliferation of human lung cancer cells through regulation of mutant P53 and YAP.

BACKGROUND: Non-small cell lung carcinoma (NSCLC) is the most common cause of cancer-associated deaths worldwide. Though recent development in targeted therapy has improved NSCLC prognosis, yet there is an unmet need to identify novel causative factors and appropriate therapeutic regimen against NSCLCs. METHODS AND RESULTS: In this study, we identify key molecular factors de-regulated in NSCLCs. Analyze their expression by real-time PCR and immunoblot; map their localization by immuno-fluorescence microscopy. We further propose an FDA approved drug, chloroquine (CQ) that affects the function of the molecular factors and hence can be repurposed as a therapeutic strategy against NSCLCs. Available NSCLC mutation data reflects a high probabilistic chance of patients harboring a p53 mutation, especially a gain of function (GOF)-R273H mutation. The GOF-P53 mutation enables the P53 protein to potentially interact with non-canonical protein partners facilitating oncogenesis. In this context, analysis of existing transcriptomic data from R273H-P53 expressing cells shows a concomitant up-regulation of Yes-associated protein (YAP) transcriptional targets and its protein partner TEAD1 in NSCLCs, suggesting a possible link between R273H-P53 and YAP. We therefore explored the inter-dependence of R273H-P53 and YAP in NSCLC cells. They were found to co-operatively regulate NSCLC proliferation. Genetic or pharmacological inhibition of YAP and GOF-P53 resulted in sensitization of NSCLC cells. Further analysis of pathways controlled by GOF-P53 and YAP showed that they positively regulate the cellular homeostatic process- autophagy to mediate survival. We hence postulated that a modulation of autophagy might be a potent strategy to curb proliferation. In accordance to above, autophagy inhibition, especially with the FDA-approved drug- chloroquine (CQ) resulted in cytoplasmic accumulation and reduced transcriptional activity of GOF-P53 and YAP, leading to growth arrest of NSCLC cells. CONCLUSION: Our study highlights the importance of GOF-P53 and YAP in NSCLC proliferation and proposes autophagy inhibition as an efficient strategy to attenuate NSCLC tumorigenesis.

Mechanical microscopy of cancer cells: TGF-ß induced epithelial to mesenchymal transition corresponds to low intracellular viscosity in cancer cells.

Viscosity is an essential parameter that regulates bio-molecular reaction rates of diffusion-driven cellular processes. Hence, abnormal viscosity levels are often associated with various diseases and malfunctions like cancer. For this reason, monitoring intracellular viscosity becomes vital. While several approaches have been developed for in vitro and in vivo measurement of viscosity, analysis of intracellular viscosity in live cells has not yet been well realized. Our research introduces a novel, natural frequency-based, non-invasive method to determine the intracellular viscosity in cells. This method can not only efficiently analyze the differences in intracellular viscosity post modulation with molecules like PEG or glucose but is sensitive enough to distinguish the difference in intra-cellular viscosity among various cancer cell lines such as Huh-7, MCF-7, and MDAMB-231. Interestingly, TGF-ß a cytokine reported to induce epithelial to mesenchymal transition (EMT), a feature associated with cancer invasiveness resulted in reduced viscosity of cancer cells, as captured through our method. To corroborate our findings with existing methods of analysis, we analyzed intra-cellular viscosity with a previously described viscosity-sensitive molecular rotor-based fluorophore-TPSII. In parity with our position sensing device (PSD)-based approach, an increase in fluorescence intensity was observed with viscosity enhancers, while, TGF-ß exposure resulted in its reduction in the cells studied. This is the first study of its kind that attempts to characterize differences in intracellular viscosity using a novel, non-invasive PSD-based method.

Tuning axial and lateral localization precision in 3D super-resolution microscopy with variable astigmatism.

Astigmatism imaging is a three-dimensional (3D) single molecule fluorescence microscopy approach that yields super-resolved spatial information on a rapid time scale from a single image. It is ideally suited for resolving structures on a sub-micrometer scale and temporal behavior in the millisecond regime. While traditional astigmatism imaging utilizes a cylindrical lens, adaptive optics enables the astigmatism to be tuned for the experiment. We demonstrate here how the precisions in x, y, and z are inter-linked and vary with the astigmatism, z-position, and photon level. This experimentally driven and verified approach provides a guide for astigmatism selection in biological imaging strategies.

Exploring the extensive crosstalk between the antagonistic cytokines- TGF-ß and TNF-α in regulating cancer pathogenesis.

A plethora of cytokines are produced in the tumor microenvironment (TME) those play a vital role in cancer prognosis. Though it is completely contextual, cytokines produced from an inflammatory micro-environment can either modulate cancer progression at early stages of tumor development or in later stages cytokine derived cues can in turn control tumor cell invasion and metastasis. Therefore, understanding the crosstalk between the key cytokines regulating cancer prognosis is critical for the development of an effective therapy. In this regard, the role of transforming growth factor-beta (TGF-ß) in cancer is controversially discussed in general inhibition of TGF-ß promotes de novo tumorigenesis whereas paradoxically, TGF-ß can promote malignancy in already established tumors. Another important cytokine, TNF-α have intense crosstalk with TGF-ß from the fact that in a non-cancer context, TGF-ß promotes fibrosis whereas TNF-α has anti-fibrotic activity. We have recently reported that TGF-ß-induced differentiation of epithelial cells to mesenchymal type is suppressed by TNF-α through regulation of cellular homeostatic machinery- autophagy. Moreover, there are also rare reports of synergy between these two cytokines as well. The crosstalk between TGF-ß and TNF-α is not only limited to regulating cancer cell differentiation and proliferation but also includes involvement in cell death. In this review, we hence summarize the molecular mechanisms by which these two important cytokines, TGF-ß and TNF-α control cancer prognosis.

Verteporfin disrupts multiple steps of autophagy and regulates p53 to sensitize osteosarcoma cells.

BACKGROUND: Osteosarcoma (OS) is a malignant tumor of the bone mostly observed in children and adolescents. The current treatment approach includes neoadjuvant and adjuvant chemotherapy; however, drug resistance often hinders therapy in OS patients. Also, the post-relapse survival of OS patients is as low as 20%. We therefore planned to understand the molecular cause for its poor prognosis and design an appropriate therapeutic strategy to combat the disease. METHODS: We analyzed OS patient dataset from Gene Expression Omnibus (GEO) and identified the differentially expressed genes and the top deregulated pathways in OS. Subsequently, drugs targeting the major de-regulated pathways were selected and the following assays were conducted- MTT assay to assess cytotoxicity of drugs in OS cells; immunoblotting and immunostaining to analyze key protein expression and localization after drug treatment; LysoTracker staining to monitor lysosomes; Acridine Orange to label acidic vesicles; and DCFDA to measure Reactive Oxygen Species (ROS). RESULTS: The differential gene expression analysis from OS patient dataset implicated the striking involvement of cellular processes linked to autophagy and protein processing in the development of OS. We therefore selected the FDA approved drugs, chloroquine (CQ) and verteporfin (VP) known for autophagy inhibitory and proteotoxic functions to explore against OS. Importantly, VP, but not CQ, showed an extensive dose-dependent cytotoxicity. It resulted in autophagy disruption at multiple steps extending from perturbation of early autophagic processes, inhibition of autophagic flux to induction of lysosomal instability. Interestingly, VP treated protein lysates showed a ROS-dependent high molecular weight (HMW) band when probed for P62 and P53 protein. Further, VP triggered accumulation of ubiquitinated proteins as well. Since VP had a pronounced disruptive effect on cellular protein homeostasis, we explored the possibility of simultaneous inhibition of the ubiquitin-proteasomal system (UPS) by MG-132 (MG). Addition of a proteasomal inhibitor significantly aggravated VP induced cytotoxicity. MG co-treatment also led to selective targeting of P53 to the lysosomes. CONCLUSION: Herein, we propose VP and MG induce regulation of autophagy and protein homeostasis which can be exploited as an effective therapeutic strategy against osteosarcoma.

A global transcriptomic pipeline decoding core network of genes involved in stages leading to acquisition of drug-resistance to cisplatin in osteosarcoma cells.

MOTIVATION: Traditional cancer therapy is focused on eradicating fast proliferating population of tumor cells. However, existing evidences suggest survival of sub-population of cancer cells that can resist chemotherapy by entering a 'persister' state of minimal growth. These cells eventually survive to produce cells resistant to drugs. The identifying of appropriate targets that can eliminate the drug-tolerant 'persisters' remains a challenge. Hence, a deeper understanding of the distinctive genetic signatures that lead to resistance is of utmost importance to design an appropriate therapy. RESULTS: In this study, deep-sequencing of mRNA was performed in osteosarcoma (OS) cells, exposed to the widely used drug, cisplatin which is an integral part of current treatment regime for OS. Transcriptomic analysis was performed in (i) untreated OS; (ii) persister sub-population of cells post-drug shock; (iii) cells which evade growth bottleneck and (iv) drug-resistant cells obtained after several rounds of drug shock and revival. The transcriptomic signatures and pathways regulated in each group were compared; the transcriptomic pipeline to the acquisition of resistance was analyzed and the core network of genes altered during the process was delineated. Additionally, our transcriptomic data were compared with OS patient data obtained from Gene Ontology Omnibus. We observed a sub-set of genes to be commonly expressed in both data sets with a high correlation (0.81) in expression pattern. To the best of our knowledge, this study is uniquely designed to understand the series of genetic changes leading to the emergence of drug-resistant cells, and implications from this study have a potential therapeutic impact. AVAILABILITY AND IMPLEMENTATION: All raw data can be accessed from GEO database (https://www.ncbi.nlm.nih.gov/geo/) under the GEO accession number GSE86053. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Time-dependent enhancement of fluorescence from Rhodobacter capsulatus SB1003 and its critical dependence on concentration temperature and static magnetic field.

Continuous exposure of 395 nm light increases the fluorescence emission intensity of photosynthetic purple non-sulphur bacteria, Rhodobacter capsulatus (SB1003). We show that such an increase in fluorescence emission of extracellular pigment complexes (PC) from these photosynthetic bacteria depends on the concentration of the pigment and temperature and can also be modulated by the static magnetic field. The time-dependent enhanced emission disappears either at or below 300 K or below a threshold sample concentration (0.1 mg/ml). The enhanced emission reappears at this condition (T < 278 K) if a static magnetic field (395 mT) is introduced during fluorescence measurement. The time dependence of emission is expressed in terms of a first-order rate constant, k = dF/(Fdt). The sign of k shifts from positive to negative as PC concentration is lowered than a threshold value, implying onset of fluorescence decay (k < 0) rather than amplification (k > 0). At PC concentration higher than a threshold, k becomes negative if the temperature is lowered. But, surprisingly, at low temperature, a static magnetic field reverts the k value to positive. We explain the logical nature of k-switching and photo-dynamics of the aforesaid microbial fluorescence emission by aggregation of protoporphyrin rings present in the PC. While the simultaneous presence of decay in fluorescence and susceptibility to static magnetic field suggests the dominance of triplet states at low temperatures, the process is reversed by SMF-induced removal of spin degeneracy. At higher temperatures, the optical excitability and lack of magnetic response suggest the dominance of singlet states. We propose that the restructuring of the singlet-triplet distribution by intersystem crossing may be the basis of this logical behaviour. In context with microbial function, time-dependent enhancement of fluorescence also implies relay of red photons to the neighbouring microbes not directly exposed to the incident radiation, thus serving as an indirect photosynthetic regulator.

Transcriptomic analysis associated with reversal of cisplatin sensitivity in drug resistant osteosarcoma cells after a drug holiday.

BACKGROUND: Resistance to chemotherapy is one of the major hurdles in current cancer therapy. With the increasing occurrence of drug resistance, a paradigm shift in treatment strategy is required. Recently "medication vacation" has emerged as a unique, yet uncomplicated strategy in which withdrawal of drug pressure for certain duration allowed tumor cells to regain sensitivity to the drug. However, little is known about the molecular alterations associated with such an outcome. METHODS: In this study, human osteosarcoma (OS) cells resistant to the extensively used drug cisplatin, were withdrawn from drug pressure, and thereafter cytotoxic response of the cells to the drug was evaluated. We further performed next-generation RNA sequencing and compared transcriptome between parental (OS), resistant (OS-R) and the drug withdrawn (OS-DW) cells. Differentially expressed transcripts were identified, and biological association network (BAN), gene ontology (GO) and pathway enrichment analysis of the differentially regulated transcripts were performed to identify key events associated with withdrawal of drug pressure. RESULTS: Following drug withdrawal, the sensitivity of the cells to the drug was found to be regained. Analysis of the expression profile showed that key genes like, IRAK3, IL6ST, RELA, AKT1, FKBP1A and ADIPOQ went significantly down in OS-DW cells when compared to OS-R. Also, genes involved in Wnt signaling, PI3K-Akt, Notch signaling, and ABC transporters were drastically down-regulated in OS-DW cells compared to OS-R. Although, a very small subset of genes maintained similar expression pattern between OS, OS-R and OS-DW, nonetheless majority of the transcriptomic pattern of OS-DW was distinctively different and unique in comparison to either the drug sensitive OS or drug resistant OS-R cells. CONCLUSION: Our data suggests that though drug withdrawal causes reversal of sensitivity, the transcriptomic pattern does not necessarily show significant match with resistant or parental control cells. We strongly believe that exploration of the molecular basis of drug holiday might facilitate additional potential alternative treatment options for aggressive and resistant cancers.

Resistance a major hindrance to chemotherapy in hepatocellular carcinoma: an insight.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer mortality, accounting for almost 90% of total liver cancer burden. Surgical resection followed by adjuvant and systemic chemotherapy are the most meticulously followed treatment procedures but the complex etiology and high metastatic potential of the disease renders surgical treatment futile in majority of the cases. Another hindrance to the scenario is the acquired resistance to drugs resulting in relapse of the disease. Hence, to provide insights into development of novel therapeutic targets and diagnostic biomarkers, this review focuses on the various molecular mechanisms underlying chemoresistance in HCC. We have provided a comprehensive summary of the various strategies adopted by HCC cells, extending from apoptosis evasion, autophagy activation, drug expulsion to epigenetic transformation as modes of therapy resistance. The role of stem cells in imparting chemoresistance is also discussed. Furthermore, the review also focuses on how this knowledge might be exploited for the development of an effective, prospective therapy against HCC.

Exploring the hidden potential of a benzothiazole-based Schiff-base exhibiting AIE and ESIPT and its activity in pH sensing, intracellular imaging and ultrasensitive & selective detection of aluminium (Al3+).

In this article, we tried to redefine the unexplored potential of a benzothiazole type of Schiff-base (OM), which was identified as an AIE-active molecule that exhibits excited-state intramolecular proton transfer (ESIPT). Interestingly, this compound shows ultra-sensitivity and selectivity in the detection of Al(iii) (12 pM; 456 ppt). The OM was capable of pH sensing and was also tested for internalization in cancerous cells for intracellular imaging. Computational modeling was performed and the results were in good agreement with the experimental UV-Vis spectrum and the energy gap obtained in basic and acidic media.

Synthesis and evaluation of bile acid amides of [Formula: see text]-cyanostilbenes as anticancer agents.

A series of amino-substituted [Formula: see text]-cyanostilbene derivatives and their bile acid (cholic and deoxycholic acid) amides were designed and synthesized. A comparative study on the anticancer and antibacterial activity evaluation on the synthesized analogs was carried against the human osteosarcoma (HOS) cancer cell line, and two gram -ve (E. coli and S. typhi) and two gram [Formula: see text]ve (B. subtilis and S. aureus) bacterial strains. All the cholic acid [Formula: see text]-cyanostilbene amides showed an [Formula: see text] in the range 2-13 [Formula: see text] against human osteosarcoma cells (HOS) with the most active analog (6g) possessing an [Formula: see text] of [Formula: see text]. One of the amino-substituted [Formula: see text]-cyanostilbene, 4e, was found to possess an [Formula: see text] of [Formula: see text]. An increase in the number of cells at the sub-[Formula: see text] phase of the cell was observed in the in vitro cell cycle analysis of two most active compounds in the series (4e, 6g) suggesting a clear indication toward induction of apoptotic cascade. With respect to antibacterial screening, amino-substituted [Formula: see text]-cyanostilbenes were found to be more active than their corresponding bile acid amides. The synthesized compounds were also subjected to in silico study to predict their physiochemical properties and drug-likeness score.

Split-ubiquitin yeast two-hybrid interaction reveals a novel interaction between a natural resistance associated macrophage protein and a membrane bound thioredoxin in Brassica juncea.

Natural resistance associated macrophage proteins (NRAMPs) are evolutionarily conserved metal transporters involved in the transport of essential and nonessential metals in plants. Fifty protein interactors of a Brassica juncea NRAMP protein was identified by a Split-Ubiquitin Yeast-Two-Hybrid screen. The interactors were predicted to function as components of stress response, signaling, development, RNA binding and processing. BjNRAMP4.1 interactors were particularly enriched in proteins taking part in photosynthetic or light regulated processes, or proteins predicted to be localized in plastid/chloroplast. Further, many interactors also had a suggested role in cellular redox regulation. Among these, the interaction of a photosynthesis-related thioredoxin, homologous to Arabidopsis HCF164 (High-chlorophyll fluorescence164) was studied in detail. Homology modeling of BjNRAMP4.1 suggested that it could be redox regulated by BjHCF164. In yeast, the interaction between the two proteins was found to increase in response to metal deficiency; Mn excess and exogenous thiol. Excess Mn also increased the interaction in planta and led to greater accumulation of the complex at the root apoplast. Network analysis of Arabidopsis homologs of BjNRAMP4.1 interactors showed enrichment of many protein components, central to chloroplastic/cellular ROS signaling. BjNRAMP4.1 interacted with BjHCF164 at the root membrane and also in the chloroplast in accordance with its proposed function related to photosynthesis, indicating that this interaction occurred at different sub-cellular locations depending on the tissue. This may serve as a link between metal homeostasis and chloroplastic/cellular ROS through protein-protein interaction.

Spectral mapping of 3D multi-cellular tumor spheroids: time-resolved confocal microscopy.

A tumor-like multi-cellular spheroid (3D) differs from a 2D cell in a number of ways. This is demonstrated using time resolved confocal microscopy. Two different tumor spheroids - HeLa (cervical cancer) and A549 (lung cancer) - are studied using 3 different fluorescent dyes - C153 (non-covalent), CPM (covalent) and doxorubicin (non-covalent, anti-cancer drug). The pattern of localization of these three fluorescent probes in the 3D tumor cell exhibits significant differences from that in the conventional 2D cells. For both the cells (HeLa and A549), the total uptake of doxorubicin in the 3D cell is much lower than that in the 2D cell. The uptake of doxorubicin molecules in the A549 spheroid is significantly different compared to the HeLa spheroid. The local polarity (i.e. emission maxima) and solvation dynamics in the 3D tumor cell differ from those in 2D cells. The covalent probe CPM exhibits intermittent fluorescence oscillations in the 1-2 s time scale. This is attributed to redox processes. These results may provide new insights into 3D tumors.

Amyloid beta peptides inside a reconstituted cell-like liposomal system: aggregation, FRET, fluorescence oscillations and solvation dynamics.

Aggregations of amyloid-beta (Aß) peptides were studied inside a reconstituted cell like liposomal system using time-resolved confocal microscopy. Fluorescence correlation spectroscopy (FCS) and confocal images indicate that Aß forms a very large aggregate in bulk and more efficiently, in the bilayer region of the liposome, respectively. The aggregates formed inside the liposome gradually migrate out to bulk water. FRET, from HiLyte Fluor 488 (covalently attached to an Aß peptide) to TRITC (tetramethylrhodamine isothiocyanate) covalently attached to a DHPE lipid present in the bilayer, reveals intermittent oscillations in the time scale of ∼0.5 s. This is attributed to the structural fluctuations of the membrane of the liposome. The solvation dynamics of Aß in monomer and in oligomeric state is studied by monitoring the emission of HiLyte Fluor 488. The solvation dynamics of the Aß monomer is similar to that of oligomeric aggregates in the liposome.

Structural relaxation of acridine orange dimer in bulk water and inside a single live lung cell.

Structural relaxation of the acridine orange (AO) dimer in bulk water and inside a single live lung cell is studied using time resolved confocal microscopy and molecular dynamics (MD) simulations. The emission maxima (λem (max)∼ 630 nm) of AO in a lung cancer cell (A549) and a non-cancer lung fibroblast cell (WI38) suggest that AO exists as a dimer inside the cell. Time-dependent red shift in emission maximum indicates dynamic relaxation of the AO dimer (in the excited state) with a time constant of 500-600 ps, both in bulk water and inside the cell. We have calculated the equilibrium relaxation dynamics of the AO dimer in the ground state using MD simulations and found a slow component of time scale ∼ 350 ps. The intra- and inter-molecular components of the total relaxation dynamics of the AO dimer reveal the presence of a slow component of the order of a few hundred picoseconds. Upon restricting intra-molecular dye dynamics by harmonic constraint between AO monomers, the slow component vanishes. Combining the experimental observations and MD simulation results, we ascribe the slow component of the dynamic relaxation of the AO dimer to the structural relaxation, namely, fluctuations in the distance between the two monomers and associated fluctuation in the number of water molecules.

Reactive nitrogen species regulate autophagy through ATM-AMPK-TSC2-mediated suppression of mTORC1.

Reactive intermediates such as reactive nitrogen species play essential roles in the cell as signaling molecules but, in excess, constitute a major source of cellular damage. We found that nitrosative stress induced by steady-state nitric oxide (NO) caused rapid activation of an ATM damage-response pathway leading to downstream signaling by this stress kinase to LKB1 and AMPK kinases, and activation of the TSC tumor suppressor. As a result, in an ATM-, LKB1-, TSC-dependent fashion, mTORC1 was repressed, as evidenced by decreased phosphorylation of S6K, 4E-BP1, and ULK1, direct targets of the mTORC1 kinase. Decreased ULK1 phosphorylation by mTORC1 at S757 and activation of AMPK to phosphorylate ULK1 at S317 in response to nitrosative stress resulted in increased autophagy: the LC3-II/LC3-I ratio increased as did GFP-LC3 puncta and acidic vesicles; p62 levels decreased in a lysosome-dependent manner, confirming an NO-induced increase in autophagic flux. Induction of autophagy by NO correlated with loss of cell viability, suggesting that, in this setting, autophagy was functioning primarily as a cytotoxic response to excess nitrosative stress. These data identify a nitrosative-stress signaling pathway that engages ATM and the LKB1 and TSC2 tumor suppressors to repress mTORC1 and regulate autophagy. As cancer cells are particularly sensitive to nitrosative stress, these data open another path for therapies capitalizing on the ability of reactive nitrogen species to induce autophagy-mediated cell death.

Fluorescence fluctuation of an antigen-antibody complex: circular dichroism, FCS and smFRET of enhanced GFP and its antibody.

The structure and dynamics of an antigen-antibody complex are monitored by circular dichroism (CD) spectroscopy, fluorescence correlation spectroscopy (FCS) and single molecule FRET (smFRET). In this work, the antigen is enhanced GFP (EGFP) and the antibody is anti-EGFP VHH-His6. From FCS measurements, the hydrodynamic radius (rH) of EGFP and its antibody (VHH-His6) is found to be 24 ± 2 Å and 18 ± 2 Å, respectively. For the antigen-antibody complex (EGFP:anti-EGFP VHH-His6), rH is 41 ± 3 Å. CD spectra indicate that the addition of guanidium hydrochloride (GdnHCl) causes unfolding of the antigen, its antibody and their complex, and a consequent increase in size is observed from FCS data. smFRET between EGFP (donor, D) and Alexa 594 (acceptor, A) bound to anti-EGFP VHH-His6 reveals a time dependent fluctuation in donor-acceptor distances. This suggests that the structure of the antigen-antibody complex is dynamic in nature and is not rigid.