AMPK activation reduces cancer cell aggressiveness via inhibition of monoamine oxidase A (MAO-A) expression/activity.

AIM: AMPK can be considered as an important target molecule for cancer for its unique ability to directly recognize cellular energy status. The main aim of this study is to explore the role of different AMPK activators in managing cancer cell aggressiveness and to understand the mechanistic details behind the process. MAIN METHODS: First, we explored the AMPK expression pattern and its significance in different subtypes of lung cancer by accessing the TCGA data sets for LUNG, LUAD and LUSC patients and then established the correlation between AMPK expression pattern and overall survival of lung cancer patients using Kaplan-Meire plot. We further carried out several cell-based assays by employing different wet lab techniques including RT-PCR, Western Blot, proliferation, migration and invasion assays to fulfil the aim of the study. KEY FINDINGS: SIGNIFICANCE: This study identifies the importance of AMPK activators as a repurposing agent for combating lung and colon cancer cell aggressiveness. It also suggests SRT-1720 as a potent repurposing agent for cancer treatment especially in NSCLC patients where a point mutation is present in LKB1.

Meta-analysis reveals obesity associated gut microbial alteration patterns and reproducible contributors of functional shift.

The majority of cohort-specific studies associating gut microbiota with obesity are often contradictory; thus, the replicability of the signature remains questionable. Moreover, the species that drive obesity-associated functional shifts and their replicability remain unexplored. Thus, we aimed to address these questions by analyzing gut microbial metagenome sequencing data to develop an in-depth understanding of obese host-gut microbiota interactions using 3329 samples (Obese, n = 1494; Control, n = 1835) from 17 different countries, including both 16S rRNA gene and metagenomic sequence data. Fecal metagenomic data from diverse geographical locations were curated, profiled, and pooled using a machine learning-based approach to identify robust global signatures of obesity. Furthermore, gut microbial species and pathways were systematically integrated through the genomic content of the species to identify contributors to obesity-associated functional shifts. The community structure of the obese gut microbiome was evaluated, and a reproducible depletion of diversity was observed in the obese compared to the lean gut. From this, we infer that the loss of diversity in the obese gut is responsible for perturbations in the healthy microbial functional repertoire. We identified 25 highly predictive species and 37 pathway associations as signatures of obesity, which were validated with remarkably high accuracy (AUC, Species: 0.85, and pathway: 0.80) with an independent validation dataset. We observed a reduction in short-chain fatty acid (SCFA) producers (several Alistipes species, Odoribacter splanchnicus, etc.) and depletion of promoters of gut barrier integrity (Akkermansia muciniphila and Bifidobacterium longum) in obese guts. Our analysis underlines SCFAs and purine/pyrimidine biosynthesis, carbohydrate metabolism pathways in control individuals, and amino acid, enzyme cofactor, and peptidoglycan biosynthesis pathway enrichment in obese individuals. We also mapped the contributors to important obesity-associated functional shifts and observed that these are both dataset-specific and shared across the datasets. In summary, a comprehensive analysis of diverse datasets unveils species specifically contributing to functional shifts and consistent gut microbial patterns associated to obesity.

Sumoylation and phosphorylation of PTEN boosts and curtails autophagy respectively by influencing cell membrane localisation.

Autophagy is involved in the entirety of cellular survival, homeostasis and death which becomes more self-evident when its dysregulation is implicated in several pathological conditions. PTEN positively regulates autophagy and like other proteins undergo post-translational modifications. It is crucial to investigate the relationship between PTEN and autophagy as it is generally observed to be negligible in PTEN deficient cancer cells. Here, we have shown that such modifications of PTEN namely sumoylation and phosphorylation upregulates and downregulates autophagy respectively. Transfection of plasmid containing full length PTEN in PTEN-negative prostate cancer cell line PC3, induced autophagy on further starvation. When a sumoylation-deficient mutant of PTEN was transfected and cells were put under similar starvation, a decline in autophagy was observed. On the other hand, cells transfected with phosphorylation-deficient mutant of PTEN showed elevated expression of autophagy. Contrarily, transfection with phosphorylation-mimicking mutant caused reduced expression of autophagy. On further analysis, it was detected that PTEN's association with the plasma membrane was under positive and negative influence from its sumoylation and phosphorylation respectively. This association is integral as it is the foremost site for PTEN to oppose PI3K/AKT pathway and consequently upregulate autophagy. Thus, this study indicates that sumoylation and phosphorylation of PTEN can control autophagy via its cell membrane association.

Characterization of pumilacidin, a lipopeptide biosurfactant produced from Bacillus pumilus NITDID1 and its prospect in bioremediation of hazardous pollutants.

Highly hydrophobic compounds like petroleum and their byproducts, once released into the environment, can persist indefinitely by virtue of their ability to resist microbial degradation, ultimately paving the path to severe environmental pollution. Likewise, the accumulation of toxic heavy metals like lead, cadmium, chromium, etc., in the surroundings poses an alarming threat to various living organisms. To remediate the matter in question, the applicability of a biosurfactant produced from the mangrove bacterium Bacillus pumilus NITDID1 (Accession No. KY678446.1) is reported here. The structural characterization of the produced biosurfactant revealed it to be a lipopeptide and has been identified as pumilacidin through FTIR, NMR, and MALDI-TOF MS. The critical micelle concentration of pumilacidin was 120 mg/L, and it showed a wide range of stability in surface tension reduction experiments under various environmental conditions and exhibited a high emulsification index of as much as 90%. In a simulated setup of engine oil-contaminated sand, considerable oil recovery (39.78%) by this biosurfactant was observed, and upon being added to a microbial consortium, there was an appreciable enhancement in the degradation of the used engine oil. As far as the heavy metal removal potential of biosurfactant is concerned, as much as 100% and 82% removal was observed for lead and cadmium, respectively. Thus, in a nutshell, the pumilacidin produced from Bacillus pumilus NITDID1 holds promise for multifaceted applications in the field of environmental remediation.

Strategic Application of Epigenetic Regulators for Efficient Neuronal Reprogramming of Human Fibroblasts.

Background and Objectives: Cellular reprogramming in regenerative medicine holds great promise for treating patients with neurological disorders. In this regard, small molecule-mediated cellular conversion has attracted special attention because of its ease of reproducibility, applicability, and fewer safety concerns. However, currently available protocols for the direct conversion of somatic cells to neurons are limited in clinical application due of their complex nature, lengthy process, and low conversion efficiency. Methods and Results: Here, we report a new protocol involving chemical-based direct conversion of human fibroblasts (HF) to matured neuron-like cells with a short duration and high conversion efficiency using temporal and strategic dual epigenetic regulation. In this protocol, epigenetic modulation by inhibition of histone deacetylase and bromodomain enabled to overcome "recalcitrant" nature of adult fibroblasts and shorten the duration of neuronal reprogramming. We further observed that an extended epigenetic regulation is necessary to maintain the induced neuronal program to generate a homogenous population of neuron-like cells. Conclusions: Therefore, our study provides a new protocol to produce neurons-like cells and highlights the need of proper epigenetic resetting to establish and maintain neuronal program in HF.

Stem Cell Aging and Regenerative Medicine.

Stem cells are a promising source for regenerative medicine to cure a plethora of diseases that are currently treated based on either palliative or symptomatic relief or by preventing their onset and progression. Aging-associated degenerative changes in stem cells, stem cell niches, and signaling pathways bring a step by step decline in the regenerative and functional potential of tissues. Clinical studies and experiments on model organisms have pointed out checkpoints that aging will inevitably impose on stem cell aiming for transplantation and hence questions are raised about the age of the donor. In the following discourse, we review the fundamental molecular pathways that are implicated in stem cell aging and the current progress in tissue engineering and transplantation of each type of stem cells in regenerative medicine. We further focus on the consequences of stem cell aging on their clinical uses and the development of novel strategies to bypass those pitfalls and improve tissue replenishment.

Targeted epigenetic modulation using a DNA-based histone deacetylase inhibitor enhances cardiomyogenesis in mouse embryonic stem cells.

The epigenome has an essential role in orchestrating transcriptional activation and modulating key developmental processes. Previously, we developed a library of pyrrole-imidazole polyamides (PIPs) conjugated with suberoylanilide hydroxamic acid (SAHA), a histone deacetylase (HDAC) inhibitor, for the purpose of sequence-specific modification of epigenetics. Based on the gene expression profile of SAHA-PIPs and screening studies using the α-myosin heavy chain promoter-driven reporter and SAHA-PIP library, we identified that SAHA-PIP G activates cardiac-related genes. Studies in mouse ES cells showed that SAHA-PIP G could enhance the generation of spontaneous beating cells, which is consistent with upregulation of several cardiac-related genes. Moreover, ChIP-seq results confirmed that the upregulation of cardiac-related genes is highly correlated with epigenetic activation, relevant to the sequence-specific binding of SAHA-PIP G. This proof-of-concept study demonstrating the applicability of SAHA-PIP not only improves our understanding of epigenetic alterations involved in cardiomyogenesis but also provides a novel chemical-based strategy for stem cell differentiation.

Exosome-Mediated Differentiation of Mouse Embryonic Fibroblasts and Exocrine Cells into ß-Like Cells and the Identification of Key miRNAs for Differentiation.

Diabetes is a concerning health malady worldwide. Islet or pancreas transplantation is the only long-term treatment available; however, the scarcity of transplantable tissues hampers this approach. Therefore, new cell sources and differentiation approaches are required. Apart from the genetic- and small molecule-based approaches, exosomes could induce cellular differentiation by means of their cargo, including miRNA. We developed a chemical-based protocol to differentiate mouse embryonic fibroblasts (MEFs) into ß-like cells and employed mouse insulinoma (MIN6)-derived exosomes in the presence or absence of specific small molecules to encourage their differentiation into ß-like cells. The differentiated ß-like cells were functional and expressed pancreatic genes such as Pdx1, Nkx6.1, and insulin 1 and 2. We found that the exosome plus small molecule combination differentiated the MEFs most efficiently. Using miRNA-sequencing, we identified miR-127 and miR-709, and found that individually and in combination, the miRNAs differentiated MEFs into ß-like cells similar to the exosome treatment. We also confirmed that exocrine cells can be differentiated into ß-like cells by exosomes and the exosome-identified miRNAs. A new differentiation approach based on the use of exosome-identified miRNAs could help people afflicted with diabetes.

Improved differentiation of human adipose stem cells to insulin-producing ß-like cells using PDFGR kinase inhibitor Tyrphostin9.

Diabetes mellitus (DM) is a metabolic syndrome where insulin secretion or the response to insulin produced by the body is compromised. The only available long-term treatment is the transplantation of pancreas or islet for procuring ß-cells. However, due to the shortage of ß-cell sources from the tissues, differentiation of pluripotent stem cells or terminally differentiated cells into ß-cell is proposed as an alternative strategy. Previously, human adipose-derived stem cells (ADSCs) were reported to be converted into ß-like cells by a stepwise treatment of chemicals and growth factors. However, due to the low conversion efficiency, the clinical application was not feasible. In this study, we developed a modified conversion protocol with improved yield and functionality, which is achieved by changing the culture method and addition of Tyrphostin9, a platelet-derived growth factor receptor (PDGFR) kinase inhibitor. Tyrphostin9 was identified from a cell-based chemical screening using the mCherry reporter under the control of the Pdx1 promoter. The ß-like cells differentiated under the new protocol showed a 3.6-fold increase in the expression of Pdx1, a marker for pancreatic differentiation, as compared to the previous protocol. We propose that Tyrphostin9 contributes to the ß-like cell differentiation by playing a dual role; enhancing the definitive endoderm generation by inhibiting the PI3K signaling and suppressing the taurine-mediated proliferation of definitive endoderm. Importantly, these differentiated cells responded well to low and high glucose stimulations compared to cells differentiated by the previous protocol, as confirmed by the 2.0-fold increase in the C-peptide release. As ADSCs are abundant, easily isolated, and autologous in nature, improved differentiation approaches to generate ß-like cells from ADSCs would provide a better opportunity for treating diabetes.

Chemical induced conversion of mouse fibroblasts and human adipose-derived stem cells into skeletal muscle-like cells.

Use of stem cells in regenerative medicine holds great promise in treating people suffering from various otherwise incurable ailments. Direct conversion of somatic cells to other lineages thereby bypassing the intermediate pluripotent state has enormous applicability with respect to time requirement for conversion as well as safety issues. Among various approaches, chemical induced cell conversion is safe yet effective, and the use of small molecules has thus increased greatly in recent years in regenerative fields due to easy applicability, efficient scalability, and consistent reproducibility. Here we report a combination of small molecules capable of converting mouse fibroblasts into skeletal muscle-like cells (SMLCs) without requiring ectopic transcription factor expression. We observed that a combination of chemicals is necessary and sufficient to convert mouse fibroblast to SMLCs that have functional similarity to skeletal muscles. In addition, we also found that cytokines responsible for modulating several key signaling pathways enhance the maturation of converted SMLCs into multinucleated myocytes. Epigenetic analysis revealed that this conversion is accomplished by an epigenetic overhaul, followed by activation of key signal pathways responsible for activating skeletal specific loci. We further observed that human adipocyte-derived stem cells can be converted into SMLCs under conditions similar to that of fibroblasts. This study not only provides an example of chemical induced direct conversion, but also underlines the key signaling pathways that are needed to induce mesodermal lineages and muscles from pleotropic type cells.

Discovery of Natural Compounds Promoting Cardiomyocyte Differentiation.

The commitment of pluripotent stem cells to the cardiac lineage has enormous potential in regenerative medicine interventions for several cardiac diseases. Thus, it is necessary to understand and regulate this differentiation process for potential clinical application. In this study, we developed defined conditions with chemical inducers for effective cardiac lineage commitment and elucidated the mechanism for high-efficiency differentiation. First, we designed a robust reporter-based platform to screen chemical inducers of cardiac differentiation in the mouse P19 teratocarcinoma cell line. Using this system, we identified two natural alkaloids, lupinine and ursinoic acid, which enhanced cardiomyocyte differentiation of P19 cells in terms of beating colony numbers with respect to oxytocin, and confirmed their activity in mouse embryonic stem cells. By analyzing the expression of key markers, we found that this enhancement can be attributed to the early and rapid induction of the Wnt signaling pathway. We also found that these natural compounds could not only supersede the action of the Wnt3a ligand but also had a very quick response time, allowing them to act as efficient cardiac mesoderm inducers that subsequently promoted cardiomyocyte differentiation. Thus, this study offers a way to develop chemical-based differentiation strategy for high-efficiency cardiac lineage commitment, which has an advantage over currently available methods with complex medium composition and parameters. Furthermore, it also provides an opportunity to pinpoint the key molecular mechanisms pivotal to the cardiac differentiation process, which are necessary to design an efficient strategy for cardiomyocyte differentiation.

Small molecule-induced cellular conversion.

Over the last decade, the development of methods to promote conversion of one type of cell to a specific type of another cell (or change of cell fate) has received great attention in basic biological research and therapeutic applications. A precise, reproducible and safe protocol for inducing this change is a prerequisite for cellular conversion. Although genetic manipulation, which relies on the introduction of specific genes into cells, is a promising approach, the results of initial investigations have highlighted serious safety concerns associated with forced ectopic gene expression with unpredictable side effects. Alternatively, a chemical approach that relies on the use of small molecules to modulate the cell fate has great potential in terms of precise control and clinical safety. In addition, the ease of application, reproducibility and scalability are features that make a small molecule-based approach an extraordinary resource for this purpose. In this review we summarize methods which have been devised to identify small molecules that induce cellular conversion and highlight recent advances made using small molecule modulators to induce changes in the fate of cells.

Examining cooperative binding of Sox2 on DC5 regulatory element upon complex formation with Pax6 through excess electron transfer assay.

Functional cooperativity among transcription factors on regulatory genetic elements is pivotal for milestone decision-making in various cellular processes including mammalian development. However, their molecular interaction during the cooperative binding cannot be precisely understood due to lack of efficient tools for the analyses of protein-DNA interaction in the transcription complex. Here, we demonstrate that photoinduced excess electron transfer assay can be used for analysing cooperativity of proteins in transcription complex using cooperative binding of Pax6 to Sox2 on the regulatory DNA element (DC5 enhancer) as an example. In this assay, (Br)U-labelled DC5 was introduced for the efficient detection of transferred electrons from Sox2 and Pax6 to the DNA, and guanine base in the complementary strand was replaced with hypoxanthine (I) to block intra-strand electron transfer at the Sox2-binding site. By examining DNA cleavage occurred as a result of the electron transfer process, from tryptophan residues of Sox2 and Pax6 to DNA after irradiation at 280 nm, we not only confirmed their binding to DNA but also observed their increased occupancy on DC5 with respect to that of Sox2 and Pax6 alone as a result of their cooperative interaction.

Combining Suppression of Stemness with Lineage-Specific Induction Leads to Conversion of Pluripotent Cells into Functional Neurons.

Sox2 is a key player in the maintenance of pluripotency and stemness, and thus inhibition of its function would abrogate the stemness of pluripotent cells and induce differentiation into several types of cells. Herein we describe a strategy that relies on a combination of Sox2 inhibition with lineage-specific induction to promote efficient and selective differentiation of pluripotent P19 cells into neurons. When P19 cells transduced with Skp protein, an inhibitor of Sox2, are incubated with a neurogenesis inducer, the cells are selectively converted into neurons that generate depolarization-induced sodium currents and action potentials. This finding indicates that the differentiated neurons are electrophysiologically active. Signaling pathway studies lead us to conclude that a combination of Skp with the neurogenesis inducer enhances neurogenesis in P19 cells by activating Wnt and Notch pathways. The present differentiation protocol could be valuable to selectively generate functionally active neurons from pluripotent cells.

Single molecule visualization and characterization of Sox2-Pax6 complex formation on a regulatory DNA element using a DNA origami frame.

We report the use of atomic force microscopy (AFM) to study Sox2-Pax6 complex formation on the regulatory DNA element at a single molecule level. Using an origami DNA scaffold containing two DNA strands with different levels of tensile force, we confirmed that DNA bending is necessary for Sox2 binding. We also demonstrated that two transcription factors bind cooperatively by observing the increased occupancy of Sox2-Pax6 on the DNA element compared to that of Sox2 alone.

Inhibition of master transcription factors in pluripotent cells induces early stage differentiation.

The potential for pluripotent cells to differentiate into diverse specialized cell types has given much hope to the field of regenerative medicine. Nevertheless, the low efficiency of cell commitment has been a major bottleneck in this field. Here we provide a strategy to enhance the efficiency of early differentiation of pluripotent cells. We hypothesized that the initial phase of differentiation can be enhanced if the transcriptional activity of master regulators of stemness is suppressed, blocking the formation of functional transcriptomes. However, an obstacle is the lack of an efficient strategy to block protein-protein interactions. In this work, we take advantage of the biochemical property of seventeen kilodalton protein (Skp), a bacterial molecular chaperone that binds directly to sex determining region Y-box 2 (Sox2). The small angle X-ray scattering analyses provided a low resolution model of the complex and suggested that the transactivation domain of Sox2 is probably wrapped in a cleft on Skp trimer. Upon the transduction of Skp into pluripotent cells, the transcriptional activity of Sox2 was inhibited and the expression of Sox2 and octamer-binding transcription factor 4 was reduced, which resulted in the expression of early differentiation markers and appearance of early neuronal and cardiac progenitors. These results suggest that the initial stage of differentiation can be accelerated by inhibiting master transcription factors of stemness. This strategy can possibly be applied to increase the efficiency of stem cell differentiation into various cell types and also provides a clue to understanding the mechanism of early differentiation.

E. coli HflX interacts with 50S ribosomal subunits in presence of nucleotides.

HflX is a GTP binding protein of unknown function. Based on the presence of the hflX gene in hflA operon, HflX was believed to be involved in the lytic-lysogenic decision during phage infection in Escherichia coli. We find that E. coli HflX binds 16S and 23S rRNA - the RNA components of 30S and 50S ribosomal subunits. Here, using purified ribosomal subunits, we show that HflX specifically interacts with the 50S. This finding is in line with the homology of HflX to GTPases involved in ribosome biogenesis. However, HflX-50S interaction is not limited to a specific nucleotide-bound state of the protein, and the presence of any of the nucleotides GTP/GDP/ATP/ADP is sufficient. In this respect, HflX is different from other GTPases. While E. coli HflX binds and hydrolyses both ATP and GTP, only the GTP hydrolysis activity is stimulated by 50S binding. This work uncovers interesting attributes of HflX in ribosome binding.

Hydrogen production by Cyanobacteria.

The limited fossil fuel prompts the prospecting of various unconventional energy sources to take over the traditional fossil fuel energy source. In this respect the use of hydrogen gas is an attractive alternate source. Attributed by its numerous advantages including those of environmentally clean, efficiency and renew ability, hydrogen gas is considered to be one of the most desired alternate. Cyanobacteria are highly promising microorganism for hydrogen production. In comparison to the traditional ways of hydrogen production (chemical, photoelectrical), Cyanobacterial hydrogen production is commercially viable. This review highlights the basic biology of cynobacterial hydrogen production, strains involved, large-scale hydrogen production and its future prospects. While integrating the existing knowledge and technology, much future improvement and progress is to be done before hydrogen is accepted as a commercial primary energy source.

Inactive enzymatic mutant proteins (phosphoglycerate mutase and enolase) as sugar binders for ribulose-1,5-bisphosphate regeneration reactors.

BACKGROUND: Carbon dioxide fixation bioprocess in reactors necessitates recycling of D-ribulose1,5-bisphosphate (RuBP) for continuous operation. A radically new close loop of RuBP regenerating reactor design has been proposed that will harbor enzyme-complexes instead of purified enzymes. These reactors will need binders enabling selective capture and release of sugar and intermediate metabolites enabling specific conversions during regeneration. In the current manuscript we describe properties of proteins that will act as potential binders in RuBP regeneration reactors. RESULTS: We demonstrate specific binding of 3-phosphoglycerate (3PGA) and 3-phosphoglyceraldehyde (3PGAL) from sugar mixtures by inactive mutant of yeast enzymes phosphoglycerate mutase and enolase. The reversibility in binding with respect to pH and EDTA has also been shown. No chemical conversion of incubated sugars or sugar intermediate metabolites were found by the inactive enzymatic proteins. The dissociation constants for sugar metabolites are in the micromolar range, both proteins showed lower dissociation constant (Kd) for 3-phosphoglycerate (655-796 muM) compared to 3-phosphoglyceraldehyde (822-966 muM) indicating higher affinity for 3PGA. The proteins did not show binding to glucose, sucrose or fructose within the sensitivity limits of detection. Phosphoglycerate mutase showed slightly lower stability on repeated use than enolase mutants. CONCLUSIONS: The sugar and their intermediate metabolite binders may have a useful role in RuBP regeneration reactors. The reversibility of binding with respect to changes in physicochemical factors and stability when subjected to repeated changes in these conditions are expected to make the mutant proteins candidates for in-situ removal of sugar intermediate metabolites for forward driving of specific reactions in enzyme-complex reactors.