Photothermal nanoblade for patterned cell membrane cutting.

We report a photothermal nanoblade that utilizes a metallic nanostructure to harvest short laser pulse energy and convert it into a highly localized and specifically shaped explosive vapor bubble. Rapid bubble expansion and collapse punctures a lightly-contacting cell membrane via high-speed fluidic flows and induced transient shear stress. The membrane cutting pattern is controlled by the metallic nanostructure configuration, laser pulse polarization, and energy. Highly controllable, sub-micron sized circular hole pairs to half moon-like, or cat-door shaped, membrane cuts were realized in glutaraldehyde treated HeLa cells

Alpha-Ketoglutarate Influences the Self-Renewal and Differentiation of Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) hold great potential for regenerative medicine due to their ability to self-renew indefinitely in in vitro culture and to differentiate into all three germ layers. However, the use of hPSCs in the clinic has been limited by the lack of differentiation protocols that produce fully mature and functional cell types. Development of more efficient differentiation strategies therefore will be key to fully realizing the therapeutic potential of hPSCs. Differentiation occurs through epigenetic changes that turn off genes important for self-renewal and activate genes required for cellular maturation and specialization. In addition, cellular metabolism shifts to address varying energetic and biosynthetic demands. Many metabolites, whose levels are influenced by the overall metabolic network, act as cofactors for enzymes that control the epigenetic state of the cell. α-Ketoglutarate (αKG), a TCA cycle metabolite, acts as a cofactor for αKG-dependent dioxygenases which included the JmjC-domain containing family of histone demethylases (JHDMs) and the Ten-eleven translocation (TET) methylcytosine oxidases. Succinate, another TCA cycle metabolite, acts as an inhibitor of the same αKG-dependent dioxygenases. Therefore, changes in the αKG-to-succinate ratio caused by changes in cellular metabolism impact the activity of αKG-dependent dioxygenases and therefore gene expression. Both JHDMs and TETs have known roles in pluripotent stem cell self-renewal and differentiation. Recently, αKG has been reported to support self-renewal in mouse embryonic stem cells (mESCs). However, mESCs differ from traditionally maintained hPSCs in numerous ways including their stages of pluripotency. Both mESCs and human embryonic stem cells are derived from the inner cell mass, but mESCs are traditionally maintained in an earlier developmental state, called the na�ve state, corresponding to the preimplantation embryo. hESCs are traditionally maintained in a more differentiated state, called the primed pluripotent state, corresponding to the post-implantation epiblast. Therefore, because the role of αKG in hPSCs has not previously been investigated, we examined the role of αKG in primed hPSC differentiation. We discovered that αKG can accelerate the differentiation of primed hPSCs likely through its action on αKG-dependent dioxygenases. Because αKG promotes self-renewal in mESCs, we investigated whether αKG promotes differentiation of primed mouse pluripotent stem cells derived from the post-implantation embryo called Epiblast stem cells (EpiSCs). αKG also promoted differentiation in mouse EpiSCs which suggests that the role of αKG is dependent on the stage of pluripotency and is not species dependent. To further confirm the role of αKG in hPSC differentiation, we decreased the αKG-to-succinate ratio by inhibition of αKG producing enzymes or succinate consuming enzymes during hPSC differentiation. Both manipulations led to a delay of hPSC differentiation. Finally, manipulation of the αKG-to-succinate ratio led to changes in DNA hydroxymethylation and histone methylation levels suggesting an epigenetic mechanism. Taken together, my data suggests that αKG plays a context specific, differentiation promoting role in primed pluripotent stem cells

Chapter Five Techniques to Monitor Glycolysis

An increased flux through glycolysis supports the proliferation of cancer cells by providing additional energy in the form of ATP as well as glucose-derived metabolic intermediates for nucleotide, lipid, and protein biosynthesis. Thus, glycolysis and other metabolic pathways that control cell proliferation may represent valuable targets for therapeutic interventions and diagnostic procedures. In this context, the measurement of glucose uptake and lactate excretion by malignant cells may be useful to detect shifts in glucose catabolism, while determining the activity of rate-limiting glycolytic enzymes can provide insights into points of metabolic regulation. Moreover, metabolomic studies can be used to generate large, integrated datasets to track changes in carbon flux through glycolysis and its collateral anabolic pathways. As discussed here, these approaches can reveal and quantify the metabolic alterations that underlie malignant cell proliferation

Pluripotent stem cell energy metabolism: an update

Recent studies link changes in energy metabolism with the fate of pluripotent stem cells (PSCs). Safe use of PSC derivatives in regenerative medicine requires an enhanced understanding and control of factors that optimize in vitro reprogramming and differentiation protocols. Relative shifts in metabolism from naïve through "primed" pluripotent states to lineage-directed differentiation place variable demands on mitochondrial biogenesis and function for cell types with distinct energetic and biosynthetic requirements. In this context, mitochondrial respiration, network dynamics, TCA cycle function, and turnover all have the potential to influence reprogramming and differentiation outcomes. Shifts in cellular metabolism affect enzymes that control epigenetic configuration, which impacts chromatin reorganization and gene expression changes during reprogramming and differentiation. Induced PSCs (iPSCs) may have utility for modeling metabolic diseases caused by mutations in mitochondrial DNA, for which few disease models exist. Here, we explore key features of PSC energy metabolism research in mice and man and the impact this work is starting to have on our understanding of early development, disease modeling, and potential therapeutic applications

The pentose phosphate pathway in health and disease.

The pentose phosphate pathway (PPP) is a glucose-oxidizing pathway that runs in parallel to upper glycolysis to produce ribose 5-phosphate and nicotinamide adenine dinucleotide phosphate (NADPH). Ribose 5-phosphate is used for nucleotide synthesis, while NADPH is involved in redox homoeostasis as well as in promoting biosynthetic processes, such as the synthesis of tetrahydrofolate, deoxyribonucleotides, proline, fatty acids and cholesterol. Through NADPH, the PPP plays a critical role in suppressing oxidative stress, including in certain cancers, in which PPP inhibition may be therapeutically useful. Conversely, PPP-derived NADPH also supports purposeful cellular generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) for signalling and pathogen killing. Genetic deficiencies in the PPP occur relatively commonly in the committed pathway enzyme glucose-6-phosphate dehydrogenase (G6PD). G6PD deficiency typically manifests as haemolytic anaemia due to red cell oxidative damage but, in severe cases, also results in infections due to lack of leucocyte oxidative burst, highlighting the dual redox roles of the pathway in free radical production and detoxification. This Review discusses the PPP in mammals, covering its roles in biochemistry, physiology and disease

Photothermal nanoblade for patterned cell membrane cutting.

We report a photothermal nanoblade that utilizes a metallic nanostructure to harvest short laser pulse energy and convert it into a highly localized and specifically shaped explosive vapor bubble. Rapid bubble expansion and collapse punctures a lightly-contacting cell membrane via high-speed fluidic flows and induced transient shear stress. The membrane cutting pattern is controlled by the metallic nanostructure configuration, laser pulse polarization, and energy. Highly controllable, sub-micron sized circular hole pairs to half moon-like, or cat-door shaped, membrane cuts were realized in glutaraldehyde treated HeLa cells

α-Ketoglutarate Accelerates the Initial Differentiation of Primed Human Pluripotent Stem Cells.

Pluripotent stem cells (PSCs) can self-renew or differentiate from naive or more differentiated, primed, pluripotent states established by specific culture conditions. Increased intracellular α-ketoglutarate (αKG) was shown to favor self-renewal in naive mouse embryonic stem cells (mESCs). The effect of αKG or αKG/succinate levels on differentiation from primed human PSCs (hPSCs) or mouse epiblast stem cells (EpiSCs) remains unknown. We examined primed hPSCs and EpiSCs and show that increased αKG or αKG-to-succinate ratios accelerate, and elevated succinate levels delay, primed PSC differentiation. αKG has been shown to inhibit the mitochondrial ATP synthase and to regulate epigenome-modifying dioxygenase enzymes. Mitochondrial uncoupling did not impede αKG-accelerated primed PSC differentiation. Instead, αKG induced, and succinate impaired, global histone and DNA demethylation in primed PSCs. The data support αKG promotion of self-renewal or differentiation depending on the pluripotent state

Acidic Methanol Treatment Facilitates Matrix-Assisted Laser Desorption Ionization-Mass Spectrometry Imaging of Energy Metabolism.

Detection of small molecule metabolites (SMM), particularly those involved in energy metabolism using MALDI-mass spectrometry imaging (MSI), is challenging due to factors including ion suppression from other analytes present (e.g., proteins and lipids). One potential solution to enhance SMM detection is to remove analytes that cause ion suppression from tissue sections before matrix deposition through solvent washes. Here, we systematically investigated solvent treatment conditions to improve SMM signal and preserve metabolite localization. Washing with acidic methanol significantly enhances the detection of phosphate-containing metabolites involved in energy metabolism. The improved detection is due to removing lipids and highly polar metabolites that cause ion suppression and denaturing proteins that release bound phosphate-containing metabolites. Stable isotope infusions of [13C6]nicotinamide coupled to MALDI-MSI (Iso-imaging) in the kidney reveal patterns that indicate blood vessels, medulla, outer stripe, and cortex. We also observed different ATP:ADP raw signals across mouse kidney regions, consistent with regional differences in glucose metabolism favoring either gluconeogenesis or glycolysis. In mouse muscle, Iso-imaging using [13C6]glucose shows high glycolytic flux from infused circulating glucose in type 1 and 2a fibers (soleus) and relatively lower glycolytic flux in type 2b fiber type (gastrocnemius). Thus, improved detection of phosphate-containing metabolites due to acidic methanol treatment combined with isotope tracing provides an improved way to probe energy metabolism with spatial resolution in vivo