Lactate Metabolism in Human Lung Tumors.

Cancer cells consume glucose and secrete lactate in culture. It is unknown whether lactate contributes to energy metabolism in living tumors. We previously reported that human non-small-cell lung cancers (NSCLCs) oxidize glucose in the tricarboxylic acid (TCA) cycle. Here, we show that lactate is also a TCA cycle carbon source for NSCLC. In human NSCLC, evidence of lactate utilization was most apparent in tumors with high 18fluorodeoxyglucose uptake and aggressive oncological behavior. Infusing human NSCLC patients with 13C-lactate revealed extensive labeling of TCA cycle metabolites. In mice, deleting monocarboxylate transporter-1 (MCT1) from tumor cells eliminated lactate-dependent metabolite labeling, confirming tumor-cell-autonomous lactate uptake. Strikingly, directly comparing lactate and glucose metabolism in vivo indicated that lactate's contribution to the TCA cycle predominates. The data indicate that tumors, including bona fide human NSCLC, can use lactate as a fuel in vivo.

Metabolic Heterogeneity in Human Lung Tumors.

Non-small cell lung cancer (NSCLC) is heterogeneous in the genetic and environmental parameters that influence cell metabolism in culture. Here, we assessed the impact of these factors on human NSCLC metabolism in vivo using intraoperative (13)C-glucose infusions in nine NSCLC patients to compare metabolism between tumors and benign lung. While enhanced glycolysis and glucose oxidation were common among these tumors, we observed evidence for oxidation of multiple nutrients in each of them, including lactate as a potential carbon source. Moreover, metabolically heterogeneous regions were identified within and between tumors, and surprisingly, our data suggested potential contributions of non-glucose nutrients in well-perfused tumor areas. Our findings not only demonstrate the heterogeneity in tumor metabolism in vivo but also highlight the strong influence of the microenvironment on this feature.

TCA Cycle and Mitochondrial Membrane Potential Are Necessary for Diverse Biological Functions.

Mitochondrial metabolism is necessary for the maintenance of oxidative TCA cycle function and mitochondrial membrane potential. Previous attempts to decipher whether mitochondria are necessary for biological outcomes have been hampered by genetic and pharmacologic methods that simultaneously disrupt multiple functions linked to mitochondrial metabolism. Here, we report that inducible depletion of mitochondrial DNA (ρ(ο) cells) diminished respiration, oxidative TCA cycle function, and the mitochondrial membrane potential, resulting in diminished cell proliferation, hypoxic activation of HIF-1, and specific histone acetylation marks. Genetic reconstitution only of the oxidative TCA cycle function specifically in these inducible ρ(ο) cells restored metabolites, resulting in re-establishment of histone acetylation. In contrast, genetic reconstitution of the mitochondrial membrane potential restored ROS, which were necessary for hypoxic activation of HIF-1 and cell proliferation. These results indicate that distinct mitochondrial functions associated with respiration are necessary for cell proliferation, epigenetics, and HIF-1 activation.

Glutamine oxidation maintains the TCA cycle and cell survival during impaired mitochondrial pyruvate transport.

Alternative modes of metabolism enable cells to resist metabolic stress. Inhibiting these compensatory pathways may produce synthetic lethality. We previously demonstrated that glucose deprivation stimulated a pathway in which acetyl-CoA was formed from glutamine downstream of glutamate dehydrogenase (GDH). Here we show that import of pyruvate into the mitochondria suppresses GDH and glutamine-dependent acetyl-CoA formation. Inhibiting the mitochondrial pyruvate carrier (MPC) activates GDH and reroutes glutamine metabolism to generate both oxaloacetate and acetyl-CoA, enabling persistent tricarboxylic acid (TCA) cycle function. Pharmacological blockade of GDH elicited largely cytostatic effects in culture, but these effects became cytotoxic when combined with MPC inhibition. Concomitant administration of MPC and GDH inhibitors significantly impaired tumor growth compared to either inhibitor used as a single agent. Together, the data define a mechanism to induce glutaminolysis and uncover a survival pathway engaged during compromised supply of pyruvate to the mitochondria.

Insights into transcriptional silencing and anti-silencing in Shigella flexneri: a detailed molecular analysis of the icsP virulence locus.

Transcriptional silencing and anti-silencing mechanisms modulate bacterial physiology and virulence in many human pathogens. In Shigella species, many virulence plasmid genes are silenced by the histone-like nucleoid structuring protein H-NS and anti-silenced by the virulence gene regulator VirB. Despite the key role that these regulatory proteins play in Shigella virulence, their mechanisms of transcriptional control remain poorly understood. Here, we characterize the regulatory elements and their relative spacing requirements needed for the transcriptional silencing and anti-silencing of icsP, a locus that requires remotely located regulatory elements for both types of transcriptional control. Our findings highlight the flexibility of the regulatory elements' positions with respect to each other, and yet, a molecular roadblock docked between the VirB binding site and the upstream H-NS binding region abolishes transcriptional anti-silencing by VirB, providing insight into transcriptional anti-silencing. Our study also raises the need to re-evaluate the currently proposed VirB binding site. Models of transcriptional silencing and anti-silencing at this genetic locus are presented, and the implications for understanding these regulatory mechanisms in bacteria are discussed.

The predictive ability of childhood animal cruelty methods for later interpersonal crimes.

Research on the topic of childhood animal cruelty methods and their link to interpersonal violence is sparse. Most of the studies that do exist focus only on the frequencies of different methods of childhood animal cruelty. Only two studies to date have examined the predictive nature of these methods for later violence toward humans. One of these previous studies found that drowning and having sex with animals were predictive of later human violence, while the other found that sex with animals and the age at which the offenders began committing animal cruelty were its only statistically significant predictors. Using data collected from 257 anonymous self-reports by male inmates at a medium-security prison in a Southern state, we investigate the predictive ability of several retrospectively identified childhood animal cruelty methods (i.e., drowning, hitting/beating, hitting with rocks, shooting, kicking, choking, burning, stabbing, having sex, and starving/neglecting) for later violent crimes toward humans. Regression analyses revealed that recurrent (i.e., more than once) childhood animal cruelty and stabbing animals were the only statistically significant variables in the model that predicted recurrent interpersonal violence in adulthood.

Midinfrared frequency comb by difference frequency of erbium and thulium fiber lasers in orientation-patterned gallium phosphide.

We generate over 60 mW of pulses with wavelengths from 6 to 11 micrometers by difference frequency mixing between erbium and thulium fiber amplifiers in orientation-patterned GaP with a photon conversion efficiency of 0.2. By stabilizing the repetition rate of the shared oscillator and adding a frequency shifter to one arm, the output becomes a frequency comb with tunable carrier envelope offset.

A nanoparticle-based strategy for the imaging of a broad range of tumours by nonlinear amplification of microenvironment signals.

Stimuli-responsive nanomaterials are increasingly important in a variety of applications such as biosensing, molecular imaging, drug delivery and tissue engineering. For cancer detection, a paramount challenge still exists in the search for methods that can illuminate tumours universally regardless of their genotypes and phenotypes. Here we capitalized on the acidic, angiogenic tumour microenvironment to achieve the detection of tumour tissues in a wide variety of mouse cancer models. This was accomplished using ultra pH-sensitive fluorescent nanoprobes that have tunable, exponential fluorescence activation on encountering subtle, physiologically relevant pH transitions. These nanoprobes were silent in the circulation, and then strongly activated (>300-fold) in response to the neovasculature or to the low extracellular pH in tumours. Thus, we have established non-toxic, fluorescent nanoreporters that can nonlinearly amplify tumour microenvironmental signals, permitting the identification of tumour tissue independently of histological type or driver mutation, and detection of acute treatment responses much more rapidly than conventional imaging approaches.

Characterization of the ospZ promoter in Shigella flexneri and its regulation by VirB and H-NS.

OspZ is an effector protein of the type III secretion system in Shigella spp. that downregulates the human inflammatory response during bacterial infection. The ospZ gene is located on the large virulence plasmid of Shigella. Many genes on this plasmid are transcriptionally repressed by the nucleoid structuring protein H-NS and derepressed by VirB, a DNA-binding protein that displays homology to the plasmid partitioning proteins ParB and SopB. In this study, we characterized the ospZ promoter and investigated its regulation by H-NS and VirB in Shigella flexneri. We show that H-NS represses and VirB partially derepresses the ospZ promoter. H-NS-mediated repression requires sequences located between -731 and -412 relative to the beginning of the ospZ gene. Notably, the VirB-dependent derepression of ospZ requires the same VirB binding sites as are required for the VirB-dependent derepression of the divergent icsP gene. These sites are centered 425 bp upstream of the ospZ gene but over 1 kb upstream of the icsP transcription start site. Although these VirB binding sites lie closer to ospZ than icsP, the VirB-dependent increase in ospZ promoter activity is lower than that observed at the icsP promoter. This indicates that the proximity of VirB binding sites to Shigella promoters does not necessarily correlate with the level of VirB-dependent derepression. These findings have implications for virulence gene regulation in Shigella and other pathogens that control gene expression using mechanisms of transcriptional repression and derepression.

Disruption of redox balance in glutaminolytic triple negative breast cancer by inhibition of glutamate export and glutaminase.

In triple-negative breast cancer (TNBC) that relies on catabolism of amino acid glutamine, glutaminase (GLS) converts glutamine to glutamate, which facilitates glutathione synthesis by mediating the enrichment of intracellular cystine via xCT antiporter activity. To overcome chemo resistant TNBC, we have tested a strategy of disrupting cellular redox balance by inhibition of GLS and xCT by CB839 and Erastin, respectively. Key findings of our study include: 1. Dual metabolic inhibition (CB839+Erastin) led to significant increases of cellular superoxide level in both parent and chemo resistant TNBC cells, but superoxide level was distinctly lower in resistant cells. 2. Dual metabolic inhibition combined with doxorubicin or cisplatin induced significant apoptosis in TNBC cells and is associated with high degrees of GSH depletion. In vivo , dual metabolic inhibition plus cisplatin led to significant growth delay of chemo resistant human TNBC xenografts. 3. Ferroptosis is induced by doxorubicin (DOX) but not by cisplatin or paclitaxel. Addition of dual metabolic inhibition to DOX chemotherapy significantly enhanced ferroptotic cell death. 4. Significant changes in cellular metabolites concentration preceded transcriptome changes revealed by single cell RNA sequencing, underscoring the potential of capturing early changes in metabolites as pharmacodynamic markers of metabolic inhibitors. Here we demonstrated that 4-(3-[ 18 F]fluoropropyl)-L-glutamic acid ([ 18 F]FSPG) PET detected xCT blockade by Erastin or its analog in mice bearing human TNBC xenografts. In summary, our study provides compelling evidence for the therapeutic benefit and feasibility of non-invasive monitoring of dual metabolic blockade as a translational strategy to sensitize chemo resistant TNBC to cytotoxic chemotherapy.

Imaging of isolated molecules with ultrafast electron pulses.

Imaging isolated molecules in three dimensions with atomic resolution is important for elucidating complex molecular structures and intermediate states in molecular dynamics. This goal has so far remained elusive due to the random orientation of molecules in the gas phase. We show that three-dimensional structural information can be retrieved from multiple electron diffraction patterns of aligned molecules. The molecules are aligned impulsively with a femtosecond laser pulse and probed with a femtosecond electron pulse two picoseconds later, when the degree of alignment reaches a maximum.

Two promoters and two translation start sites control the expression of the Shigella flexneri outer membrane protease IcsP.

The Shigella flexneri outer membrane protease IcsP proteolytically cleaves the actin-based motility protein IcsA from the bacterial surface. The icsP gene is monocistronic and lies downstream of an unusually large intergenic region on the Shigella virulence plasmid. In silico analysis of this region predicts a second transcription start site 84 bp upstream of the first. Primer extension analyses and beta-galactosidase assays demonstrate that both transcription start sites are used. Both promoters are regulated by the Shigella virulence gene regulator VirB and both respond similarly to conditions known to influence Shigella virulence gene expression (iron concentration, pH, osmotic pressure, and phase of growth). The newly identified promoter lies upstream of a Shine-Dalgarno sequence and second 5'-ATG-3', which is in frame with the annotated icsP gene. The use of either translation start site leads to the production of IcsP capable of proteolytically cleaving IcsA. A bioinformatic scan of the Shigella genome reveals multiple occurrences of in-frame translation start sites associated with putative Shine-Dalgarno sequences, immediately upstream and downstream of annotated open reading frames. Taken together, our observations support the possibility that the use of in-frame translation start sites may generate different protein isoforms, thereby expanding the proteome encoded by bacterial genomes.

Does Tumor FDG-PET Avidity Represent Enhanced Glycolytic Metabolism in Non-Small Cell Lung Cancer?

BACKGROUND: In non-small cell lung cancer (NSCLC), 18fluoro-2-deoxyglucose-positron emission tomography (FDG-PET) assists in diagnosis, staging, and evaluating treatment response. One variable of FDG-PET, the maximum standard uptake value (SUVm), is considered an objective measure of glucose uptake. However, little is known about the fate of glucose in FDG-avid lung tumors in vivo. This study used stable glucose isotope tracing to determine whether the SUVm predicts glycolytic metabolism or other glucose fates in tumors. METHODS: In this prospective Institutional Review Board-approved clinical trial, 52 untreated potentially resectable confirmed NSCLC patients underwent FDG-PET computed tomography. During the surgical procedure, the patients were infused with 13C-labeled glucose. Blood, tumor, and normal lung samples were analyzed by mass spectrometry to determine 13C enrichment in glycolytic intermediates. These values were compared with clinical variables, including SUVm, maximum tumor diameter, stage, grade, and MIB-1/Ki67 proliferation index. RESULTS: For each patient, 13C enrichment in each metabolite was compared between tumor and adjacent lung. Although all tumors metabolized glucose, SUVm did not correlate with glycolytic intermediate labeling. Rather, SUVm correlated with markers indicating the use of other respiratory substrates, including lactate, and with the proliferation index. CONCLUSIONS: SUVm does not correlate with glycolytic metabolism in human NSCLC but does correlate with the proliferation index, suggesting that SUVm predicts glucose use by pathways other than glycolysis. These pathways may offer alternative therapeutic targets, including biosynthetic pathways required for cell proliferation. The research techniques in this study offer the opportunity to understand the relationships between SUVm, tumor metabolism, and therapeutic vulnerabilities in human NSCLCs.

Highly-efficient coupling of linearly- and radially-polarized femtosecond pulses in hollow-core photonic band-gap fibers.

We demonstrate extremely efficient excitation of linearly-, radially-, and azimuthally-polarized modes in a hollow-core photonic band-gap fiber with femtosecond laser pulses. We achieve coupling efficiencies as high as 98% with linearly polarized input Gaussian beams and with high-power pulses we obtain peak intensities greater than 10(14) W/cm(2) inside and transmitted through the fiber. With radially polarized pulses, we achieve 91% total transmission through the fiber while maintaining the polarization state. Alternatively with azimuthally-polarized pulses, the mode is degraded in the fiber, and the pure polarization state is not maintained.

Childhood and adolescent animal cruelty methods and their possible link to adult violent crimes.

Few researchers have investigated the potentially predictive power of childhood and adolescent animal cruelty methods as they are associated with subsequent interpersonal violence in adulthood. Based on a sample of 261 inmates at medium- and maximum-security prisons in a southern state, the present study examines the relationship between several retrospectively reported animal cruelty methods (drowned, hit or kicked, shot, choked, burned, and had sex) and violent criminal acts committed against humans (assault, rape, and murder). More than half of the sample reported they had shot animals, and almost half had either kicked or hit them. About one in five said they had choked animals, and about one in seven said they had either drowned, burned, or had sex with them. Regression analyses revealed that drowning and having sex with an animal was predictive of later interpersonal violence as adults.

The effect of inmates' self-reported childhood and adolescent animal cruelty: motivations on the number of convictions for adult violent interpersonal crimes.

Few researchers have investigated the potentially predictive power of motives for childhood and adolescent animal cruelty as it is associated with interpersonal violence in adulthood. Based on a sample of 261 inmates at medium- and maximum-security prisons in a southern state, the present study examines the relationship among several retrospectively reported motives (anger, fun, dislike, and imitation) for animal cruelty and violent crime convictions (assault, rape, and murder). Almost half reported abusing animals out of anger, whereas more than one third did so for fun. Dislike for the animal and imitation were less frequently occurring motives. Participants who abused animals at an earlier age and those who did so out of anger or for fun were more likely to repeat the offense. Regression analyses revealed that abusing an animal out of fun in their youth was the most statistically salient motive for predicting later interpersonal violence as adults.

Recurrent Childhood Animal Cruelty and Its Link to Recurrent Adult Interpersonal Violence.

In the early 1960s, researchers began to examine the potential link between childhood animal cruelty and future interpersonal violence. Findings since then have been inconsistent in establishing a relationship between the two. This may be due to researchers failing to measure the recurrency of childhood animal abuse and the recurrency of later violent acts committed in adulthood. The current study, using data from 257 inmates at a medium-security prison in a Southern state, is a replication of research conducted by Tallichet and Hensley, and Hensley, Tallichet, and Dutkiewicz, which examined this recurrency issue. The only statistically significant predictor of recurrent adult interpersonal violence in this study was recurrent childhood animal cruelty. Inmates who engaged in recurrent childhood animal cruelty were more likely to commit recurrent adult interpersonal violence. Respondents' race, education, and childhood residence were not significant predictors of the outcome variable.

Photonic band-gap fiber gas cell fabricated using femtosecond micromachining.

Femtosecond laser drilling is used to produce a variablepressure fiber gas cell. Tightly focused laser pulses are used to produce micrometer-diameter radial channels in a hollow-core photonic band-gap fiber (HC-PBGF), and through these microchannels the core of the fiber is filled with a gas. The fiber cell is formed by fusion splicing and sealing the ends of the HC-PBGF to standard step-index fiber. As a demonstration, acetylene is introduced into an evacuated fiber at multiple backing pressures and spectra are measured.

Silica-glass contribution to the effective nonlinearity of hollow-core photonic band-gap fibers.

We measure the effective nonlinearity of various hollow-core photonic band-gap fibers. Our findings indicate that differences of tens of nanometers in the fiber structure result in significant changes to the power propagating in the silica glass and thus in the effective nonlinearity of the fiber. These results show that it is possible to engineer the nonlinear response of these fibers via small changes to the glass structure.

Does Witnessing Animal Cruelty and Being Abused During Childhood Predict the Initial Age and Recurrence of Committing Childhood Animal Cruelty?

The goal of the current study was to examine the association between demographic characteristics and childhood experiences on the respondents' age of committing childhood animal cruelty and its recurrency. Using data collected from 257 male inmates at a Southern medium-security state prison, the current study seeks to replicate a study by Hensley, Tallichet, and Dutkiewicz. Results revealed that those respondents who were physically abused as children reported engaging in recurrent animal cruelty. The younger the age of respondent for first witnessing animal cruelty, the sooner his initiation to hurting and killing animals occurred. In addition, those who reported witnessing a parent commit acts of animal abuse reported that they committed animal abuse themselves at an older age, while those who witnessed a brother/sister commit animal abuse reported engaging in it at an earlier age. Therefore, physical abuse and witnessing primary socializers engage in animal abuse seem to be important in understanding the respondents' age of onset and repeated childhood animal cruelty.