Lysosome inhibition sensitizes pancreatic cancer to replication stress by aspartate depletion.

Functional lysosomes mediate autophagy and macropinocytosis for nutrient acquisition. Pancreatic ductal adenocarcinoma (PDAC) tumors exhibit high basal lysosomal activity, and inhibition of lysosome function suppresses PDAC cell proliferation and tumor growth. However, the codependencies induced by lysosomal inhibition in PDAC have not been systematically explored. We performed a comprehensive pharmacological inhibition screen of the protein kinome and found that replication stress response (RSR) inhibitors were synthetically lethal with chloroquine (CQ) in PDAC cells. CQ treatment reduced de novo nucleotide biosynthesis and induced replication stress. We found that CQ treatment caused mitochondrial dysfunction and depletion of aspartate, an essential precursor for de novo nucleotide synthesis, as an underlying mechanism. Supplementation with aspartate partially rescued the phenotypes induced by CQ. The synergy of CQ and the RSR inhibitor VE-822 was comprehensively validated in both 2D and 3D cultures of PDAC cell lines, a heterotypic spheroid culture with cancer-associated fibroblasts, and in vivo xenograft and syngeneic PDAC mouse models. These results indicate a codependency on functional lysosomes and RSR in PDAC and support the translational potential of the combination of CQ and RSR inhibitors.

Non-small cell lung cancer clinical trials requiring biopsies with biomarker-specific results for enrollment provide unique challenges.

BACKGROUND: Clinical trials in lung cancer increasingly require patients to provide fresh tumor tissue as a prerequisite to enrollment. The effects of this requirement on enrollment rates, enrollment durations, and patient selection have not been fully elucidated. METHODS: The authors retrospectively reviewed data generated by patients who consented to 1 or more interventional lung cancer clinical trials at the University of California-Los Angeles Jonsson Comprehensive Cancer Center between January 2013 and December 2014. Trials were considered to require a biopsy when enrollment was conditional on the procurement of tissue without intervening therapy between procurement and enrollment. RESULTS: In total, 311 patients underwent 368 screening incidents for 1 or more of 19 trials. Trials that required a new biopsy had a longer median screening duration (34 vs 14 days) than trials that did not require a biopsy (P < .001). Trials that required a biopsy had a greater screen failure rate (49.1% vs 26.5%; P < .001), which was largely driven by patients who did not undergo the required biopsy or lacked the required biomarker. Worsening performance status led to the majority of screen failures (56.5%) among biomarker-eligible patients. CONCLUSIONS: Although the scientific benefits of obtaining a new biopsy and requiring specific results for trial enrollment are clear, these requirements lead to a lengthening of the screening period, which, in some patients, is associated with clinical decline before enrollment. Implications for the interpretation of data from studies of this design should be explored. Cancer 2017;123:4800-7. © 2017 American Cancer Society.

Prevention of neural tube defects in the UK: a missed opportunity.

OBJECTIVE: In 1991, the Medical Research Council (MRC) Vitamin Study demonstrated that folic acid taken before pregnancy and in early pregnancy reduced the risk of a neural tube defect (NTD). We aimed to estimate the number of NTD pregnancies that would have been prevented if flour had been fortified with folic acid in the UK from 1998 as it had been in the USA. DESIGN: Estimates of NTD prevalence, the preventive effect of folic acid and the proportion of women taking folic acid supplements before pregnancy were used to predict the number of NTD pregnancies that would have been prevented if folic acid fortification had been implemented. SETTING: Eight congenital anomaly registers in England and Wales. MAIN OUTCOME MEASURES: The prevalence of pregnancies with an NTD in the UK and the number of these pregnancies that would have been prevented if folic acid fortification had been implemented. RESULTS: From 1991 to 2012, the prevalence of NTD pregnancies was 1.28 (95% CI 1.24 to 1.31) per 1000 total births (19% live births, 81% terminations and 0.5% stillbirths and fetal deaths ≥20 weeks' gestation). If the USA levels of folic acid fortification from 1998 onwards had been adopted in the UK, an estimated 2014 fewer NTD pregnancies would have occurred. CONCLUSIONS: Failure to implement folic acid fortification in the UK has caused, and continues to cause, avoidable terminations of pregnancy, stillbirths, neonatal deaths and permanent serious disability in surviving children.

Towards normalization of soybean somatic embryo maturation.

Soybean (Glycine max L. Merrill) somatic embryos have been useful for assaying seed-specific traits prior to plant recovery. Such traits could be assessed more accurately if somatic embryos more closely mimicked seed development. Amino acid supplements, carbon source, and abscisic acid and basal salt formulations were tested in an effort to modify existing soybean embryogenesis histodifferentiation/maturation media to further normalize the development of soybean somatic embryos. The resultant liquid medium, referred to as soybean histodifferentiation and maturation medium (SHaM), consists of FNL basal salts, 3% sucrose, 3% sorbitol, filter-sterilized 30 mM glutamine and 1 mM methionine. SHaM-derived somatic embryos are more similar to seed in terms of protein and fatty acid/lipid composition, and conversion ability, than somatic embryos obtained from traditional soybean histodifferentiation and maturation media.

EMG biofeedback: the effects of CRF, FR, VR, FI, and VI schedules of reinforcement on the acquisition and extinction of increases in forearm muscle tension.

Biofeedback was used to increase forearm-muscle tension. Feedback was delivered under continuous reinforcement (CRF), variable interval (VI), fixed interval (FI), variable ratio (VR), and fixed ratio (FR) schedules of reinforcement when college students increased their muscle tension (electromyograph, EMG) above a high threshold. There were three daily sessions of feedback, and Session 3 was immediately followed by a session without feedback (extinction). The CRF schedule resulted in the highest EMG, closely followed by the FR and VR schedules, and the lowest EMG scores were produced by the FI and VI schedules. Similarly, the CRF schedule resulted in the greatest amount of time-above-threshold and the VI and FI schedules produced the lowest time-above-threshold. The highest response rates were generated by the FR schedule, followed by the VR schedule. The CRF schedule produced relatively low response rates, comparable to the rates under the VI and FI schedules. Some of the data are consistent with the partial-reinforcement-extinction effect. The present data suggest that different schedules of feedback should be considered in muscle-strengthening-contexts such as during the rehabilitation of muscles following brain damage or peripheral nervous-system injury.

Dense sensor array topography of the event-related potential to task-relevant auditory stimuli.

High spatial density recording and better topographic mapping algorithms have improved the spatial resolving power of the event-related potential (ERP), adding to its already excellent temporal resolution. This study used a 64 channel recording array and spherical spline interpolation to create topographic descriptions of the voltage and current density scalp distributions of the ERP in an auditory oddball paradigm. Frequent (standard) and infrequent (target) tones were presented at a rate of one every approximately 2500 ms to a group of 20 college undergraduates in passive listening and active (count the infrequent tones) task blocks. ANOVAs and topographic analyses were performed on the primary deflections in the 'late' portion of the ERP: the P1, N1, P2, N2 and P3. A target minus standard difference wave was also created for each task. The difference wave contained a mismatch negativity (MMN), an N2b and a P3d. The MMN did not differ between the passive and active tasks and had a topography similar to the N1; also the difference wave P3d was topographically similar to the target P3. The N2b, which occurred only to targets in the active condition, and was the first index of target detection, had a scalp distribution consistent with generation in frontal and superior temporal cortex, suggesting activity in cortical areas of selective attention and auditory stimulus representation.

Social and emotional self-regulation.

In humans, frontal lesions result in deficits of social and emotional behavior that are often surprising in the presence of intact language and other cognitive skills. The connections between the motivation and memory functions of limbic cortex and the motor planning functions of frontal neocortex must be fundamental to meeting the daily challenges of self-regulation. The connectional architecture of limbic and neocortical networks suggests a model of function. The densely interconnected paralimbic cortices may serve to maintain a global motivational context within which specific actions are articulated and sequenced within frontal neocortical networks. The paralimbic networks represent the visceral and kinesthetic information that is integral to the representation of the bodily self. In a general sense, the implicit self-representation within paralimbic networks may shape the significance of perceptions and the motivational context for developing actions. The network architecture of the frontal lobe reflects the dual limbic origins of frontal cortex, in the dorsal archicortical and ventral paleocortical structures. In this paper, we speculated that these two limbic-cortical pathways apply different motivational biases to direct the frontal lobe representation of working memory. The dorsal limbic mechanisms projecting through the cingulate gyrus may be influenced by hedonic evaluations, social attachments, and they may initiate a mode of motor control that is holistic and impulsive. In contrast, the ventral limbic pathway from the amygdala to orbital frontal cortex may implement a tight, restricted mode of motor control that reflects adaptive constraints of self-preservation. In the human brain, hemispheric specialization appears to have led to asymmetric elaborations of the dorsal and ventral pathways. Understanding the inherent asymmetries of corticolimbic architecture may be important in interpreting the increasing evidence that the left and right frontal lobes contribute differently to normal and pathological forms of self-regulation.

A high capacity assay for inhibitors of human papillomavirus DNA replication.

The discovery of antiviral compounds against human papillomaviruses (HPV) has been hindered by the difficulties in culturing virus in vitro or assaying stable HPV DNA replication. However, plasmids containing the HPV replication origin replicate transiently upon co-transfection with HPV E1 and E2 expression vectors. We have adapted this assay using secreted alkaline phosphatase (SAP) as a reporter for rapid analysis of DNA copy number. Use of the SV40 early promoter in controlling SAP expression was critical in ensuring both a strong signal and copy number dependence: the stronger beta-actin promotor inhibited replication, while the weaker SV40 late promoter yielded very low levels of SAP. The precise configuration of the E1 and E2 expression vectors also was critical, most pre-existing vectors did not support efficient replication and SAP secretion. The extent of DNA replication and SAP secretion were both proportional to the amount of E1/E2 vector used in transfections; under optimal conditions SAP increased 100-fold during replication. The assay has been developed for compound screening in 96-well plates and several inhibitors have been identified. Quantitative Southern blot analysis has shown that most of these inhibit HPV DNA replication rather than SAP accumulation or activity, and several are under test in models of viral replication. The assay also provides a rapid system for functional analysis of the HPV E1, E2 genes and the replication origin.

Organization of central adrenergic pathways: I. Relationships of ventrolateral medullary projections to the hypothalamus and spinal cord.

We studied the organization of projections from the C1 adrenergic and A1 noradrenergic cell groups in the ventrolateral medulla (VLM) to the hypothalamus and the spinal cord by using a combination of retrograde transport of fluorescent tracers and immunocytochemistry. Three issues were addressed. Neurons in the VLM that stain immunohistochemically for phenylethanolamine N-methyltransferase (PNMT) have been assumed to be adrenergic. However, the presence of PNMT-immunoreactive neurons in the hypothalamus that do not stain for tyrosine hydroxylase (TH) prompted us to re-evaluate the VLM by an elution-restaining immunohistochemical procedure. We confirmed that nearly all of the rostral medullary PNMT-immunoreactive neurons also stained for TH. By contrast, in the caudal medulla, very few TH-positive neurons stained for PNMT. Neurons of the C1 group in the rostral VLM project both to the thoracic spinal cord and to the hypothalamus. To determine whether individual C1 neurons send collaterals to the hypothalamus and spinal cord, we injected different-colored fluorescent dyes (diamidino yellow or fast blue) into the thoracic spinal gray matter and either the median preoptic (MnPO) or paraventricular (PVH) nuclei of the hypothalamus. Very few double-labeled neurons were found in the VLM, indicating that hypothalamic and spinal cord projections arise from almost completely independent populations of cells. Approximately half of the neurons projecting to the spinal cord from rostral VLM were not immunoreactive for TH or PNMT, indicating that a substantial part of this projection is noncatecholaminergic. The MnPO and the PVH both receive extensive catecholaminergic inputs from the VLM. We also used fluorescent retrograde tracers to determine whether individual VLM neurons send collaterals to both hypothalamic sites. Approximately 20% of neurons projecting to the MnPO in the rostral two thirds of the VLM also sent collaterials to the PVH, nearly all of these neurons being TH-positive. The collateralization of the VLM catecholaminergic projection to the hypothalamus may provide an anatomical substrate for integration of fore-brain participation in cardiovascular regulation. In contrast, the adrenergic projection from the VLM to the intermediolateral column of the spinal cord arises from a separate population of neurons.

Specificity of spinal projections from hypothalamic and brainstem areas which innervate sympathetic preganglionic neurons.

The specificity and topographic organization of afferent projections to the intermediolateral column (IML) were examined using retrograde transport of fluorescent tracers injected into pairs of thoracic spinal segments. Neurons within the hypothalamus (parvocellular paraventricular nucleus, dorsomedial nucleus and lateral hypothalamus), pons (Kolliker-Fuse and A5 nuclei) and medulla (ventrolateral nucleus of the solitary tract and rostral ventrolateral medulla) each appeared to innervate only a single spinal segment. Neurons in each cell group projecting to different spinal segments were intermixed and showed no evidence of topographic organization. These results provide a potential anatomical substrate for organ-specific autonomic responses to physiological and psychological stimuli.

Regional changes in EEG power and coherence during cognition: intensive study of two individuals.

Two men underwent weekly electroencephalogram (EEG) recordings while living for several months in a controlled laboratory environment. Data collected from an eight-channel EEG during a resting period and during performance of two cognitive tasks (word fluency and mental imagery) were subjected to spectral analysis. Statistical analyses on power and coherence were conducted for each subject separately, to determine whether that individual showed a characteristic pattern of EEG activity for a given cognitive task which was stable over time. Although substantial individual differences were observed, particularly for the theta band, both subjects showed changes in the spectral information over the anterior left hemisphere during the word fluency task.

Multivariate analysis of EEG coherence: stability of the metric, individual differences in patterning and response to arousal.

When coherence is computed among all pairs of a multichannel EEG, the resultant matrix can be de-structured with conventional multivariate analysis procedures to characterize the patterning of electrophysiologic information at each frequency band of the EEG across brain regions. Six right-handed young men had EEG data recorded from left and right frontal, temporal, parietal, and occipital locations during three visits to the laboratory at one week intervals. During each session, data were recorded during a relaxation period, an alert resting period, and as loud white noise was presented. Factor analysis of coherence matrices showed a model pattern of a large posterior factor and an anterior factor, both of which were right-lateralized, plus a residual factor that was most often left-lateralized. Four of the six men showed this pattern on each occasion, particularly for the delta and the theta bands. Two men showed variant patterns, which were also stable over time. A priori de-structuring of the matrix into partial multiple intrahemispheric and inter-hemispheric coherences also showed generally higher coherences on the right side, with the exception of left occipital inter-hemispheric values. Effects of arousal manipulations were not striking, but the factor patterns suggested increases in right hemisphere coherence for the alpha and beta bands during high arousal.