High-resolution X-Ray imaging of small animal samples based on Commercial-Off-The-Shelf CMOS image sensors.

 An automated system for acquiring microscopic-resolution radiographic images of biological samples was developed. Mass-produced, low-cost, and easily automated components were used, such as Commercial-Off-The-Self CMOS image sensors (CIS), stepper motors, and control boards based on Arduino and RaspberryPi. System configuration, imaging protocols, and Image processing (filtering and stitching) were defined to obtain high-resolution images and for successful computational image reconstruction.

Timing of Novel Drug 1A-116 to Circadian Rhythms Improves Therapeutic Effects against Glioblastoma.

Unlabelled: The Ras homologous family of small guanosine triphosphate-binding enzymes (GTPases) is critical for cell migration and proliferation. The novel drug 1A-116 blocks the interaction site of the Ras-related C3 botulinum toxin substrate 1 (RAC1) GTPase with some of its guanine exchange factors (GEFs), such as T-cell lymphoma invasion and metastasis 1 (TIAM1), inhibiting cell motility and proliferation. Knowledge of circadian regulation of targets can improve chemotherapy in glioblastoma.

Differential Thermoregulatory and Inflammatory Patterns in the Circadian Response to LPS-Induced Septic Shock.

Sepsis is caused by a dysregulated host response to infection, and characterized by uncontrolled inflammation together with immunosuppression, impaired innate immune functions of phagocytes and complement activation. Septic patients develop fever or hypothermia, being the last one characteristic of severe cases. Both lipopolysaccharide (LPS) and Tumor Necrosis Factor (TNF)-α- induced septic shock in mice is dependent on the time of administration.

Circadian Alterations in a Murine Model of Hypothalamic Glioma.

The mammalian circadian system is controlled by a central oscillator located in the suprachiasmatic nuclei (SCN) of the hypothalamus, in which glia appears to play a prominent role. Gliomas originate from glial cells and are the primary brain tumors with the highest incidence and mortality. Optic pathway/hypothalamic gliomas account for 4-7% of all pediatric intracranial tumors.

Spontaneous circadian rhythms in a cold-adapted natural isolate of Aureobasidium pullulans.

Circadian systems enable organisms to synchronize their physiology to daily and seasonal environmental changes relying on endogenous pacemakers that oscillate with a period close to 24 h even in the absence of external timing cues. The oscillations are achieved by intracellular transcriptional/translational feedback loops thoroughly characterized for many organisms, but still little is known about the presence and characteristics of circadian clocks in fungi other than Neurospora crassa. We sought to characterize the circadian system of a natural isolate of Aureobasidium pullulans, a cold-adapted yeast bearing great biotechnological potential.

Cell-intrinsic, Bmal1-dependent Circadian Regulation of Temozolomide Sensitivity in Glioblastoma.

The safety and efficacy of chemotherapeutics can vary as a function of the time of their delivery during the day. This study aimed to improve the treatment of glioblastoma (GBM), the most common brain cancer, by testing whether the efficacy of the DNA alkylator temozolomide (TMZ) varies with the time of its administration. We found cell-intrinsic, daily rhythms in both human and mouse GBM cells.

Vasoactive intestinal polypeptide mediates circadian rhythms in mammalian olfactory bulb and olfaction.

Accumulating evidence suggests that the olfactory bulbs (OBs) function as an independent circadian system regulating daily rhythms in olfactory performance. However, the cells and signals in the olfactory system that generate and coordinate these circadian rhythms are unknown. Using real-time imaging of gene expression, we found that the isolated olfactory epithelium and OB, but not the piriform cortex, express similar, sustained circadian rhythms in PERIOD2 (PER2).

Modulation of mammalian circadian rhythms by tumor necrosis factor-α.

Unlabelled: Systemic low doses of the endotoxin lipopolysaccharide (LPS, 100 µg/kg) administered during the early night induce phase-delays of locomotor activity rhythms in mice. Our aim was to evaluate the role of tumor necrosis factor (Tnf)-alpha and its receptor 1/p55 (Tnfr1) in the modulation of LPS-induced circadian effects on the suprachiasmatic nucleus (SCN). We observed that Tnfr1-defective mice (Tnfr1 KO), although exhibiting similar circadian behavior and light response to that of control mice, did not show LPS-induced phase-delays of locomotor activity rhythms, nor LPS-induced cFos and Per2 expression in the SCN and Per1 expression in the paraventricular hypothalamic nucleus (PVN) as compared to wild-type (WT) mice.

Forkhead box O1 (FOXO1) protein, but not p53, contributes to robust induction of p21 expression in fasted mice.

Reporter mice that enable the activity of the endogenous p21 promoter to be dynamically monitored in real time in vivo and under a variety of experimental conditions revealed ubiquitous p21 expression in mouse organs including the brain. Low light bioluminescence microscopy was employed to localize p21 expression to specific regions of the brain. Interestingly, p21 expression was observed in the paraventricular, arcuate, and dorsomedial nuclei of the hypothalamus, regions that detect nutrient levels in the blood stream and signal metabolic actions throughout the body.

Role of proinflammatory cytokines on lipopolysaccharide-induced phase shifts in locomotor activity circadian rhythm.

We previously reported that early night peripheral bacterial lipopolysaccharide (LPS) injection produces phase delays in the circadian rhythm of locomotor activity in mice. We now assess the effects of proinflammatory cytokines on circadian physiology, including their role in LPS-induced phase shifts. First, we investigated whether differential systemic induction of classic proinflammatory cytokines could explain the time-specific behavioral effects of peripheral LPS.

In vitro circadian rhythms: imaging and electrophysiology.

In vitro assays have localized circadian pacemakers to individual cells, revealed genetic determinants of rhythm generation, identified molecular players in cell-cell synchronization and determined physiological events regulated by circadian clocks. Although they allow strict control of experimental conditions and reduce the number of variables compared with in vivo studies, they also lack many of the conditions in which cellular circadian oscillators normally function. The present review highlights methods to study circadian timing in cultured mammalian cells and how they have shaped the hypothesis that all cells are capable of circadian rhythmicity.

Bioluminescence imaging captures the expression and dynamics of endogenous p21 promoter activity in living mice and intact cells.

To interrogate endogenous p21(WAF1/CIP1) (p21) promoter activity under basal conditions and in response to various forms of stress, knock-in imaging reporter mice in which expression of firefly luciferase (FLuc) was placed under the control of the endogenous p21 promoter within the Cdkn1a gene locus were generated. Bioluminescence imaging (BLI) of p21 promoter activity was performed noninvasively and repetitively in mice and in cells derived from these mice. We demonstrated that expression of FLuc accurately reported endogenous p21 expression at baseline and under conditions of genotoxic stress and that photon flux correlated with mRNA abundance and, therefore, bioluminescence provided a direct readout of p21 promoter activity in vivo.

Circadian regulation of ATP release in astrocytes.

Circadian clocks sustain daily oscillations in gene expression, physiology, and behavior, relying on transcription-translation feedback loops of clock genes for rhythm generation. Cultured astrocytes display daily oscillations of extracellular ATP, suggesting that ATP release is a circadian output. We hypothesized that the circadian clock modulates ATP release via mechanisms that regulate acute ATP release from glia.

Diurnal variation in endotoxin-induced mortality in mice: correlation with proinflammatory factors.

Many immune parameters exhibit daily and circadian oscillations, including the number of circulating cells and levels of cytokines in the blood. Mice also have a differential susceptibility to lipopolysaccharide (LPS or endotoxin)-induced endotoxic shock, depending on the administration time in the 24 h light-dark (LD) cycle. We replicated these results in LD, but we did not find temporal differences in LPS-induced mortality in constant darkness (DD).

Vasoactive intestinal polypeptide entrains circadian rhythms in astrocytes.

Many mammalian cell types show daily rhythms in gene expression driven by a circadian pacemaker. For example, cultured astrocytes display circadian rhythms in Period1 and Period2 expression. It is not known, however, how or which intercellular factors synchronize and sustain rhythmicity in astrocytes.

Stably integrated luxCDABE for assessment of Salmonella invasion kinetics.

Salmonella Typhimurium is a common cause of gastroenteritis in humans and also localizes to neoplastic tumors in animals. Invasion of specific eukaryotic cells is a key mechanism of Salmonella interactions with host tissues. Early stages of gastrointestinal cell invasion are mediated by a Salmonella type III secretion system, powered by the adenosine triphosphatase invC.

Suprachiasmatic astrocytes as an interface for immune-circadian signalling.

The hypothalamic suprachiasmatic nuclei (SCN), the site of a mammalian circadian clock, exhibit a dense immunoreactivity for glial fibrillary acidic protein (GFAP), a specific marker for astrocytes. Although there is evidence of a circadian variation in GFAP-IR in the hamster SCN and of the participation of glial cells in input and output mechanisms of the clock, the role of these cells within the circadian system is not clearly understood. The fact that astroglia can express and respond to cytokines suggests that they could work as mediators of immune signals to the circadian system.

Circadian responses to endotoxin treatment in mice.

We tested the ability of Escherichia coli lipopolysaccharide (LPS) to phase-shift the activity circadian rhythm in C57Bl/6J mice. Intraperitoneal administration of 25 microg/kg LPS induced photic-like phase delays (-43+/-10 min) during the early subjective night. These delays were non-additive to those induced by light at CT 15, and were reduced by the previous administration of sulfasalazine, a NF-kappaB activation inhibitor.

Participation of transcription factors from the Rel/NF-kappa B family in the circadian system in hamsters.

We have studied the presence and activity of components of the nuclear factor-kappaB (NF-kappaB) transcription factor in the hamster circadian system analyzing wheel-running activity, protein expression and DNA binding activity by electrophoresis mobility shift assays (EMSA). Non-rhythmic specific immunoreactive bands corresponding to a NF-kappaB subunit (p65) were found in hamster suprachiasmatic nuclei (SCN) homogenates. The active form of NF-kappaB evidenced by EMSA was clear and specific in SCN nuclear extracts.

From light to genes: moving the hands of the circadian clock.

Mammalian circadian rhythms are generated by the hypothalamic suprachiasmatic nuclei and finely tuned to environmental periodicities by neurochemical responses to the light-dark cycle. Light reaches the clock through a direct retinohypothalamic tract, primarily through glutamatergic innervation, and its action is probably regulated by a variety of other neurotransmitters. A key second messenger in circadian photic entrainment is calcium, mobilized through membrane channels or intracellular reservoirs, which triggers the activation of several enzymes, including a calcium/calmodulin-dependent protein kinase and nitric oxide synthase.