Luciano Marpegan

In the last decades, the use of camera traps for wildlife studies has increased significantly due to advancements in technology leading to cost reduction and improved reliability. They facilitate the study of wild animals in their natural environment in a minimally invasive manner, eliminating the need for researchers to remain in the field for long periods of time and to stay alert for extended hours. While their use is not too complex, data analysis often represents a bottleneck that demands considerable effort. When cameras are used to obtain videos, the first step upon retrieving information from them is to sort the videos, name them in a standardized manner, identify the content of each file, and label them to organize the information for subsequent data analysis. This process is laborious and repetitive, often leading to human errors that later hinder analysis. In this work an easy to use, customizable semi‐automated video processing and tagging software is presented alongside camera trap data analysis from sites used by an endangered otter species Lontra provocax . With its use, we were able to process over 3000 video files in a short time, finding that in the area under study (Nahuel Huapi National Park) all otter sites were also used by American minks, foxes, domestic dogs, and cats. In a particular site a detailed analysis provided data showing a nocturnal pattern of use for the site. When compared to manual data processing the semi‐automated process improved efficiency and overall user experience, and reduced the cognitive load associated with manual tagging. The aim of this work was to develop a versatile and user‐friendly yet powerful solution to the typical tasks associated with the analysis of camera trap videos.

This paper presents a system to obtain X-ray phase contrast images using a Commercial-Off-The-Shelf, low cost, CMOS Image Sensor as a direct radiation detector. The 5.2 μm pixel pitch allows the detection of Fresnel diffraction fringes, which enables the observation of borders and interfaces in samples. A simple experimental set-up comprised of a microfocus, tungsten anode, X-ray source, an image sensor, and the sample placed at tens of centimeters from the detector is used to measure Fresnel diffraction patterns in opaque and semi-transparent samples on semi-infinite planes. Additionally, two biological samples are presented as case studies. The sensor’s spectral resolution allows for a better understanding of the results.

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. Thus, circadian regulation in the efficacy of 1A-116 was studied in LN229 human glioblastoma cells and tumor-bearing nude mice. Methods. Wild-type LN229 and BMAL1-deficient (i.e., lacking a functional circadian clock) LN229E1 cells were assessed for rhythms in TIAM1, BMAL1, and period circadian protein homolog 1 (PER1), as well as Tiam1, Bmal1, and Rac1 mRNA levels. The effects of 1A-116 on proliferation, apoptosis, and migration were then assessed upon applying the drug at different circadian times. Finally, 1A-116 was administered to tumor-bearing mice at two different circadian times. Results. In LN229 cells, circadian oscillations were found for BMAL1, PER1, and TIAM1 (mRNA and protein), and for the effects of 1A-116 on proliferation, apoptosis, and migration, which were abolished in LN229E1 cells. Increased survival time was observed in tumor-bearing mice when treated with 1A-116 at the end of the light period (zeitgeber time 12, ZT12) compared either to animals treated at the beginning (ZT3) or with vehicle. Conclusions. These results unveil the circadian modulation in the efficacy of 1A-116, likely through RAC1 pathway rhythmicity, suggesting that a chronopharmacological approach is a feasible strategy to improve glioblastoma treatment.

Circadian disruption negatively affects physiology, posing a global health threat that manifests in proliferative, metabolic, and immune diseases, among others. Because outputs of the circadian clock regulate daily fluctuations in the immune response, we determined whether circadian disruption results in tumor-associated immune cell remodeling, facilitating tumor growth. Our findings show that tumor growth rate increased and latency decreased under circadian disruption conditions compared to normal light-dark (LD) schedules in a murine melanoma model. Circadian disruption induced the loss or inversion of daily patterns of M1 (proinflammatory) and M2 (anti-inflammatory) macrophages and cytokine levels in spleen and tumor tissues. Circadian disruption also induced (i) deregulation of rhythmic expression of clock genes and (ii) of cyclin genes in the liver, (iii) increased CcnA2 levels in the tumor, and (iv) dampened expression of the cell cycle inhibitor p21^WAF/CIP1, all of which contribute to a proliferative phenotype.

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. In this study, we aimed to further characterize the circadian response to high doses of LPS. First, we found that mice injected with LPS at ZT11 developed a higher hypothermia than those inoculated at ZT19. This response was accompanied by higher neuronal activation of the preoptic, suprachiasmatic, and paraventricular nuclei of the hypothalamus. However, LPS-induced Tnf-α and Tnf-α type 1 receptor (TNFR1) expression in the preoptic area was time-independent. We also analyzed peritoneal and spleen macrophages, and observed an exacerbated response after ZT11 stimulation. The serum of mice inoculated with LPS at ZT11 induced deeper hypothermia in naïve animals than the one coming from ZT19-inoculated mice, related to higher TNF-α serum levels during the day. We also analyzed the response in TNFR1-deficient mice, and found that both the daily difference in the mortality rate, the hypothermic response and neuronal activation were lost. Moreover, mice subjected to circadian desynchronization showed no differences in the mortality rate throughout the day, and developed lower minimum temperatures than mice under light-dark conditions. Also, those injected at ZT11 showed increased levels of TNF-α in serum compared to standard light conditions. These results suggest a circadian dependency of the central thermoregulatory and peripheral inflammatory response to septic-shock, with TNF-α playing a central role in this circadian response.

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. Given the anatomical location, which compromises both the circadian pacemaker and its photic input pathway, we decided to study whether the presence of gliomas in the hypothalamic region could alter circadian behavioral outputs. Athymic nude mice implanted with LN229 human glioma cells showed an increase in the endogenous period of the circadian clock, which was also less robust in terms of sustaining the free running period throughout 2 weeks of screening. We also found that implanted mice showed a slower resynchronization rate after an abrupt 6 h advance of the light-dark (LD) cycle, advanced phase angle, and a decreased direct effect of light in general activity (masking), indicating that hypothalamic tumors could also affect photic sensitivity of the circadian clock. Our work suggests that hypothalamic gliomas have a clear impact both on the endogenous pacemaking of the circadian system, as well as on the photic synchronization of the clock. These findings strongly suggest that the observation of altered circadian parameters in patients might be of relevance for glioma diagnosis.

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. A. pullulans formed daily concentric rings that were synchronized by light/dark cycles and were also formed in constant darkness with a period of 24.5 h. Moreover, these rhythms were temperature compensated, as evidenced by experiments conducted at temperatures as low as 10 °C. Finally, the expression of clock-essential genes, frequency, white collar-1, white collar-2 and vivid was confirmed. In summary, our results indicate the existence of a functional circadian clock in A. pullulans, capable of sustaining rhythms at very low temperatures and, based on the presence of conserved clock-gene homologues, suggest a molecular and functional relationship to well-described circadian systems.

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. Circadian time of treatment affected TMZ sensitivity of murine GBM tumor cells in vitro. The maximum TMZ-induced DNA damage response, activation of apoptosis, and growth inhibition occurred near the daily peak in expression of the core clock gene Bmal1. Deletion of Bmal1 (Arntl) abolished circadian rhythms in gene expression and TMZ-induced activation of apoptosis and growth inhibition. These data indicate that tumor cell-intrinsic circadian rhythms are common to GBM tumors and can regulate TMZ cytotoxicity. Optimization of GBM treatment by timing TMZ administration to daily rhythms should be evaluated in prospective clinical trials.

The suprachiasmatic nuclei, the main circadian clock in mammals, are entrained by light through glutamate released from retinal cells. Astrocytes are key players in glutamate metabolism but their role in the entrainment process is unknown. We studied the time dependence of glutamate uptake and glutamine synthetase (GS) activity finding diurnal oscillations in glutamate uptake (high levels during the light phase) and daily and circadian fluctuations in GS activity (higher during the light phase and the subjective day). These results show that glutamate-related astroglial processes exhibit diurnal and circadian variations, which could affect photic entrainment of the circadian system.

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).In vivo, PER2 expression in the OB of mice is circadian, approximately doubling with a peak around subjective dusk. Furthermore, mice exhibit circadian rhythms in odor detection performance with a peak at approximately subjective dusk. We also found that circadian rhythms in gene expression and odor detection performance require vasoactive intestinal polypeptide (VIP) or its receptor VPAC2R. VIP is expressed, in a circadian manner, in interneurons in the external plexiform and periglomerular layers, whereas VPAC2R is expressed in mitral and external tufted cells in the OB. Together, these results indicate that VIP signaling modulates the output from the OB to maintain circadian rhythms in the mammalian olfactory system.