Dark Cycle Research Articles

Microglia undergo molecular and functional adaptations to dark and light phases in male laboratory mice

Microglia are increasingly recognized to contribute to brain health and disease. Preclinical studies using laboratory rodents are essential to advance our understanding of the physiological and pathophysiological roles of these cells in the central nervous system. Rodents are nocturnal animals, and they are mostly maintained in a defined light–dark cycle within animal facilities, with many laboratories investigating the molecular and functional profiles of microglia exclusively during the animals’ light (sleep) phase. However, only a few studies have considered possible differences in microglial functions between the active and sleep phases. Based on initial evidence suggesting that microglial intrinsic clock genes can affect their phenotypes, we sought to investigate differences in transcriptional, proteotype and functional profiles of microglia between light (sleep) and dark (active) phases, and how these changes are affected in pathological models. We found marked transcriptional and proteotype differences between microglia harvested from male mice during the light or dark phase. Amongst others, these differences related to genes and proteins associated with immune responses, motility, and phagocytosis, which were reflected by functional alterations in microglial synaptic pruning and response to bacterial stimuli. Possibly accounting for such changes, we found RNA and protein regulation in SWI/SNF and NuRD chromatin remodeling complexes between light and dark phases. Importantly, we also show that the time of microglial sample collection influences the nature of microglial transcriptomic changes in a model of immune-mediated neurodevelopmental disorders. Our findings emphasize the importance of considering diurnal factors in studying microglial cells and indicate that implementing a circadian perspective is pivotal for advancing our understanding of their physiological and pathophysiological roles in brain health and disease.

The primary carbon metabolism in cyanobacteria and its regulation.

Cyanobacteria are the only prokaryotes capable of performing oxygenic photosynthesis. Many cyanobacterial strains can live in different trophic modes, ranging from photoautotrophic and heterotrophic to mixotrophic growth. However, the regulatory mechanisms allowing a flexible switch between these lifestyles are poorly understood. As anabolic fixation of CO2 in the Calvin-Benson-Bassham (CBB) cycle and catabolic sugar-degradation pathways share intermediates and enzymatic capacity, a tight regulatory network is required to enable simultaneous opposed metabolic fluxes. The Entner-Doudoroff (ED) pathway was recently predicted as one glycolytic route, which cooperates with other pathways in glycogen breakdown. Despite low carbon flux through the ED pathway, metabolite analyses of mutants deficient in the ED pathway revealed a distinct phenotype pointing at a strong regulatory impact of this route. The small Cp12 protein downregulates the CBB cycle in darkness by inhibiting phosphoribulokinase and glyceraldehyde 3-phosphate dehydrogenase. New results of metabolomic and redox level analyses on strains with Cp12 variants extend the known role of Cp12 regulation towards the acclimation to external glucose supply under diurnal conditions as well as to fluctuations in CO2 levels in the light. Moreover, carbon and nitrogen metabolism are closely linked to maintain an essential C/N homeostasis. The small protein PirC was shown to be an important regulator of phosphoglycerate mutase, which identified this enzyme as central branching point for carbon allocation from CBB cycle towards lower glycolysis. Altered metabolite levels in the mutant ΔpirC during nitrogen starvation experiments confirm this regulatory mechanism. The elucidation of novel mechanisms regulating carbon allocation at crucial metabolic branching points could identify ways for targeted redirection of carbon flow towards desired compounds, and thus help to further establish cyanobacteria as green cell factories for biotechnological applications with concurrent utilization of sunlight and CO2.

Optimal growth conditions and heavy metal removal of Coelastrella sp. CORE-3 isolated from wastewater

This study was investigated to optimize growth conditions of native microalgae (Coelastrella sp. CORE-3) from wastewater, evaluate the removal of Cu2+, Zn2+, Cd2+ and Ni2+ and compare both isolated microalgae and Ankistrodesmus bibraianus, and describe their sorption mechanisms on the cell surface. The effects of the pH, light: dark cycle and temperature on the growth of the isolated Coelastrella sp. CORE-3 was optimized. The optimal L:D cycle, temperature, and pH conditions were 14:10 hr, 25℃, pH 7. The growth of isolated microalgae, Coelastrella sp. CORE-3. was compared with A. bibraianus. The removal efficiency of heavy metals by Coelastrella sp. CORE-3 and A. bibraianus were found as follows, respectively: (live cell) Zn2+ > Cd2+ > Cu2+ > Ni2+, (dead cell) Cu2+ > Cd2+ > Zn2+ > Ni2+. SEM analysis indicated that more cell surface deformation occurred in A. bibraianus compared to Coelastrella sp. after exposure to heavy metals. This result suggests that Coelastrella sp. CORE-3 has more resistant to heavy metal toxicity. In addition, based on the FT-IR analysis, the live cells of Coelastrella sp. CORE-3 and A. bibraianus showed the interactions of heavy metals with the functional groups: alcohol, alkane, amide, amine on their cell surfaces.

Transcriptome Analysis on the ALAN-Induced Zebrafish Ovary: Desynchronization of Life Processes and Initiation of Diseases

Objective: The effect of artificial light at night (ALAN) on “transcriptome” is prominent owing to its capacity for “desynchronization” of organismal physiology. Light influences the circadian rhythm. This study aims to explore the ALAN-induced ovarian transcriptome of zebrafish for desynchronization of life processes. Methods: Four experimental conditions were set up for female zebrafish: one normal 12-hour light and 12-hour dark (LD) cycle, and three continuous exposures to ALAN for one week (LLW), one month (LLM), and one year (LLY). The whole transcriptome data analysis of the ALAN-exposed samples was then compared with the normal sample using RNA-Seq, followed by exploratory analyses. Results: The analysis revealed two different patterns of expression of genes where LLW and LLM differ with LLY samples in comparison to LD. Compared to LD, downregulation of the predicted hub genes was observed in all treatments; ribosome and oxidative phosphorylation pathways were enriched. LLY vs. LD contrast depicts the enrichment of three more pathways—RNA polymerase, adhesion junction, and signaling. The gene ontology (GO) enrichment portrays more prevalent biological processes in LLW vs. LD and LLM vs. LD than in LLY vs. LD. Contrast-wise disease annotation represents neoplasms as the most prevalent; disease enrichment denotes the major class of neoplasm, carcinoma, coupled with intellectual disability, global developmental delay, and seizures. Conclusion: Our study displayed desynchronization of various genes and pathways leading to the initiation of diseases, for the first time in zebrafish. Even though, this data shows that ALAN is a serious threat, further research is needed to determine the intensity and the duration of ALAN which might cause potential repercussions.

Investigation of the structural changes in the hippocampus and prefrontal cortex using FTIR spectroscopy in sleep deprived mice

Sleep is a basic, physiological requirement for living things to survive and is a process that covers one third of our lives. Melatonin is a hormone that plays an important role in the regulation of sleep. Sleep deprivation affect brain structures and functions. Sleep deprivation causes a decrease in brain activity, with particularly negative effects on the hippocampus and prefrontal cortex. Despite the essential role of protein and lipids vibrations, polysaccharides, fatty acid side chains functional groups, and ratios between amides in brain structures and functions, the brain chemical profile exposed to gentle handling sleep deprivation model versus Melatonin exposure remains unexplored. Therefore, the present study, aims to investigate a molecular profile of these regions using FTIR spectroscopy measurement’s analysis based on lipidomic approach with chemometrics and multivariate analysis to evaluate changes in lipid composition in the hippocampus, prefrontal regions of the brain. In this study, C57BL/6J mice were randomly assigned to either the control or sleep deprivation group, resulting in four experimental groups: Control (C) (n = 6), Control + Melatonin (C + M) (n = 6), Sleep Deprivation (S) (n = 6), and Sleep Deprivation + Melatonin (S + M) (n = 6). Interventions were administered each morning via intraperitoneal injections of melatonin (10 mg/kg) or vehicle solution (%1 ethanol + saline), while the S and S + M groups underwent 6 h of daily sleep deprivation from using the Gentle Handling method. All mice were individually housed in cages with ad libitum access to food and water within a 12-hour light–dark cycle. Results presented that the brain regions affected by insomnia. The structure of phospholipids, changed. Yet, not only changes in lipids but also in amides were noticed in hippocampus and prefrontal cortex tissues. Additionally, FTIR results showed that melatonin affected the lipids as well as the amides fraction in cortex and hippocampus collected from both control and sleep deprivation groups.

Adult Inception of Ketogenic Diet Therapy Increases Sleep during the Dark Cycle in C57BL/6J Wild Type and Fragile X Mice.

Sleep problems are a significant phenotype in children with fragile X syndrome. Our prior work assessed sleep-wake cycles in Fmr1KO male mice and wild type (WT) littermate controls in response to ketogenic diet therapy where mice were treated from weaning (postnatal day 18) through study completion (5-6 months of age). A potentially confounding issue with commencing treatment during an active period of growth is the significant reduction in weight gain in response to the ketogenic diet. The aim here was to employ sleep electroencephalography (EEG) to assess sleep-wake cycles in mice in response to the Fmr1 genotype and a ketogenic diet, with treatment starting at postnatal day 95. EEG results were compared with prior sleep outcomes to determine if the later intervention was efficacious, as well as with published rest-activity patterns to determine if actigraphy is a viable surrogate for sleep EEG. The data replicated findings that Fmr1KO mice exhibit sleep-wake patterns similar to wild type littermates during the dark cycle when maintained on a control purified-ingredient diet but revealed a genotype-specific difference during hours 4-6 of the light cycle of the increased wake (decreased sleep and NREM) state in Fmr1KO mice. Treatment with a high-fat, low-carbohydrate ketogenic diet increased the percentage of NREM sleep in both wild type and Fmr1KO mice during the dark cycle. Differences in sleep microstructure (length of wake bouts) supported the altered sleep states in response to ketogenic diet. Commencing ketogenic diet treatment in adulthood resulted in a 15% (WT) and 8.6% (Fmr1KO) decrease in body weight after 28 days of treatment, but not the severe reduction in body weight associated with starting treatment at weaning. We conclude that the lack of evidence for improved sleep during the light cycle (mouse sleep time) in Fmr1KO mice in response to ketogenic diet therapy in two studies suggests that ketogenic diet may not be beneficial in treating sleep problems associated with fragile X and that actigraphy is not a reliable surrogate for sleep EEG in mice.

Effects of chronic light cycle disruption during adolescence on circadian clock, neuronal activity rhythms, and behavior in mice.

The advent of artificial lighting, particularly during the evening and night, has significantly altered the predictable daily light and dark cycles in recent times. Altered light environments disrupt the biological clock and negatively impact mood and cognition. Although adolescents commonly experience chronic changes in light/dark cycles, our understanding of how the adolescents' brain adapts to altered light environments remains limited. Here, we investigated the impact of chronic light cycle disruption (LCD) during adolescence, exposing adolescent mice to 19 h of light and 5 h of darkness for 5 days and 12 L:12D for 2 days per week (LCD group) for 4 weeks. We showed that LCD exposure did not affect circadian locomotor activity but impaired memory and increased avoidance response in adolescent mice. Clock gene expression and neuronal activity rhythms analysis revealed that LCD disrupted local molecular clock and neuronal activity in the dentate gyrus (DG) and in the medial amygdala (MeA) but not in the circadian pacemaker (SCN). In addition, we characterized the photoresponsiveness of the MeA and showed that somatostatin neurons are affected by acute and chronic aberrant light exposure during adolescence. Our research provides new evidence highlighting the potential consequences of altered light environments during pubertal development on neuronal physiology and behaviors.

Impact of Serotonin Deficiency on Circadian Dopaminergic Rhythms.

Physiology and behavior are structured temporally to anticipate daily cycles of light and dark, ensuring fitness and survival. Neuromodulatory systems in the brain-including those involving serotonin and dopamine-exhibit daily oscillations in neural activity and help shape circadian rhythms. Disrupted neuromodulation can cause circadian abnormalities that are thought to underlie several neuropsychiatric disorders, including bipolar mania and schizophrenia, for which a mechanistic understanding is still lacking. Here, we show that genetically depleting serotonin in Tph2 knockout mice promotes manic-like behaviors and disrupts daily oscillations of the dopamine biosynthetic enzyme tyrosine hydroxylase (TH) in midbrain dopaminergic nuclei. Specifically, while TH mRNA and protein levels in the Substantia Nigra (SN) and Ventral Tegmental Area (VTA) of wild-type mice doubled between the light and dark phase, TH levels were high throughout the day in Tph2 knockout mice, suggesting a hyperdopaminergic state. Analysis of TH expression in striatal terminal fields also showed blunted rhythms. Additionally, we found low abundance and blunted rhythmicity of the neuropeptide cholecystokinin (Cck) in the VTA of knockout mice, a neuropeptide whose downregulation has been implicated in manic-like states in both rodents and humans. Altogether, our results point to a previously unappreciated serotonergic control of circadian dopamine signaling and propose serotonergic dysfunction as an upstream mechanism underlying dopaminergic deregulation and ultimately maladaptive behaviors.

Associations between circadian alignment and cognitive functioning in a nationally representative sample of older adults

Proper alignment of activity-rest and light–dark patterns allows for healthy bodily functions to occur at optimal times of the day. Disruptions to this alignment may cause poor sleep as well as physical, mental, and cognitive problems. The purpose of this cross-sectional study was to determine if poorer circadian alignment was associated with decreased cognitive functioning among older (> 60 years) participants in the National Health and Nutrition Examination Survey. We utilized actigraphy-based rest-activity and dark-light measurements to calculate phasor magnitude (strength of circadian alignment coupling) and phasor angle (phase difference between activity-rest and light–dark cycles). Multiple linear regression models were used to determine associations of phasor magnitude and angle with performance in various cognitive tests, including Digit Symbol Substitution Test score (DSSS), CERAD Savings Percentage (CSP), and Animal Fluency Test (AFT) score. The results showed that a lower phasor magnitude (which indicates decreased strength of alignment coupling between rest-activity and dark–light cycles) was significantly associated with decreased DSSS (indicating slower processing speed and poorer working memory) when controlling for many important sociodemographic factors. However, this association became non-significant when accounting for sleep duration and total physical activity. Phasor angle did not have a significant association with any of the cognitive scores. Overall, we provided evidence indicating that circadian alignment may be a predictor of cognitive performance. Future studies should investigate whether improving circadian alignment may improve cognitive function and prevent cognitive decline.

Altered Metabolism during the Dark Period in Drosophila Short Sleep Mutants.

Sleep is regulated via circadian mechanisms, but effects of sleep disruption on physiological rhythms, in particular metabolic cycling, remain unclear. To examine this question, we probed diurnal metabolic alterations of two Drosophila short sleep mutants, fumin and sleepless. Samples were collected with high temporal sampling (every 2 h) over 24 h under a 12:12 light:dark cycle, and profiling was done using an ion-switching LCMS/MS method. Fewer metabolites with 24 h oscillations were noted with short sleep (50 and 46 in fumin and sleepless, BH. Q < 0.2 by RAIN analysis) compared to a wild-type control (iso31, 63 with BH. Q < 0.2), and peak phases of the sleep mutants were consolidated into two major phase peaks at mid-day and middle of night. Overall, altered nicotinate/nicotinamide, alanine/aspartate/glutamate, acetylcholine, glyoxylate/dicarboxylate, and TCA cycle metabolism were observed in the short sleep mutants, indicative of increased energetic demand and oxidative stress compared to wild type. Both changes in cycling and discriminant models suggest unique alterations in the dark period indicative of constrained metabolic networks. Thus, we conclude that sleep loss alters metabolic function uniquely throughout the day, and further examination of specific mechanisms is warranted.

Time-restricted feeding protects against cisplatin-induced acute kidney injury in mice.

Time-restricted feeding (TRF), devoid of calorie restriction, is acknowledged for promoting metabolic health and mitigating various chronic metabolic diseases. While TRF exhibits widespread benefits across multiple tissues, there is limited exploration into its impact on kidney function. In this study, our aim was to investigate the potential ameliorative effects of TRF on kidney damage in a mouse model of cisplatin-induced acute kidney injury (AKI). Cisplatin-induced AKI was induced through intraperitoneal injection of cisplatin into C57BL/6 male mice. Mice undergoing TRF were provided unrestricted access to standard chow daily but were confined to an 8-hour feeding window during the dark cycle for 2 weeks before cisplatin injection. The mice were categorized into four groups: control, TRF, cisplatin, and TRF + cisplatin. The tubular damage score and serum creatinine levels were significantly lower in the TRF + cisplatin group compared to the cisplatin group. The TRF + cisplatin group exhibited reduced expression of phosphorylated nuclear factor kappa B, inflammatory cytokines, and F4/80-positive macrophages compared to the cisplatin group. Furthermore, oxidative stress markers for DNA, protein, and lipid were markedly decreased in the TRF + cisplatin group compared to the cisplatin group. TUNEL-positive tubular cells, cleaved caspase-3 expression, and the Bax/Bcl-2 ratio in the TRF + cisplatin group were lower than those in the cisplatin group. TRF, without calorie restriction, effectively mitigated kidney damage by suppressing inflammatory reactions, oxidative stress, and tubular apoptosis in a mouse model of cisplatin-induced AKI. TRF holds promise as a novel dietary intervention for preventing cisplatin-induced AKI.

Sleep in the East African root rat, Tachyoryctes splendens.

The present study reports the results of an electrophysiological analysis of sleep in the East African root rat, Tachyoryctes splendens, belonging to the rodent subfamily Spalacinae. Telemetric electroencephalographic (EEG) and electromyographic recordings, with associated video recording, on three root rats over a continuous 72 h period (12 h light/12 h dark cycle) were analyzed. The analysis revealed that the East African root rat has a total sleep time (TST) of 8.9 h per day. Despite this relatively short total sleep time in comparison to fossorial rodents, nonrapid eye movement (non-REM) sleep and rapid eye movement (REM) sleep states showed similar physiological signatures to that observed in other rodents and no unusual sleep states were observed. REM occupied 19.7% of TST, which is within the range observed in other rodents. The root rats were extremely active during the dark period, and appeared to spend much of the light period in quiet wake while maintaining vigilance (as determined from both EEG recordings and behavioral observation). These recordings were made under normocapnic environmental conditions, which contrasts with the hypercapnic environment of their natural burrows.

Chronic hyperactivation of midbrain dopamine neurons causes preferential dopamine neuron degeneration.

Parkinson's disease (PD) is characterized by the death of substantia nigra (SNc) dopamine (DA) neurons, but the pathophysiological mechanisms that precede and drive their death remain unknown. The activity of DA neurons is likely altered in PD, but we understand little about if or how chronic changes in activity may contribute to degeneration. To address this question, we developed a chemogenetic (DREADD) mouse model to chronically increase DA neuron activity, and confirmed this increase using ex vivo electrophysiology. Chronic hyperactivation of DA neurons resulted in prolonged increases in locomotor activity during the light cycle and decreases during the dark cycle, consistent with chronic changes in DA release and circadian disturbances. We also observed early, preferential degeneration of SNc projections, recapitulating the PD hallmarks of selective vulnerability of SNc axons and the comparative resilience of ventral tegmental area axons. This was followed by eventual loss of midbrain DA neurons. Continuous DREADD activation resulted in a sustained increase in baseline calcium levels, supporting an important role for increased calcium in the neurodegeneration process. Finally, spatial transcriptomics from DREADD mice examining midbrain DA neurons and striatal targets, and cross-validation with human patient samples, provided insights into potential mechanisms of hyperactivity-induced toxicity and PD. Our results thus reveal the preferential vulnerability of SNc DA neurons to increased neural activity, and support a potential role for increased neural activity in driving degeneration in PD.

First report of Colletotrichum karstii causing anthracnose on soybean in China.

Soybean (Glycine max) is a significant grain and oil crop. Among the various challenges faced by soybean cultivation, anthracnose stands out as one of the most prevalent diseases. In June 2023, anthracnose symptoms on leaves characterized by irregular disease spots featuring gray-white centers and brown edges, along with many small black dots on their surface, were observed in a 20-hectare soybean (variety "Liu Yuehuang") field located in Luodian County (25°40'20″ N, 106°53'50″ E, 575 m), Guizhou Province, China. Around 30% of the 300 soybean plants examined were symptomatic, and a total of ten leaves were collected. Fragments (5×5 mm) from the edge of disease spots were sheared and surface-sterilized with 3% sodium hypochlorite and 75% ethanol for 60 s and 30 s, respectively. They were then flushed twice with sterile water, dried using sterile filter papers, finally placed on potato dextrose agar (PDA) and incubated at 28°C for two days. In total, 11 isolates with identical morphological characteristics were obtained. The colonies grown with white aerial mycelia on their surface; conidia were cylindrical, both ends are rounded, aseptate, hyaline, 11.0-14.0 (12.5) × 4.5-6.0 (5.0) μm (n = 30); appressoria were nearly ovoid, brown to black, 8.5-10.5 (9.5) × 5.5-7.5 (6.0) μm (n = 30). The morphological characteristics closely resembled the description of C. karstii (Damm et al., 2012). To further identify the isolates, chitin synthase (CHS-1), actin (ACT), beta-tubulin (TUB2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the internal transcribed spacer (ITS) loci were amplified by using CHS-79F/CHS-345R, ACT-512F/ACT-783R (Carbone and Kohn, 1999), Bt2F/Bt2R (Woudenberg et al., 2009), GDF/GDR (Guerber et al., 2003) and ITS1/ITS4 (White et al., 1990) PCR primers, respectively. The BLAST results showed that the sequences of two representative strains, LD 2023048-1 and LD 2023048-2, were highly similar to those of strain C. karstii CGMCC3.14194 (ITS: OR342620 (99%) and OR342621 (99%) with HM585409, ACT: OR412337 (97%) and OR423341 (100%) with HM581995, CHS-1: OR423344 (99%,) and OR423345 (100%) with HM582023, GAPDH: OR423348 (98%) and OR423349 (98%) with HM585391, and TUB: OR423352 (99%) and OR423353 (99%) with HM585428). The phylogenetic tree combined five sequences showed that the two strains clustered into a branch of C. karstii CGMCC3.14194 with high support values. Thirty-day-old soybean plants (n = 10) (variety Liu Yuehuang) were separately sprayed with 1 × 105 spore suspensions/mL of the two strains by spray method, and plants sprayed with sterile distilled water were used as the negative control (n = 5). All the plants were then covered with plastic bags and cultured in the greenhouse (28℃, 80% humidity, 12 h light dark cycle). After ten days of inoculation, plants inoculated with C. karstii began to produce typical anthracnose symptoms, while the control remained asymptomatic. The confirmation of the reisolated pathogen as C. karstii was established through a comprehensive analysis of morphology and five sequencing loci. Pathogenicity tests were repeated three times. Anthracnose on soybean is caused by Colletotrichum spp. reported in China including C. truncatum (Hu et al., 2015), C. brevisporum (Shi et al., 2021) and C. fructicola (Xu et al., 2023). As far as we know, this study is the initial report of C. karstii inducing anthracnose on soybean to date, which establishes a fundamental reference for preventing and controlling this disease.

What accounts for the difference in the emergence times of Drosophila melanogaster between the first and second eclosion days?

AbstractOne of the characteristics of the adult eclosion rhythm in Drosophila melanogaster is that adult emergence time differs greatly between the first and second eclosion days. The emergence time is in the middle of the light period on the first eclosion day, but immediately after light‐on on the second day. We hypothesized that incomplete entrainment of the endogenous pacemaker to the light–dark (LD) cycle is responsible for the daily variation. Due to the very short pupal period of this species, adult emergence may occur before complete synchronization of the pacemaker with the external cycle on the first eclosion day. Therefore, the peak time on the first eclosion day may differ significantly from that on the second day. To verify this hypothesis, using pupae that had pupariated within 24 h, the time difference between the first and second peaks was compared in LD 12:12 or constant darkness at five different temperatures from 30 to 15°C. In both light regimes, the time difference decreased with decreasing temperature and extended pupal duration. The interval between the eclosion peaks approached 24 h, supporting this hypothesis. These results can be interpreted by using a two‐oscillator model.

Abstract PO3-25-02: Circadian Rhythm Disruption Accelerates Aggressive Mammary Tumorigenesis by Altering Tumor Microenvironment

Abstract \Breast cancer is the most common cancer in women worldwide, leading to over 650,000 deaths worldwide. The circadian rhythm is a 24-hour biological rhythm that controls most cellular, hormonal, and behavioral processes in living organisms, which is why perturbation of these rhythms is linked to several devastating diseases such as cardiovascular diseases, diabetes, and cancer. In the mammary gland, circadian clocks regulate the rhythmic expression of numerous genes. Circadian rhythm disruption (CRD), which has been termed a "carcinogen" by the International Agency for Research in Cancer, is caused by different factors, one of them being shift work. Several epidemiological studies have revealed that women who work night shifts are predisposed to breast cancer. The aim of this study is to investigate the effect of circadian rhythm disruption on aggressive mammary tumorigenesis. To do this, we used genetically engineered mouse models of mammary tumors (HER2-tumor and triple-negative breast tumor, or TNBC). Chronic jetlag, which mimics shift work in mice and is known to cause CRD, was introduced in one group of mice by perturbing the normal light and dark cycles with an 8-hour advance every three days. Wheel-running, which is represented by an actogram, was used to confirm the circadian disruption in the CRD mice. CRD was found to accelerate tumor initiation with a significant increase in weight in the CRD group of mice. CRD enhanced tumor burden significantly compared to LD mice. Our results also showed high metastatic foci in the lungs of CRD mice compared to LD mice. A flow cytometry study showed a higher cancer stem cell population in CRD tumors. Mammosphere assays with tumor-derived cells showed that CRD increased the number and size of the mammospheres compared to the control tumors. The tumor cells were analyzed using single-cell RNA sequencing, which showed that CRD alters the tumor microenvironment by developing more immunosuppressive and "cold" tumors. The tumor-derived organoids (tumoroid) showed resistance to chemotherapeutics. Finally, bioinformatics analysis using single-cell RNA seq data from human breast tumors (TNBC, ER/PR, and HER2) showed that the malignant cells in TNBC tumors had a significantly higher circadian rhythm disrupted score (CRD score) compared to ER/PR tumors. Taken together, our data showed how circadian rhythm disruption affects TNBC tumorigenesis and drug response by altering the tumor microenvironment. Citation Format: Olajumoke Ogunlusi, Mrinmoy Sarkar, Tristan Nguyen, Devon Boland, James Cai, Arhit Chakrabarti, Bani Mallick, Alex Keene, Tapasree Roy Sarkar. Circadian Rhythm Disruption Accelerates Aggressive Mammary Tumorigenesis by Altering Tumor Microenvironment [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-25-02.

Potencial de Verrucodesmus verrucosus en la remoción de estructuras de nitrógeno y fósforo de aguas residuales de granjas porcinas

Verrucodesmus verrucosus in residual water from a pig farm located in Suchiapa, Chiapas, Mexico, was evaluated as a mixotrophic culture medium. The evaluation was carried out for 40 days, under the 12:12 light / dark cycle, two growth stages were evaluated (piglet and fattening) and the 50:50 mixture of these, as well as to the residual water two pre-treatments were applied, this consisted of a filtration process using a 15 µm diameter nylon filter and a sterilization process. The microalgal species Verrucodesmus verrucosus was shown to have bioremediation potential by growing in wastewater and producing biomass, demonstrating high efficiency in removing contaminants. The maximum Chemical Oxygen Demand (COD) removal was in the Ps treatment (sterile piglet) where the removal of 96.8 % was reached, while the Biochemical Oxygen Demand (BOD) had a maximum removal of 96.7 % in the Pf treatment (filtered piglet). The removal of total nitrogen, ammonia and nitrate was demonstrated with a percentage of 85.5 %, 74 % and 91 % respectively. As for the maximum removal of phosphorus and phosphate, they reach values of 97.9 % and 82 % respectively. On the other hand, it was possible to demonstrate the antagonistic capacity of this microalga with respect to Escherichia coli, where 100 % elimination was achieved.

A pilot exploring the impact of paternal puberty blocking on offspring voluntary wheel running

Previous work from our lab has shown puberty blocking treatment in young female rats followed by discontinued use (or detransition) leads to decreased voluntary wheel running activity in their offspring. The purpose of the present study was to determine if puberty blocking and detransition in young male rats affects offspring voluntary wheel running activity. We hypothesized that offspring of previously puberty blocked male rats will have lower wheel running activity than offspring of control rats. Four week old, male Sprague-Dawley rats received either 100 μg of the gonadotropin releasing hormone agonist triptorelin daily as a puberty blocker or 0.9% saline daily as a control. At the end of the 4 week treatment period, injections ceased, and each male rat was paired with an age-matched female rat for breeding. The breeding period lasted approximately 8 weeks and resulted in pregnancy of each paired female. At 6 weeks of age, a portion of the offspring were randomly assigned to the following voluntary wheel running groups: male offspring of previously puberty blocked males (M-PBO, n=2), female offspring of previously puberty blocked males (F-PBO, n=2), male offspring of control males (M-CON, n=5), and female offspring of control males (F-CON, n=5). Offspring were housed in cages outfitted with voluntary running wheels, received food and water ad libitum, and were maintained on a12:12 hour light:dark cycle. Voluntary wheel running activity was monitored for 8 weeks, and between-group comparisons were made via Welch’s t-test. Voluntary wheel running activity is presented as number of wheel running rotations, and data are shown as mean ± standard error. For total wheel running activity over 8 weeks, M-PBO had significantly lower voluntary wheel running activity than M-CON (107,453 ± 50,683 rotations vs. 456,011 ± 62,970 rotations, respectively, p=0.0122). There were no differences observed, however, between F-PBO and F-CON for total 8-week wheel running activity (376,582 ± 1,847 rotations vs. 472,271 ± 116,017 rotations, p=0.4559). Additionally, M-PBO ran significantly less than M-CON in weeks 1, 2, 3, 6, 7, and 8 (Week 1: 5,606 ± 225 rotations vs. 17,824 ± 2,544 rotations, p=0.0084; Week 2: 11,537 ± 3,129 rotations vs. 51,882 ± 6,494 rotations, p=0.0025; Week 3: 10,157 ± 2,639 rotations vs. 79,257 ± 5,842 rotations, p=0.0001; Week 6: 15,816 ± 7,865 rotations vs. 64,425 ± 11,755 rotations, p=0.0207; Week 7: 14,068 ± 6,999 rotations vs. 58,112 ± 9,781 rotations, p=0.0178; Week 8: 13,234 ± 6351 rotations vs. 67,349 ± 16,142 rotations, p=0.0272, respectively). No differences were observed in weekly wheel running activity between F-PBO and F-CON. Based on the observed voluntary wheel running activity, puberty blocking male rats prior to breeding negatively impacted physical activity of male offspring but not female offspring. This is contrary to our previous work showing that puberty blocking treatment followed by detransition in female rats prior to breeding negatively impacted physical activity of both male and female offspring. Our hypothesis was therefore partially supported as M-PBO had significantly less wheel running activity than M-CON, but F-PBO and F-CON had similar wheel running activity. Supported by a University of Northern Colorado Provost's Seed Grant awarded to DSH. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

First Report of Neoscytalidium dimidiatum causing leaf zonate spot disease of Aloe vera L. in China.

In February 2022, leaf zonate spot disease afflicted Aloe vera L. in Yunnan, China, endangering the $39 billion industry with 0.33ha under cultivation (Wan 2015). The disease manifested with watery spots progressing into oval or circular necrosis lesions, characterized by a dark center surrounded by a gray-brown zone. In the late stage of the disease, lesions regress in size and several small dark picnidia dots appeared on the gray-brown zone. The disease incidence ranged from 10% to 15% in three commercial plantations. If left uncontrolled, the disease could diminish the commercial value of Aloe vera plants. Eighteen symptomatic leaf samples underwent morphological and genetic identification. The samples were carefully washed with distilled water and 1×1 cm2 sections of tissue were excised using a sterile scalpel. The sections underwent surface-disinfection with 3% NaOCl for 3 min and 75% ethanol for 30 s. After three sterile water rinses the sections were air-dried. Subsequently, they were transferred to potato dextrose agar (PDA) before being incubated at 25 ℃ in the dark. Of the 18 samples, eight produced the colonies with similar morphological characteristics, named LH7. Isolate LH7 had downy to woolly aerial mycelia, initially pinkish white on the surface, and gradually turned greenish-olivaceous from the middle, and eventually turned dark brown to black after seven days. The fungus formed arthric chains in the aerial mycelium on PDA but did not produce conidiomata. The conidia, which occurred in arthric chains were 5.50-9.9 × 4.08-7.51 μm (mean 7.09× 5.26 μm, n=50) in size, cylindrical, brown, and 0-1 septate. To ascertain LH7's pathogenicity, three healthy one-year old aloe plants were surface-sanitized with a 1% aqueous chlorine solution, rinsed with sterile water, and dried. Three leaves from each plant were punctuated and inoculated using conidial suspension (100 μl of 1x 106 conidial mL-1), while three control plants were inoculated with sterile distilled water. The pathogenicity tests were repeated twice. The inoculated plants were kept at 25 ℃ with a 12-hour light/12-hour dark cycle. After seven days, symptoms observed in the field appeared in the plants, while no disease occurred in the control plants. After 21 days, conidiomata formed on the inoculated leaves, averaging 116.92 μm (n=20) in diameter. These conidiomata were globose to subglobose, and brown to sub-brown. The fungus was successfully re-isolated from symptomatic tissue and the resulting colonies were morphologically consistent with isolate LH7. Based on the characteristics, the fungus was identified as Neoscytalidium dimidiatum (Philips et al. 2013). The specimen was deposited in China Center for Type Culture Collection ( CCTCC AF 2024001). This identification was confirmed through sequencing of ITS gene region of rDNA using ITS1/ITS4 (Imran et al. 2022). The sequence was submitted into GenBank database (ON878059). BLAST analysis of the LH7's ITS amplicon showed 100% similarity with that of JN093303.1. A phylogenetic tree constructed using the maximum likelihood method revealed that ON878059 was clustered with JN093303.1. Previous studies have documented that pathogens such as Colletotrichum gloeosporioides (Penz.), Fusarium spp. and Rhizopus oryzae can also cause diseases in A. vera in China (Zhou et al. 2008; Ding et al. 2015). Additinonally, Cladosporium sphaerospermum, Pseudopestalotiopsis theae, and Lasiodiplodia theobromae have been identified as causal agents of aloe leaf spot diseases in India, Bangladesh and Malaysia (Avasthi et al. 2016; Ahmmed et al. 2022; Khoo et al. 2022). To our knowledge, this is the first report of N. dimidiatum causing leaf necrosis of aloe in China. Vigilant surveillance and disease control measures are imperative to mitigate potential losses in this region.

Urothelial Piezo1 channels do not alter voiding or afferent nerve sensitivity in the mouse

The mechanosensitive piezo ion channels, Piezo1 and Piezo2, are expressed in tissues of the lower urinary tract, including the urothelium and sensory nerves. Interest in a role of piezo mechanosensitive ion channels in mediating sensory physiology in the lower urinary tract has been highlighted by the recent reports of putative voiding dysfunction in mice lacking Piezo2 channels in the lower extremities and sensory nerves (Marshall et al 2020) and Piezo1/2 in urothelium (Dalghi et al 2022). To investigate the potential role of urothelial Piezo1 channels in mediating normal function in the lower urinary tract, we generated a mouse line in which the Piezo1 gene can be conditionally knocked out in the urothelium (Piezo1UPKcKO mice). We examined spontaneous voiding behavior in male and female Piezo1UPKcKO and control mice during the light and dark phases of the day/night cycle. There were no gender differences in voiding parameters in our study (p=0.659, 3-way ANOVA), thus we did not isolate gender as a variable for further analysis. Piezo1UPKcKO mice (n=9) voided 6 ± 1 times per 24 hours, while the control mice (n=11) voided 7 ± 1 times per 24 hours (p = 0.7281). A clear nocturnal voiding pattern was observed with both Piezo1UPKcKO and control mice exhibiting more frequent voids during the dark cycle, but there were no differences in the number of voids, intermicturition intervals, or void volumes in any cycle between Piezo1UPKcKO and control mice. These results indicate that spontaneous voiding behavior remains fully intact in both male and female Piezo1UPKcKO mice during the light and dark phases of the day/night cycle. Furthermore, in an ex vivo bladder model where we measure afferent nerve output from the bladder during filling cycles, sensory nerve activity in the Piezo1UPKcKO mice was similar to that in bladders from control animals. The maximum frequency of afferent nerve activity during bladder filling reached 295 ± 28 Hz in control mice (n=6) and 336 ± 34 Hz in Piezo1UPKcKO mice (n=6; p = 0.368). Functional Piezo1 activity could be demonstrated in bladders from both Piezo1UPKcKO and control mice, as the pharmacological activator for Piezo1 channels, Yoda1 (5 μM) increased maximum afferent nerve activity during bladder filling by 60 ± 15 Hz in control mice (P = 0.0029) and 33 ± 15 Hz in Piezo1UPKcKO mice (P = 0.0567). In Ca2+ imaging experiments, we found that Yoda1 induced an increase in Ca2+ signaling events in urothelial tissue from control mice. Thus, it appears that urothelial cells express functional Piezo1 ion channels, but activity of these channels is not required for maintaining normal sensory nerve activity and voiding behavior. This work was supported by a grant from the National Institute of Diabetes and Digestive and Kidney Diseases (R01DK125543). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.