Myopia (short-sightedness) is the most common ocular disorder. It generally develops after over-exposure to aberrant visual environments, disrupting emmetropization mechanisms that should match eye growth with optical power. A pre-screening of strongly associated myopia-risk genes identified through human genome-wide association studies implicates efemp1 in myopia development, but how this gene impacts ocular growth remains unclear. Here, we modify efemp1 expression specifically in the retina of zebrafish. We found that under normal lighting, efemp1 mutants developed axial myopia, enlarged eyes, reduced spatial vision and altered retinal function. However, under myopia-inducing dark-rearing, compared to control fish, mutants remained emmetropic and showed changes in retinal function. Efemp1 modification changed the expression of efemp1, egr1, tgfb1a, vegfab and rbp3 genes in the eye, and changed the inner retinal distributions of myopia-associated EFEMP1, TIMP2 and MMP2 proteins. Efemp1 modification also impacted dark-rearing-induced responses of vegfab and wnt2b genes and above-mentioned myopia-associated proteins. Together, we provided robust evidence that light-dependent ocular growth is regulated by efemp1.

The 17th annual meeting of the Centre for Trophoblast Research (CTR) took place at the University of Cambridge, UK, on 1-2 July 2024. This year's meeting provided an opportunity to reflect on the significant advancements made recently in modelling the human placenta in vitro. The meeting featured 12 invited speakers and attracted 260 participants from 25 countries. Many of the speakers were leading figures who have developed methods to derive human trophoblast stem cells or organoids from first trimester and term placentas, and from pluripotent stem cells. Accompanying the invited presentations were flash talks selected from the abstract submissions and poster presentations. The meeting concluded with a stimulating panel discussion to evaluate the current human trophoblast models. This Meeting Review aims to capture the spirit of the event and highlight the key themes and take-home messages that emerged.

The hypothalamic-pituitary-adrenal (HPA) axis in mammals and the hypothalamic-pituitary-interrenal (HPI) axis in fish are open systems that adapt to the environment during development. Little is known about how this adaptation begins and regulates early stress responses. We used larval zebrafish to examine the impact of prolonged forced swimming at 5 days post-fertilization (dpf), termed early-life challenge (ELC), on cortisol responses, neuropeptide expression in the nucleus preopticus (NPO), and gene transcript levels. At 6 dpf, ELC-exposed larvae showed normal baseline cortisol but reduced reactivity to an initial stressor. Conversely, they showed increased reactivity to a second stressor within the 30-min refractory period, when cortisol responses are typically suppressed. ELC larvae had fewer corticotropin-releasing hormone (crh), arginine vasopressin (avp), and oxytocin (oxt)-positive cells in the NPO, with reduced crh and avp co-expression. Gene expression analysis revealed upregulation of genes related to cortisol metabolism (hsd11b2, cyp11c1), steroidogenesis (star), and stress modulation (crh, avp, oxt). These results suggest that early environmental challenge initiates adaptive plasticity in the HPI axis, tuning cortisol regulation to balance responsiveness and protection during repeated stress. Future studies should explore the broader physiological effects of prolonged forced swimming and its long-term impact on cortisol regulation and stress-related circuits.

The role of nitric oxide as a neurotransmitter in the olfactory and chemoreception systems of invertebrates has been well documented. This suggests an early and efficient sensory detection system that is evolutionarily preserved in various species, including vertebrates and invertebrates. Additionally, the presence of a nitric oxide neurotransmitter system has been reported in molluscs, particularly in octopus species. In this work, we present evidence for the existence of nitric oxide synthase in neurons and fibers, as well as its anatomical localization in various nuclei involved in chemosensory integration and the motor responses associated with these processes in Octopus maya.

AMBRA1 has critical roles in autophagy, mitophagy, cell cycle regulation, neurogenesis and apoptosis. Dysregulation of these processes are hallmarks of various neurodegenerative diseases and therefore AMBRA1 represents a potential therapeutic target. The flexibility of its intrinsically disordered regions allows AMBRA1 to undergo conformational changes and thus to perform its function as an adaptor protein for various different complexes. Understanding the relevance of these multiple protein-protein interactions will allow us to gain information about which to target pharmacologically. To compare potential AMBRA1 activation strategies, we have designed and validated several previously described mutant constructs in addition to characterising their effects on proliferation, apoptosis, autophagy and mitophagy in SHSY5Y cells. AMBRA1TAT, which is a mutant form of AMBRA1 that cannot interact with DLC1 at the microtubules, produced the most promising results. Overexpression of this mutant protected cells against apoptosis and induced autophagy/mitophagy in SHSY5Y cells in addition to enhancing the switch from quiescence to proliferation in mouse neural stem cells. Future studies should focus on designing compounds that inhibit the protein-protein interaction between AMBRA1/DLC1 and thus have potential to be used as a drug strategy for neurodegeneration.

Cigarette smoking negatively impacts mesenchymal stem cell functionality, including proliferation, viability, and differentiation potential. Adipose-derived mesenchymal stem cells (ADMSCs) are increasingly used for therapeutic purposes, but the specific effects of smoking in vivo on these cells are poorly understood. This study investigates the effects of cigarette smoke on the proliferation, viability, gene expression, and cellular functions of ADMSCs from smoking and non-smoking donors. In this study, ADMSCs were isolated from healthy smokers and non-smokers, and cell proliferation was assessed using the MTT assay, viability with apoptosis assays, mitochondrial membrane potential (MMP), and gene expression related to oxidative stress and cellular functions. Cell cycle analysis was also conducted. Our findings reveal a significant decrease in the proliferation of ADMSCs from smokers. Apoptosis assays showed reduced viable cells in smokers without a significant change in MMP, suggesting alternative pathways contributing to decreased viability. Gene expression analysis indicated the upregulation of genes associated with oxidative stress response and cellular defense mechanisms and the downregulation of genes related to inflammatory signaling, detoxification, and cellular metabolism. Cell cycle analysis indicates cycle arrest or delay in smokers, possibly due to stress and potential DNA damage. Smoking negatively affects ADMSCs' proliferation, viability, and function through oxidative stress and gene expression alterations. These findings highlight the importance of considering smoking status in ADMSC therapies and the need for further research to mitigate the effect of smoking on stem cells.

Benthic fish, such as the round goby (Neogobius melanostomus Pallas, 1814) tend to swim near the bottom, especially at increased water velocities. To test whether these fish have a hydraulic advantage from swimming near the bottom and how the substrate affects the forces experienced, we measured the hydraulic forces experienced by preserved fish in a flow channel. The fish were tested 5.0 mm above the bottom at smooth and rough surface, and in the water column (10.0 cm elevation) above smooth and rough surface at 0.95 m/s water velocity. No significant effect among the mean hydraulic forces was observed between both fish positions, whereas the mean hydraulic forces in the water column were significantly higher (P<0.05) above the rough surface (mean 0.077 N±0.025 s.d.) than above the smooth surface (mean 0.068 N±0.021 s.d.). A convolutional neural network (CNN) predicted the column smooth treatment was the most characteristic force data time series (mean F1=0.88±0.03 s.d.). We conclude that the body posture and body movements of the fish are more relevant for the hydraulic forces experienced by the fish than the vertical position in the water column. Further factors explaining the affinity to swimming near the bottom are discussed.

Exosomes are small extracellular vesicles (sEVs) secreted via multivesicular bodies (MVBs)/late endosomes and mediators of cell-cell communication. We previously reported a novel post-translational modification by ubiquitin-like 3 (UBL3). UBL3 is localized in MVBs and the plasma membrane and released outside as sEVs, including exosomes. Approximately 60% of proteins sorted in sEVs are affected by UBL3 and localized in various organelles, the plasma membrane, and the cytosol, suggesting that its dynamic movement in the cell before entering the MVBs. To examine the intracellular dynamics of UBL3, we constructed a sophisticated visualization system via fusing fluorescent timers that changed from blue to red form over time with UBL3 and by its expression under Tet-on regulation. Intriguingly, we found that after synthesis, UBL3 was initially distributed within the cytosol. Subsequently, UBL3 was localized to MVBs and the plasma membrane and finally showed predominant accumulation in MVBs. Furthermore, by super-resolution microscopy analysis, UBL3 was found to be associated with one of its substrates, α-tubulin, in the cytosol, and the complex was subsequently transported to MVBs. This spatiotemporal visualization system for UBL3 will form a basis for further studies to elucidate when and where UBL3 associates with its substrates/binding proteins before localization in MVBs.

During neurogenesis, excessive numbers of neurons are produced in most regions of the central and peripheral nervous systems. Nonessential neurons are eliminated by apoptosis, or programmed cell death. This has been most thoroughly characterized in the peripheral nervous system (PNS) where targets of innervation play a key role in this process. As maturing neurons project axons towards their targets of innervation, they become dependent upon these targets for survival. Survival factors, also called neurotrophic factors, are produced by targets, inhibit apoptosis cascades, and promote further growth and differentiation. Because neurotrophic factors are limited, as is target size, neurons that do not correctly and efficiently innervate targets undergo apoptosis ( Levi-Montalcini, 1987; Davies, 1996). Thus, excessive neurogenesis acts to ensure that sufficient numbers of neurons are produced during development. In the superior cervical ganglion (SCG), this process of neurogenesis and subsequent apoptosis is reported to be complete by postnatal day 3-4 (P3-P4) in mice. Surprisingly, we observed significant numbers of apoptotic neurons out to P14, and neurogenesis was still present at P14 as well. In both the SCG and geniculate ganglion (GG), postnatal neurogenesis was dependent on apoptosis because little or no postnatal neurogenesis was observed in Bax-/- mice, in which apoptosis is eliminated. These results indicate that both neurogenesis and apoptosis continue to occur well after birth in peripheral ganglia, and that neurogenesis depends on apoptosis, suggesting that neurogenesis continues postnatally to replace neurons that are eliminated during synaptic refinement.

Drosophila oogenesis has long been an important model for understanding myriad cellular processes controlling development, RNA biology and patterning. Flies are easily fed drugs to disrupt various molecular pathways. However, this is often done under poor nutrient conditions that adversely affect oogenesis, thus making analysis challenging. Cycloheximide is a widely used compound that binds to and stalls the ribosome, therefore reducing protein synthesis. As egg production is a highly nutrient-dependent process, we developed a method to feed female Drosophila a rich diet of yeast paste supplemented with cycloheximide to better determine the effect of cycloheximide treatment on oogenesis. We found that flies readily consumed cycloheximide-supplemented yeast paste. Males and females had reduced lifespans when maintained on cycloheximide, with males exhibiting a dose-dependent decrease. Although females did not exhibit decreased egg laying, their ovaries were smaller and the number of progeny reduced, indicating substandard egg quality. Finally, females fed cycloheximide had disrupted oogenesis, with smaller ovaries, missing ovariole stages, and an increase in apoptotic follicles. Together, these data support that reduced protein synthesis adversely affects oogenesis with a rich diet that provides optimal nutrient conditions. In addition, this method could be used more broadly to test the effect of other drugs on Drosophila oogenesis without the confounding effects caused by poor nutrition.

Saccharomyces cerevisiae is a powerful model for aging research due to its short lifespan and genetic malleability. Microfluidic devices offer an attractive approach enabling rapid monitoring of hundreds of cells during their entire replicative lifespan (RLS). Yet, key operational issues such as contaminations, cell loss, and cell-aggregates-dependent flow obstruction can hinder RLS experiments. We report the development of a microfluidic device configuration that effectively prevents flow blockage. We conducted comprehensive performance characterization, evaluating trapping efficiency, cell retention, budding orientation, and cell aggregate formation. The optimized device successfully supported long-term culturing and reliable RLS measurements of budding yeast strains. For accurate lifespan determination, a detailed workflow is provided that includes device fabrication, live microscopy setup, and characterization of cell age distribution. This work describes an accessible and reliable microfluidic device for yeast RLS studies, promoting further exploration in aging research.

Reproduction requires high amounts of energy, and challenging environments during breeding can force parents to prioritize their current reproductive bout over self-maintenance or vice versa. However, little is known about how common stressors, such as food restriction, can influence these trade-offs during breeding, and the physiological mechanisms for these trade-off decisions. In this study, adult zebra finches (Taeniopygia castanotis) were subjected to a control diet (ad libitum) or a 40% food restriction while raising nestlings and fledglings, and we measured body mass, furculum fat, plasma corticosterone (CORT) and blood glucose levels of the parents at the time of pairing, when their offspring fledged, and when their offspring reached nutritional independence. We also measured body mass and growth rate in the offspring from hatching until the end of the treatment period. Food-restricted parents had lower body mass when their offspring fledged and reached nutritional independence and higher baseline CORT when their offspring fledged compared to controls. Offspring did not differ in body mass or growth rate between treatment groups. However, there was no effect of food restriction on parents' furculum fat, baseline glucose, the adrenocortical response, or the glucose response. Furthermore, path analysis results suggest that alterations in baseline glucose is the primary driver of changes in body mass in parents and offspring brood mass. Taken together, these results suggest that food restriction during chick rearing in a short-lived passerine drives parents to prioritize their current reproductive bout over self-maintenance, and glucose could potentially be a mechanism for diverting energy toward parental effort.

Joubert Syndrome (JBTS) is a neurodevelopmental ciliopathy defined by a highly specific midbrain-hindbrain malformation, variably associated with additional neurological features. JBTS displays prominent genetic heterogeneity with >40 causative genes that encode proteins localising to the primary cilium, a sensory organelle that is essential for transduction of signalling pathways during neurodevelopment, among other vital functions. JBTS proteins localise to distinct ciliary subcompartments, suggesting diverse functions in cilium biology. Currently, there is no unifying pathomechanism to explain how dysfunction of such diverse primary cilia-related proteins results in such a highly specific brain abnormality. To identify the shared consequence of JBTS gene dysfunction, we carried out transcriptomic analysis using zebrafish mutants for the JBTS-causative genes cc2d2aw38, cep290fh297, inpp5ezh506, talpid3i264 and togaram1zh510 and the Bardet-Biedl syndrome-causative gene bbs1k742. We identified no commonly dysregulated signalling pathways in these mutants and yet all mutants displayed an enrichment of altered gene sets related to central nervous system function. We found that JBTS mutants have altered primary cilia throughout the brain but do not display abnormal brain morphology. Nonetheless, behavioural analyses revealed reduced locomotion and loss of postural control which, together with the transcriptomic results, hint at underlying abnormalities in neuronal activity and/or neuronal circuit function. These zebrafish models therefore offer the unique opportunity to study the role of primary cilia in neuronal function beyond early patterning, proliferation and differentiation.

Routine histochemical techniques are capable of producing vast amount of information from diverse sample types, but these techniques are limited in their ability to generate 3D information. Autofluorescence imaging can be used to analyse samples in 3D but it suffers from weak/low signal intensities. Here, we describe a simple chemical treatment with glutaraldehyde to enhance autofluorescence for 3D fluorescence imaging and to generate detailed morphological images on whole-mount samples. This methodology is straightforward and cost-effective to implement, suitable for a wide range of organisms and sample types. Furthermore, it can be readily integrated with standard confocal and fluorescence microscopes for analysis. This approach has the potential to facilitate the analysis of biological 3D structures and research in developmental biology, including studies on model and non-model organisms.

Insects exhibit a remarkable ability to interact with inanimate objects to facilitate essential behaviors such as foraging, reproduction, shelter building, and defense. In this study, we assessed whether Drosophila interacted with inanimate objects when they were suspended on their wings and provided with a thermocol ball (foam ball). Drosophila indeed exhibited ball rolling behavior. We further examined the sexual dimorphism in this ball rolling-associated locomotor behavior. We carried out a ball rolling assay using 3-day-old male and female w1118 flies and measured the duration for which the flies could roll the ball without dropping it within a 10 min period. The ball was returned to the flies whenever they dropped it, and we calculated the number of times the ball was dropped within the 10 min duration. Females exhibited a longer ball holding duration than males. We also observed a decrease in ball holding duration and an increase in the number of times the ball was dropped by 15-day-old male and female flies than their younger counterparts. These results suggest sexual dimorphism and age-dependent alterations in Drosophila ball rolling-associated locomotor behavior.

The generation of lung epithelial cells through the directed differentiation of human pluripotent stem cells (hPSCs) in vitro provides a platform to model both embryonic lung development and adult airway disease. Here, we describe a robust differentiation protocol that closely recapitulates human embryonic lung development. Differentiating cells progress through obligate intermediate stages, beginning with definitive endoderm formation and then patterning into anterior foregut endoderm that yields lung progenitors (LPs) with extended culture. These LPs can be purified using the cell surface marker CD166 (also known as ALCAM), and further matured into proximal airway epithelial cells including basal cells, secretory cells and multiciliated cells using either an organoid platform or culture at the air-liquid interface (ALI). We additionally demonstrate that these hPSC-derived airway epithelial cells can be used to model Influenza A infection. Collectively, our results underscore the utility of CD166 expression for the efficient enrichment of LPs from heterogenous differentiation cultures and the ability of these isolated cells to mature into more specialized, physiologically relevant proximal lung cell types.

The multiprotein complexes known as the complex of proteins associated with Set1 (COMPASS) play a crucial role in the methylation of histone 3 lysine 4 (H3K4). In Drosophila, the COMPASS series complexes comprise core subunits Set1, Trx, and Trr, which share several common subunits such as ash2, Dpy30-L1, Rbbp5, and wds, alongside their unique subunits: Wdr82 for Set1/COMPASS, Mnn1 for Trx/COMPASS-like, and Ptip for Trr/COMPASS-like. Our research has shown that flies deficient in any of these common or unique subunits exhibited high lethality at eclosion (the emergence of adult flies from their pupal cases) and significantly shortened lifespans of the few adults that do emerge. Silencing these common or unique subunits led to severe heart morphological and functional defects. Moreover, specifically silencing the unique subunits of the COMPASS series complexes, Wdr82, Mnn1, and Ptip, in the heart results in decreased levels of H3K4 monomethylation and dimethylation, consistent with effects observed from silencing the core subunits Set1, Trx, and Trr. These findings underscore the critical roles of each subunit of the COMPASS series complexes in regulating histone methylation during heart development and provide valuable insights into their potential involvement in congenital heart diseases, thereby informing ongoing research in heart disease.

This Review delves into the mechanisms behind drug resistance in colorectal cancer (CRC), particularly examining the role of nutrient depletion and its contribution to multidrug resistance (MDR). The study highlights metabolic adaptations of cancer cells as well as metabolic adaptations of cancer cells under low nutrient availability, including shifts in glycolysis and lipid metabolism. It emphasizes the significance of MDR1 and its encoded efflux transporter, P-glycoprotein (P-gp/B1), in mediating drug resistance and how pathways such as HIF1α, AKT, and mTOR influence the expression of P-gp/B1 under limited nutrient availability. Additionally, the Review explores the dual roles of autophagy in drug sensitivity and resistance under nutrient limited conditions. It further investigates the involvement of lysosomes and mitochondria, focusing on their roles in drug sequestration and the challenges posed by lysosomal entrapment facilitated by non-enzymatic processes and ABC transporters like P-gp/B1. Finally, the Review underscores the importance of understanding the interplay between drug sequestration, lysosomal functions, nutrient depletion, and MDR1 gene modulation. It suggests innovative strategies, including structural modifications and nanotechnology, as promising approaches to overcoming drug resistance in cancer therapy.

Zebrafish larvae show a rapid increase in cortisol in response to acute stressors, followed by a decline. While these responses are documented, both the duration of the refractory period to repeated stressors and the role of glucocorticoid receptors (GR) in specific phases of the glucocorticoid negative feedback are still being clarified. We explored these questions using water vortices as stressors, combined with GR blockage and measurements of whole-body cortisol in zebrafish larvae subjected to single and repeated stress protocols. Cortisol levels were elevated 10 min after stress onset and returned to baseline within 30-40 min, depending on the stressor strength. In response to homotypic stress, cortisol levels rose above baseline if the second stressor occurred 60 or 120 min after the first, but not with a 30-min interval. This suggests a rapid cortisol-mediated feedback loop with a refractory period of at least 30 min. Treatment with a GR blocker delayed the return to baseline and suppressed the refractory period, indicating GR-dependent early-phase feedback regulation. These findings are consistent with mammalian models and provide a framework for further analyses of early-life cortisol responses and feedback in zebrafish larvae, ideal for non-invasive imaging and high-throughput screening.