The ability to visualize and track multiple biological processes in vivo in real time is highly desirable. Bioluminescence imaging (BLI) has emerged as an attractive modality for non-invasive cell tracking, with various luciferase reporters enabling parallel monitoring of several processes. However, simultaneous multiplexed imaging in vivo is challenging due to suboptimal reporter intensities and the need to image one luciferase at a time. We report a multiplexed BLI approach using a single substrate that leverages bioluminescence resonance energy transfer (BRET)-based reporters with distinct spectral profiles for triple-color BLI. These luciferase-fluorophore fusion reporters address light transmission challenges and use optimized coelenterazine substrates. Comparing BRET reporters across two substrate analogs identified a green-yellow-orange combination that allows simultaneous imaging of three distinct cell populations in vitro and in vivo. These tools provide a template for imaging other biological processes in vivo during a single BLI session using a single reporter substrate.

Mitochondria-endoplasmic reticulum (ER) contact sites (MERCs) emerged to play critical roles in numerous cellular processes, and their dysregulation has been associated to neurodegenerative disorders. Mutations in the SPG4 gene coding for spastin are among the main causes of hereditary spastic paraplegia (HSP). Spastin binds and severs microtubules, and the long isoform of this protein, namely M1, spans the outer leaflet of ER membrane where it interacts with other ER-HSP proteins. Here, we showed that overexpressed M1 spastin localizes in ER-mitochondria intersections and that endogenous spastin accumulates in MERCs. We demonstrated in different cellular models that downregulation of spastin enhances the number of MERCs, alters mitochondrial morphology, and impairs ER and mitochondrial calcium homeostasis. These effects are associated with reduced mitochondrial membrane potential, oxygen species levels, and oxidative metabolism. These findings extend our knowledge on the role of spastin in the ER and suggest MERCs deregulation as potential causes of SPG4-HSP disease.

Chemical synaptic transmission is modulated to accommodate different activity levels, thus enabling homeostatic scaling in pre- and postsynaptic compartments. In nematodes, cholinergic neurons use neuropeptide signaling to modulate synaptic vesicle content. To explore if this mechanism is conserved in vertebrates, we studied the involvement of neuropeptides in cholinergic transmission at the neuromuscular junction of larval zebrafish. Optogenetic stimulation by photoactivated adenylyl cyclase evoked locomotion. We generated mutants lacking the neuropeptide-processing enzyme carboxypeptidase E (cpe), and the most abundant neuropeptide precursor in motor neurons, tachykinin (tac1). Both mutants showed exaggerated locomotion after photostimulation. Recording excitatory postsynaptic currents demonstrated overall larger amplitudes in the wild type. Exaggerated locomotion in the mutants thus reflected upscaling of postsynaptic excitability. Both mutant muscles expressed more nicotinic acetylcholine receptors (nAChRs) on their surface; thus, neuropeptide signaling regulates synaptic transmitter output in zebrafish motor neurons, and muscle cells homeostatically regulate nAChR surface expression, compensating reduced presynaptic input.

Visual perception is enacted and constrained by the constantly moving eyes. Although it is well known that the first two fixations are crucial for face recognition, the function of each fixation remains unspecified. Here we demonstrate a central-to-divergent pattern of the two fixations and specify their functions: Fix I clustered along the nose bridge to cover the broad facial information; Fix II diverged to eyes, nostrils, and lips to get the local information. Fix II correlated more than Fix I with the differentiating information between faces and contributed more to recognition responses. While face categories can be significantly discriminated by Fix II\'s but not Fix I\'s patterns alone, the combined patterns of the two yield better discrimination. Our results suggest a functional division and collaboration of the two fixations in sampling the general-to-specific facial information and add to understanding visual perception as an active process undertaken by structural motor programs.

A key step of Canonical Nonhomologous End Joining (C-NHEJ) is synapsis of DNA double strand break (DSB) ends for ligation. The DNA-PKcs dimer mediates synapsis in a long-range complex with DSB ends remaining apart, whereas the XLF homodimer can mediate synapsis in both long-range and short-range complexes. Recent structural studies found the PAXX homodimer may also facilitate synapsis in long-range complexes with DNA-PKcs via its interactions with Ku70. Thus, we examined the influence of PAXX in C-NHEJ of chromosomal DSBs, which we compared to another Ku-binding factor, MRI. Using EJ of blunt DSBs with Cas9 reporters as a readout for C-NHEJ, we found that PAXX and/or MRI are dispensable. However, when combined with disruption of DNA-PKcs, particularly with DNA-PKcs kinase inhibition, PAXX becomes important for blunt DSB EJ. In contrast, while DNA-PKcs is also important to suppress short deletion mutations with microhomology, this effect is not magnified with PAXX loss. MRI loss had no effect combined with DNA-PKcs disruption, but becomes important for blunt DSB EJ when combined with disruption of XLF, as is PAXX. Finally, XLF loss causes an increase in larger deletions compared to DNA-PKcs inhibition, which is magnified with combined loss of MRI. Altogether, we suggest that PAXX promotes DSB end synapsis during C-NHEJ in a manner that is partially redundant with DNA-PKcs and XLF, whereas MRI appears to be mainly important in the context of XLF disruption.

Sepsis-induced arrhythmia, linked to sudden cardiac death, is associated with gut microbiota, though the exact relationship is unclear. This study aimed to elucidate the relationship between Cronobacter sakazakii (C. sakazakii) and arrhythmia. The relative abundance of C. sakazakii was increased in cecal ligation and puncture (CLP)-induced septic mice. Live C. sakazakii, supernatant, and outer membrane vesicles (OMVs) resulted in premature ventricular beat (PVB), sinus arrhythmia (SA), and increased arrhythmia and mortality in sepsis model through dysregulated ion channel proteins. Moreover, short-chain fatty acids (SCFAs) showed antibacterial effects in vitro. We confirmed sodium acetate (C2) and sodium butyrate (C4) protect from C. sakazakii-induced arrhythmia, and C2 and C4 protected from septic arrhythmia by activating free fatty acid receptor 2 and 3 (FFAR2 and FFAR3) in mice. These findings point to how C. sakazakii\'s OMVs trigger arrhythmia, and SCFAs may be a treatment for septic arrhythmia.

Endocrine glands secrete hormones into the circulation to target distant tissues and regulate their functions. The qualitative relationship between hormone-secreting organs and their target tissues is well established, but a quantitative approach is currently limited. Quantification is important, as it could allow us to study the endocrine system using engineering concepts of optimality and tradeoffs. In this study, we collected literature data on 24 human hormones secreted from dedicated endocrine cells. We find that the number of endocrine cells secreting a hormone is proportional to the number of its target cells. A single endocrine cell serves approximately 2,000 target cells, a relationship that spans 6 orders of magnitude of cell numbers. This suggests an economic principle of cells working near their maximal capacity, and glands that are no bigger than they need to be.

Ferroptosis is implicated in several diseases, including iron overload-induced osteoarthritis (IOOA), which is marked by oxidative stress, iron imbalance, and lipid peroxidation. Given rosiglitazone\'s (RSG) ability to inhibit lipid peroxidation and ferroptosis, this study aims to assess its therapeutic potential for treating IOOA. Our in vitro results show that RSG targets acyl-CoA synthetase long-chain family member 4 to mitigate impairments induced by interleukin-1 beta and ferric ammonium citrate, including cell apoptosis, senescence, inflammatory responses, extracellular matrix degradation, and ferroptosis. RSG reduced intracellular iron content, alleviated oxidative stress and lipid peroxidation, mitigated damage to membrane-bound organelles, and enhanced glucose transport. Additionally, pre-treatment with RSG imparted anti-ferroptotic properties to chondrocytes. In vivo, RSG alleviated cartilage degradation, inflammatory responses, and ferroptosis in mice with IOOA. In conclusion, RSG exhibits chondroprotective and anti-ferroptotic effects by suppressing lipid peroxidation and restoring iron homeostasis, highlighting its potential for treating IOOA.

Antibodies represent a primary mediator of protection against respiratory viruses. Serum neutralizing antibodies (NAbs) are often considered a primary correlate of protection. However, detailed antibody profiles including characterization of antibody functions in different anatomic compartments are poorly understood. Here we show that antibody correlates of protection against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) challenge are different in systemic versus mucosal compartments in rhesus macaques. In serum, NAbs were the strongest correlate of protection and linked to spike-specific binding antibodies and other extra-NAb functions that create a larger protective network. In bronchiolar lavage (BAL), antibody-dependent cellular phagocytosis (ADCP) proved the strongest correlate of protection rather than NAbs. Within BAL, ADCP was linked to mucosal spike-specific immunoglobulin (Ig)G, IgA/secretory IgA, and Fcγ-receptor binding antibodies. Our results support a model in which antibodies with different functions mediate protection at different anatomic sites.

Articular cartilage degeneration may lead to osteoarthritis (OA) during the aging process, but its underlying mechanism remains unknown. Here, we found that chondrocytes exhibited an energy metabolism shift from glycolysis to oxidative phosphorylation (OXPHOS) during aging. Parkin regulates various cellular metabolic processes. Reprogrammed cartilage metabolism by Parkin ablation decreased OXPHOS and increased glycolysis, with ameliorated aging-related OA. Metabolomics analysis indicated that lauroyl-L-carnitine (LLC) was decreased in aged cartilage, but increased in Parkin-deficient cartilage. In vitro, LLC improved the cartilage matrix synthesis of aged chondrocytes. In vivo, intra-articular injection of LLC in mice with anterior cruciate ligament transaction (ACLT) ameliorated OA progression. These results suggest that metabolic changes are regulated by Parkin-impaired cartilage during aging, and targeting this metabolomic changes by supplementation with LLC is a promising treatment strategy for ameliorating OA.