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Peroxisomes in rat sympathetic ganglia and adrenal medulla.
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Design and optimization of in situ self-functionalizing stress sensors.
Mechanical contributions are crucial regulators of diverse biological processes, yet their in vivo measurement remains challenging due to limitations of current techniques that can be destructive or require complex dedicated setups. This study introduces a novel method to synthesize biocompatible, self-functionalizing stress sensors based on inverted emulsions that can be used to probe stresses inside tissues but can also locally perturb the biological environment through specific binder presentation or drug delivery. We engineered an optimal design for these inverted emulsions, focusing on finding the balance between the two contradictory constraints: achieving low surface tension for deformability while maintaining emulsion instability for efficient self-functionalization and drug release. Proof-of-concept experiments in both agarose gels and complex biological systems, including brain organoids and zebrafish embryos, confirm the droplets ability to deform in response to mechanical stress applied within the tissue, to self-functionalize and to release encapsulated molecules locally. These versatile sensors offer a method for noninvasive stress measurements and targeted chemical delivery within living biological tissues, giving the potential to overcome current technical barriers in biophysical studies.
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Activity and stability of centrosomes in Chinese hamster ovary cells in nucleation of microtubules in vitro.
Mitotic centrosomes were prepared from Chinese hamster ovary cells and their capacity to nucleate microtubules in vitro was demonstrated by incubation with exogenous brain microtubule protein. The number of microtubules polymerized onto centrosomes was directly counted by electron microscopy of whole-mount preparations. This simple and accurate quantitative assay has permitted characterization of the microtubule nucleating activity of centrosomes in vitro. The number of microtubules polymerized onto centrosomes varied according to the structure of the centrosome. The activity was roughly proportional to the centriole number. The number and length of microtubules nucleated by centrosomes depended both on the concentration of tubulin and the incubation time with tubulin. Under saturating conditions, an average of 200-250 microtubules were initiated by single centrosomes. Centrosomal activity is unstable (t 1/2 = 8 h) and could easily be irreversibly disrupted by a medium of high ionic strength. The activity is stabilized by the addition of glycerol. Centrosomes can be stored at -80 degrees C. The optimum pH for microtubule nucleation is 6.8. Activity is sensitive to protease digestion, but neither DNase or RNase affected the nucleating activity of centrosomes. The activity is temperature-sensitive, but addition of phenylmethylsulphonyl fluoride (PMSF) induces thermal stability. At an optimal concentration of 0.5 mg/ml, this drug increased the half-life of the activity (t 1/2 = 95 h) and made it resistant to salt extraction. Protease inhibitors other than PMSF or dansyl fluoride did not have any stabilizing effect on the activity. The difference between the centrosomal structure of polymerized microtubules in vivo and in vitro is discussed.
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Integrated, automated maintenance, expansion and differentiation of 2D and 3D patient-derived cellular models for high throughput drug screening.
Patient-derived cellular models become an increasingly powerful tool to model human diseases for precision medicine approaches. The identification of robust cellular disease phenotypes in these models paved the way towards high throughput screenings (HTS) including the implementation of laboratory advanced automation. However, maintenance and expansion of cells for HTS remains largely manual work. Here, we describe an integrated, complex automated platform for HTS in a translational research setting also designed for maintenance and expansion of different cell types. The comprehensive design allows automation of all cultivation steps and is flexible for development of methods for variable cell types. We demonstrate protocols for controlled cell seeding, splitting and expansion of human fibroblasts, induced pluripotent stem cells (iPSC), and neural progenitor cells (NPC) that allow for subsequent differentiation into different cell types and image-based multiparametric screening. Furthermore, we provide automated protocols for neuronal differentiation of NPC in 2D culture and 3D midbrain organoids for HTS. The flexibility of this multitask platform makes it an ideal solution for translational research settings involving experiments on different patient-derived cellular models for precision medicine.
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3D bioprinting complex models of cancer.
Cancer is characterized by the uncontrolled division of cells, resulting in the formation of tumors. The tumor microenvironment (TME) consists of a variety of cell types present within a heterogeneous extracellular matrix (ECM). Current 2D culture methods for mimicking this microenvironment remain limited due to spatial constraints. Many different types of 3D cancer models have been developed in recent years using spheroids/organoids, biomaterial scaffolds, and cancer-on-chip systems. However, these models cannot precisely control the organization of multiple cell types inside of complex architectures. Bioprinted cancer models can incorporate both stromal and cancer cells inside of 3D constructs to generate custom models of this complex disease. 3D bioprinting can generate complex, multicellular, and reproducible constructs where the matrix composition and rigidity are tailored locally to the tumor. These capabilities make 3D bioprinting an attractive method for reproducing the tumor TME found in vivo. Recent advancements in biomaterial-based bioinks enable the generation of 3D bioprinted cancer models that accurately mimic the TM. Here we discuss recent examples of such 3D-bioprinted cancer models, including those of the lungs, prostate, skin, brain, and colon. We then highlight the advantages of using 3D bioprinting compared to other in vitro modeling techniques and detail its limitations.
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Human-Derived Bifidobacterium dentium Modulates the Mammalian Serotonergic System and Gut-Brain Axis.
BACKGROUND & AIMS: The human gut microbiota can regulate production of serotonin (5-hydroxytryptamine [5-HT]) from enterochromaffin cells. However, the mechanisms underlying microbial-induced serotonin signaling are not well understood. METHODS: Adult germ-free mice were treated with sterile media, live Bifidobacterium dentium, heat-killed B dentium, or live Bacteroides ovatus. Mouse and human enteroids were used to assess the effects of B dentium metabolites on 5-HT release from enterochromaffin cells. In vitro and in vivo short-chain fatty acids and 5-HT levels were assessed by mass spectrometry. Expression of tryptophan hydroxylase, short-chain fatty acid receptor free fatty acid receptor 2, 5-HT receptors, and the 5-HT re-uptake transporter (serotonin transporter) were assessed by quantitative polymerase chain reaction and immunostaining. RNA in situ hybridization assessed 5-HT-receptor expression in the brain, and 5-HT-receptor-dependent behavior was evaluated using the marble burying test. RESULTS: B dentium mono-associated mice showed increased fecal acetate. This finding corresponded with increased intestinal 5-HT concentrations and increased expression of 5-HT receptors 2a, 4, and serotonin transporter. These effects were absent in B ovatus-treated mice. Application of acetate and B dentium-secreted products stimulated 5-HT release in mouse and human enteroids. In situ hybridization of brain tissue also showed significantly increased hippocampal expression of 5-HT-receptor 2a in B dentium-treated mice relative to germ-free controls. Functionally, B dentium colonization normalized species-typical repetitive and anxiety-like behaviors previously shown to be linked to 5-HT-receptor 2a. CONCLUSIONS: These data suggest that B dentium, and the bacterial metabolite acetate, are capable of regulating key components of the serotonergic system in multiple host tissues, and are associated with a functional change in adult behavior.
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Loss of function of the mitochondrial peptidase PITRM1 induces proteotoxic stress and Alzheimer's disease-like pathology in human cerebral organoids.
Mutations in pitrilysin metallopeptidase 1 (PITRM1), a mitochondrial protease involved in mitochondrial precursor processing and degradation, result in a slow-progressing syndrome characterized by cerebellar ataxia, psychotic episodes, and obsessive behavior, as well as cognitive decline. To investigate the pathogenetic mechanisms of mitochondrial presequence processing, we employed cortical neurons and cerebral organoids generated from PITRM1-knockout human induced pluripotent stem cells (iPSCs). PITRM1 deficiency strongly induced mitochondrial unfolded protein response (UPRmt) and enhanced mitochondrial clearance in iPSC-derived neurons. Furthermore, we observed increased levels of amyloid precursor protein and amyloid β in PITRM1-knockout neurons. However, neither cell death nor protein aggregates were observed in 2D iPSC-derived cortical neuronal cultures. On the other hand, over time, cerebral organoids generated from PITRM1-knockout iPSCs spontaneously developed pathological features of Alzheimer's disease (AD), including the accumulation of protein aggregates, tau pathology, and neuronal cell death. Single-cell RNA sequencing revealed a perturbation of mitochondrial function in all cell types in PITRM1-knockout cerebral organoids, whereas immune transcriptional signatures were substantially dysregulated in astrocytes. Importantly, we provide evidence of a protective role of UPRmt and mitochondrial clearance against impaired mitochondrial presequence processing and proteotoxic stress. Here, we propose a novel concept of PITRM1-linked neurological syndrome whereby defects of mitochondrial presequence processing induce an early activation of UPRmt that, in turn, modulates cytosolic quality control pathways. Thus, our work supports a mechanistic link between mitochondrial function and common neurodegenerative proteinopathies.
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A morphometric analysis of pyramidal tract structures during postnatal undernourishment and recovery.
Rats were postnatally undernourished during the suckling period (up to 20 days) and the brainstems of the perfused rats were dissected and prepared for electronmicroscopy at 21, 35 and 63 days of age. The effects on myelin were relatively mild and consisted primarily of a slight reduction in the relative numbers of myelinated fibers, most likely caused by a lag in the rate of loss of non-myelinated fibers, and fewer lamellae in myelinated axons of less than 2.5 micron circumference. Organelles were examined in the interfasicular oligodendroglia and in paragigantocellular reticular neurons immediately dorsal to the pyramidal tract. The numbers of mitochondrial particles in neuronal perikarya were significantly increased by postnatal undernourishment, although the numbers of other organelles appeared normal. Increased numbers of mitochondria persisted in nutritionally rehabilitated rats. Mitochondrial particles in oligodendroglia were not altered.
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Experimental Models for the Study of Central Nervous System Infection by SARS-CoV-2.
INTRODUCTION: The response to the SARS-CoV-2 coronavirus epidemic requires increased research efforts to expand our knowledge of the disease. Questions related to infection rates and mechanisms, the possibility of reinfection, and potential therapeutic approaches require us not only to use the experimental models previously employed for the SARS-CoV and MERS-CoV coronaviruses but also to generate new models to respond to urgent questions. DEVELOPMENT: We reviewed the different experimental models used in the study of central nervous system (CNS) involvement in COVID-19 both in different cell lines that have enabled identification of the virus' action mechanisms and in animal models (mice, rats, hamsters, ferrets, and primates) inoculated with the virus. Specifically, we reviewed models used to assess the presence and effects of SARS-CoV-2 on the CNS, including neural cell lines, animal models such as mouse hepatitis virus CoV (especially the 59 strain), and the use of brain organoids. CONCLUSION: Given the clear need to increase our understanding of SARS-CoV-2, as well as its potential effects on the CNS, we must endeavor to obtain new information with cellular or animal models, with an appropriate resemblance between models and human patients.
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Studies on the morphologic alterations of axonal membraneous organelles in neurofilamentous neuropathies.
Alterations in the membraneous organelles were studied in two experimental neuropathies: 2,5-hexanedione (2,5-HD) and beta,beta'-iminodiproprionitrile (IDPN), both of which exhibit massive neurofilamentous accumulation. The membranous organelles were stained using the diaminobenzidine potassium ferrocyanide (DPF) method. In 2,5-HD, where there is a progressive impairment of fast axoplasmic transport, studies of the smooth endoplasmic reticulum (SER) revealed a fragmentation and disruption of the continuity of the longitudinally oriented interconnecting tubules and a significant increase in the number of free vesicles. In IDPN, where studies of fast transport have been normal, the longitudinal orientation of the smooth SER tubules was not disturbed although channels of longitudinally oriented tubules of SER were clustered with microtubules and mitochondria. This study suggests that in 2,5-HD the accumulation of vesicles in association with fragmentation and disorganization of the SER is one of the morphologic correlates of the impaired fast anterograde transport system.