Cellular Diversity and Differential Subcellular Localization of the G-Protein Gαo Subunit in the Mouse Cerebellum

Heterotrimeric guanine nucleotide-binding proteins (G proteins) transduce signals from G protein-coupled receptors (GPCRs) to effector ion channels and enzymes Gαo, a member of the pertussis toxin-sensitive Gi/o family, is widely expressed in the brain, although its role within a neuronal context remains largely unknown. Using immunohistochemical and quantitative immunoelectron microscopy techniques, we have investigated the expression, cellular and subcellular localization of Gαo in the cerebellar cortex. Histoblot revealed that Gαo is expressed in many brain regions, including the cerebellum. At the cellular level, Gαo protein was distributed in Purkinje cells, basket cells, stellate cells, granule cells and Golgi cells. At the subcellular level, pre-embedding immunoelectron microscopy revealed mainly a postsynaptic localization of Gαo along the extrasynaptic plasma membrane of Purkinje cell dendritic shafts and spines, and dendrites of basket, stellate and granule cells. To a lesser extent, immunolabeling for Gαo was localized in different types of axon terminals establishing excitatory synapses. Moreover, post-embedding immunoelectron microscopy revealed the synaptic localization of Gαo on PSDs of glutamatergic synapses between Purkinje cell spines and parallel fiber terminals and its co-localization with GABAB1 in the same spines. Quantitative analysis of Gαo immunoparticles revealed they preferentially localized on the cytoplasmic face of the plasma membrane. Furthermore, the analysis revealed a high concentration of Gαo around excitatory synapses on Purkinje cell dendritic spines, but a uniform distribution in granule cell dendrites. These molecular-anatomical findings suggest that Gαo is a major signal transducer of specific GPCRs in different neuronal populations in the cerebellum.

The Effect of Talaromyces Marneffei Infection on CD86 Expression in THP-1 Cells

Background: Talaromyces Marneffei (T.marneffei) is an destructive opportunistic dimorphic fungal which can cause lethiferous Talaromycosis, but the clearance of T.marneffei mainly depends on the innate immune response. Objectives: To investigate the effect of T.marneffei on CD86 expression in THP-1 cells after infection and discuss the potential mechanisms. Methods: Western blot and immunoelectron microscopy were used to detect the CD86 expression on T.marneffei cultured on BHI medium at 37℃. Western blot、enzyme-linked immunoassay and immunofluorescence were used to detect the change of CD86 expression on macrophages incubating with T.marneffei. Enzyme-linked immunoassay was used to detect the content of CD86 in supernatant in the co-culture system. Immunohistochemistry and immunoelectron microscopy were used to detect the expression of CD86 on T.marneffei incubating with macrophages. Results: T.marneffei didn’t express CD86 when cultured separately at 37℃ detected by western blot and immunoelectron microscopy, but it did express CD86 when incubated with macrophages detected by immunohistochemistry and immunoelectron microscopy. The CD86 expression of macrophages significantly decreased at 72 hours when infected with T.marneffei while the content of CD86 in supernatant significantly increased at 72 hours compared with the control group which were detected by western blot, enzyme-linked immunoassay and immunofluorescence. Conclusion: 1.After T.marneffei infection，CD86 expression on THP-1 decreased，and with the progression of infection, insufficient polarization of M1 macrophages gradually appeared；2.T.marneffei may adsorb or uptake CD86 in supernatant produced by macrophages during the contact with THP-1 cells, thus leading to the consumption of CD86 in macrophages.

Negative bone scintigraphy in wild-type transthyretin cardiac amyloidosis

Background Amyloidosis is a rare systemic disease due to the extracellular tissue deposition of a fibrillar-shaped misfolded protein, called amyloid. Only two types of proteins commonly affect the heart leading to an infiltrative cardiomyopathy: immunoglobulin light chain and transthyretin (TTR) cardiac amyloidosis (CA). Despite the promising role of emerging imaging modalities, such as strain echocardiography, cardiac magnetic resonance and bone scintigraphy, its diagnosis is still often missed or delayed due to their inherent limitations and to a nonspecific clinical scenario with frequent concomitance of cardiac comorbidities. The gold standard for a definite diagnosis still remains endomyocardial biopsy, but in rare cases Congo Red staining could provide false negative results, as in our case, requiring immunoelectron microscopy. Case presentation A middle-aged male adult presented to the emergency department for relapse of heart failure. Echocardiography and cardiac magnetic resonance, along with the history of bilateral carpal tunnel syndrome, were suspicious for TTR-CA. The diagnosis, however, was hampered by concomitant cardiac comorbidities and conflicting results of imaging modalities. In fact bone scintigraphy was negative, as well as Congo Red Staining on myocardial tissue samples obtained by endomyocardial biopsy. Given the high clinical suspicion, immunoelectron microscopy was performed, showing TTR amyloid fibrils deposits, that confirmed the diagnosis. A genetic analysis excluded and hereditary form. The patient was then referred to a specialist center for specific treatment. Conclusions This is a rare case of a TTR-CA with a negative Bone Scintigraphy and Congo red staining, which demonstrated that CA is frequently misdiagnosed because of the low specific clinical manifestations and the results of imaging modalities that sometimes could be misleading, with subsequent delayed diagnosis and correct treatment.

Novel function for AP-1B during cell migration

Kell and Ang et al. show that adaptor protein (AP)-1B expression in epithelial cells decreased the speed of collective cell migration. Consistent with this novel function, they demonstrate that AP-1B localized in membrane ruffles during cell migration using high-end fluorescence microcopy, stochastic optical reconstruction microscopy, and immunoelectron microscopy.

Effect of Postmortem Degradation on the Preservation of Viral Particles and Rabies Antigens in Mice Brains. Light and Electron Microscopic Study

Rabies diagnosis is mainly made on fresh brain tissue postmortem by means of the direct immunofluorescence test. However, in some cases, it is not possible to use this technique, given that the affected nervous tissue goes through a postmortem degradation process, due to problems in the handling and transport of the samples. For this reason, the preservation in time of the rabies virus inclusions was assessed, as well as the immunoreactivity and the ultrastructure of viral particles in tissue with postmortem degradation. Brains of mice inoculated with rabies virus and control mice were processed for conventional histology, immunohistochemistry, electron microscopy, and immunoelectron microscopy in different postmortem times. In the processed tissues for hematoxylin and eosin, the presence of eosinophilic inclusions was not observed beyond 12 h postmortem. Surprisingly, the immunoreactivity of the viral antigens increased with time, at least until 30 h postmortem. It was possible to easily recognize the viral particles by means of conventional electron microscopy until 12 h postmortem. Immunoelectron microscopy allowed us to identify the presence of viral antigens disseminated in the neuronal cytoplasm until 30 h postmortem, but immunoreactive viral particles were not observed. The rabies infection did not cause histological or ultrastructural alterations different from those in the control group as a result of the postmortem degradation. In conclusion, the immunohistochemistry is a reliable test for rabies diagnosis in samples with postmortem degradation and that have been fixed with aldehydes.