The application of electron microscopic immunolabeling techniques to the identification and analysis of degenerating processes in neural tissue has greatly enhanced the ability of researchers to examine apoptosis and other degenerative disease mechanisms. This is particularly true for the early stages of such mechanisms. Traditionally, degenerating processes could only be identified at the ultrastructural level after significant cellular atrophy had occurred, when subcellular detail was obscured and synaptic relationships altered. Using immunocytochemical labeling procedures, degenerating neural and glial processes are first identified through the use of antibodies directed against a variety of degenerative markers, such as proapoptotic effectors (i.e., cytoplasmic cytochrome c), pathological components (i.e., beta amyloid deposits), or inflammatory agents (i.e., Iba1). Both the subcellular distribution of the marker within the process and the relationship of the labeled process to surrounding elements can then be carefully characterized. The information obtained can be further refined through the use of dual immunolabeling, which can provide additional data on the phenotype of the degenerating process and inputs to the process.

BacT/Alert Virtuo is an advanced, automated blood culture system incorporating improved automation and an enhanced detection algorithm to shorten time to detection. A multicenter study of the investigational Virtuo system (bioMérieux, Inc., Durham, NC) compared to BacT/Alert 3D (BTA3D) for detection of bacteremia/fungemia in four bottle types, SA and FA Plus (aerobic) and SN and FN Plus (anaerobic), was performed in a clinical setting with patient samples in a matched system design clinical trial. Blood was added to paired aerobic or anaerobic bottles, with the volume in each bottle in each pair required to be ≤10 ml and with the volumes required to be within 30% of each other. Of 5,709 bottle sets (52.5% aerobic pairs and 47.5% anaerobic pairs), 430 (7.5%) were positive for bacterial or fungal growth, with 342 (6.0%) clinically significant and 83 (1.5%) contaminated. A total of 3,539 sets (62.0%) were volume compliant, with 203 sets (5.7%) clinically significant. The positivity rates for volume-compliant bottle pairs determined by the two systems were comparable, with 68.7% of clinically significant isolates detected by both instruments, 15.7% by Virtuo only, and 15.7% by BTA3D only. Virtuo detected microbial growth nearly 2 h sooner overall than BTA3D (mean, 15.9 h versus 17.7 h). Shorter time to detection by Virtuo was related to organism group, with the time to detection being significantly shorter for enteric Gram-negative bacilli and enterococci (means, 3.6 h and 2.3 h shorter, respectively). This large clinical study demonstrated that the Virtuo blood culture system produced results comparable to those seen with the long-established BTA3D system, with significantly shorter time to detection.

The alpha-7 nicotinic acetylcholine receptor (α7nAChR) and the dopamine D2 receptor (D2R) are both implicated in attentional processes and cognition, mediated in part through the prefrontal cortex (PFC). We examined the dual electron microscopic immunolabeling of α7nAChR and either D2R or the vesicular acetylcholine transporter (VAChT) in rodent PFC to assess convergent functional activation sites. Immunoreactivity (ir) for α7nAChR and/or D2R was seen in the same as well as separate neuronal and glial profiles. At least half of the dually labeled profiles were somata and dendrites, while most labeled axon terminals expressed only D2R-ir. The D2R-labeled terminals were without synaptic specializations or formed inhibitory or excitatory-type synapses with somatodendritic profiles, some of which expressed the α7nAChR and/or D2R. Astrocytic glial processes comprised the majority of nonsomatodendritic α7nAChR or α7nAChR and D2R-labeled profiles. Glial processes containing α7nAChR-ir were frequently located near VAChT-labeled terminals and also showed perisynaptic and perivascular associations. We conclude that in rodent PFC α7nACh and D2R activation can dually modulate (1) postsynaptic dendritic responses within the same or separate but synaptically linked neurons in which the D2R has the predominately presynaptic distribution, and (2) astrocytic signaling that may be crucial for synaptic transmission and functional hyperemia.

The nucleus accumbens (Acb) contains subpopulations of neurons defined by their receptor content and potential involvement in sensorimotor gating and other behaviors that are dysfunctional in schizophrenia. In Acb neurons, the NMDA NR1 (NR1) subunit is co-expressed not only with the dopamine D1 receptor (D1R), but also with the μ-opioid receptor (μ-OR), which mediates certain behaviors that are adversely impacted by schizophrenia. The NMDA-NR1 subunit has been suggested to play a role in the D1R trafficking and behavioral dysfunctions resulting from systemic administration of apomorphine, a D1R and dopamine D2 receptor agonist that impacts prepulse inhibition (PPI) to auditory-evoked startle (AS). Together, this evidence suggests that the NMDA receptor may regulate D1R trafficking in Acb neurons, including those expressing μ-OR, in animals exposed to auditory startle and apomorphine. We tested this hypothesis by combining spatial-temporal gene deletion technology, dual labeling immunocytochemistry, and behavioral analysis. Deleting NR1 in Acb neurons prevented the increase in the dendritic density of plasma membrane D1Rs in single D1R and dual (D1R and μ-OR) labeled dendrites in the Acb in response to apomorphine and AS. Deleting NR1 also attenuated the decrease in AS induced by apomorphine. In the absence of apomorphine and startle, deletion of Acb NR1 diminished social interaction, without affecting novel object recognition, or open field activity. These results suggest that NR1 expression in the Acb is essential for apomorphine-induced D1R surface trafficking and reduction in AS, but also plays an independent role in controling social behaviors that are impaired in multiple psychiatric disorders.

The PI 3-kinase Vps34 (Pik3c3) synthesizes phosphatidylinositol 3-phosphate (PI3P), a lipid critical for both endosomal membrane traffic and macroautophagy. Human genetics have implicated PI3P dysregulation, and endosomal trafficking in general, as a recurring cause of demyelinating Charcot-Marie-Tooth (CMT) peripheral neuropathy. Here, we investigated the role of Vps34, and PI3P, in mouse Schwann cells by selectively deleting Vps34 in this cell type. Vps34-Schwann cell knockout (Vps34SCKO) mice show severe hypomyelination in peripheral nerves. Vps34−/− Schwann cells interact abnormally with axons, and there is a delay in radial sorting, a process by which large axons are selected for myelination. Upon reaching the promyelinating stage, Vps34−/− Schwann cells are significantly impaired in the elaboration of myelin. Nerves from Vps34SCKO mice contain elevated levels of the LC3 and p62 proteins, indicating impaired autophagy. However, in the light of recent demonstrations that autophagy is dispensable for myelination, it is unlikely that hypomyelination in Vps34SCKO mice is caused by impaired autophagy. Endosomal trafficking is also disturbed in Vps34−/− Schwann cells. We investigated the activation of the ErbB2/3 receptor tyrosine kinases in Vps34SCKO nerves, as these proteins, which play essential roles in Schwann cell myelination, are known to traffic though endosomes. In Vps34SCKO nerves, ErbB3 was hyperphosphorylated on a tyrosine known to be phosphorylated in response to Nrg1 exposure. ErbB2 protein levels were also decreased during myelination. Our findings suggest that the loss of Vps34 alters the trafficking of ErbB2/3 through endosomes. Abnormal ErbB2/3 signaling to downstream targets may contribute to the hypomyelination observed in Vps34SCKO mice.

Cellular processes rely on the precise orchestration of signaling and effector molecules in space and time, yet it remains challenging to gain a comprehensive picture of the molecular organization underlying most basic biological functions. This organization often takes place at length scales below the resolving power of conventional microscopy. In recent years, several “superresolution” fluorescence microscopic techniques have emerged that can surpass the diffraction limit of conventional microscopy by a factor of two to twenty. These methods have been used to reveal previously unknown organization of macromolecular complexes and cytoskeletal structures. The resulting high-resolution view of molecular organization and dynamics is already changing our understanding of cellular processes at the systems level. However, current subdiffractive microscopic techniques are not without limitations; challenges remain to be overcome before these techniques achieve their full potential. Here, we introduce three primary types of subdiffractive microscopic techniques, consider their current limitations and challenges, and discuss recent biological applications.

Today’s scientific data are primarily stored and accessed via centralized Web-based infrastructure. Centralization has advantages but also carries risks such as link rot and content drift, which can hinder scientific progress. It is time to ask whether traditional, centralized Web architecture aligns with scholarly priorities and values, and to collaboratively move towards new approaches that do.

Distributed computing systems (dcs) are increasingly becoming an integral and strategic part of organisations. Research in dcs has concentrated on languages and operating systems which address the problems of implementing distributed systems, but has not considered the problems of long-term management of distributed systems. This paper presents a new approach to distributed system management. The central concept is the Domain Model, which allows administrators to specify and structure management policies. As such, it provides a uniform interface across different functions of management while being able to scale and handle multiple organisation systems.