RANBP2 and USP9x regulate nuclear import of adenovirus minor coat protein IIIa.

As intracellular parasites, viruses exploit cellular proteins at every stage of infection. Adenovirus outbreaks are associated with severe acute respiratory illnesses and conjunctivitis, with no specific antiviral therapy available. An adenoviral vaccine based on human adenovirus species D (HAdV-D) is currently in use for COVID-19. Herein, we investigate host interactions of HAdV-D type 37 (HAdV-D37) protein IIIa (pIIIa), identified by affinity purification and mass spectrometry (AP-MS) screens. We demonstrate that viral pIIIa interacts with ubiquitin-specific protease 9x (USP9x) and Ran-binding protein 2 (RANBP2). USP9x binding did not invoke its signature deubiquitination function but rather deregulated pIIIa-RANBP2 interactions. In USP9x-knockout cells, viral genome replication and viral protein expression increased compared to wild type cells, supporting a host-favored mechanism for USP9x. Conversely, RANBP2-knock down reduced pIIIa transport to the nucleus, viral genome replication, and viral protein expression. Also, RANBP2-siRNA pretreated cells appeared to contain fewer mature viral particles. Transmission electron microscopy of USP9x-siRNA pretreated, virus-infected cells revealed larger than typical paracrystalline viral arrays. RANBP2-siRNA pretreatment led to the accumulation of defective assembly products at an early maturation stage. CRM1 nuclear export blockade by leptomycin B led to the retention of pIIIa within cell nuclei and hindered pIIIa-RANBP2 interactions. In-vitro binding analyses indicated that USP9x and RANBP2 bind to C-terminus of pIIIa amino acids 386-563 and 386-510, respectively. Surface plasmon resonance testing showed direct pIIIa interaction with recombinant USP9x and RANBP2 proteins, without competition. Using an alternative and genetically disparate adenovirus type (HAdV-C5), we show that the demonstrated pIIIa interaction is also important for a severe respiratory pathogen. Together, our results suggest that pIIIa hijacks RANBP2 for nuclear import and subsequent virion assembly. USP9x counteracts this interaction and negatively regulates virion synthesis. This analysis extends the scope of known adenovirus-host interactions and has potential implications in designing new antiviral therapeutics.

Clinicopathological Analysis of miRNA Expression in Breast Cancer Tissues by Using miRNA In Situ Hybridization.

In this article, we describe a detailed protocol for miRNA detection in breast cancer tissue using in situ hybridization with a digoxigenin-labelled LNA (Locked Nucleic Acid) probe. The probe was recognized by anti-DIG alkaline phosphatase antibodies and later developed using alkaline phosphatase substrate producing fluorescence signals. Here we utilized miRNA in situ hybridization (MISH) technique to analyze expression of miR-489 in tissues from breast cancer patients. This technique can detect the localization of miRNA of interest in individual tissue samples. This technique can be used to compare the expression of desired miRNA in tumor tissue with that in adjacent normal tissue and to identify the specific structures responsible for expressing this miRNA. This technique can be very useful in answering certain clinical questions, such as role of specific miRNA in the development of cancer. Our results indicate that mammary epithelial cells express significantly higher levels of miR-489 than adjacent tumor cells.

Hypothalamic, feeding/arousal-related peptidergic projections to the paraventricular thalamic nucleus in the rat.

Based on the importance of paraventricular thalamic nucleus (PVT) as a relay station of energy balance, arousal, and food reward, we aimed in the present study to determine projection patterns of neuropeptide Y (NPY), cocaine- and amphetamine-regulated transcript (CART), melanin-concentrating hormone (MCH), and orexin (ORX)-ergic fibers to the PVT. First, the distribution of peptidergic axon terminals within the PVT was examined. NPY and CART terminals were confined within the boundary of the thalamic nucleus, exhibiting almost identical distribution. MCH terminals were rarely observed. In contrast, ORX terminals were as extensive as NPY/CART terminals, but spread into the peri-PVT region. Second, neuronal origin of feeding/arousal-related peptides projecting to the PVT was investigated. NPY neurons were observed in the medial subdivision of the arcuate nucleus (Arc), whereas CART cells were in the lateral Arc as well as other hypothalamic regions including the paraventricular hypothalamic nucleus, lateral hypothalamus (LH), dorsal hypothalamic area, and zona incerta. Both NPY- and CART-fiber projections to the PVT were bilateral; ipsilateral proportion was 54.0% ± 3.6% (n = 6) for NPY and 57.1% ± 2.5% (n = 6) for CART. The total number of CART neurons projecting to the PVT exceeded that of NPY cells; the ratio of labeled CART neurons to NPY cells was 2.4 ± 0.2 (n = 6). In contrast, ORX-ergic fiber projection to the PVT exhibited a slight ipsilateral dominance (62.7% ± 1.6%, n = 6), with majority of labeled cells located in the LH medial to the fornix (72.2% ± 2.3%, n = 6). Third, based on heavy projection from the PVT to the nucleus accumbens shell (NAcSh), the convergence of NPY and CART terminals on a single PVT neuron was identified; the proportion of labeled PVT neurons that received converging NPY/CART terminals compared with the total PVT neurons projecting to the NAcSh was 2.7% ± 0.6% (n = 3). Finally, PVT cells receiving NPY, CART, or ORX terminals provided divergent axon collaterals to NAcSh and medial prefrontal cortex. The present observations provided the anatomical evidence that the PVT might play an essential role in the integration of antagonistically-acting, feeding/arousal-related peptidergic inputs on their way to the cortical reward circuit.

Symptomatic steno-occlusion of cerebral arteries and subsequent ischemic events in patients with acute ischemic stroke.

BACKGROUND: We aimed to assess the impact of symptomatic steno-occlusion (SYSO) of cerebral arteries and its characteristics on subsequent ischemic event (SIE) in patients with acute ischemic stroke. METHODS: Using a prospective stroke registry database, we identified consecutive patients with ischemic stroke who were hospitalized within 48 hours of symptom onset. SYSO denoted significant stenosis or occlusion of major cerebral arteries with ischemic lesions at the corresponding arterial territories and was characterized by its location and severity. Primary outcome was SIE that was defined as ischemic progression or recurrence within 1 year. RESULTS: In total, 1546 patients (age, 67.4 ± 13.0 years; median National Institutes of Health Stroke Scale score, 4) were enrolled in this study. The cumulative risk of SIE was 14.5% at 7 days, 14.9% at 14 days, 15.5% at 90 days, and 16.9% at 1 year. Patients with SYSO had significantly higher SIE rates compared with those without SYSO (23.0% versus 11.6%). Of the characteristics of SYSO, the location, not the severity, was significantly associated with SIE (P < .001 and P = .186, respectively). Multiple (adjusted hazard ratio, 5.85; 95% confidence interval, 1.81-18.85), intracranial internal carotid artery (ICA) (3.54; 1.21-8.21), and extracranial ICA SYSO (2.88; 1.01-8.21) raised the risk of SIE. CONCLUSIONS: Subsequent cerebral ischemic events (progression or recurrence) after an acute ischemic stroke occur mostly within several days of stroke onset and is associated with the location, but not the severity, of symptomatic steno-occlusion of cerebral arteries.

Reciprocal connections between CART-immunoreactive, hypothalamic paraventricular neurons and serotonergic dorsal raphe cells in the rat: Light microscopic study.

Based on the overlapping physiological roles of cocaine- and amphetamine-regulated transcript (CART) peptides and serotonin, the present study examined the anatomical connection between the hypothalamic paraventricular nucleus (PVN) and the dorsal raphe (DR). The first series of experiments were performed to investigate descending projections from the CART-immunoreactive (CART-ir) PVN to serotonergic DR cells. CART-ir varicosities made contact with serotonergic DR neurons. An anterograde tracing study revealed that varicosities originating from the PVN formed close appositions to serotonergic neuronal profiles along the entire rostro-caudal extent of the DR. A retrograde study demonstrated that CART neurons projecting to the DR were mainly localized in the caudal parvicellular PVN, comprising approximately 3.0%±0.4% (n=8) of total CART cells. A second series of experiments was performed to investigate ascending projections from the DR to CART-ir PVN cells. Serotonin transporter-ir boutons made contact with CART-ir PVN neurons. Anterograde tracing revealed that varicosities originating from the DR formed close appositions to CART-ir PVN cells. Retrograde examination demonstrated that serotonergic neurons projecting to the parvicellular PVN were located along the entire rostro-caudal extent of the DR. The present observation provided an anatomical basis for accumulating evidence in the literature that suggests a functional interaction between the CART and serotonin systems during the regulation of energy balance, emotional behavior, and arousal.

Retrograde study of CART- or NPY-neuronal projection from the hypothalamic arcuate nucleus to the dorsal raphe and/or the locus coeruleus in the rat.

The present study was designed to reveal cocaine- and amphetamine-regulated transcript (CART)- or neuropeptide Y (NPY)-immunoreactive neuronal projections from the hypothalamic arcuate nucleus (Arc) to the dorsal raphe (DR) and/or the locus coeruleus (LC) in the rat. Our results demonstrated that CART or NPY axon terminals formed close appositions to the neuronal profiles in the DR and the LC. Thus, arcuate sections were immunostained for the CART or NPY after the injections of green RetroBeads(™) into the DR and red tracer into the LC (or vice versa). First, retrogradely-labeled CART cells were mainly observed in the lateral Arc without colchicine. Of the total population of arcuate CART neurons, DR- and LC-projecting cells were 5.7% ± 0.9% and 6.6% ± 0.7%, respectively. In addition, a subset (3.3% ± 0.7%) of CART neurons provided divergent axon collaterals to the DR and the LC. Second, retrogradely-labeled NPY cells were observed in lateral or ventral borders of the medial Arc only after colchicine injection. Of the entire NPY cell population, DR- and LC-projecting neurons were 1.5% ± 0.3% and 1.3% ± 0.3%, respectively. Only a scanty proportion (0.1% ± 0.0%) sent axon collaterals to the DR and the LC. These observations suggested that arcuate CART or NPY system might have a potential influence on the brainstem monoaminergic nuclei, modulating their roles in feeding, nociception, emotional behaviors, arousal, and stress responses. Furthermore, a portion of arcuate CART neurons (along with only a few NPY cells) sending divergent axon collaterals to the DR/LC might have a simultaneous (and possibly more efficient) way to exert their specific influences on the monoaminergic nuclei.

Mitochondrial respiratory complex I deficiency simulating spinal muscular atrophy.

Two female patients with clinical features resembling spinal muscular atrophy were presented. Patient 1 presented with hypotonia and proximal weakness of extremities at age 4 months. Electromyography revealed motor neuronopathy suggestive of spinal muscular atrophy. Patient 2 presented with severe hypotonia, motor weakness, and joint contractures since birth. Muscle biopsy findings were consistent with spinal muscular atrophy. However, deletions in the survival motor neuron gene and the neuronal apoptosis inhibitor protein gene were not found in both patients. They finally manifested clinical features unlike spinal muscular atrophy: epileptic seizure, cardiomyopathy, and spasticity. The clinical course of each patient was not like that of spinal muscular atrophy type I. Mitochondrial respiratory chain complex enzyme activities in cultured skin fibroblasts were measured. Respiratory complex I enzyme activity was decreased, suggestive of isolated complex I deficiency in both patients. In conclusion, in patients who have clinical features resembling spinal muscular atrophy but no deletions in the spinal muscular atrophy gene, the possibility of the mitochondrial respiratory chain complex I deficiency should be considered.