Non-destructive and efficient method for obtaining miRNA information in cells by artificial extracellular vesicles.

In recent years, research has explored the use of microRNA (miRNA) analysis in extracellular vesicles (EVs) as a minimally invasive strategy for the diagnosis and prediction of diseases. This is because miRNAs in EVs partly reflect the miRNA information and cellular status of the origin cells. However, not all intracellular miRNAs are internalized into EVs. Therefore, the miRNA information obtained from EVs is limited. To get more miRNA information, we aimed to produce artificial EVs (aEVs) encapsulating Argonaute 2 (Ago2) miRNA-binding protein, which actively incorporate miRNAs within themselves. In this study, we utilized the protein EPN-01, which is capable of releasing aEVs encapsulating it and associated proteins. This system enables us to obtain more miRNA species and increase each miRNA's yield in the EV fraction. Furthermore, we examined whether miRNAs in the EV fraction using our system reflect the cellular condition. In cells treated with CoCl2, a reagent for inducing a hypoxia-mimic state, we detected a change in the level of hypoxia marker miR-210 with aEVs. To the best of our knowledge, this is the first report on a method to increase the yield and variety of endogenous miRNAs in the EV fraction. This approach leads to improved accuracy of cell status assessment using miRNAs in EVs.

Role of the Orphan Transporter SLC35E1 in the Nuclear Egress of Herpes Simplex Virus 1.

This study developed a system consisting of two rounds of screening cellular proteins involved in the nuclear egress of herpes simplex virus 1 (HSV-1). Using this system, we first screened cellular proteins that interacted with the HSV-1 nuclear egress complex (NEC) consisting of UL34 and UL31 in HSV-1-infected cells, which are critical for the nuclear egress of HSV-1, by tandem affinity purification coupled with mass spectrometry-based proteomics technology. Next, we performed CRISPR/Cas9-based screening of live HSV-1-infected reporter cells under fluorescence microscopy using single guide RNAs targeting the cellular proteins identified in the first proteomic screening to detect the mislocalization of the lamin-associated protein emerin, which is a phenotype for defects in HSV-1 nuclear egress. This study focused on a cellular orphan transporter SLC35E1, one of the cellular proteins identified by the screening system. Knockout of SLC35E1 reduced HSV-1 replication and induced membranous invaginations containing perinuclear enveloped virions (PEVs) adjacent to the nuclear membrane (NM), aberrant accumulation of PEVs in the perinuclear space between the inner and outer NMs and the invagination structures, and mislocalization of the NEC. These effects were similar to those of previously reported mutation(s) in HSV-1 proteins and depletion of cellular proteins that are important for HSV-1 de-envelopment, one of the steps required for HSV-1 nuclear egress. Our newly established screening system enabled us to identify a novel cellular protein required for efficient HSV-1 de-envelopment. IMPORTANCE The identification of cellular protein(s) that interact with viral effector proteins and function in important viral procedures is necessary for enhancing our understanding of the mechanics of various viral processes. In this study, we established a new system consisting of interactome screening for the herpes simplex virus 1 (HSV-1) nuclear egress complex (NEC), followed by loss-of-function screening to target the identified putative NEC-interacting cellular proteins to detect a defect in HSV-1 nuclear egress. This newly established system identified SLC35E1, an orphan transporter, as a novel cellular protein required for efficient HSV-1 de-envelopment, providing an insight into the mechanisms involved in this viral procedure.

ESCRT-III controls nuclear envelope deformation induced by progerin.

Hutchinson-Gilford progeria syndrome (HGPS) is a premature aging disorder, caused by mutation in the gene encoding lamin A/C, which produces a truncated protein called progerin. In cells from HGPS patients, progerin accumulates at the nuclear membrane (NM), where it causes NM deformations. In this study, we investigated whether progerin-induced NM deformation involved ESCRT-III, a protein complex that remodels nuclear and cytoplasmic membranes. The ESCRT-III protein CHMP4B was recruited to sites of aberrant NM proliferation in human cells ectopically expressing progerin and in patient-derived HGPS fibroblasts. Derepression of NM deformation in these cells was observed following depletion of CHMP4B or an ESCRT-III adaptor, ALIX. Treatment with rapamycin (which induce autophagic clearance of progerin and reverse progerin-induced cellular phenotypes) down-regulated progerin-induced NM deformation, whereas treatment with bafilomycin A1 (an inhibitor of autophagy and lysosome-based degradation) or CHMP4B depletion antagonized the effects of rapamycin. These results indicate that the ALIX-mediated ESCRT-III pathway plays a suppressive role in progerin-induced NM deformation and suggest that autophagy down-regulates progerin-induced NM deformation in a manner dependent on ESCRT-III machinery.

ESCRT-III mediates budding across the inner nuclear membrane and regulates its integrity.

Vesicle-mediated nucleocytoplasmic transport is a nuclear pore-independent mechanism for the nuclear export of macromolecular complexes, but the molecular basis for this transport remains largely unknown. Here we show that endosomal sorting complex required for transport-III (ESCRT-III) is recruited to the inner nuclear membrane (INM) during the nuclear export of herpes simplex virus 1 (HSV-1). Scission during HSV-1 budding through the INM is prevented by depletion of ESCRT-III proteins. Interestingly, in uninfected human cells, the depletion of ESCRT-III proteins induces aberrant INM proliferation. Our results show that HSV-1 expropriates the ESCRT-III machinery in infected cells for scission of the INM to produce vesicles containing progeny virus nucleocapsids. In uninfected cells, ESCRT-III regulates INM integrity by downregulating excess INM.

Herpes Simplex Virus 1 UL34 Protein Regulates the Global Architecture of the Endoplasmic Reticulum in Infected Cells.

Upon herpes simplex virus 1 (HSV-1) infection, the CD98 heavy chain (CD98hc) is redistributed around the nuclear membrane (NM), where it promotes viral de-envelopment during the nuclear egress of nucleocapsids. In this study, we attempted to identify the factor(s) involved in CD98hc accumulation and demonstrated the following: (i) the null mutation of HSV-1 UL34 caused specific dispersion throughout the cytoplasm of CD98hc and the HSV-1 de-envelopment regulators, glycoproteins B and H (gB and gH); (ii) as observed with CD98hc, gB, and gH, wild-type HSV-1 infection caused redistribution of the endoplasmic reticulum (ER) markers calnexin and ERp57 around the NM, whereas the UL34-null mutation caused cytoplasmic dispersion of these markers; (iii) the ER markers colocalized efficiently with CD98hc, gB, and gH in the presence and absence of UL34 in HSV-1-infected cells; (iv) at the ultrastructural level, wild-type HSV-1 infection caused ER compression around the NM, whereas the UL34-null mutation caused cytoplasmic dispersion of the ER; and (v) the UL34-null mutation significantly decreased the colocalization efficiency of lamin protein markers of the NM with CD98hc and gB. Collectively, these results indicate that HSV-1 infection causes redistribution of the ER around the NM, with resulting accumulation of ER-associated CD98hc, gB, and gH around the NM and that UL34 is required for ER redistribution, as well as for efficient recruitment to the NM of the ER-associated de-envelopment factors. Our study suggests that HSV-1 induces remodeling of the global ER architecture for recruitment of regulators mediating viral nuclear egress to the NM.IMPORTANCE The ER is an important cellular organelle that exists as a complex network extending throughout the cytoplasm. Although viruses often remodel the ER to facilitate viral replication, information on the effects of herpesvirus infections on ER morphological integrity is limited. Here, we showed that HSV-1 infection led to compression of the global ER architecture around the NM, resulting in accumulation of ER-associated regulators associated with nuclear egress of HSV-1 nucleocapsids. We also identified HSV-1 UL34 as a viral factor that mediated ER remodeling. Furthermore, we demonstrated that UL34 was required for efficient targeting of these regulators to the NM. To our knowledge, this is the first report showing that a herpesvirus remodels ER global architecture. Our study also provides insight into the mechanism by which the regulators for HSV-1 nuclear egress are recruited to the NM, where this viral event occurs.

Subjective effect of magnetic resonance-guided focused ultrasound surgery for uterine fibroids.

AIM: We treated 69 patients with symptomatic uterine fibroids using a magnetic resonance-guided focused ultrasound surgery (MRgFUS) system. Our objective was to determine the clinical outcome of MRgFUS, focusing on symptom improvement, with emphasis on the time and extent of improvement. METHODS: Patients who would have been otherwise offered conventional surgery were considered for eligibility. They were asked to report their symptom severities before and after treatment on the same query form. The questionnaire, given 6 months after treatment, included a question asking when the patients' symptoms started to improve. Their fibroids were classified into three types according to the signal intensity on T2-weighted magnetic resonance images: type 1, low intensity as skeletal muscle, type 2, intermediate intensity, lower than myometrium but higher than skeletal muscle; and type 3, high intensity, the same as or higher than myometrium. RESULTS: No severe adverse events occurred in any of the patients. Seven patients required alternative treatment after MRgFUS, with five of them having type 3 fibroids. Mean symptom scores were all reduced after MRgFUS, regardless of the symptom types. Frequent urination improved first, while heavy bleeding took longer to resolve than the other symptoms. CONCLUSION: MRgFUS is an effective and safe method for treating symptomatic uterine fibroids, especially for type 1 and type 2 fibroids. Type 3 fibroids, however, are difficult to treat using the current MRgFUS system.