Intravenously engrafted human multilineage-differentiating stress-enduring (Muse) cells rescue erectile function after rat cavernous nerve injury.

OBJECTIVE: To evaluate the effect of intravenous administration of human multilineage-differentiating stress-enduring (Muse) cells on rat postoperative erectile dysfunction (ED) with cavernous nerve (CN) injury without an immunosuppressant. MATERIALS AND METHODS: Male Sprague-Dawley rats were randomised into three groups after CN crush injury. Either human-Muse cells, non-Muse mesenchymal stem cells (MSCs) (both 1.0 × 105 cells), or vehicle was infused intravenously at 3 h after CN injury without immunosuppressant. Erectile function was assessed by measuring intracavernous pressure (ICP) and arterial pressure (AP) during pelvic nerve electrostimulation 28 days after surgery. At 48 h and 28 days after intravenous infusion of Muse cells, the homing of Muse cells and non-Muse MSCs was evaluated in the major pelvic ganglion (MPG) after CN injury. In addition, expressions of C-X-C motif chemokine ligand (Cxcl12) and glial cell line-derived neurotrophic factor (Gdnf) in the MPG were examined by real-time polymerase chain reaction. Statistical analyses and comparisons among groups were performed using one-way analysis of variance followed by the Tukey test for parametric data and Kruskal-Wallis test followed by the Dunn-Bonferroni test for non-parametric data. RESULTS: The mean (SEM) ICP/AP values at 28 days were 0.51 (0.02) in the Muse cell group, 0.37 (0.03) in the non-Muse MSC group, and 0.36 (0.04) in the vehicle group, showing a significant positive response in the Muse cell group compared with the non-Muse and vehicle groups (P = 0.013 and P = 0.010, respectively). In the MPG, Muse cells were observed to be engrafted at 48 h and expressed Schwann cell markers S100 (~46%) and glial fibrillary acidic protein (~24%) at 28 days, while non-Muse MSCs were basically not engrafted at 48 h. Higher gene expression of Cxcl12 (P = 0.048) and Gdnf (P = 0.040) was found in the MPG of the Muse group than in the vehicle group 48 h after infusion. CONCLUSION: Intravenously engrafted human Muse cells recovered rat erectile function after CN injury in a rat model possibly by upregulating Cxcl12 and Gdnf.

Construction and Stability of All-in-One Adenovirus Vectors Simultaneously Expressing Four and Eight Multiplex Guide RNAs and Cas9 Nickase.

CRISPR/Cas9 technology is expected to offer novel genome editing-related therapies for various diseases. We previously showed that an adenovirus vector (AdV) possessing eight expression units of multiplex guide RNAs (gRNAs) was obtained with no deletion of these units. Here, we attempted to construct "all-in-one" AdVs possessing expression units of four and eight gRNAs with Cas9 nickase, although we expected obstacles to obtain complete all-in-one AdVs. The first expected obstacle was that extremely high copies of viral genomes during replication may cause severe off-target cleavages of host cells and induce homologous recombination. However, surprisingly, four units in the all-in-one AdV genome were maintained completely intact. Second, for the all-in-one AdV containing eight gRNA units, we enlarged the E3 deletion in the vector backbone and shortened the U6 promoter of the gRNA expression units to shorten the AdV genome within the adenovirus packaging limits. The final size of the all-in-one AdV genome containing eight gRNA units still slightly exceeded the reported upper limit. Nevertheless, approximately one-third of the eight units remained intact, even upon preparation for in vivo experiments. Third, the genome editing efficiency unexpectedly decreased upon enlarging the E3 deletion. Our results suggested that complete all-in-one AdVs containing four gRNA units could be obtained if the problem of the low genome editing efficiency is solved, and those containing even eight gRNA units could be obtained if the obstacle of the vector size is also removed.

Visual Impairment due to a large paraclinoid aneurysm treated with parent artery occlusion and bypass: A case report.

INTRODUCTION: Paraclinoid internal carotid artery (ICA) aneurysms can sometimes cause visual field disturbances due to their size, and it is challenging to treat either surgically or using endovascular techniques. Flow diverters generally have positive outcomes, but sometimes in symptomatic aneurysms, we see the thrombosed section becomes enlarged. Therefore, optimal treatment strategies are difficult to determine. CASE: A 68-year-old woman presented with a chief complaint of vision loss in the left eye. A large wide-necked saccular aneurysm was found on the left ICA paraclinoid portion. Under general anesthesia, a Pipeline Flex was inserted along with coil embolization. After treatment, the aneurysm showed thrombotic expansion, and the visual impairment worsened. One year later, aneurysm recanalization was evident; therefore, another Pipeline was inserted to overlap the stent. However, her visual impairment worsened again, and parent artery occlusion with high flow bypass was performed 20 months after her first treatment. Two weeks postoperatively, improved peripheral vision was confirmed. Further, no enlargement of the aneurysm was observed using magnetic resonance imaging 6 months later. CONCLUSION: This case examined a symptomatic, large paraclinoid aneurysm in a patient, which continued to enlarge after Pipeline stent placement, but was later treated successfully using direct parent artery occlusion in combination with high-flow bypass.

Adenovirus Vectors Expressing Eight Multiplex Guide RNAs of CRISPR/Cas9 Efficiently Disrupted Diverse Hepatitis B Virus Gene Derived from Heterogeneous Patient.

Hepatitis B virus (HBV) chronically infects more than 240 million people worldwide, causing chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). Genome editing using CRISPR/Cas9 could provide new therapies because it can directly disrupt HBV genomes. However, because HBV genome sequences are highly diverse, the identical target sequence of guide RNA (gRNA), 20 nucleotides in length, is not necessarily present intact in the target HBV DNA in heterogeneous patients. Consequently, possible genome-editing drugs would be effective only for limited numbers of patients. Here, we show that an adenovirus vector (AdV) bearing eight multiplex gRNA expression units could be constructed in one step and amplified to a level sufficient for in vivo study with lack of deletion. Using this AdV, HBV X gene integrated in HepG2 cell chromosome derived from a heterogeneous patient was cleaved at multiple sites and disrupted. Indeed, four targets out of eight could not be cleaved due to sequence mismatches, but the remaining four targets were cleaved, producing irreversible deletions. Accordingly, the diverse X gene was disrupted at more than 90% efficiency. AdV containing eight multiplex gRNA units not only offers multiple knockouts of genes, but could also solve the problems of heterogeneous targets and escape mutants in genome-editing therapy.

Cell surface molecule, Klingon, mediates the refinement of synaptic specificity in the Drosophila visual system.

In the Drosophila brain, neurons form genetically specified synaptic connections with defined neuronal targets. It is proposed that each central nervous system neuron expresses specific cell surface proteins, which act as identification tags. Through an RNAi screen of cell surface molecules in the Drosophila visual system, we found that the cell adhesion molecule Klingon (Klg) plays an important role in repressing the ectopic formation of extended axons, preventing the formation of excessive synapses. Cell-specific manipulation of klg showed that Klg is required in both photoreceptors and the glia, suggesting that the balanced homophilic interaction between photoreceptor axons and the glia is required for normal synapse formation. Previous studies suggested that Klg binds to cDIP and our genetic analyses indicate that cDIP is required in glia for ectopic synaptic repression. These data suggest that Klg play a critical role together with cDIP in refining synaptic specificity and preventing unnecessary connections in the brain.

Efficacy of Direct Revascularization Surgery for Hemorrhagic Moyamoya Syndrome As a Late Complication of Cranial Irradiation for Childhood Craniopharyngioma.

Moyamoya syndrome (MMS) is an uncommon late complication after cranial irradiation. Its hemorrhagic presentation from the associated pseudo-aneurysm is extremely rare, and the optimal management strategy is undetermined. We herein report a 36-year-old man who developed intraventricular hemorrhage from a pseudo-aneurysm at the extended left anterior choroidal artery as an abnormal collateral of MMS 30 years after surgical removal and cranial irradiation for childhood craniopharyngioma. Catheter angiography confirmed the diagnosis of MMS, and multiple pseudo-aneurysms were evident at the ipsilateral abnormal choroidal collateral, one of which was considered to be a source of bleeding. The patient underwent left superficial temporal artery (STA)-middle cerebral artery (MCA) anastomosis with indirect pial synangiosis based on the observation that the development of choroidal collateral may be associated with a high rebleeding risk in hemorrhagic moyamoya disease. The patient was discharged without neurological deficit, and postoperative magnetic resonance angiography confirmed the STA-MCA bypass to be patent. Catheter angiography 1 year after revascularization surgery revealed the complete disappearance of the pseudoaneurysms with the apparently patent STA-MCA bypass. The patient did not exhibit any cerebrovascular events during the follow-up period of 16 months. In conclusion, hemorrhagic MMS with choroidal collateral as a dangerous anastomosis was effectively managed by STA-MCA anastomosis. Although long-term follow-up is necessary to evaluate our strategy, the favorable disappearance of pseudoaneurysms after revascularization surgery in the present case strongly suggests that STA-MCA anastomosis has a potential role for preventing rebleeding in MMS after cranial irradiation.

[Anticoagulation Therapy for Prevention of Acute Pulmonary Thromboembolism in Patients with Intracerebral Hemorrhage in Acute Phase].

Pulmonary thromboembolism(PTE)can be a lethal complication in patients with intracerebral hemorrhage(ICH), and the early detection of deep venous thrombosis(DVT)is important for prevention of PTE. Anticoagulation therapy is effective for prevention of PTE; however, in ICH patients, the safety of anticoagulants is not established because of concern about ICH expansion. We investigated the clinical data of patients who developed ICH and assessed risk factors for DVT and the safety of anticoagulation therapy. Our study included 250 patients between 2014 and 2016. We performed weekly screening of D-dimer and ultrasonography of lower limb veins was performed when levels gradually increased or reached 10 µg/mL. In patients with DVT, we started anticoagulation therapy after systolic blood pressures were controlled at ≤140 mmHg. DVT was detected in 35(14.0%)patients, and 29(11.6%)underwent anticoagulation therapy. A hemorrhagic complication was observed in 1 case as gastrointestinal bleeding. Expansion of ICH was not detected in any cases. Symptomatic PTE occurred in 1 case with DVT, just before initiation of anticoagulants. Univariate logistic regression analysis revealed hemorrhage volume ≥30 mL and modified Rankin Scale score ≥5 at discharge were associated with increased risk of DVT, with odds ratios of 2.69 and 2.51, respectively. Our study suggests that DVT tends to occur in patients with severe ICH and that periodic measurement of D-dimer is useful for early detection of DVT. Anticoagulation therapy can be safely started in ICH patients under strict control of blood pressure.

"Doping" of Polyyne with an Organometallic Fragment Leads to Highly Conductive Metallapolyyne Molecular Wire.

Exploration of highly conductive molecules is essential to achieve single-molecule electronic devices. The present paper describes the results on single-molecule conductance study of polyyne wires doped with the organometallic Ru(dppe)2 fragment, X-(C≡C) n-Ru(dppe)2-(C≡C) n-X. The metallapolyyne wires end-capped with the gold fragments (X = AuL) are subjected to single-molecule conductance measurements with the STM break junction technique, which reveal the high conductance (10-3-10-2 G0; n = 2-4) with the low attenuation factor (0.25 Å-1) and the low contact resistance (33 kΩ). A unique "'doping'" effect of Ru(dppe)2 fragment was found to lead to the high performance as suggested by the hybrid density functional theory-nonequilibrium green function calculation.

Defective interferon signaling in the circulating monocytes of type 2 diabetic mice.

Type 2 diabetes mellitus (T2DM) is associated with poor outcome after stroke. Peripheral monocytes play a critical role in the secondary injury and recovery of damaged brain tissue after stroke, but the underlying mechanisms are largely unclear. To investigate transcriptome changes and molecular networks across monocyte subsets in response to T2DM and stroke, we performed single-cell RNA-sequencing (scRNAseq) from peripheral blood mononuclear cells and bulk RNA-sequencing from blood monocytes from four groups of adult mice, consisting of T2DM model db/db and normoglycemic control db/+ mice with or without ischemic stroke. Via scRNAseq we found that T2DM expands the monocyte population at the expense of lymphocytes, which was validated by flow cytometry. Among the monocytes, T2DM also disproportionally increased the inflammatory subsets with Ly6C+ and negative MHC class II expression (MO.6C+II-). Conversely, monocytes from control mice without stroke are enriched with steady-state classical monocyte subset of MO.6C+II+ but with the least percentage of MO.6C+II- subtype. Apart from enhancing inflammation and coagulation, enrichment analysis from both scRNAseq and bulk RNAseq revealed that T2DM specifically suppressed type-1 and type-2 interferon signaling pathways crucial for antigen presentation and the induction of ischemia tolerance. Preconditioning by lipopolysaccharide conferred neuroprotection against ischemic brain injury in db/+ but not in db/db mice and coincided with a lesser induction of brain Interferon-regulatory-factor-3 in the brains of the latter mice. Our results suggest that the increased diversity and altered transcriptome in the monocytes of T2DM mice underlie the worse stroke outcome by exacerbating secondary injury and potentiating stroke-induced immunosuppression. Significance Statement: The mechanisms involved in the detrimental diabetic effect on stroke are largely unclear. We show here, for the first time, that peripheral monocytes have disproportionally altered the subsets and changed transcriptome under diabetes and/or stroke conditions. Moreover, genes in the IFN-related signaling pathways are suppressed in the diabetic monocytes, which underscores the immunosuppression and impaired ischemic tolerance under the T2DM condition. Our data raise a possibility that malfunctioned monocytes may systemically and focally affect the host, leading to the poor outcome of diabetes in the setting of stroke. The results yield important clues to molecular mechanisms involved in the detrimental diabetic effect on stroke outcome.

Identification of genes regulating stimulus-dependent synaptic assembly in Drosophila using an automated synapse quantification system.

Neural activity-dependent synaptic plasticity is an important physiological phenomenon underlying environmental adaptation, memory and learning. However, its molecular basis, especially in presynaptic neurons, is not well understood. Previous studies have shown that the number of presynaptic active zones in the Drosophila melanogaster photoreceptor R8 is reversibly changed in an activity-dependent manner. During reversible synaptic changes, both synaptic disassembly and assembly processes were observed. Although we have established a paradigm for screening molecules involved in synaptic stability and several genes have been identified, genes involved in stimulus-dependent synaptic assembly are still elusive. Therefore, the aim of this study was to identify genes regulating stimulus-dependent synaptic assembly in Drosophila using an automated synapse quantification system. To this end, we performed RNAi screening against 300 memory-defective, synapse-related or transmembrane molecules in photoreceptor R8 neurons. Candidate genes were narrowed down to 27 genes in the first screen using presynaptic protein aggregation as a sign of synaptic disassembly. In the second screen, we directly quantified the decreasing synapse number using a GFP-tagged presynaptic protein marker. We utilized custom-made image analysis software, which automatically locates synapses and counts their number along individual R8 axons, and identified cirl as a candidate gene responsible for synaptic assembly. Finally, we present a new model of stimulus-dependent synaptic assembly through the interaction of cirl and its possible ligand, ten-a. This study demonstrates the feasibility of using the automated synapse quantification system to explore activity-dependent synaptic plasticity in Drosophila R8 photoreceptors in order to identify molecules involved in stimulus-dependent synaptic assembly.