Genetic and Functional Analyses Point to FAN1 as the Source of Multiple Huntington Disease Modifier Effects.

A recent genome-wide association study of Huntington disease (HD) implicated genes involved in DNA maintenance processes as modifiers of onset, including multiple genome-wide significant signals in a chr15 region containing the DNA repair gene Fanconi-Associated Nuclease 1 (FAN1). Here, we have carried out detailed genetic, molecular, and cellular investigation of the modifiers at this locus. We find that missense changes within or near the DNA-binding domain (p.Arg507His and p.Arg377Trp) reduce FAN1's DNA-binding activity and its capacity to rescue mitomycin C-induced cytotoxicity, accounting for two infrequent onset-hastening modifier signals. We also idenified a third onset-hastening modifier signal whose mechanism of action remains uncertain but does not involve an amino acid change in FAN1. We present additional evidence that a frequent onset-delaying modifier signal does not alter FAN1 coding sequence but is associated with increased FAN1 mRNA expression in the cerebral cortex. Consistent with these findings and other cellular overexpression and/or suppression studies, knockout of FAN1 increased CAG repeat expansion in HD-induced pluripotent stem cells. Together, these studies support the process of somatic CAG repeat expansion as a therapeutic target in HD, and they clearly indicate that multiple genetic variations act by different means through FAN1 to influence HD onset in a manner that is largely additive, except in the rare circumstance that two onset-hastening alleles are present. Thus, an individual's particular combination of FAN1 haplotypes may influence their suitability for HD clinical trials, particularly if the therapeutic agent aims to reduce CAG repeat instability.

Population-specific genetic modification of Huntington's disease in Venezuela.

Modifiers of Mendelian disorders can provide insights into disease mechanisms and guide therapeutic strategies. A recent genome-wide association (GWA) study discovered genetic modifiers of Huntington's disease (HD) onset in Europeans. Here, we performed whole genome sequencing and GWA analysis of a Venezuelan HD cluster whose families were crucial for the original mapping of the HD gene defect. The Venezuelan HD subjects develop motor symptoms earlier than their European counterparts, implying the potential for population-specific modifiers. The main Venezuelan HD family inherits HTT haplotype hap.03, which differs subtly at the sequence level from European HD hap.03, suggesting a different ancestral origin but not explaining the earlier age at onset in these Venezuelans. GWA analysis of the Venezuelan HD cluster suggests both population-specific and population-shared genetic modifiers. Genome-wide significant signals at 7p21.2-21.1 and suggestive association signals at 4p14 and 17q21.2 are evident only in Venezuelan HD, but genome-wide significant association signals at the established European chromosome 15 modifier locus are improved when Venezuelan HD data are included in the meta-analysis. Venezuelan-specific association signals on chromosome 7 center on SOSTDC1, which encodes a bone morphogenetic protein antagonist. The corresponding SNPs are associated with reduced expression of SOSTDC1 in non-Venezuelan tissue samples, suggesting that interaction of reduced SOSTDC1 expression with a population-specific genetic or environmental factor may be responsible for modification of HD onset in Venezuela. Detection of population-specific modification in Venezuelan HD supports the value of distinct disease populations in revealing novel aspects of a disease and population-relevant therapeutic strategies.

Novel allele-specific quantification methods reveal no effects of adult onset CAG repeats on HTT mRNA and protein levels.

Huntington's disease (HD) reflects dominant consequences of a CAG repeat expansion mutation in HTT. Expanded CAG repeat size is the primary determinant of age at onset and age at death in HD. Although HD pathogenesis is driven by the expanded CAG repeat, whether the mutation influences the expression levels of mRNA and protein from the disease allele is not clear due to the lack of sensitive allele-specific quantification methods and the presence of confounding factors. To determine the impact of CAG expansion at the molecular level, we have developed novel allele-specific HTT mRNA and protein quantification methods based on principles of multiplex ligation-dependent probe amplification and targeted MS/MS parallel reaction monitoring, respectively. These assays, exhibiting high levels of specificity and sensitivity, were designed to distinguish allelic products based upon expressed polymorphic variants in HTT, including rs149 109 767. To control for other cis-haplotype variations, we applied allele-specific quantification assays to a panel of HD lymphoblastoid cell lines, each carrying the major European disease haplotype (i.e. hap.01) on the mutant chromosome. We found that steady state levels of HTT mRNA and protein were not associated with expanded CAG repeat length. Rather, the products of mutant and normal alleles, both mRNA and protein, were balanced, thereby arguing that a cis-regulatory effect of the expanded CAG repeat is not a critical component of the underlying mechanism of HD. These robust allele-specific assays could prove valuable for monitoring the impact of allele-specific gene silencing strategies currently being explored as therapeutic interventions in HD.

Full sequence of mutant huntingtin 3'-untranslated region and modulation of its gene regulatory activity by endogenous microRNA.

Huntington's disease (HD) is caused by an expanded CAG trinucleotide repeat in the first exon of the huntingtin gene (HTT). Since the entire course of the disease starts from this dominant gain-of-function mutation, lowering total or mutant huntingtin mRNA/protein has emerged as an appealing therapeutic strategy. We reasoned that endogenous mechanisms underlying HTT gene regulation may inform strategies to target the source of the disease. As part of our investigation to understand how the expression of HTT is controlled, we performed (1) complete sequencing analysis for mutant HTT 3'-UTR and (2) unbiased screening assays to identify naturally-occurring miRNAs that could lower the HTT mRNA levels. By sequencing HD families inheriting the major European mutant haplotype, we determined the full sequence of HTT 3'-UTRs of the most frequent mutant (i.e., hap.01) and normal (i.e., hap.08) haplotypes, revealing 5 sites with alternative alleles. In subsequent miRNA activity assays using the full-length hap.01 and hap.08 3'-UTR reporter vectors and follow-up validation experiments, hsa-miR-4324 and hsa-miR-4756-5p significantly reduced HTT 3'-UTR reporter activity and endogenous HTT protein levels. However, those miRNAs did not show strong haplotype-specific effects. Nevertheless, our data highlighting full sequences of HTT 3'-UTR haplotypes, effects of miRNAs on HTT levels, and potential interaction sites provide rationale and promising targets for total and mutant-specific HTT lowering intervention strategies using endogenous and artificial miRNAs, respectively.

8 W 240 fs diode-pumped Yb:Y2O3 ceramic thin-rod femtosecond amplifier.

A diode-pumped Yb:Y2O3 ceramic thin-rod amplifier which operates in the femtosecond regime is studied here. In a single-stage and direct four-pass amplification scheme, the amplifier delivers maximum output power of 8.1 W at a center wavelength of 1030.5 nm and spectral bandwidth of 4.8 nm. Assume a sech2-shaped pulse, a pulse duration of 239 fs is measured, exhibiting a time-bandwidth product value of 0.324. To the best of our knowledge, our Yb:Y2O3 ceramic thin-rod femtosecond amplifier exhibits the shortest pulse duration with Watt-level output power among all Yb:Y2O3-based femtosecond amplifiers.

Comparative study of Yb:KYW planar waveguide lasers Q-switched by direct- and evanescent-field interaction with carbon nanotubes.

Both direct- and evanescent-field interactions with carbon nanotubes (CNTs) are applied to achieve stable Q-switched operation of Yb:KYW planar waveguide lasers. The performance characteristics were investigated in a same cavity configuration and analyzed in detail in the following three cases, CNTs deposited onto end mirror (M-coating), output coupler (OC-coating) and top surface of the planar waveguide (WG-coating). Maximum output powers, repetition rates, and minimum pulse durations are 61 mW, 1103 kHz and 215 ns for OC-coating, 39 mW, 1052 kHz and 275 ns for WG-coating, and 26 mW, 1119 kHz and 217 ns for M-coating, respectively. From the calculation of the configuration-dependent stability range, the beam size and the electric field distribution in the Yb:KYW planar waveguide, it is confirmed that the evanescent-field interaction scheme makes stable Q-switching possible with much lower intensities at saturable absorber compared to the direct-field interaction scheme in the presented waveguide laser operation.

Permanent inactivation of Huntington's disease mutation by personalized allele-specific CRISPR/Cas9.

A comprehensive genetics-based precision medicine strategy to selectively and permanently inactivate only mutant, not normal allele, could benefit many dominantly inherited disorders. Here, we demonstrate the power of our novel strategy of inactivating the mutant allele using haplotype-specific CRISPR/Cas9 target sites in Huntington's disease (HD), a late-onset neurodegenerative disorder due to a toxic dominant gain-of-function CAG expansion mutation. Focusing on improving allele specificity, we combined extensive knowledge of huntingtin (HTT) gene haplotype structure with a novel personalized allele-selective CRISPR/Cas9 strategy based on Protospacer Adjacent Motif (PAM)-altering SNPs to target patient-specific CRISPR/Cas9 sites, aiming at the mutant HTT allele-specific inactivation for a given diplotype. As proof-of-principle, simultaneously using two CRISPR/Cas9 guide RNAs (gRNAs) that depend on PAM sites generated by SNP alleles on the mutant chromosome, we selectively excised ∼44 kb DNA spanning promoter region, transcription start site, and the CAG expansion mutation of the mutant HTT gene, resulting in complete inactivation of the mutant allele without impacting the normal allele. This excision on the disease chromosome completely prevented the generation of mutant HTT mRNA and protein, unequivocally indicating permanent mutant allele-specific inactivation of the HD mutant allele. The perfect allele selectivity with broad applicability of our strategy in disorders with diverse disease haplotypes should also support precision medicine through inactivation of many other gain-of-function mutations.

Point-of-Care Ultrasound-Guided Percutaneous Cannulation of Extracorporeal Membrane Oxygenation: Make it Simple.

BACKGROUND: Cannulation of the great vessels is required for extracorporeal membrane oxygenation (ECMO). Currently, there is no guideline for optimal imaging modalities during percutaneous cannulation of ECMO. OBJECTIVE: The purpose of this study was to describe percutaneous cannulation guided by point-of-care ultrasound (POCUS) for ECMO and compare it with fluoroscopy and landmark guidance. METHODS: Three groups (POCUS-, fluoroscopy-, and landmark-guided) of percutaneous cannulation for ECMO were analyzed retrospectively in a tertiary academic hospital. In the POCUS-guided group, visual confirmation of guidewire and cannula by ultrasound in both the access and return cannula were essential for successful cannulation. Fluoroscopy- and landmark-guided groups were cannulated with the conventional technique. RESULTS: A total of 128 patients were treated by ECMO during the study period, of which 94 (73.4%) cases were venoarterial ECMO. This included 56 cases of extracorporeal cardiopulmonary resuscitation. Also, there were 30 (23.4%) cases of venovenous ECMO and 4 (3.1%) cases of venoarteriovenous ECMO. A total of 71 (55.5%) patients were cannulated under POCUS guidance, and 43 (33.6%) patients were cannulated under fluoroscopy guidance and 14 (10.9%) patients were cannulated by landmark guidance. No surgical cut downs were required. Misplacement of cannula occurred in 3 (2.3%) cases. All three occurred in the landmark-guided group. CONCLUSIONS: POCUS-guided cannulation is comparable to fluoroscopy-guided cannulation in terms of avoiding cannula misplacement. In our experience, POCUS-guided cannulation is a useful strategy over fluoroscopy- and landmark-guided cannulation during peripheral ECMO.

[Analyses on distribution and structure of blaCARB-2 in Klebsiella pneumoniae].

In order to characterize the structure of the beta-lactamase gene and its corresponding mobile genetic elements in Klebsiella pneumoniae, the beta-lactamase genes from 240 clinical Klebsiella pneumoniae isolates were studied. blaCARB-2, a newly characterized gene, was extensively investigated utilizing next-generation sequencing, PCR, molecular cloning, conjugation, and comparative genomics analysis. We identified 11 beta-lactamase genes among the 240 clinical Klebsiella pneumoniae isolates; the blaCARB-2 gene exists only in one specific isolate (Klebsiella pneumoniae KP1276) (1/240, 0.42%). The blaCARB-2 gene lies on a conjugative plasmid pKP1276-82, a 182,450-bp plasmid, which encodes 222 open reading frames. The plasmid has seven resistance genes, termed blaCARB-2, blaKLUC, aadA1, aadA2, cmlA1, dfrA1, and sul2. Among these genes, blaCARB-2 was identified for the first time in Klebsiella pneumoniae. Four of these resistance genes and an int gene form a class 1 integron (int-blaCARB-2-aadA2-cmlA1-aadA1). Further studies show that the blaCARB-2, aadA2, and cmlA1 genes are resistant to their corresponding antibiotics and the blaCARB-2 exhibits higher resistance activities to penicillin beta-lactams. These results reveal the possibility of horizontal transfer of the resistance genes and dissemination of resistance among bacteria of different genera or species of Enterobacteriaceae.

Extracorporeal CPR and intra-aortic balloon pumping in tachycardia-induced cardiomyopathy complicating cardiac arrest.

Although tachycardia-induced cardiomyopathy (TIC) due to atrial fibrillation occurs frequently, it is under-recognized in clinical settings. TIC has a wide range of clinical manifestations, from asymptomatic tachycardia to cardiomyopathy leading to end stage heart failure. We present a case of a 48year-old-woman who presented as cardiogenic shock, and rapidly progressed to cardiac arrest from recently diagnosed but undertreated atrial fibrillation, resulting TIC in the emergency department (ED). She was rescued by extracorporeal cardiopulmonary resuscitation (E-CPR) for refractory cardiac arrest in the ED, and received concomitant intra-aortic balloon counterpulsation (IABP) support for severe left ventricular failure. Cardiogenic shock can present as an initial manifestation of TIC, and E-CPR and subsequent IABP support can be a valuable rescue therapy for severe TIC.

Graphene mode-locked femtosecond Cr2+:ZnS laser with ~300 nm tuning range.

Graphene has proved to be an excellent broadband saturable absorber for mode-locked operation of ultrafast lasers. However, for the mid-infrared (mid-IR) range where broadly tunable sources are in great needs, graphene-based broadly tunable ultrafast mid-IR lasers have not been demonstrated so far. Here, we report on passive mode-locking of a mid-IR Cr:ZnS laser by utilizing a transmission-type monolayer graphene saturable absorber and broad spectral tunability between 2120 nm and 2408 nm, which is the broadest tuning bandwidth ever reported for graphene mode-locked mid-IR solid-state lasers. The recovery time of the saturable absorber is measured to be ~2.4 ps by pump-probe technique at a wavelength of 2350 nm. Stably mode-locked Cr:ZnS laser delivers Fourier transform-limited 220-fs pulses with a pulse energy of up to 7.8 nJ.

Phrenic Arterial Injury Presenting as Delayed Hemothorax Complicating Simple Rib Fracture.

Delayed hemothorax after blunt torso injury is rare, but might be associated with significant morbidity and mortality. We present a case of delayed hemothorax bleeding from phrenic artery injury in a 24-year-old woman. The patient suffered from multiple rib fractures on the right side, a right hemopneumothorax, thoracic vertebral injury and a pelvic bone fracture after a fall from a fourth floor window. Delayed hemothorax associated with phrenic artery bleeding, caused by a stab injury from a fractured rib segment, was treated successfully by a minimally invasive thoracoscopic surgery. Here, we have shown that fracture of a lower rib or ribs might be accompanied by delayed massive hemothorax that can be rapidly identified and promptly managed by thoracoscopic means.

Extracorporeal cardiopulmonary resuscitation in bedside echocardiography-diagnosed massive pulmonary embolism.

Acute pulmonary embolism (PE) is one of the major causes of inhospital cardiac arrest as well as out-of-hospital cardiac arrest. Bedside diagnosis of acute PE in the emergency department (ED) can be challenging, especially in a cardiac arrest setting. Even if the early diagnosis of an acute massive PE had been made, hemodynamic instability may be worsened unless obstructive shock gets resolved. We present a case of a 46-year-old woman who developed pulseless electrical activity (PEA) after complaining of weakness and dyspnea in an ambulance, presumptively diagnosed as acute PE by bedside focused echocardiography. She received thrombolytic therapy and was rescued by extracorporeal cardiopulmonary resuscitation for recurrent PEA arrest in the ED. Focused bedside echocardiography provides a rapid diagnostic adjunct, and extracorporeal cardiopulmonary resuscitation can be a valuable rescue therapy for PEA arrest from massive PE.

Carbon nanostructure-based saturable absorber mirror for a diode-pumped 500-MHz femtosecond Yb:KLu(WO4)2 laser.

We report a diode-pumped Yb:KLu(WO(4))(2) (Yb:KLuW) laser passively mode-locked by employing a carbon nanostructure-based multi-functional saturable absorber mirror. Two types of carbon nanostructures, single-walled carbon nanotubes (SWCNTs) and graphene, were deposited on a single dielectric mirror and applied both for stable mode-locking of the Yb:KLuW laser near 1050 nm in a compact cavity configuration. The carbon nanostructure mode-locked laser delivers 157-fs pulses with output powers of up to 85 mW at a repetition rate of 500 MHz.

Personalized allele-specific CRISPR-Cas9 strategies for myofibrillar myopathy 6.

Myofibrillar myopathy 6 (MFM6) is a rare childhood-onset myopathy characterized by myofibrillar disintegration, muscle weakness, and cardiomyopathy. The genetic cause of MFM6 is p.Pro209Leu mutation (rs121918312-T) in the BAG3 gene, which generates the disease outcomes in a dominant fashion. Since the consequences of the BAG3 mutation are strong and rapidly progressing, most MFM6 patients are due to de novo mutation. There are no effective treatments for MFM6 despite its well-known genetic cause. Given p.Pro209Leu mutation is dominant, regenerative medicine approaches employing orthologous stem cells in which mutant BAG3 is inactivated offer a promising avenue. Here, we developed personalized allele-specific CRISPR-Cas9 strategies capitalizing on PAM-altering SNP and PAM-proximal SNP. In order to identify the disease chromosome carrying the de novo mutation in our two affected individuals, haplotype phasing through cloning-sequencing was performed. Based on the sequence differences between mutant and normal BAG3, we developed personalized allele-specific CRISPR-Cas9 strategies to selectively inactivate the mutant allele 1) by preventing the transcription of the mutant BAG3 and 2) by inducing nonsense-mediated decay (NMD) of mutant BAG3 mRNA. Subsequent experimental validation in patient-derived induced pluripotent stem cell (iPSC) lines showed complete allele specificities of our CRISPR-Cas9 strategies and molecular consequences attributable to inactivated mutant BAG3. In addition, mutant allele-specific CRISPR-Cas9 targeting did not alter the characteristics of iPSC or the capacity to differentiate into cardiomyocytes. Together, our data demonstrate the feasibility and potential of personalized allele-specific CRISPR-Cas9 approaches to selectively inactivate the mutant BAG3 to generate cell resources for regenerative medicine approaches for MFM6.

Base editing strategies to convert CAG to CAA diminish the disease-causing mutation in Huntington's disease.

An expanded CAG repeat in the huntingtin gene (HTT) causes Huntington's disease (HD). Since the length of uninterrupted CAG repeat, not polyglutamine, determines the age-at-onset in HD, base editing strategies to convert CAG to CAA are anticipated to delay onset by shortening the uninterrupted CAG repeat. Here, we developed base editing strategies to convert CAG in the repeat to CAA and determined their molecular outcomes and effects on relevant disease phenotypes. Base editing strategies employing combinations of cytosine base editors and guide RNAs (gRNAs) efficiently converted CAG to CAA at various sites in the CAG repeat without generating significant indels, off-target edits, or transcriptome alterations, demonstrating their feasibility and specificity. Candidate BE strategies converted CAG to CAA on both expanded and non-expanded CAG repeats without altering HTT mRNA and protein levels. In addition, somatic CAG repeat expansion, which is the major disease driver in HD, was significantly decreased in the liver by a candidate BE strategy treatment in HD knock-in mice carrying canonical CAG repeats. Notably, CAG repeat expansion was abolished entirely in HD knock-in mice carrying CAA-interrupted repeats, supporting the therapeutic potential of CAG-to-CAA conversion strategies in HD and potentially other repeat expansion disorders.

Genetic modifiers of somatic expansion and clinical phenotypes in Huntington's disease reveal shared and tissue-specific effects.

Huntington's disease (HD), due to expansion of a CAG repeat in HTT , is representative of a growing number of disorders involving somatically unstable short tandem repeats. We find that overlapping and distinct genetic modifiers of clinical landmarks and somatic expansion in blood DNA reveal an underlying complexity and cell-type specificity to the mismatch repair-related processes that influence disease timing. Differential capture of non-DNA-repair gene modifiers by multiple measures of cognitive and motor dysfunction argues additionally for cell-type specificity of pathogenic processes. Beyond trans modifiers, differential effects are also illustrated at HTT by a 5'-UTR variant that promotes somatic expansion in blood without influencing clinical HD, while, even after correcting for uninterrupted CAG length, a synonymous sequence change at the end of the CAG repeat dramatically hastens onset of motor signs without increasing somatic expansion. Our findings are directly relevant to therapeutic suppression of somatic expansion in HD and related disorders and provide a route to define the individual neuronal cell types that contribute to different HD clinical phenotypes.

Thymoquinone inhibits phorbol ester-induced activation of NF-κB and expression of COX-2, and induces expression of cytoprotective enzymes in mouse skin in vivo.

Thymoquinone (TQ), the active ingredient of Nigella sativa, has been reported to possess anti-inflammatory and chemopreventive properties. The present study was aimed at elucidating the molecular mechanisms of anti-inflammatory and antioxidative activities of thymoquinone in mouse skin. Pretreatment of female HR-1 hairless mouse skin with TQ attenuated 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced expression of cyclooxygenase-2 (COX-2). TQ diminished nuclear translocation and the DNA binding of nuclear factor-kappaB (NF-κB) via the blockade of phosphorylation and subsequent degradation of IκBα in TPA-treated mouse skin. Pretreatment with TQ attenuated the phosphorylation of Akt, c-Jun-N-terminal kinase and p38 mitogen-activated protein kinase, but not that of extracellular signal-regulated kinase-1/2. Moreover, topical application of TQ induced the expression of heme oxygenase-1, NAD(P)H-quinoneoxidoreductase-1, glutathione-S-transferase and glutamate cysteine ligase in mouse skin. Taken together, the inhibitory effects of TQ on TPA-induced COX-2 expression and NF-κB activation, and its ability to induce the expression of cytoprotective proteins provide a mechanistic basis of anti-inflammatory and antioxidative effects of TQ in hairless mouse skin.

Yb:KYW planar waveguide laser Q-switched by evanescent-field interaction with carbon nanotubes.

We report Q-switched operation of a planar waveguide laser by evanescent-field interaction with single-walled carbon nanotubes deposited on top of the waveguide. The saturable-absorber-integrated gain medium, which operates based on evanescent-field interaction, enables the realization of a diode-pumped 2.5-cm-long Q-switched Yb:KYW waveguide laser emitting at 1030 nm. With such a compact cavity design, we achieve maximum output powers of up to 30 mW, corresponding to a single-pulse energy of 124 nJ, at 241 kHz repetition rate. The shortest pulse duration of 433 ns is generated at a repetition rate of 231 kHz.

Optimal Hyperspectral Band Selection for Tissue Oxygenation Mapping with Generative Adversarial Network.

Tissue oxygenation assessment using hyperspectral imaging is an emerging technique for the diagnosis and pre- and post-treatment monitoring of ischemic patients. However, the high spectral resolution of hyperspectral imaging leads to large data sizes and a long imaging time. In this study, we propose a method that utilizes multi-objective evolutionary algorithms to determine the optimal hyperspectral band combination when developing a deep learning model for predicting tissue oxygenation from hyperspectral images. Our results confirm that the deep learning model effectively predicts tissue oxygenation images for various oxygenation states. Moreover, we demonstrate that a high-performance prediction model can be developed using only a small number of spectral bands, indicating the potential for more efficient non-contact tissue oxygenation mapping with the proposed method.Clinical Relevance- The proposed method allows for the non-contact and efficient acquisition of two-dimensional tissue oxygenation information in various oxygenation states.