The development of CRISPR-based gene-editing technologies has brought an unprecedented revolution in the field of genome engineering. Cas12a, a member of the Class 2 Type V CRISPR-associated ...endonuclease family distinct from Cas9, has been repurposed and developed into versatile gene-editing tools with distinct PAM recognition sites and multiplexed gene targeting capability. However, with current CRISPR/Cas12a technologies, it remains a challenge to perform efficient and precise genome editing of long sequences in mammalian cells. To address this limitation, we utilized phage recombination enzymes and developed an efficient CRISPR/Cas12a tool for multiplexed precision editing in mammalian cells. Through protein engineering, we were able to recruit phage recombination proteins to Cas12a to enhance its homology-directed repair efficiencies. Our phage-recombination-assisted Cas12a system achieved up to 3-fold improvements for kilobase-scale knock-ins in human cells without compromising the specificity of the enzyme. The performance of this system compares favorably against Cas9 references, the commonly used enzyme for gene-editing tasks, with improved specificity. Additionally, we demonstrated multi-target editing with similar improved activities thanks to the RNA-processing activity of the Cas12a system. This compact, multi-target editing tool has the potential to assist in understanding multi-gene interactions. In particular, it paves the way for a gene therapy method for human diseases that complements existing tools and is suitable for polygenic disorders and diseases requiring long-sequence corrections.
ABSTRACT
Recently, noncoding RNAs have been thought to play important roles in the sporadic occurrence of spinal deformity of adolescent idiopathic scoliosis (AIS). As a prognostic factor for curve ...progression, low bone mass has been hypothesized to crosstalk with AIS pathogenesis. Abnormal osteoblasts activities are reported in AIS without a clear mechanism. In this study, bone biopsies from patients with AIS and control subjects and the primary osteoblasts derived from those samples were used to identify the potential microRNA (miRNA) candidates that interfere with osteoblasts and osteocytes function. Microarray analysis identified miRNA‐145–5p (miR‐145) as a potential upstream regulator. miR‐145 and β‐catenin mRNA (CTNNB1) were overexpressed in AIS bone tissues and primary osteoblasts, and their expression correlated positively in AIS. Knockdown of miR‐145 restored impaired osteocyte activity through the down‐regulation of active β‐catenin expression and its transcriptional activity. Significant negative correlations between circulating miR‐145 and serum sclerostin, osteopontin, and osteoprotegerin were noted in patients with AIS, which was in line with our cellular findings. This is the first study to demonstrate the effect of aberrant miRNA expression and its effect on osteocyte function in AIS, which may contribute to the low bone mass. Our findings also provide insight into the development of circulating microRNAs as a bone quality biomarker or even a prognostic biomarker for AIS.—Zhang, J., Chen, H., Leung, R. K. K., Choy, K. W., Lam, T.‐P., Ng, B. K. W., Qiu, Y., Feng, J. Q., Cheng, J. C. Y., Lee, W. Y. W. Aberrant miR‐145–5p/β‐catenin signal impairs osteocyte function in adolescent idiopathic scoliosis. FASEB J. 32, 6537–6549 (2018). www.fasebj.org
A prospective cohort study.
The aim of this study was to compare the somatosensory-evoked potential (SEP) findings of adolescent idiopathic scoliosis (AIS) subjects of different curve severity with ...age- and gender-matched controls and to evaluate any correlation between the site of the SEP abnormality with cerebellar tonsillar level measured by magnetic resonance imaging (MRI).
Our previous studies showed that a higher percentage of SEP abnormality and cerebellar tonsillar ectopia was present in AIS patients than in normal controls. However, the relationship between the anatomical site of the neurophysiological abnormality and the severity in AIS patients has not been defined.
SEP measurement was conducted on 91 Chinese AIS girls with major right thoracic curve of different curve severity (mild, moderate, severe) and 49 matched normal controls. Waveform characteristics (latency and amplitude) were compared among groups. Specific location of SEP abnormality was identified from tibial to cortical levels. Cerebellar tonsillar ectopia was defined by the previously established reference line between basion and opisthion on MRI.
Significant prolonged P37 latency was found on the right side between severe AIS patients and normal controls, while increased inter-side P37 latency difference was found between severe versus moderate, and severe versus normal controls. Cerebellar tonsillar ectopia was detected in 27.3% of severe group, 5.8% to 6.7% in mild and moderate group, but none in normal controls. Abnormal SEP occurred superior to C5 region in all surgical (severe) patients, of whom 58% had cerebellar tonsillar ectopia.
AIS patients showed significant prolonged latency and increased latency difference on the side of major curvature. The incidence of SEP abnormality increased with curve severity and occurred above the C5 level. The findings suggested that there was a subgroup of progressive AIS with subclinical neurophysiological dysfunction, associated with underlying neuromorphological abnormalities, which were only detectable by SEP and MRI.
3.
Open surgical repair for infected thoracic aortic pseudoaneurysms carries significant mortality and morbidity. Endovascular stent graft repair has been our preferred approach, although its role ...remains controversial because persistent infection is always a concern. We aimed to assess the efficacy and durability of endovascular stent graft repair in these patients.
Between August 2000 and November 2005, seven consecutive patients with eight infected pseudoaneurysms of the thoracic aorta were treated with endovascular stent graft repair. Patients were diagnosed based on a typical appearance of an infected pseudoaneurysm on imaging together with a positive bacteriology culture or clinical evidence of sepsis. The follow-up protocol included regular clinical examination, hematologic tests, and computed tomography scans.
There were six men and one woman with a median age of 68 years at operation. Three patients presented with an aortoenteric fistula. The operations were performed in the operating room with the image guidance of a mobile C-arm. Endovascular stent grafts were deployed successfully in all patients, with complete exclusion of the pseudoaneurysms. Intravenous antibiotics were continued for 1 to 6 weeks and followed by lifelong maintenance oral antibiotics. The median hospital stay was 27 days, with no hospital deaths. No paraplegia or other major complications occurred. Two patients with aortoesophageal fistula where the fistula tracts were persistent died during follow-up. The other five patients remained well, with no evidence of graft infection at a median follow-up of 34 months. A significant reduction in the diameter of the pseudoaneurysm (>5 mm) was noted on computed tomography scans after 12 months.
Endovascular stent graft repair is effective and may be a durable option for infected pseudoaneurysms of the thoracic aorta.
Background: Adverse drug events (ADE) have been studied widely in hospitalised and emergency department (ED) patients. Less is known about the ED visits of drug‐related injury in Taiwan. This study ...seeks to determine the incidence, risk and patient outcomes of ADE in an ED population.
Methods: We conducted a prospective observational cohort study of patients 18 years and older presenting to the ED of an urban, tertiary medical centre. ED visits between 1 March 2009 and 28 February 2010 identified by investigators for suspected ADE were further assessed by using the Naranjo Adverse Drug Reaction probability scale. Outcomes (ED disposition, injury severity and preventability) and associated variables (triage, gender, drug category, number of drugs, Charlson comorbidity index score and ADE mechanism) were measured.
Results: Of 58 569 ED visits, 452 patients (0.77%) had physician‐documented ADE. 24% of patients with ADE were hospitalised with life‐threatening conditions, with a mortality rate of 10.0%. The majority of ADE were considered preventable (73.4%), and the unintentional overdose was the most common cause. Cardiovascular agents accounted for the most ADE (25.8%) and consisted of 65.3% of ADE in patients aged 65 years and older. Risk factors for ADE‐related hospitalisation were elderly age (odds ratio (OR) 1.9, 95% confidence interval (CI) 1.1–3.4), severity of ADE (OR 6.9, 95% CI 3.3–14.5) and higher Charlson comorbidity index scores (OR 3.4, 95% CI 2.0–5.7).
Conclusion: ADE‐related ED visits are not uncommon in Taiwan and many cases are preventable. ED‐based surveillance may provide useful information for monitoring outpatient ADE.
Tumour suppressor genes (TSGs) were frequently inactivated through promoter hypermethylation in gastric carcinoma as well as pre-malignant gastric lesions, suggesting that promoter hypermethylation ...can be used as a marker to define novel TSGs and also biomarkers for early detection of gastric cancer. In an effort to search for such genes aberrantly methylated in gastric cancer development, fibulin 1 (FBLN1) was found as a candidate TSG epigenetically downregulated in gastric cancer. FBLN1 expression was downregulated in all of gastric cancer cell lines used (100%, 7 out of 7) and the primary gastric carcinoma tissues (84%, 86 out of 102) and significantly restored after pharmacological demethylation. Hypermethylation of the FBLN1 promoter was frequently (71%, 5 out of 7) detected in gastric cancer cell lines and primary gastric carcinoma tissues. Ectopic expression of FBLN1 led to the growth inhibition of gastric cancer cells through the induction of apoptosis. In summary, FBLN1 was identified as a novel candidate TSG epigenetically downregulated in gastric cancer.
Background Hybrid technique with open surgical supra-aortic extra-anatomical bypasses (to provide a suitable proximal landing zone) and simultaneous or staged thoracic endovascular stent grafting is ...less invasive than open surgery in the treatment of complex aortic arch pathology. The aim of this study is to report our hybrid experience. Methods Retrospective review of prospectively collected computerized database. All patients had regular clinical and radiological computed tomography follow-up. Results Sixteen patients (13 males and 3 females) with mean age of 64.8 (range 51-79) were treated with hybrid techniques between May 2005 and September 2007. There were nine elective, two urgent (within 2 weeks of presentation), and five emergency cases. Thirteen patients had arch or proximal descending thoracic aortic aneurysms, and six patients had aortic dissections. All extra-anatomical bypasses were performed simultaneously with stent grafts, with mean operating time of 331.2 (range 195-540) minutes. Eight patients had right to left carotid-carotid cross-over graft, five had bifurcating grafts from ascending aorta to innominate and left carotid artery, two had left carotid to left subclavian artery, and one had left carotid to aberrant right subclavian bypass graft. All patients had Cook Zenith thoracic stent grafts. Deployment success was 100%, with no endoleak on completion angiogram. There was no mortality. Three patients had postoperative nondebilitating stroke and no paraplegia. The mean follow-up was 14 (range 1-27) months. One reintervention was required, and two patients had type II endoleak treated conservatively. Conclusion Supra-aortic hybrid procedures in treating aortic arch pathology are safe and effective, and early- to midterm results are encouraging.
A major consequence of traumatic brain injury (TBI) is the rapid proteolytic degradation of structural cytoskeletal proteins. This process is largely reflected by the interruption of axonal transport ...as a result of extensive axonal injury leading to neuronal cell injury. Previous work from our group has described the extensive degradation of the axonally enriched cytoskeletal αII-spectrin protein which results in molecular signature breakdown products (BDPs) indicative of injury mechanisms and to specific protease activation both in vitro and in vivo. In the current study, we investigated the integrity of βII-spectrin protein and its proteolytic profile both in primary rat cerebrocortical cell culture under apoptotic, necrotic, and excitotoxic challenge and extended to in vivo rat model of experimental TBI (controlled cortical impact model). Interestingly, our results revealed that the intact 260-kDa βII-spectrin is degraded into major fragments (βII-spectrin breakdown products (βsBDPs)) of 110, 108, 85, and 80 kDa in rat brain (hippocampus and cortex) 48 h post-injury. These βsBDP profiles were further characterized and compared to an in vitro βII-spectrin fragmentation pattern of naive rat cortex lysate digested by calpain-2 and caspase-3. Results revealed that βII-spectrin was degraded into major fragments of 110/85 kDa by calpain-2 activation and 108/80 kDa by caspase-3 activation. These data strongly support the hypothesis that in vivo activation of multiple protease system induces structural protein proteolysis involving βII-spectrin proteolysis via a specific calpain and/or caspase-mediated pathway resulting in a signature, protease-specific βsBDPs that are dependent upon the type of neural injury mechanism. This work extends on previous published work that discusses the interplay spectrin family (αII-spectrin and βII-spectrin) and their susceptibility to protease proteolysis and their implication to neuronal cell death mechanisms.
The novel switched reluctance generator (SRG) with hybrid magnetic paths is developed for wind power generation in this paper. For the proposed SRG, there is no mutual coupling between phase ...windings. The proposed SRG has the longitudinal magnetic structure at the completely aligned position. However, it has the transverse and longitudinal magnetic structure at the unaligned position. Thus, three-dimensional finite element computation is needed in the design. The computed results based on three-dimensional finite-element analysis (FEA) are shown in the paper. Furthermore, the experimental results of the prototype demonstrate that the proposed SRG is feasible and effective.
PDF
