CRISPR applications for Duchenne muscular dystrophy: From animal models to potential therapies.

CRISPR gene-editing technology creates precise and permanent modifications to DNA. It has significantly advanced our ability to generate animal disease models for use in biomedical research and also has potential to revolutionize the treatment of genetic disorders. Duchenne muscular dystrophy (DMD) is a monogenic muscle-wasting disease that could potentially benefit from the development of CRISPR therapy. It is commonly associated with mutations that disrupt the reading frame of the DMD gene that encodes dystrophin, an essential scaffolding protein that stabilizes striated muscles and protects them from contractile-induced damage. CRISPR enables the rapid generation of various animal models harboring mutations that closely simulates the wide variety of mutations observed in DMD patients. These models provide a platform for the testing of sequence-specific interventions like CRISPR therapy that aim to reframe or skip DMD mutations to restore functional dystrophin expression. This article is categorized under: Congenital Diseases > Genetics/Genomics/Epigenetics.

[Two cases of herpes simplex keratitis after trans-epithelial photorefractive keratectomy].

We herein report 2 cases of herpes simplex keratitis after trans-epithelial photorefractive keratectomy. Patients' medical histories, symptoms, signs, clinical examination results, diagnosis and treatment were showed in detail. Following precision diagnosis and medical intervention, including topical and systemic antiviral treatmented for 1 to 2 weeks. The two patients were cured with full reepithelialization without corneal scar.

[Efficacy of mitomycin C 0.02% for prevention of haze after transepithelial photorefractive keratectomy for mild and moderate myopia].

Objective: To investigate the efficacy of mitomycin C (MMC) 0.02% for prevention of haze after transepithelial photorefractive keratectomy (Trans-PRK) for mild and moderate myopia. Methods: Retrospective cohort study. We reviewed medical records of 295 patients (588 eyes) who underwent Trans-PRK with or without use of MMC. There were 45 patients (90 eyes) in the mild myopia group (aged between 18 and 41 years; 37 males and 8 females; myopia diopter <3.00 D) and 250 patients (498 eyes) in the moderate myopia group (aged between 18 and 46 years; 168 males and 82 females; myopia diopter: 3.00 to 6.00 D). The two groups were divided into subgroups with MMC 0.02% and without MMC, respectively. The time of intraoperative application of MMC, if there was, was 15 s and 30 s in the mild myopia group and the moderate myopia group, respectively. The mean follow-up time was 6 months. Postoperative best corrected visual acuity (BCVA), spherical equivalent (SE) and haze were analyzed and compared using an independent Student t-test or Mann-Whitney U test between subgroups. Haze variables were compared using chi-square statistics. Results: Haze was quantified with Fantes from grade 0.5 to 4. In the mild myopia group, all haze grades were 0.5 within 3 months. The incidence of haze was 6.25% (2/32) in eyes treated with MMC and 8.62% (5/58) in eyes treated without MMC; there was no statistical significance (χ²=0.00, P>0.999). In the moderate myopia group, the incidence of haze was 9.19% (24/261) in eyes treated with MMC within 3 months; the grade was 0.5 in 91.67% (22/24) of eyes with haze and 1 in 8.33% (2/24). The incidence of haze was 29.53% (70/237) in eyes treated without MMC; the grade was 0.5 in 60.00% (42/70) of eyes with haze, 1 in 18.57% (13/70), and 2 in 5.71% (4/70) within 3 months, and 0.5 in 15.71% (11/70) after 3 months (χ²=12.36, P=0.002). In the mild myopia group, BCVA was 5.0(5.0, 5.1) versus 5.0(5.0, 5.1) in the subgroups with MMC and without MMC (Z=-0.34, P=0.733). In the moderate myopia group, BCVA was 5.0(5.0, 5.1) versus 5.0(5.0, 5.1) in the subgroups with and without MMC (Z=-2.05, P=0.040). In the mild myopia group, SE was (0.33±1.07) D versus (0.32±0.57) D in the subgroups with and without MMC (t=0.25, P=0.805). In the moderate myopia group, SE was (0.66±0.85) D versus (0.53±0.67) D in the subgroups with and without MMC (t=2.97, P=0.003). Conclusions: MMC 0.02% was effective in preventing haze after Trans-PRK in the treatment of moderate myopia. However, it was not effective in mild myopia.

First-line nivolumab + ipilimumab in advanced NSCLC: CheckMate 227 subpopulation analyses in Asian patients.

BACKGROUND: Nivolumab plus ipilimumab demonstrated clinically meaningful improvement in efficacy versus chemotherapy with a manageable safety profile in patients with advanced non-small cell lung cancer (NSCLC) and tumor programmed death-ligand 1 (PD-L1) expression ≥1% or <1% in Part 1 of CheckMate 227. Here we report efficacy and safety results for the Asian subpopulation. METHODS: Patients with stage IV/recurrent NSCLC were randomized 1 : 1 : 1 to nivolumab plus ipilimumab, nivolumab monotherapy, or chemotherapy (PD-L1 ≥1%) or nivolumab plus ipilimumab, nivolumab plus chemotherapy, or chemotherapy (PD-L1 <1%). Overall survival (OS), progression-free survival, objective response rate, duration of response, and safety were evaluated among patients in Japan, South Korea, and Taiwan. RESULTS: In the Asian subpopulation with PD-L1 ≥1%, 81 patients received nivolumab plus ipilimumab and 81 received chemotherapy. Median OS was not reached with nivolumab plus ipilimumab versus 24.8 months with chemotherapy; 3-year OS rate was 53% versus 37% [hazard ratio (HR), 0.72; 95% confidence interval (CI) 0.47-1.11]. The 3-year progression-free survival rate was 26% versus 7% (HR, 0.65; 95% CI 0.45-0.96), objective response rate was 56% versus 37%, and median duration of response was 29.0 months (95% CI 15.0 months-not reached) versus 6.9 months (95% CI 3.9-11.1 months). Similar results were observed regardless of tumor PD-L1 expression and in Japanese patients. Grade 3-4 treatment-related adverse events occurred in 40% of patients receiving nivolumab plus ipilimumab and 36% receiving chemotherapy, in the overall Asian subpopulation (tumor PD-L1 expression ≥1% and <1%); no new safety signals were identified. CONCLUSIONS: At 3-year follow-up, nivolumab plus ipilimumab provided durable long-term efficacy benefits versus chemotherapy regardless of tumor PD-L1 expression in the Asian subpopulation, including Japanese patients. Consistent with findings for all randomized patients, these data support the use of nivolumab plus ipilimumab as first-line treatment of Asian patients with advanced NSCLC.

Optimized nickase- and nuclease-based prime editing in human and mouse cells.

Precise genomic modification using prime editing (PE) holds enormous potential for research and clinical applications. In this study, we generated all-in-one prime editing (PEA1) constructs that carry all the components required for PE, along with a selection marker. We tested these constructs (with selection) in HEK293T, K562, HeLa and mouse embryonic stem (ES) cells. We discovered that PE efficiency in HEK293T cells was much higher than previously observed, reaching up to 95% (mean 67%). The efficiency in K562 and HeLa cells, however, remained low. To improve PE efficiency in K562 and HeLa, we generated a nuclease prime editor and tested this system in these cell lines as well as mouse ES cells. PE-nuclease greatly increased prime editing initiation, however, installation of the intended edits was often accompanied by extra insertions derived from the repair template. Finally, we show that zygotic injection of the nuclease prime editor can generate correct modifications in mouse fetuses with up to 100% efficiency.

Nivolumab with carboplatin, paclitaxel, and bevacizumab for first-line treatment of advanced nonsquamous non-small-cell lung cancer.

BACKGROUND: This international, randomized, double-blind phase III study (ONO-4538-52/TASUKI-52) evaluated nivolumab with bevacizumab and cytotoxic chemotherapy as first-line treatment for nonsquamous non-small-cell lung cancer (NSCLC). PATIENTS AND METHODS: Between June 2017 and July 2019, this study enrolled treatment-naïve patients with stage IIIB/IV or recurrent nonsquamous NSCLC without sensitizing EGFR, ALK, or ROS1 alterations. They were randomly assigned in a 1 : 1 ratio to receive nivolumab or placebo in combination with carboplatin, paclitaxel, and bevacizumab every 3 weeks for up to six cycles, followed by nivolumab/placebo with bevacizumab until progressive disease or unacceptable toxicity. The primary endpoint was progression-free survival (PFS) assessed by an independent radiology review committee (IRRC). RESULTS: Overall, 550 patients from Japan, Korea, and Taiwan were randomized; of these patients, 273 and 275 received the nivolumab and placebo combinations, respectively. In the present preplanned interim analysis with a median follow up of 13.7 months, the IRRC-assessed median PFS was significantly longer in the nivolumab arm than in the placebo arm (12.1 versus 8.1 months; hazard ratio 0.56; 96.4% confidence interval 0.43-0.71; P < 0.0001). The PFS benefit was observed across all patients with any programmed death-ligand 1 (PD-L1) expression levels including PD-L1-negative patients. The IRRC-assessed objective response rates were 61.5% and 50.5% in the nivolumab and placebo arms, respectively. The incidence of treatment-related adverse events of grade 3 or 4 was comparable between the two arms; treatment-related adverse events leading to death were observed in five and four patients in the nivolumab and placebo arms, respectively. CONCLUSION: The TASUKI-52 regimen should be considered a viable new treatment strategy for treatment-naïve patients with advanced nonsquamous NSCLC.

LINC00887 aggravates the malignant progression of glioma via upregulating CCND1.

OBJECTIVE: This study aims to explore the impact of LINC00887 on the malignant progression of glioma via upregulating CCND1. PATIENTS AND METHODS: LINC00887 and CCND1 levels in glioma patients in different tumor grades or metastasis statuses were detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Kaplan-Meier curves were depicted for analyzing the prognostic potential of LINC00887 in glioma patients. Meanwhile, Pearson correlation test was conducted to assess the expression correlation between LINC00887 and CCND1 in glioma tissues. After knockdown of LINC00887 in LN229 and U251 cells, proliferative abilities were examined by cell counting kit-8 (CCK-8) and 5-Ethynyl-2'- deoxyuridine (EdU) assays. Subcellular distribution of LINC00887 was determined. Thereafter, RNA Binding Protein Immunoprecipitation (RIP) was performed to uncover the interaction between LINC00887 and CCND1. After α-amanitin induction in glioma cells overexpressing LINC00887, RNA degradation of CCND1 was examined at 0, 6, 12 and 24 h, respectively. Finally, the synergistic regulation of both LINC00887 and CCND1 on glioma proliferation was explored by CCK-8 assay. RESULTS: It was found that LINC00887 was upregulated in glioma tissues, especially in stage III+IV or metastatic glioma cases. Overall survival was remarkably worse in glioma patients expressing a high level of LINC00887 than those with a low level. CCND1 was upregulated in glioma tissues as well, showing a positive correlation to LINC00887. In addition, LINC00887 was mainly distributed in the cytoplasm and interacted with CCND1, and it shortened the half-life of CCND1. Moreover, the knockdown of LINC00887 inhibited glioma cell proliferation, and this inhibitory effect was abolished by overexpression of CCND1. CONCLUSIONS: LINC00887 is upregulated in glioma tissues, and it aggravates the malignant progression of glioma by upregulating CCND1.

The diagnosis of lung cancer in the era of interventional pulmonology.

Advances in bronchoscopic and other interventional pulmonology technologies have expanded the sampling procedures pulmonologist can use to diagnose lung cancer and accurately stage the mediastinum. Among the modalities available to the interventional pulmonologist are endobronchial ultrasound-guided transbronchial needles aspiration (EBUS-TBNA) and transoesophageal bronchoscopic ultrasound-guided fine-needle aspiration (EUS-B-FNA) for sampling peribronchial/perioesophageal central lesions and for mediastinal lymph node staging, as well as navigational bronchoscopy and radial probe endobronchial ultrasound (RP-EBUS) for the diagnosis of peripheral lung cancer. The role of the interventional pulmonologist in this setting is to apply these procedures based on the correct interpretation of clinical and radiological findings in order to maximise the chances of achieving the diagnosis and obtaining sufficient tissue for molecular biomarker testing to guide targeted therapies for advanced non-small cell lung cancer. The safest and the highest diagnosis-yielding modality should be chosen to avoid a repeat sampling procedure if the first one is non-diagnostic. The choice of site and biopsy modality are influenced by tumour location, patient comorbidities, availability of equipment and local expertise. This review provides a concise state-of-the art account of the interventional pulmonology procedures in the diagnosis and staging of lung cancer.

Impact of different subspecies on disease progression in initially untreated patients with Mycobacterium avium complex lung disease.

OBJECTIVE: Disease progression is a strong indicator of treatment for Mycobacterium avium complex lung disease (MAC-LD). The impact of MAC subspecies on the risk of disease progression remains uncertain in MAC-LD patients. METHODS: In this cohort study, we included MAC-LD patients from 2013 to 2018 and classified them into M. intracellulare, M. avium, M. chimaera and other subspecies groups by genotype. We observed the disease progression of MAC-LD, indicated by antibiotic initiation and/or radiographic progression. We used Cox regression analysis to assess predictors for disease progression. RESULTS: Of 105 MAC isolates from unique MAC-LD patients, 35 (33%) were M. intracellulare, 41 (39%) M. avium, 16 (15%) M. chimaera and 13 (12%) other subspecies. After a mean follow-up time of 1.3 years, 56 (53%) patients developed disease progression: 71% (25/35), 54% (22/41), 31% (4/13) and 31% (5/16) in patients with M. intracellulare, M. avium, others and M. chimaera, respectively. The independent predictors for disease progression were M. chimaera subspecies (HR 0.356, 95% CI (0.134-0.943)), compared with the reference group of M. intracellulare, body mass index ≤20 kg/m2 (HR 1.788 (1.022-3.130)) and initial fibrocavitary pattern (HR 2.840 (1.190-6.777)) after adjustment for age, sex and sputum smear positivity. Among patients without fibrocavitary lesions (n = 94), the risk of disease progression significantly decreased in patients with other subspecies (HR 0.217 (0.050-0.945)) and remained low in those with M. chimaera (HR 0.352 (0.131-0.947)). CONCLUSIONS: Mycobacterium chimaera was not uncommon in this study; unlike M. intracellulare, it was negatively correlated with disease progression of MAC-LD, suggesting a role of MAC subspecies identification in prioritizing patients.

[Research progress in therapeutic action of mesenchymal stem cell in allergic rhinitis].

Allergic rhinitis (AR) is an allergic disease of nasal mucosa, currently the consensus has been reached that there is a Th1/Th2 imbalance in AR's pathogenesis, meanwhile there are defects in the number and/or function of Treg cells. Mesenchymal stem cells (MSCs) have characteristics of multiple differentiation potential, low immunogenicity and immunomodulation function, which can be migrated and implanted into the nasal inflammatory mucosal, recovering the Th1/Th2 immune balance and up-regulate the Treg cells by immunomodulatory function to improve AR. For the present, the treatment of AR by MSCs is basically at the animal experiment stage. This paper reviews the progress of MSCs in AR's treatment and provides a basis for the clinical treatment of MSCs.

[Clinical significance of continuous transfixion suture for nasal septum in septoplasty].

Objective:To systemically evaluate the clinical significance of continuous transfixion suture for nasal septum in septoplasty. Method:Forty patients with nasal septum deviation were randomly assigned to the suture group including 20 patients who underwent endoscopic septoplasty followed by continuous transfixion suture for nasal septum, or the nasal packing group including 20 patients who underwent endoscopic septoplasty followed by nasal packing. Two groups were compared for the visual analogue scale (VAS) scores for postoperative rhinalgia, headache, lacrimation, dysphagia and sleep disorder, changes in mucociliary transport time (MTT) before and after surgery, and postoperative capillary hemorrhage. Patients were followed up for 2 weeks to observe the short-term postoperative complications. Result:Mean VAS scores for rhinalgia, headache, lacrimation, dysphagia and sleep disorder were all higher in nasal packing group than those in suture group (P<0.05); compared to suture group, there was greater prolongation of MTT before and after surgery in packing group (P<0.05); there was significant difference between two groups in postoperative capillary hemorrhage volume (P<0.05); in the nasal packing group, nasal synechia, nasal dryness and hyposmia were observed in 1, 3 and 2 patients, respectively, within 2 weeks postoperatively, whereas no short-term complications were observed in the suture group. Conclusion:Use of continuous transfixion suture in place of nasal packing following septoplasty can significantly improve the postoperative symptoms, protect nasal mucociliary clearance, and reduce short-term postoperative complications.

[Value discussion of radical sinus surgery for difficult-to-treat rhinosinusitis].

Objective:To investigate the efficacy of radical sinus surgery (RSS) on difficult-to-treat rhinosinusitis(DTRS) with nasal polyps. Method: We retrospectively analyzed the clinical data of 26 patients with DTRS that treated with RSS and patients who were not treated with RSS in our hospital from August 2013 to June 2017.The effect of RSS were evaluated by comparing the CT Lund-Kennery score,endoscope Lund-Kennery score,global VAS score and dysosmia VAS score before operation and 6 months after operation. Result: RSS group had more severe Lund-Kennery score,global VAS score and dysosmia VAS score than non-RSS group,and also had more previous surgeries,higher ratio of olfactory region polyps,and higher ratio of co-existing asthma and allergic rhinitis. However RSS group had a lower ratio with pus anot than non-RSS group. The eosinophil count in periheral blood between two groups had no statistical significance. Six months after RSS,the score of endoscope Lund-Kennery,global VAS and dysosmia VAS dependence. 14 patients were successfully cured(53.8%), 12 patients showed improvement(46.2%), no invalid cases. Conclusion: The global symptoms and olfaction of DTRS patients can be improved by RSS combine individual perioperative drug therapy.

The Factor Inhibiting HIF Asparaginyl Hydroxylase Regulates Oxidative Metabolism and Accelerates Metabolic Adaptation to Hypoxia.

Animals require an immediate response to oxygen availability to allow rapid shifts between oxidative and glycolytic metabolism. These metabolic shifts are highly regulated by the HIF transcription factor. The factor inhibiting HIF (FIH) is an asparaginyl hydroxylase that controls HIF transcriptional activity in an oxygen-dependent manner. We show here that FIH loss increases oxidative metabolism, while also increasing glycolytic capacity, and that this gives rise to an increase in oxygen consumption. We further show that the loss of FIH acts to accelerate the cellular metabolic response to hypoxia. Skeletal muscle expresses 50-fold higher levels of FIH than other tissues: we analyzed skeletal muscle FIH mutants and found a decreased metabolic efficiency, correlated with an increased oxidative rate and an increased rate of hypoxic response. We find that FIH, through its regulation of oxidation, acts in concert with the PHD/vHL pathway to accelerate HIF-mediated metabolic responses to hypoxia.

Large-scale screening and characterisation of Lemna aequinoctialis and Spirodela polyrhiza strains for starch production.

Duckweed is considered a promising feedstock for bioethanol production due to its high biomass and starch production. Selection of duckweed strains with high starch accumulation is essential for application of duckweeds to bioethanol production. Geographic differentiation had a large influence on genetic diversity of duckweeds. Biomass production, starch content and starch amount in geographically isolated strains of 20 Lemna aequinoctialis and Spirodela polyrhiza were calculated to evaluate their potential for bioethanol production. The influence of different collection time, culture medium and NaCl concentration on starch accumulation of the best strains were analysed. The results showed that biomass production, starch content and starch production of duckweeds demonstrated clonal dependency. The best strain was L. aequinoctialis 6000, with biomass production of 15.38 ± 1.47 g m-2 , starch content of 28.68 ± 1.10% and starch production of 4.39 ± 0.25 g m-2 . Furthermore, starch content of L. aequinoctialis 6000 was highest after 8 h of light, tap water was the best medium for starch induction, and NaCl did not induce starch accumulation. This study suggests duckweed biomass production and starch production demonstrate clonal dependency, indicating that extensive clonal comparisons will be required to identify the most suitable isolates for duckweed selective breeding for bioethanol.

[RNA interference targeting DNA-PKcs inhibits glioma cells malignancies and enhances temozolomide sensitivity].

Objective: To investigate the effect of DNA dependent protein kinase catalytic subunit (DNA-PKcs) on glioma proliferation, invasion and temozolomide sensitivity, and also to explore the potential mechanisms. Methods: Human glioma cell lines H4 and U87 were chosen to carry out RNA interference transfection, and then divided into negative control group (blank group) and siRNA group (test group). The knockdown efficacy of DNA-PKcs siRNA was tested by quantitative PCR and Western blot. The MTS assay and Transwell assay were used to investigate the effect of DNA-PKcs knockdown on glioma cell growth and invasion, respectively. We also used MTS assay to investigate the IC(50) value of temozolomide in negative control group and siRNA groups. Result: Compared with blank group, DNA-PKcs specific siRNA significantly downregulated both mRNA and protein level of DNA-PKcs. MTS assay results demonstrated that 72-hours proliferation of test group were only 52.48%, 54.70% (H4) and 52.98%, 50.45% (U87) of the blank group's counterpart. Transwell assay results showed that the invasiveness abilities of blank and test groups were 1.00±0.03, 0.41±0.05, 0.39±0.04 (H4) and 1.00±0.02, 0.28±0.04, 0.27±0.04 (U87). Moreover, knockdown of DNA-PKcs significantly decreased the temozolomide IC(50) value (H4: 249±27, 97±39, 88±35; U87: 485±41, 86±49, 73±38). Further we applied the Western blot to reveal the mechanism of inhibitory effect of DNA-PKcs knockdown on glioma malignancies and temozolomide sensitivity. We found that downregulation of DNA-PKcs reduced the activity of AKT signal and the expression of its downstream effectors, such as c-Myc, MMP9, and Survivin. Conclusion: RNA interference targeting DNA-PKcs could inhibit glioma malignancies and enhance temozolomide sensitivity. The inhibitory effect of DNA-PKcs knockdown on those biological activities were mainly through inhibition of AKT signal and its downstream effectors.

[Distribution and compensation mechanism of aberrations between anterior and posterior surface of the cornea in myopia and myopic astigmatism eyes].

Objective: To study the compensation mechanism of aberrations between anterior and posterior corneal surface, and to investigate the correlations between corneal aberrations and K values, intraocular pressure and central corneal thickness. Methods: One hundred and sixty-one subjects (300 eyes) with myopia and myopic astigmatism were recruited randomly. Corneal aberrations (anterior, posterior and total) in three different optical zones (2 mm, 4 mm, and 6 mm) centered on the corneal vertex were assessed with a corneal topographer (Sirius). We also calculated compensation factors (CFs) as a measure of the relative efficiency of the aberration compensation mechanism. Astigmatism was divided into corneal astigmatism and non-corneal astigmatism. Mann-Whitney U test was utilized for the comparison of different aberrations and CFs between the two astigmatism groups. Spearman correlation was applied to analyze the correlations between corneal aberrations and K values, intraocular pressure and central corneal thickness. Results: As the order of the aberrations elevated from the second to the seventh, the RMS values decreased significantly. The larger the optical zone, the greater the values of aberrations and the lower the percentage of the compensation mechanism among all the Zernike terms. At the same time, as the order of the aberrations increased, the predominance of the compensation mechanism was increasingly obvious. Slight compensation of spherical aberration (Z40) was observed in the peripheral. In the center of the analyzing zones (2 mm), compensation mechanism represented in coma (Z3±1), and it disappeared in the peripheral. We detected slight compensation of the corneal astigmatism (Z2±2) in the surrounding zone, although the compensation factors were closed to zero in diverse optical zones. Superposition was found in trefoil (Z3±3) straightly. Nevertheless, a tendency towards compensation was discovered with the enlargement of the optical zones. And the secondary spherical aberration (Z60) behaved compensation continuously. However, similar compensation was discovered between the right eye and left eye in different analyzing optical zones. There were significant correlations between the flattest K values and Z2±2, Z3±3and Z40 in the anterior, posterior and total cornea. It was similar with the steepest K values. When the optical zone expanded to 6 mm, Z3±1 and Z40 significantly decreased with intraocular pressure (rcoma=-0.188, P<0.05. rspherical=-0.147, P<0.05). No correlation was found between various aberrations and central corneal thickness (P>0.05). Conclusions: Compensation dominated in the corneal center, while the percentage decreased gradually as the optical zone extended. Slight compensation in astigmatism and spherical aberration between the anterior and posterior cornea may be benificial to the scotopic visual quality. Corneal aberrations were significantly related to K values and intraocular pressure. (Chin J Ophthalmol, 2016, 52: 840-849).