Human MuStem cells are competent to fuse with nonhuman primate myofibers in a clinically relevant transplantation context: A proof-of-concept study.

We previously reported that human muscle-derived stem cells (hMuStem cells) contribute to tissue repair after local administration into injured skeletal muscle or infarcted heart in immunodeficient rodent models. However, extrapolation of these findings to a clinical context is problematic owing to the considerable differences often seen between in vivo findings in humans versus rodents. Therefore, we investigated whether the muscle regenerative behavior of hMuStem cells is maintained in a clinically relevant transplantation context. Human MuStem cells were intramuscularly administered by high-density microinjection matrices into nonhuman primates receiving tacrolimus-based immunosuppression thereby reproducing the protocol that has so far produced the best results in clinical trials of cell therapy in myopathies. Four and 9 weeks after administration, histological analysis of cell injection sites revealed large numbers of hMuStem cell-derived nuclei in all cases. Most graft-derived nuclei were distributed in small myofiber groups in which no signs of a specific immune response were observed. Importantly, hMuStem cells contributed to simian tissue repair by fusing mainly with host myofibers, demonstrating their capacity for myofiber regeneration in this model. Together, these findings obtained in a valid preclinical model provide new insights supporting the potential of hMuStem cells in future cell therapies for muscle diseases.

Totally minimally invasive laparoscopic robot-assisted Ivor Lewis esophagectomy: improved technique and outcomes over 200 cases.

Background: Surgical resection of esophageal and gastroesophageal junction cancers is a very complex procedure with step learning curve. New technologies have made minimally invasive surgery possible, but challenges still remain for wide spread adoption of these techniques. This article aims to describe the outcomes and salient technical points of a totally minimally invasive, laparoscopic, robot-assisted Ivor Lewis esophagectomy (LRAMIE). Methods: Retrospective observational cohort study performed at a specialty cancer center using a prospectively maintained institutional database. Patients undergoing LRAMIE (laparoscopic abdomen, robotic chest) from 2014-2023 were included. Patients undergoing transhiatal and three-field esophagectomy were excluded. Operative and postoperative outcomes were compared over the study period to identify potential associations between outcomes over time. Results: Two-hundred patients were identified who underwent LRAMIE. Median age was 65 years and most were male (87.5%). The open conversion rate was 1% (n=2), which occurred within the first 30 cases. Operative time and blood loss were improved at the 60-case mark (P<0.001). Anastomotic stricture rate improved after 50 cases, and leak rate improved after 80 cases. Postoperative length of stay improved at both 50 and 100 cases with a median LOS of 6 days after 100 cases. Rate of postoperative pneumonia, 30- and 90-day mortality were reduced after 100 cases, although not statistically significant for mortality due to too few events. Conclusions: Totally minimally invasive Ivor Lewis esophagectomy at a high-volume center is a safe procedure. Operative outcomes improved significantly after 50-80 cases, followed by improvement in anastomotic results and postoperative outcomes, with corresponding excellent oncologic outcomes.

Reconstruction of Remodeling units reveals positive effects after 2 and 12 months of Romosozumab treatment.

Romosozumab treatment results in a transient early increase in bone formation and sustained decrease in bone resorption. Histomorphometric analyses revealed that the primary bone-forming effect of romosozumab is transient early stimulation of modeling-based bone formation on cancellous and endocortical surfaces; preclinical studies have demonstrated that romosozumab may affect changes in the remodeling unit resulting in positive bone balance. To further investigate the effects of romosozumab on bone balance, month 12 (M12) and M2 (to analyze early effects) unpaired bone biopsies from the FRAME clinical trial were analyzed using remodeling site reconstruction to assess whether positive changes in bone balance on cancellous/endocortical surfaces may contribute to the progressive improvement in bone mass/structure and reduced fracture risk in osteoporotic women at high fracture risk. At M12, bone balance was higher with romosozumab vs placebo on cancellous (+6.1 µm vs +1.5 µm; p = 0.012) and endocortical (+5.2 µm vs -1.7 µm; p = 0.02) surfaces; higher bone balance was due to lower final erosion depth (40.7 µm vs 43.7 µm; p = 0.05) on cancellous surfaces and higher completed wall thickness (50.8 µm vs 47.5 µm; p = 0.037) on endocortical surfaces. At M2, final erosion depth was lower on the endocortical surfaces (42.7 µm vs 50.7 µm; p = 0.021) and slightly lower on the cancellous surfaces (38.5 µm vs 44.6 µm; p = 0.11) with romosozumab vs placebo. Sector analysis of early endocortical formative sites revealed higher osteoid thickness (29.9 µm vs 19.2 µm; p = 0.005) and mineralized wall thickness (18.3 µm vs 11.9 µm; p = 0.004) with romosozumab vs placebo. These evolving bone packets may reflect early stimulation of bone formation that contributes to the increase in completed wall thickness at M12. These data suggest that romosozumab induces a positive bone balance due to its effects on bone resorption and formation at the level of the remodeling unit, contributing to the positive effects on bone mass, structure, and fracture risk.

Romosozumab treatment has a dual effect on bone, adding new bone and reducing bone loss. In the FRAME clinical trial, romosozumab increased bone mass and strength, and reduced fracture risk in postmenopausal women with osteoporosis. Addition of new bone occurs early in treatment and rapidly on cancellous and endocortical bone surfaces where bone resorption is not ongoing. However, it remains unclear if romosozumab affects bone loss or gain in areas where bone resorption is ongoing (remodeling units), contributing to a further positive bone balance. Here, we examined whether changes at the remodeling unit occur early (2 months) and/or late (12 months) in treatment, using bone biopsies from patients treated with romosozumab or placebo in FRAME. At month 2, a combination of lower bone resorption and higher bone gain on endocortical surfaces resulted in a positive bone balance with romosozumab versus placebo. At month 12, bone balance was positive with romosozumab versus placebo due to lower bone resorption on cancellous surfaces and greater bone gain on endocortical surfaces. This demonstrates that romosozumab induces a positive bone balance at remodeling units early in treatment leading to overall gains observed later, contributing to the positive effects of romosozumab on bone mass and structure.

Postfracture survival in a population-based study of adults aged ≥66 yr: a call to action at hospital discharge.

Postfracture survival rates provide prognostic information but are rarely reported along with other mortality outcomes in adults aged ≥50 yr. The timing of survival change following a fracture also needs to be further elucidated. This population-based, matched-cohort, retrospective database study examined 98 474 patients (73% women) aged ≥66 yr with an index fracture occurring at an osteoporotic site (hip, clinical vertebral, proximal non-hip non-vertebral [pNHNV], and distal non-hip non-vertebral [dNHNV]) from 2011 to 2015, who were matched (1:1) to nonfracture individuals based on sex, age, and comorbidities. All-cause 1- and 5-yr overall survival and relative survival ratios (RSRs) were assessed, and time trends in survival changes were characterized starting immediately after a fracture. In both sexes, overall survival was markedly decreased over 6 yr of follow-up after hip, vertebral, and pNHNV fractures, and as expected, worse survival rates were observed in older patients and males. The lowest 5-yr RSRs were observed after hip fractures in males (66-85 yr, 51.9%-63.9%; ≥86 yr, 34.5%), followed by vertebral fractures in males (66-85 yr, 53.2%-69.4%; ≥86 yr, 35.5%), and hip fractures in females (66-85 yr, 69.8%-79.0%; ≥86 yr, 52.8%). Although RSRs did not decrease as markedly after dNHNV fractures in younger patients, relatively low 5-yr RSRs were observed in females (75.9%) and males (69.5%) aged ≥86 yr. The greatest reduction in survival occurred within the initial month after hip, vertebral, and pNHNV fractures, indicating a high relative impact of short-term factors, with survival-reduction effects persisting over time. Therefore, the most critical period for implementing interventions aimed at improving post-fracture prognosis appears to be immediately after a fracture; however, considering the immediate need for introducing such interventions, primary fracture prevention is also crucial to prevent the occurrence of the initial fracture in high-risk patients.

Association between the Preoperative Standard Uptake Value (SUV) and Survival Outcomes after Robotic-Assisted Segmentectomy for Resectable Non-Small Cell Lung Cancer (NSCLC).

BACKGROUND: Lung-sparing procedures, specifically segmentectomies and wedge resections, have increased over the years to treat early-stage non-small cell lung cancer (NSCLC). We investigate here the perioperative and long-term outcomes of patients who underwent robotic-assisted segmentectomy (RAS) at an NCI-designated cancer center and aim to show associations between the preoperative standard update value (SUV) to tumor stage, recurrence patterns, and overall survival. METHODS: A retrospective analysis was performed on 166 consecutive patients who underwent RAS at a single institution from 2010 to 2021. Of this number, 121 robotic-assisted segmentectomies were performed for primary NSCLC, and a total of 101 patients were evaluated with a PET-CT scan. The SUV from the primary tumor was determined from the PET-CT. The clinical, surgical, and pathologic profiles and perioperative outcomes were summarized via descriptive statistics. Numerical variables were described as the median and interquartile range because all numerical variables were not normally distributed as assessed by the Shapiro-Wilk test of normality. Categorical variables were described as the count and proportion. Chi-square or Fisher's exact test was used for association. The main outcomes were overall survival (OS) and recurrence-free survival (RFS). Kaplan-Meier (KM) curves were constructed to visualize the OS and RFS, which were also stratified according to tumor histology, the pathologic stage, and standard uptake value. A log-rank test for the equality of survival curves was performed to determine significant differences between groups. RESULTS: The most common postoperative complications were atrial fibrillation (8.8%, 9/102), persistent air leak (7.84%, 8/102), and pneumonia (4.9%, 5/102). The median operative duration was 168.5 min (IQR 59), while the median estimated blood loss was 50 mL (IQR 125). The conversion rate to thoracotomy in this cohort was 3.9% (4/102). Intraoperative complications occurred in 2.9% (3/102). The median hospital length of stay was 3 days (IQR 3). The median chest tube duration was 3 days (IQR 2), but 4.9% (5/102) of patients were sent home with a chest tube. The recurrence for this cohort was 28.4% (29/102). The time to recurrence was 353 days (IQR 504), while the time to mortality was 505 days (IQR 761). The NSCLC patients were divided into the following two groups: low SUV (<5, n = 55) and high SUV (≥5, n = 47). Statistically significant associations were noted between SUV and the tumor histology (p = 0.019), tumor grade (p = 0.002), lymph-vascular invasion (p = 0.029), viscera-pleural invasion (p = 0.008), recurrence (p < 0.001) and the site of recurrence (p = 0.047). KM survival analysis showed significant differences in the curves for OS (log-rank p-value 0.0204) and RFS (log-rank p-value 0.0034) between the SUV groups. CONCLUSION: Robotic-assisted segmentectomy for NSCLC has reasonable perioperative and oncologic outcomes. Furthermore, we demonstrate here the prognostic implication of preoperative SUV to pathologic outcomes, recurrence-free survival, and overall survival.

Outcomes of robotic lobectomy for non-small cell lung cancer in a National Cancer Institute-Comprehensive Cancer Center vs. National Cancer Database.

Background: There continues to be a rise in the proportion of resectable non-small cell lung cancer (NSCLC) with the recent expansion of criteria for low-dose lung cancer screening. These are increasingly being treated with minimally invasive techniques. Our study aims to compare outcomes of robotic lobectomy (RL) for NSCLC at a National Cancer Institute-designated Comprehensive Cancer Center (NCI-CCC) to those of open lobectomy (OL), video-assisted thoracoscopic lobectomy (VL), or RL as reported in the National Cancer Database (NCDB). Methods: The first 1,021 patients with NSCLC who underwent RL between 2010 and 2020 were matched with peers from the NCDB who had OL, VL, or RL. Matching was performed based on a propensity score calculated by logistic regression using multiple variables. Surgical outcomes included numbers of examined lymph nodes, performance of mediastinal lymphadenectomy, length of stay (LOS), and 30-day mortality. Kaplan-Meier curves and overall survival (OS) were analyzed using log-rank tests. Results: Most common postoperative complications were persistent air leak, atrial fibrillation, and pneumonia. Median LOS was 4 days, and the 30-day mortality rate was 1% (n=10/1,021). Compared to NCDB patients who underwent OL, NCI-CCC patients had a higher mean number of retrieved lymph nodes (P=0.001), higher rate of mediastinal lymphadenectomy (P<0.001), and shorter median LOS (4 vs. 6 days; P<0.001). There was no difference in 30-day mortality (P=0.176). Kaplan-Meier analyses showed no differences in median OS (log-rank P=0.953) or 5-year OS (P=0.774). Compared to NCDB VL, NCI-CCC patients had a higher nodal yield (P<0.001), higher rates of mediastinal lymphadenectomy (P<0.001), and lower conversion rates (4.1% vs. 13.8%, P<0.001). There were no differences in 30-day mortality (P=0.379) or in median LOS (P=0.351). Kaplan-Meier analyses showed no differences in median OS (P=0.720) or 5-year OS (P=0.735). NCI-CCC patients were also matched with NCDB RL patients and had a higher nodal yield (P<0.001), higher rates of mediastinal lymphadenectomy (P<0.001), and lower conversion rates (4.1% vs. 9.5%; P <0.001). There were no differences in 30-day mortality (P=0.899) or in median LOS (P=0.252). Kaplan-Meier analyses showed no differences in median OS (P=0.484) or 5-year OS (P=0.524). Conclusions: RL for NSCLC performed in an NCI-CCC appears to have improved perioperative outcomes with comparable long-term OS compared to national benchmarks in OL and VL.

Proceedings of the 2023 Santa Fe Bone Symposium: Progress and Controversies in the Management of Patients with Skeletal Diseases.

The Santa Fe Bone Symposium (SFBS) held its 23rd annual event on August 5-6, 2023, in Santa Fe, New Mexico, USA. Attendees participated in-person and remotely, representing many states and countries. The program included plenary presentations, panel discussions, satellite symposia, a Project ECHO workshop, and a session on healthcare policy and reimbursement for fracture liaison programs. A broad range of topics were addressed, including transitions of osteoporosis treatments over a lifetime; controversies in vitamin D; update on Official Positions of the International Society for Clinical Densitometry; spine surgery and bone health; clinical applications of bone turnover markers; basic bone biology for clinicians; premenopausal-, pregnancy-, and lactation-associated osteoporosis; cancer treatment induced bone loss in patients with breast cancer and prostate cancer; genetic testing for skeletal diseases; and an update on nutrition and bone health. There were also sessions on rare bone diseases, including managing patients with hypophosphatasia; treatment of X-linked hypophosphatemia; and assessment and treatment of patients with hypoparathyroidism. There were oral presentations of abstracts by endocrinology fellows selected from those who participated in the Santa Fe Fellows Workshop on Metabolic Bone Diseases, held the 2 days prior to the SFBS. These proceedings of the 2023 SFBS present the clinical highlights and insights generated from many formal and informal discussions in Santa Fe.

CRISPR-Cas9 delivery strategies with engineered extracellular vesicles.

Therapeutic genome editing has the potential to cure diseases by directly correcting genetic mutations in tissues and cells. Recent progress in the CRISPR-Cas9 systems has led to breakthroughs in gene editing tools because of its high orthogonality, versatility, and efficiency. However, its safe and effective administration to target organs in patients is a major hurdle. Extracellular vesicles (EVs) are endogenous membranous particles secreted spontaneously by all cells. They are key actors in cell-to-cell communication, allowing the exchange of select molecules such as proteins, lipids, and RNAs to induce functional changes in the recipient cells. Recently, EVs have displayed their potential for trafficking the CRISPR-Cas9 system during or after their formation. In this review, we highlight recent developments in EV loading, surface functionalization, and strategies for increasing the efficiency of delivering CRISPR-Cas9 to tissues, organs, and cells for eventual use in gene therapies.

Induction Fluorouracil-Based Chemotherapy and PET-Adapted Consolidation Chemoradiation with Esophagectomy for High-Risk Gastroesophageal Adenocarcinoma.

Background: Neoadjuvant chemoradiation with esophagectomy is standard management for locally advanced esophageal adenocarcinoma. Induction chemotherapy with a tailored approach to chemoradiation based on metabolic response to therapy on PET was explored as an alternative strategy in the CALGB 80803 trial. We sought to describe real-world institutional experience implementing this approach outside of a clinical trial. Methods: Patients who were treated with induction fluorouracil-leucovorin-oxaliplatin (FOLFOX) or fluorouracil-leucovorin-oxaliplatin-docetaxel (FLOT) with tailored chemoradiation based on PET response and subsequent esophagectomy were identified from a prospectively maintained database. Primary outcomes were pathologic complete response (pCR) and overall survival (OS) following completion of all therapy. Results: There were 35 patients who completed induction chemotherapy, chemoradiation, and esophagectomy. Thirty-three completed restaging PET following induction chemotherapy with metabolic response seen in 76% (n = 25/33). The pCR rate was 31% (n = 11/35) and the ypN0 rate was 71% (n = 25/35). Among the patients who demonstrated metabolic response to induction FOLFOX/FLOT and subsequently continued fluorouracil-based chemoradiation, the pCR rate was 39% (n = 9/23). The rate of pathologically negative lymph nodes in this group was high (n = 19/23, 83%) with 100% R0 resection rate (n = 23/23). With the median follow-up of 43 months, the median OS was not reached for this group and was significantly longer than the OS for the remainder of the cohort (p = 0.027, p = 0.046 adjusted for clinical stage). Conclusions: Induction FOLFOX/FLOT chemotherapy with evaluation of sensitivity via metabolic response and tailored chemoradiation seems to lead to high pCR and ypN0 rates in high-risk patients with adenocarcinoma of the esophagus and GE junction. This approach in clinical practice seems to recapitulate encouraging results in clinical trials.

Prevalence and incidence of Paget's disease of bone: Temporal trend over 20 years in the province of Quebec, Canada.

BACKGROUND: Paget's disease of bone (PDB) is a focal bone disorder characterized by an increased bone remodeling and an anarchic bone structure. A decline of prevalence and incidence of PDB has been observed in some countries. No epidemiological data are available on PDB in Canada. AIMS: We aimed at examining the evolution of the prevalence and incidence of PDB in Quebec (Canada) by analyzing health administrative databases. METHODS: PDB case definition relied on one or more hospitalizations, or one or more physician-billing claims with a diagnosis code of PDB. To identify incident cases, a 'run-in' period of four years (1996-1999) was used to exclude prevalent cases. For each fiscal year from 2000 to 2001 to 2019-2020 (population size 2,914,480), crude age and sex-specific prevalence and incidence rates of PDB among individuals aged ≥55 years were determined, and sex-specific rates were also standardized to the 2011 age structure of the Quebec population. Generalized linear regressions were used to test for linear changes in standardized prevalence and incidence rates. RESULTS: Over the study period, standardized prevalence of PDB has remained stable in Quebec, from 0.44 % in 2000/2001 to 0.43 % in 2019/2020 (mean change -0.002, p-value = 0.0935). For the 2019-2020 fiscal year, 13,165 men and women had been diagnosed with PDB and prevalence of PDB increased with age. Standardized incidence of PDB has decreased over time from 0.77/1000 in 2000/2001 to 0.28/1000 in 2019-2020 (mean change -0.228/year, p-value<0.0001), the incidence decreasing from 0.82/1000 to 0.37/1000 in men and from 0.76/1000 to 0.22/1000 in women, respectively. This decrease was observed in all age categories. CONCLUSION: With the exception of a slight increase in PDB prevalence up to 0.55 % in years 2005 to 2007, the prevalence of PDB has remained stable in Quebec over the past 20 years, 13,160 men and women being currently diagnosed with PDB. The incidence has decreased over time. Our results support the epidemiological changes of PDB reported in other countries.

Portrait of Dysferlinopathy: Diagnosis and Development of Therapy.

Dysferlinopathy is a disease caused by a dysferlin deficiency due to mutations in the DYSF gene. Dysferlin is a membrane protein in the sarcolemma and is involved in different functions, such as membrane repair and vesicle fusion, T-tubule development and maintenance, Ca2+ signalling, and the regulation of various molecules. Miyoshi Myopathy type 1 (MMD1) and Limb-Girdle Muscular Dystrophy 2B/R2 (LGMD2B/LGMDR2) are two possible clinical presentations, yet the same mutations can cause both presentations in the same family. They are therefore grouped under the name dysferlinopathy. Onset is typically during the teenage years or young adulthood and is characterized by a loss of Achilles tendon reflexes and difficulty in standing on tiptoes or climbing stairs, followed by a slow progressive loss of strength in limb muscles. The MRI pattern of patient muscles and their biopsies show various fibre sizes, necrotic and regenerative fibres, and fat and connective tissue accumulation. Recent tools were developed for diagnosis and research, especially to evaluate the evolution of the patient condition and to prevent misdiagnosis caused by similarities with polymyositis and Charcot-Marie-Tooth disease. The specific characteristic of dysferlinopathy is dysferlin deficiency. Recently, mouse models with patient mutations were developed to study genetic approaches to treat dysferlinopathy. The research fields for dysferlinopathy therapy include symptomatic treatments, as well as antisense-mediated exon skipping, myoblast transplantation, and gene editing.

Finding an Appropriate Mouse Model to Study the Impact of a Treatment for Friedreich Ataxia on the Behavioral Phenotype.

Friedreich ataxia (FRDA) is a progressive neurodegenerative disease caused by a GAA repeat in the intron 1 of the frataxin gene (FXN) leading to a lower expression of the frataxin protein. The YG8sR mice are Knock-Out (KO) for their murine frataxin gene but contain a human frataxin transgene derived from an FRDA patient with 300 GAA repeats. These mice are used as a FRDA model but even with a low frataxin concentration, their phenotype is mild. We aimed to find an optimized mouse model with a phenotype comparable to the human patients to study the impact of therapy on the phenotype. We compared two mouse models: the YG8sR injected with an AAV. PHP.B coding for a shRNA targeting the human frataxin gene and the YG8-800, a new mouse model with a human transgene containing 800 GAA repeats. Both mouse models were compared to Y47R mice containing nine GAA repeats that were considered healthy mice. Behavior tests (parallel rod floor apparatus, hanging test, inverted T beam, and notched beam test) were carried out from 2 to 11 months and significant differences were noticed for both YG8sR mice injected with an anti-FXN shRNA and the YG8-800 mice compared to healthy mice. In conclusion, YG8sR mice have a slight phenotype, and injecting them with an AAV-PHP.B expressing an shRNA targeting frataxin does increase their phenotype. The YG8-800 mice have a phenotype comparable to the human ataxic phenotype.

Integrative-interpersonal dynamic therapy for poststroke depression (INID): study protocol of a randomised controlled pilot trial.

INTRODUCTION: Depression is the most frequent psychiatric disorder following stroke, affecting about one-third of stroke survivors. Patients experience poorer recovery, lower quality of life and higher mortality compared with stroke survivors without depression. Despite these well-known malign consequences, poststroke depression (PSD) is regarded underdiagnosed and undertreated. Evidence of beneficial effects of psychotherapy to treat PSD remains scarce and inconclusive and is limited by heterogeneity in design, content and timing of the intervention. This pilot study aims to assess the feasibility of a newly developed integrative-interpersonal dynamic PSD intervention in an outpatient setting and provide a first estimation of the potential effect size as basis for the sample size estimation for a subsequent definite trial. METHOD AND ANALYSIS: Patients will be recruited from two German stroke units. After discharge from inpatient rehabilitation, depressed stroke survivors will be randomised to short-term psychotherapy (12 weeks, ≤16 sessions) or enhanced treatment as usual. The manualised psychotherapy integrates key features of the Unified Psychodynamic and Cognitive-Behavioural Unified Protocol for emotional disorders and was adapted for PSD. Primary endpoints are recruitment feasibility and treatment acceptability, defined as a recruitment rate of ≥20% for eligible patients consenting to randomisation and ≥70% completion-rate of patients participating in the treatment condition. A preliminary estimation of the treatment effect based on the mean difference in Patient Health Questionnaire-9 (PHQ-9) scores between intervention and control group six months poststroke is calculated. Secondary endpoints include changes in depression (PHQ-9/Hamilton Depression Scale) and anxiety (Generalised Anxiety Disorder 7) of all participants across all follow-ups during the first year poststroke. ETHICS AND DISSEMINATION: The INID pilot study received full ethical approval (S-321/2019; 2022-2286_1). Trial results will be published in a peer-reviewed journal in the first half of 2025. One-year follow-ups are planned to be carried out until summer 2025. TRIAL REGISTRATION NUMBER: DRKS00030378.

Gene therapy for Duchenne muscular dystrophy: an update on the latest clinical developments.

INTRODUCTION: Duchenne muscular dystrophy (DMD) is one of the most severe and devastating neuromuscular hereditary diseases with a male newborn incidence of 20 000 cases each year. The disease caused by mutations (exon deletions, nonsense mutations, intra-exonic insertions or deletions, exon duplications, splice site defects, and deep intronic mutations) in the DMD gene, progressively leads to muscle wasting and loss of ambulation. This situation is painful for both patients and their families, calling for an emergent need for effective treatments. AREAS COVERED: In this review, the authors describe the state of the gene therapy approach in clinical trials for DMD. This therapeutics included gene replacement, gene substitution, RNA-based therapeutics, readthrough mutation, and the CRISPR approach. EXPERT OPINION: Only a few drug candidates have yet been granted conditional approval for the treatment of DMD. Most of these therapies have only a modest capability to restore the dystrophin or improve muscle function, suggesting an important unmet need in the development of DMD therapeutics. Complementary genes and cellular therapeutics need to be explored to both restore dystrophin, improve muscle function, and efficiently reconstitute the muscle fibers in the advanced stage of the disease.

Limb-Girdle Muscular Dystrophies Classification and Therapies.

Limb-girdle muscular dystrophies (LGMDs) are caused by mutations in multiple genes. This review article presents 39 genes associated with LGMDs. Some forms are inherited in a dominant fashion, while for others this occurs recessively. The classification of LGMDs has evolved through time. Lately, to be considered an LGMD, the mutation has to cause a predominant proximal muscle weakness and must be found in two or more unrelated families. This article also presents therapies for LGMDs, examining both available treatments and those in development. For now, only symptomatic treatments are available for patients. The goal is now to solve the problem at the root of LGMDs instead of treating each symptom individually. In the last decade, multiple other potential treatments were developed and studied, such as stem-cell transplantation, exon skipping, gene delivery, RNAi, and gene editing.

Rapid and Technically Simple Detection of SARS-CoV-2 Variants Using CRISPR Cas12 and Cas13.

The worldwide proliferation of the SARS-CoV-2 virus in the past 3 years has allowed the virus to accumulate numerous mutations. Dangerous lineages have emerged one after another, each leading to a new wave of the pandemic. In this study, we have developed the THRASOS pipeline to rapidly discover lineage-specific mutation signatures and thus advise the development of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based diagnostic tests. We also optimized a strategy to modify loop-mediated isothermal amplification amplicons for downstream use with Cas12 and Cas13 for future multiplexing. The close ancestry of the BA.1 and BA.2 variants of SARS-CoV-2 (Omicron) made these excellent candidates for the development of a first test using this workflow. With a quick turnaround time and low requirements for laboratory equipment, the test we have created is ideally suited for settings such as mobile clinics lacking equipment such as Next-Generation Sequencers or Sanger Sequencers and the personnel to run these devices.

Prime editing strategies to mediate exon skipping in DMD gene.

Duchenne muscular dystrophy is a rare and lethal hereditary disease responsible for progressive muscle wasting due to mutations in the DMD gene. We used the CRISPR-Cas9 Prime editing technology to develop different strategies to correct frameshift mutations in DMD gene carrying the deletion of exon 52 or exons 45 to 52. With optimized epegRNAs, we were able to induce the specific substitution of the GT nucleotides of the splice donor site of exon 53 in up to 32% of HEK293T cells and 28% of patient myoblasts. We also achieved up to 44% and 29% deletion of the G nucleotide of the GT splice site of exon 53, as well as inserted 17% and 5.5% GGG between the GT splice donor site of exon 51 in HEK293T cells and human myoblasts, respectively. The modification of the splice donor site for exon 51 and exon 53 provoke their skipping and allowed exon 50 to connect to exon 53 and allowed exon 44 to connect to exon 54, respectively. These corrections restored the expression of dystrophin as demonstrated by western blot. Thus, Prime editing was used to induce specific substitutions, insertions and deletions in the splice donor sites for exons 51 and 53 to correct the frameshift mutations in DMD gene carrying deletions of exon 52 and exons 45 to 52, respectively.

Changes in Perioperative Outcomes after Robotic-Assisted Pulmonary Lobectomy during the COVID-19 Era.

Background: The COVID-19 pandemic presented patients with barriers to receiving healthcare. We sought to determine whether changes in healthcare access and practice during the pandemic affected perioperative outcomes after robotic-assisted pulmonary lobectomy (RAPL). Methods: We retrospectively analyzed 721 consecutive patients who underwent RAPL. With March 1st, 2020, defining the start of the COVID-19 pandemic, we grouped 638 patients as "PreCOVID-19" and 83 patients as "COVID-19-Era" based on surgical date. Demographics, comorbidities, tumor characteristics, intraoperative complications, morbidity, and mortality were analyzed. Variables were compared utilizing Student's t-test, Wilcoxon rank-sum test, and Chi-square (or Fisher's exact) test, with significance at p ≤ 0.05 . Multivariable generalized linear regression was used to investigate predictors of postoperative complication. Results: COVID-19-Era patients had significantly higher preoperative FEV1%, lower cumulative smoking history and higher incidences of preoperative atrial fibrillation, peripheral vascular disease (PVD), and bleeding disorders compared to PreCOVID-19 patients. COVID-19-Era patients had lower intraoperative estimated blood loss (EBL), reduced incidence of new-onset postoperative atrial fibrillation (POAF), but higher incidence of effusion or empyema postoperatively. Overall postoperative complication rates between the groups were similar. Older age, increased EBL, lower preoperative FEV1%, and preoperative COPD are all predictive of an increased risk for postoperative complication. Conclusions: COVID-19-Era patients having lower EBL and less new-onset POAF, despite greater incidences of multiple preoperative comorbidities, demonstrates that RAPL is safe during the COVID-19 era. Risk factors for development of postoperative effusion should be determined to minimize risk of empyema in COVID-19-Era patients. Age, preoperative FEV1%, COPD, and EBL should all be considered when planning for complication risk.

Precisely timed regulation of enhancer activity defines the binary expression pattern of Fushi tarazu in the Drosophila embryo.

The genes that drive development each typically have many different enhancers. Properly coordinating the action of these different enhancers is crucial to correctly specifying cell-fate decisions, yet it remains poorly understood how their activity is choregraphed in time. To shed light on this question, we used recently developed single-cell live imaging tools to dissect the regulation of Fushi tarazu (Ftz) in Drosophila melanogaster embryos. Ftz is a transcription factor that is expressed in asymmetric stripes by two distinct enhancers: autoregulatory and zebra. The anterior edge of each stripe needs to be sharply defined to specify essential lineage boundaries. Here, we tracked how boundary cells commit to either a high-Ftz or low-Ftz fate by measuring Ftz protein traces in real time and simultaneously quantifying transcription from the endogenous locus and individual enhancers. This revealed that the autoregulatory enhancer does not establish this fate choice. Instead, it perpetuates the decision defined by zebra. This is contrary to the prevailing view that autoregulation drives the fate decision by causing bi-stable Ftz expression. Furthermore, we showed that the autoregulatory enhancer is not activated based on a Ftz-concentration threshold but through a timing-based mechanism. We hypothesize that this is regulated by several ubiquitously expressed pioneer-like transcription factors, which have recently been shown to act as timers in the embryo. Our work provides new insight into how precisely timed enhancer activity can directly regulate the dynamics of gene regulatory networks, which may be a general mechanism for ensuring that embryogenesis runs like clockwork.