Transcription activator-like effector nucleases (TALENs) are a new class of engineered nucleases that are easier to design to cleave at desired sites in a genome than previous types of nucleases. We ...report here the use of TALENs to rapidly and efficiently generate mutant alleles of 15 genes in cultured somatic cells or human pluripotent stem cells, the latter for which we differentiated both the targeted lines and isogenic control lines into various metabolic cell types. We demonstrate cell-autonomous phenotypes directly linked to disease—dyslipidemia, insulin resistance, hypoglycemia, lipodystrophy, motor-neuron death, and hepatitis C infection. We found little evidence of TALEN off-target effects, but each clonal line nevertheless harbors a significant number of unique mutations. Given the speed and ease with which we were able to derive and characterize these cell lines, we anticipate TALEN-mediated genome editing of human cells becoming a mainstay for the investigation of human biology and disease.
► A system for efficient and rapid genome editing with TALENs ► Generation of isogenic human cellular models of disease ► Identification of disease-associated phenotypes in multiple human cell types ► Minimal TALEN off-target effects, but significant clone-to-clone sequence variation
Efficient TALEN genome editing of human somatic and pluripotent stem cells to model metabolic disease.
XTEN™ is a class of unstructured hydrophilic, biodegradable protein polymers designed to increase the half-lives of therapeutic peptides and proteins. XTEN polymers and XTEN fusion proteins are ...typically expressed in Escherichia coli and purified by conventional protein chromatography as monodisperse polypeptides of exact length and sequence. Unstructured XTEN polypeptides have hydrodynamic volumes significantly larger than typical globular proteins of similar mass, thus imparting a bulking effect to the therapeutic payloads attached to them. Since their invention, XTEN polypeptides have been utilized to extend the half-lives of a variety of peptide- and protein-based therapeutics. Multiple clinical and preclinical studies and related drug discovery and development efforts are in progress. This review details the most current understanding of physicochemical properties and biological behavior of XTEN and XTENylated molecules. Additionally, the development path and status of several advanced drug discovery and development efforts are highlighted.
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Abstract
Nearly 350 IgG-based therapeutics are approved for clinical use or are under development for many diseases lacking adequate treatment options. These include molecularly engineered ...biologicals comprising the IgG Fc-domain fused to various effector molecules (so-called Fc-fusion proteins) that confer the advantages of IgG, including binding to the neonatal Fc receptor (FcRn) to facilitate in vivo stability, and the therapeutic benefit of the specific effector functions. Advances in IgG structure-function relationships and an understanding of FcRn biology have provided therapeutic opportunities for previously unapproachable diseases. This article discusses approved Fc-fusion therapeutics, novel Fc-fusion proteins and FcRn-dependent delivery approaches in development, and how engineering of the FcRn-Fc interaction can generate longer-lasting and more effective therapeutics.
Periacetabular osteotomy (PAO) is the most common treatment for symptomatic acetabular dysplasia, or developmental dysplasia of the hip (DDH), in skeletally mature patients. The purpose of this ...multicenter cohort study was to delineate the long-term radiographic natural history of the dysplastic hip following PAO.
We evaluated all patients undergoing PAO from 1996 to 2012 at 3 academic institutions in the United States. Inclusion criteria were PAO for DDH with a minimum 5-year radiographic follow-up. Exclusion criteria were PAO for isolated acetabular retroversion, neurogenic dysplasia, Legg-Calvé-Perthes disease, and prior hip surgery including osteotomies and arthroscopy. There were 288 patients, 83% of whom were women; the mean age and body mass index (BMI) were 29 years and 25 kg/m, respectively. The mean clinical and radiographic follow-up was 9.2 years (range, 5.0 to 21.1 years). Every preoperative and postoperative hip radiograph was assessed to determine the degree of osteoarthritis according to the Tönnis classification. Survivorship was analyzed by multistate modeling, enabling assessment of progression through the Tönnis grades rather than just individual transitions as with Kaplan-Meier techniques.
At the time of final follow-up, 144 patients (50%) had progressed at least 1 Tönnis grade, with 42 patients (14.6%) undergoing total hip arthroplasty. The mean number of years spent in each Tönnis grade following PAO was 19 for Tönnis grade 1, 8 for Tönnis grade 2, and 4 for Tönnis grade 3. The probability of progression to total hip arthroplasty increased significantly on the basis of a higher initial Tönnis grade (p < 0.001). The most marked difference occurred between Tönnis grade 0 or 1 and Tönnis grade 2; for Tönnis grade 1, the probability of progression to total hip arthroplasty at 5 and 10 years was 2% and 11%, respectively, compared with 23% and 53%, respectively, for Tönnis grade 2.
PAO effectively alters the natural history of DDH. Precise radiographic progression based on the Tönnis grade can now be used to ascribe prognosis for the native hip. Importantly, this investigation demonstrates a stark increase in progression to total hip arthroplasty within 10 years of PAO for patients with preoperative Tönnis grade-2 osteoarthritis compared with those with Tönnis grade-0 or 1 osteoarthritis.
Therapeutic Level IV. See Instructions for Authors for a complete description of levels of evidence.
Energy has been called the “golden thread” that connects economic growth, social equity and environmental sustainability, but important knowledge gaps exist on the impacts of low- and middle-income ...country energy interventions and transitions. This study offers perhaps the broadest characterization to date of the patterns and consistency in quantitative and peer-reviewed social science literature considering such impacts. Starting from approximately 80,000 papers identified using a search procedure organized along energy services, technology, and impact dimensions, and structured to achieve breadth and replicability, articles were first screened to yield a relevant subset of 3,000 quantitative papers. Relevance is defined as providing one or more types of impacts on intra-household, household, firm, public service, national economy, or environmental outcomes. A set of heat maps highlights areas of concentration in the literature, namely work that emphasizes the negative health and pollution effects of traditional cooking and fossil fuel use. The extent and consistency of evidence for different types of impacts (in terms of direction and statistical significance) is also discussed, which reveals considerable heterogeneity and highlights important knowledge gaps that remain despite rapidly expanding energy scholarship. The patterns of evidence are also surprisingly consistent across methods. The article concludes by articulating several research challenges that should motivate current and future generations of energy and development scholars.
•Review covers impacts of energy, along services, technology, and impact dimensions.•About 80,000 articles were screened, and 3000 quantitative articles coded.•Heat maps highlight focus on traditional cooking, fossil fuels, and air pollution.•The direction/significance of many impacts is surprisingly consistent across methods.•Key research gaps: Understudied countries and policies, off-grid technology, productive use.
•Laparoscopic inguinal herniorrhaphy is associated with higher recurrence rates compared to open herniotomy.•Our laparoscopic herniotomy (LIH) cohort shows low recurrence (1%), despite risk ...factors.•Our data supports growing favourable evidence on laparoscopic sac division techniques.
Recent evidence suggests simple laparoscopic inguinal herniorrhaphy is associated with higher rates of recurrence and testicular ascent. We instigated a standardised approach to laparoscopic inguinal herniotomy (LIH), with circumferential sac division and ‘purse-string’ closure (4/0 monofilament polypropylene). An active follow-up programme was pursued. We reviewed our outcomes of this technique and compared them to an open herniotomy (OIH) cohort.
LIH patients were identified prospectively (2017–2021): OIH retrospectively from 2016. Risk factors for complications were defined: extremely to very preterm (< 32 weeks), emergency presentation with incarceration, and redo surgery for recurrence. Data are presented as median IQR. Comparisons used Fisher's exact and Mann-Whitney U tests: significance defined as p < 0.05.
192 inguinal herniae in 140 patients were included in the LIH group and 214 herniae in 179 patients in the OIH group. Groups were similar in age and gender. The LIH group had a significantly larger proportion of cases that were premature, had emergency surgery, or had redo surgery after previous OIH. Follow-up was 24.4 months 10.8–33.6 vs. 66.4 64.5–68.5 (LIH vs. OIH). Hernia recurrence occurred in 2/192 (1.0%) vs. 4/214 (1.9%) (LIH vs. OIH), p = 0.69. There was one known case of testicular ascent after OIH but none in the LIH group.
Recreation of the open herniotomy laparoscopically appears to confer excellent outcomes, with low rates of recurrence despite a high proportion of patients having known risk factors. Further long-term data on rates of testicular ascent after active follow-up are required.
Periacetabular osteotomy (PAO) remains the gold-standard treatment for acetabular dysplasia in skeletally mature patients with preserved cartilage. The purpose of this multicenter cohort study was to ...delineate the long-term radiographic natural history of the dysplastic hip following PAO based on the final position of the acetabular fragment.
We evaluated patients who underwent PAO performed by 4 hip preservation surgeons to treat acetabular dysplasia with or without concomitant retroversion from 1996 to 2012 at 3 academic institutions. There were 288 patients with a mean clinical and radiographic follow-up of 9 years (range, 5 to 21 years). Postoperative radiographs made at the first clinical visit were used to determine if the acetabular fragment fell into a safe zone according to the absence of retroversion, a lateral center-edge angle (LCEA) of 25° to 40°, an anterior center-edge angle (ACEA) of 25° to 40°, and a Tönnis angle of 0° to 10°. Every available subsequent radiograph was assessed for degenerative changes by the Tönnis classification (grades 0 to 3). The time to progression was analyzed using Cox proportional hazards regression and multistate modeling.
Only the absence of retroversion was independently associated with a decreased risk of progressing at least 1 Tönnis grade during follow-up: hazard ratio (HR), 0.60 (95% confidence interval CI, 0.38 to 0.94; p = 0.025). Achieving the ACEA safe zone yielded the greatest time increase for remaining in Tönnis grade 0 or 1 (43 years for having an ACEA in the safe zone compared with 28 years for not having an ACEA in the safe zone), followed by the absence of retroversion (34 years for the absence of retroversion compared with 24 years for the presence of retroversion). However, attaining the Tönnis angle or LCEA safe zones did not delay progression. The achievement of additional safe zones generally increased the length of time that patients spent in Tönnis grade 0 or 1: 25 years for 0 safe zones, 36 years for 1 safe zone, 29 years for 2 safe zones, 37 years for 3 safe zones, and 44 years for 4 safe zones.
This study demonstrates the importance of achieving appropriate acetabular reorientation to enhance the longevity of the native hip following PAO. Although the LCEA and the Tönnis angle are the most common metrics used to assess appropriate acetabular correction, this study shows that adequately addressing retroversion and the ACEA has a greater impact on improving the natural history.
Therapeutic Level IV. See Instructions for Authors for a complete description of levels of evidence.
Abstract
Invasive fungal infections are a leading global cause of human mortality. Only three major classes of antifungal drugs are widely used, and resistance to all three classes can arise rapidly. ...The most widely prescribed antifungal drug, fluconazole, disseminates rapidly and reaches a wide range of concentrations throughout the body. The impact of drug concentration on the spectrum and effect of mutations acquired during adaptation is not known for any fungal pathogen, and how the specific level of a given stress influences the distribution of beneficial mutations has been poorly explored in general. We evolved 144 lineages from three genetically distinct clinical isolates of Candida albicans to four concentrations of fluconazole (0, 1, 8, and 64 μg/ml) and performed comprehensive phenotypic and genomic comparisons of ancestral and evolved populations. Adaptation to different fluconazole concentrations resulted in distinct adaptive trajectories. In general, lineages evolved to drug concentrations close to their MIC50 (the level of drug that reduces growth by 50% in the ancestor) tended to rapidly evolve an increased MIC50 and acquired distinct segmental aneuploidies and copy number variations. By contrast, lineages evolved to drug concentrations above their ancestral MIC50 tended to acquire a different suite of mutational changes and increased in drug tolerance (the ability of a subpopulation of cells to grow slowly above their MIC50). This is the first evidence that different concentrations of drug can select for different genotypic and phenotypic outcomes in vitro and may explain observed in vivo drug response variation.
Association with the D′D3 domain of von Willebrand factor (VWF) stabilizes factor VIII (FVIII) in the circulation and maintains it at a level sufficient to prevent spontaneous bleeding. We used ...negative-stain electron microscopy (EM) to visualize complexes of FVIII with dimeric and monomeric forms of the D′D3 domain. The EM averages show that FVIII interacts with the D′D3 domain primarily through its C1 domain, with the C2 domain providing a secondary attachment site. Hydrogen-deuterium exchange mass spectrometry corroborated the importance of the C1 domain in D′D3 binding and implicates additional surface regions on FVIII in the interaction. Together, our results establish that the C1 domain is the major binding site on FVIII for VWF, reiterate the importance of the a3 acidic peptide in VWF binding, and suggest that the A3 and C2 domains play ancillary roles in this interaction.
•Electron microscopy and hydrogen-deuterium exchange establish the C1 domain as the major binding site for the VWF D′D3 domain on FVIII.•Additional sites implicated in the FVIII-VWF interaction are located within the a3 acidic peptide and the A3 and C2 domains of FVIII.
Purpose
The use of laparoscopy for paediatric inguinal hernia repairs has increased significantly over the past 2 decades. However, there is significant variation in the reported recurrence rates in ...the literature, with many studies reporting higher rates than the open operation. This may be explained by the range of different techniques currently included under the term laparoscopic inguinal hernia repair. The purpose of this study is to determine whether dividing the hernia sac before ligation improves surgical outcomes following a paediatric laparoscopic inguinal hernia repair compared to ligation alone.
Methods
A systematic review of the literature was performed following PRISMA guidelines of all studies reporting the outcomes following paediatric laparoscopic inguinal hernia repair where the technique was recorded as laparoscopic suture ligation alone (LS) or laparoscopic sac division and suture ligation (LSDS). Studies were assessed for risk of bias and exclusion criteria included reported follow-up of less than 6 months.
Results
A total of 8518 LS repairs and 6272 LSDS repairs were included in the final analysis. LSDS repair was associated with a significantly lower recurrence rate (odds ratio 0.51, 95% CI 0.36–0.71,
p
= 0.001). There was no significant difference in the rates of testicular ascent or atrophy.
Conclusion
Recreating the open operation by hernia sac division followed by suture ligation significantly reduces the risk of hernia recurrence.
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