Precision genome editing using synthesis-dependent repair of Cas9-induced DNA breaks.

The RNA-guided DNA endonuclease Cas9 has emerged as a powerful tool for genome engineering. Cas9 creates targeted double-stranded breaks (DSBs) in the genome. Knockin of specific mutations (precision genome editing) requires homology-directed repair (HDR) of the DSB by synthetic donor DNAs containing the desired edits, but HDR has been reported to be variably efficient. Here, we report that linear DNAs (single and double stranded) engage in a high-efficiency HDR mechanism that requires only ∼35 nucleotides of homology with the targeted locus to introduce edits ranging from 1 to 1,000 nucleotides. We demonstrate the utility of linear donors by introducing fluorescent protein tags in human cells and mouse embryos using PCR fragments. We find that repair is local, polarity sensitive, and prone to template switching, characteristics that are consistent with gene conversion by synthesis-dependent strand annealing. Our findings enable rational design of synthetic donor DNAs for efficient genome editing.

Atypical Mycobacterial Infections After Plastic Surgery Procedures Abroad: A Multidisciplinary Algorithm for Diagnosis and Treatment.

BACKGROUND: The recent rise in medical tourism, especially for cosmetic procedures, has been mirrored by an increase in the incidence of infections with Mycobacterium abscessus, which is an atypical mycobacterium that is ubiquitous in aquatic environments. M. abscessus soft tissue infections arise from the use of improperly sterilized water and surgical equipment during surgical procedures, and these infections have devastating consequences if not promptly treated. M. abscessus infections are notoriously difficult to diagnose and properly treat, and therefore, we illustrate a typical case presentation and provide a comprehensive diagnostic and treatment algorithm. METHODS: Of the patients who have presented to our hospital for treatment of cutaneous M. abscessus infections, a representative patient's story was included to illustrate the typical presentation and treatment timeline. The current literature on M. abscessus infections was reviewed, and this literature and the clinical experience of our plastic surgery and infectious disease teams were used in the creation of a diagnostic and treatment algorithm for M. abscessus infections. RESULTS: M. abscessus infections can have an incubation period of months, and the classic presenting signs include purulent drainage, violaceous nodules, and subcutaneous abscesses at the site of a recent surgery. A key finding is persistence of the infection despite debridement and empiric antibiotic treatment. Cultures grown on mycobacterial-specific growth media are considered the diagnostic criterion standard, but high clinical suspicion is enough to warrant the initiation of treatment. Treatment itself consists of surgical drainage and debridement in combination with multidrug antibiotic regimens that typically include amikacin, a macrolide, and a carbapenem or cephalosporin antibiotic, with the option for macrolide and fluoroquinolone maintenance therapy. CONCLUSIONS: M. abscessus cutaneous infections present with unique history and physical examination findings and often require complex diagnostic workups and treatment plans. Increased provider awareness of the management and potential complications of M. abscessus is crucial to the improvement patient outcomes, as is a multidisciplinary approach that incorporates primary care providers, pathologists, plastic surgeons, and infectious disease specialists.

Surgical outcomes in adults with purpura fulminans: a systematic review and patient-level meta-synthesis.

BACKGROUND: Cutaneous manifestations of purpura fulminans (PF) present many challenges for clinicians and surgeons. In a state of septic shock complicated by limb ischemia, surgical interventions are necessary to control the pathological cascade and improve patient outcomes. The objective of this article was to report etiologies and surgical outcomes associated with cutaneous manifestations in adults. METHODS: This systematic review and meta-analysis compared 190 adult patients with etiologies, signs and symptoms, and surgical outcomes associated with cutaneous manifestations of PF. The PubMed, EMBASE, Cochrane Library, Web of Science, and Scopus databases were systematically and independently searched. Patient and clinical characteristics, surgical interventions, outcomes, and complications were recorded. RESULTS: Seventy-nine studies were eligible for the systematic review, and 77 were eligible for meta-analysis using Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) and Cochrane guidelines. A total of 71/190 (38%) cases reported surgical debridement. Fasciotomies were reported in 12/190 (6%) cases and 20 procedures. Amputations were reported in 154/190 (81%) cases. Reconstruction was reported in 45 cases. Skin grafts were applied in 31 cases. Flaps were used for reconstruction in 28 cases. Median (IQR) surgical procedures per patient were 4 (4, 5) procedures. Infectious organisms causing PF were 32% Neisseria meningitidis (n = 55) and 32% Streptococcus pneumonia (n = 55). Coagulase-negative Staphylococcus (95% confidence interval (CI)(8.2-177.9), p = 0.032), Haemophilus influenza (95%CI (7.2-133), p = 0.029), Streptococcus pneumonia (95% CI (13.3-75.9), p = 0.006), and West Nile Virus (95%CI (8.2-177.9), p = 0.032) were associated with significantly more extensive amputations compared to other organisms. CONCLUSION: This systematic review and patient-level meta-analysis found the most common presentation of PF was septic shock from an infectious organism. Neisseria meningitidis and Streptococcus pneumonia were equally the most common organisms associated with PF. The majority of cases were not treated in a burn center. The most common surgeries were amputations, with below-the-knee-amputations being the most common procedure. Skin grafting was the most commonly performed reconstructive procedure. The most common complications were secondary infections. Organisms with significantly more extensive amputations were coagulase-negative Staphylococcus, Haemophilus influenza, Streptococcus pneumonia, and West Nile Virus. Interpretation of findings should be cautioned due to limited sample data.

A Skin Rejection Grading System for Vascularized Composite Allotransplantation in a Preclinical Large Animal Model.

BACKGROUND: The Banff Criteria have been accepted as a system for grading histological rejection in graft skin in human vascularized composite allotransplantation (VCA). Preclinical swine hindlimb transplantation models have an important role in translational studies in VCA. However, unified grading criteria for rejection in swine skin have not yet been established. METHODS: Two hundred fourteen swine skin biopsy specimens were reviewed, including 88 native skin biopsies and 126 specimens from the skin component of heterotopic swine hindlimb transplants. Thorough review was performed in a blinded fashion by an expert veterinary pathologist with attention paid to the applicability of the Banff criteria as well as specific histologic characteristics and trends. Clinical and histopathologic rejection scores were then directly compared. RESULTS: Two hundred fourteen specimens reviewed showed significant similarities between swine and human skin, as previously published. Notable swine-specific characteristics, including paucicellular infiltration with rare epidermal cell infiltration or necrosis, were accounted for in a proposed grading system that parallels the Banff Criteria. CONCLUSIONS: This comprehensive grading system, based on the Banff Classification for skin rejection in VCA, provides a standardized system for more accurate comparison of rejection in preclinical swine VCA models.

International Medical Graduates in the US Plastic Surgery Residency: Characteristics of Successful Applicants.

Objectives: Within the United States, plastic surgery is a difficult field to match into for both US and international medical graduates. While the number of available residency positions has grown in recent years, this has not been mirrored by an equal increase in the number of international medical graduates who match. Furthermore, there are few reliable resources to guide international medical graduates who are interested in matching into US-based programs, so the process is often even more difficult and unpredictable than for US applicants. Methods: An anonymous survey was distributed electronically to international medical graduates who successfully matched into independent and integrated US plastic surgery residency programs. The survey assessed qualities such as medical school performance, test scores, research experience, and other relevant applicant information, and χ2 analysis was done to compare the survey results for integrated and independent track international medical graduates. Results: International medical graduates who successfully match tend to rank high and score well in their medical school classes, score between 230 and 250 on USMLE step 1 and 2CK tests, and have a mean of 2 years of research experience before applying to the match. International medical graduates in the independent track tend to have higher step 1 scores, whereas international medical graduates in the integrated track tend to have more research experience and additional nonmedical degrees. Conclusions: This is a survey-based overview that describes the characteristics of successfully matched international medical graduates. Limitations of this study include the inability to identify and survey the unsuccessful applicants as well as poor response rate of the successful candidates in the independent pathway who successfully matched.