Advanced cell-based modeling of the royal disease: characterization of the mutated F9 mRNA.

Essentials The Royal disease (RD) is a form of hemophilia B predicted to be caused by a splicing mutation. We generated an iPSC-based model of the disease allowing mechanistic studies at the RNA level. F9 mRNA analysis in iPSC-derived hepatocyte-like cells showed the predicted abnormal splicing. Mutated F9 mRNA level was very low but we also found traces of wild type transcripts. SUMMARY: Background The royal disease is a form of hemophilia B (HB) that affected many descendants of Queen Victoria in the 19th and 20th centuries. It was found to be caused by the mutation F9 c.278-3A>G. Objective To generate a physiological cell model of the disease and to study F9 expression at the RNA level. Methods Using fibroblasts from skin biopsies of a previously identified hemophilic patient bearing the F9 c.278-3A>G mutation and his mother, we generated induced pluripotent stem cells (iPSCs). Both the patient's and mother's iPSCs were differentiated into hepatocyte-like cells (HLCs) and their F9 mRNA was analyzed using next-generation sequencing (NGS). Results and Conclusion We demonstrated the previously predicted aberrant splicing of the F9 transcript as a result of an intronic nucleotide substitution leading to a frameshift and the generation of a premature termination codon (PTC). The F9 mRNA level in the patient's HLCs was significantly reduced compared with that of his mother, suggesting that mutated transcripts undergo nonsense-mediated decay (NMD), a cellular mechanism that degrades PTC-containing mRNAs. We also detected small proportions of correctly spliced transcripts in the patient's HLCs, which, combined with genetic variability in splicing and NMD machineries, could partially explain some clinical variability among affected members of the European royal families who had lifespans above the average. This work allowed the demonstration of the pathologic consequences of an intronic mutation in the F9 gene and represents the first bona fide cellular model of HB allowing the study of rare mutations at the RNA level.

Biochemical, Histopathologic, Physiologic, and Behavioral Effects of Nonsteroidal Antiinflammatory Drugs in Rainbow Trout (Oncorhynchus mykiss).

Because the number of fish being used in research is increasing rapidly, evaluating the analgesic and pathologic effects of NSAID in fish is essential. To determine the biochemical, histopathologic, physiologic and behavioral effects of 3 NSAID, 48 rainbow trout underwent anesthesia with tricaine methanesulfonate and exploratory celiotomy and were randomly assigned to receive flunixin (0.5 mg/kg IM), ketorolac (0.5 mg/kg IM), ketoprofen (2 mg/kg IM), or saline. Clinical pathologic variables were assessed 1 wk before surgery and 48 h after surgery. Histopathology was performed to evaluate the healing of the incision, tissue reaction at the injection site, and potential organ toxicity. Physiologic and behavioral parameters, including weight, feeding, opercular rate, and vertical position in the water, were measured to establish parameters for identifying pain in fish. The difference between the pre- and postoperative phosphorus concentrations was greater in the flunixin group than the saline group and was the only pathologic difference between treatment groups. Histopathology of incision site, injection site, and internal organs appeared normal, and healing did not appear to be inhibited by the drugs used. The physiologic parameters of opercular rate and weight were consistent and may be helpful in identifying pain in fish in future studies, whereas feeding and vertical position in the water were unhelpful as indicators of pain in this rainbow trout surgical model. Overall, according to clinical pathology and histopathology, the use of ketoprofen, ketorolac, and flunixin at the dosages used in this study lack negative effects in rainbow trout undergoing surgery.

Impact of a short exposure to levofloxacin on faecal densities and relative abundance of total and quinolone-resistant Enterobacteriaceae.

Emergence of resistant Enterobacteriaceae in the intestinal microbiota during antibiotic treatment is well documented but its early dynamic is not. Here, we compared the densities of total Enterobacteriaceae and relative abundance (RA) of quinolone-resistant Enterobacteriaceae (QRE) in the first stool passed by patients who had a short exposure to levofloxacin (levofloxacin, n=12) or not (control, n=8). Mean densities (SD) (log CFU/g stool) of total Enterobacteriaceae were lower in the levofloxacin group than in the control group-3.4 (1.6) versus 6.7 (1.7), respectively, p <0.001. Conversely, mean RA (SD) of QRE was significantly higher in the levofloxacin group than in the control group-49.7% (23.4) versus 0.1% (3.2), respectively, p <0.05). In conclusion, even a short exposure to levofloxacin has a profound impact on the densities of total Enterobacteriaceae and the QRE-RA.

General surgery, the general surgical subspecialties, and prerequisite training.

Currently, several specialties (cardiothoracic, pediatric, and colorectal) require a full general surgery residency before specialty training. Plastic surgery has, in essence, a de facto requirement since most residents have had at least 5 years of general surgery training before initiation of a plastic surgery residency. Forces within both general surgery and plastic surgery will dictate that such a de facto requirement will not persist in the future. The pressures that compel plastic surgery to modify the traditional role of general surgery as preparatory training for requisite or specialty residency training can be extended by analogy to the other specialties as well. This article will hypothesize that the premises and the conclusions about the relationship between plastic surgery and general surgery apply equally well to the other specialties. If the traditional and historical role of full general surgery residency as prerequisite training for a specialty residency is discarded, careful thought must be given to any substitute system to graduate surgical education. In particular, attention must be directed to the three components of clinical skills to be acquired and to an expectation of operative or technical experience.

Total abdominal wall reconstruction.

A patient had full-thickness loss of abdominal wall from clostridial myonecrosis. Initial care consisted of resuscitation, debridement, and transfer to a hyperbaric chamber facility. After control of sepsis, multiple enteric fistulas were managed by enterotomies, gastric and duodenal defunctionalization with closed-loop gastrojejunostomy, gastrostomy, and end jejunostomy. Good nutritional status was maintained with total parenteral nutrition over a three-month period. Total abdominal wall reconstruction was accomplished by rotation of bilateral tensor fascia lata musculocutaneous flaps. Reconstruction was successful as the patient was able to return to an active life.

Repair of chest wall defects with prosthetic material.

This report summarizes our experience during a four-year period with the repair of 8 thoracic cage and 3 diaphragmatic defects requiring reinforcement with prosthetic material. Defects as large as the entire left hemidiaphragm or the right anterior chest wall including ribs two through six from the midsternum to the midaxillary line were adequately repaired. The technical approach utilized to obtain a secure, nonmobile thoracic cage involved the placement of sutures through drill holes or around ribs, rather than through the periosteum or pericostal soft tissues. Successful diaphragmatic repair was dependent on proper anchoring of the medial border of the prosthesis, placing sutures in the pericardium as necessary. Skin coverage for thoracic cage defects was achieved with widely undermined and advanced local tissue or previously delayed pedicle flaps. All patients had good evidence of chest wall stabilization after operation, and all were removed from mechanical ventilation within three days. One patient died of myocardial infarction twenty days after operation, and a second patient died later of metastatic disease. On the basis of our experience, we conclude that the range of chest wall lesions that can be surgically corrected or palliated is increased by the use of prosthetics implanted with techniques described here.

Closure of myelorachischisis defects with reverse latissimus dorsi myocutaneous flaps.

The closure of myelorachischisis defects has been difficult and at times complicated. Over the past 2 decades, the use of rotational advancement flaps has been increasingly popular, but can be attended by instances of skin ischemia and necrosis. The development of the myocutaneous and muscle flap concept has aided reconstructive surgery. Extensive experience with the useful latissimus dorsi flap with and without overlying skin has been obtained for the reconstruction of a variety of truncal defects. We describe the use of bilateral "reverse" latissimus dorsi muscle flaps based on the paraspinal perforators for the closure of myelorachischisis defects in two patients. Coverage was uneventful in both instances. A 2-year follow-up revealed no compromise of upper extremity function due to sacrifice of the latissimus dorsi muscles. We offer an approach to the problem of closure of large myelorachischisis defects: bilateral reverse latissimus dorsi muscle flaps and delayed skin coverage. This approach seems to offer a good muscle layer over the dural closure and to provide long-standing durable coverage without apparent compromise of upper extremity function.

Optimizing primary treatment for advanced laryngeal and pyriform sinus carcinoma.

Advocates of chemotherapy plus radiation as the definitive treatment for patients with advanced laryngeal cancer often cite older studies that attribute cure rates of less than 50% to laryngectomy plus radiation. The outcomes of patients with stage III and IV laryngeal and pyriform sinus carcinoma from 1980 to 1989 (96 patients) were compared with those of patients treated from 1962 to 1977 (84 patients). Demographics, the extent of disease, and nodal involvement were similar between the groups. There were more operative complications (45% versus 22%; p < 0.01) and deaths (10% versus 2%; p < 0.01) in the patients who underwent irradiation preoperatively. Overall survival was improved in the recent group compared with the early group (73% versus 54% at 5 years; p < 0.03), as was disease-free survival (64% versus 38% at 5 years; p < 0.02). Results of treatment for advanced laryngeal and pyriform sinus carcinoma have improved significantly. These modern results should be used to evaluate newer treatment modalities.

Carcinoma of the lower lip.

Carcinoma of the lower lip is sufficiently common in occurrence that a practitioner should be cognizant of the requisite diagnostic and therapeutic guidelines. This article discusses the pertinent anatomy, the management of the local or primary lesion, and some prognostic criteria for surgical decisions directed toward the regional lymph nodes. Reconstruction is discussed from the standpoint of magnitude of the defect as a percentage of the total lip transverse diameter.

The irradiated wound.

Radiation and its effects on normal tissue are important to an understanding of the alteration of healing that occurs in a radiated wound. The surgical management of irradiated tissue is discussed regarding surgical procedures in previously irradiated tissue and regarding management of the nonhealing irradiated wound, the radiation ulcer.

Oncologic and reconstructive considerations in nonmelanotic skin and lip cancers.

The conceptual approach to the reconstruction of skin and lip cancer defects is no different than the reconstruction of other tumors in other locations. To plan and execute the reconstructive effort properly requires a careful assessment of the anticipated extirpative defect, the consideration of additional postoperative therapy, if any, and the restoration of function and aesthetic form. Similar to other malignancies, a working knowledge of tumor behavior is an integral ingredient of a successful reconstructive plan.

Analysis of complex postextirpative deformity.

Reconstruction in the oncologic setting requires of the plastic surgeon a careful and concise thought process. Analysis of a complex deformity/dysfunction is problem solving, and postextirpative reconstruction is simply a variation of the same theme. An analysis model has been developed ("Here-There") that can be useful as a guide for the process of analysis as well as a teaching model for residents.

Reconstruction of the lower lip.

Reconstruction of the lower lip requires a reconstitution of the sphincter functions of oral competence, provision of articulation, as well as a satisfactory aesthetic result. A step-by-step approach based on the magnitude of the defect provides the surgeon with a consistent result.

Oncologic considerations in nonmelanotic skin cancer.

Although excision of nonmelanotic skin cancer carries a high cure rate, a potential exists in a small percentage of patients for not only significant morbidity, but mortality as well. Some prognostic variables and clinical guidelines have been developed that can be applied to individual patients.

The ordeal of Sigmund Freud. A postscript.

A number of questions about Freud's illness and treatment remain unanswered. The author ponders the duration of Freud's survival, the histologic diagnosis of the tumor, the contemporary management of this cancer, and the doctor-patient relationships involved.

The acute and subacute management of the burned hand.

Management of the severely burned upper extremity remains a significant challenge to the most experienced clinician. An understanding of the underlying mechanism that uncorrected could culminate in a negative outcome is the key to formulation of a successful treatment plan. Initial proper splinting, avoidance of edema, the appropriate sequencing and integration of physical therapy, and judicious surgical intervention, all considered within the framework of the individual patient, are the components of the treatment plan that yields the most consistently good results.

Electrical burns.

Electrical burns can be divided into flash or typical thermal injury and high-tension injury. The latter is usually caused by greater than 1000 volts and produces a clinically characteristic entry and exit wound. The optimal management of patients with high-tension electrical injury has evolved into a plan of urgent exploration and debridement, aggressive redebridement, and early wound closure.

Advanced and recurrent nonmelanoma skin cancer.

Advanced skin cancer presents a substantial challenge to the surgeon, who must incorporate sound oncologic principles and carefully considered reconstruction into the treatment plan. Using an anatomical format, this article discusses the treatment plans for advanced skin cancer and outlines various pitfalls. The appropriateness and advisability of other modalities are also discussed.

Developing concepts and treatment of complex maxillary fractures.

The last decade has witnessed a tremendous evolution in the management of maxillary fractures--an evolution that mirrors an overall more aggressive contemporary approach to complex maxillofacial trauma. This article briefly reviews past contributions to the current management of these fractures, the author's approach to management, and the future of this continually improving field.

The difficult scalp and skull wound.

Scalp and skull defects can be very difficult to reconstruct. Whereas small defects may require only primary closure, skin graft, or small rotation flaps, large defects involving full-thickness scalp can be much more problematic. These large defects may require free-tissue transfer for adequate soft-tissue coverage. The calvaria also may require reconstruction. If the underlying bone is not infected, it can be left in situ and covered with well-vascularized tissue. The bone should be removed only if it is infected. Calvarial reconstruction should then be delayed for 3 to 6 months. If the tissue has been irradiated, only selected defects are appropriate for coverage with local scalp flaps because adjacent tissues may have underlying damage. With a methodic, accurate assessment of the defect, successful reconstruction can be expected.