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.

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.

Current considerations for myelomeningocele repair.

Important advances have been made in the reconstruction of myelomeningocele during the last few years. Techniques of closure that use diverse tissues available in the back range from very simple to very complex. Most of them have become essential to the plastic surgeon. To facilitate a more efficient interdisciplinary approach, recent advances in knowledge of the vascular anatomy of the skin and specific clinical surgical considerations concerning skin grafts, cutaneous flaps, and musculocutaneous flaps are reviewed. A topographical division of the lesion that will permit a methodologically adequate treatment plan for congenital defects in the back, specifically in the thoracic, thoracolumbar, and lumbosacral regions is suggested.

Incidence and sequelae of nocturnal respiratory obstruction following posterior pharyngeal flap operation.

A competent velopharyngeal mechanism is important for the production of normal speech, and the secondary procedure of a posterior pharyngeal flap (PPF) may be necessary in some patients to achieve this goal. A number of complications have been described in the literature following pharyngeal flap surgery. The purpose of this study was to examine short- and long-term complications after PPF surgery, and in particular the incidence and the end effect of nocturnal respiratory obstruction (NRO). All PPFs over a 17-year period performed at one institution and by the same surgeon were examined retrospectively. All medical records from the Commission of Handicapped Children of patients who had a PPF were reviewed. Patients with NRO were identified clinically, and sleep studies were administered with two or more of the clinical triad. During a 17-year period, 111 patients underwent a PPF to treat velopharyngeal incompetence. Twelve patients were identified with a syndromic association in addition to a clefting disorder, of which most (N = 9) consisted of Pierre Robin syndrome. The median age at PPF performance was 6.0 years and the average follow-up was 7.4 years. The early postoperative complication rate was 10%, including a 7.2% incidence of respiratory obstruction and 0.9% postoperative bleeding. Twenty-one patients (19%) had late complications or unsatisfactory results. Twelve patients (10.5%) developed NRO, and patients with Pierre Robin syndrome were particularly prone-4 of 9 patients developed this complication. Nine of 12 patients with NRO had sleep studies performed with a minimum interval of 6 months postoperatively. Eight of the nine studies were normal. Of the NRO group, 3 patients had takedown of their PPF, including the patient with an abnormal sleep study. All 3 patients improved markedly and none developed recurrence of velopharyngeal insufficiency. NRO is not an uncommon finding in PPF patients, but NRO does not necessarily imply the presence of obstructive sleep apnea. The consequences of persistent NRO over the long term deserve further study.

Early excision and grafting versus conservative management of burns in the elderly.

Elderly burn patients have significantly higher mortality rates than younger patients with similar burns over the total body surface area. Two theories exist regarding treatment of burns in the elderly: a traditional approach to limit physiologic stress by avoidance of operative intervention in the early post-burn stage and eschar excision and wound closure within the first week of hospitalization. We examined retrospectively the outcome in patients 70 years or older, hospitalized in the University of Kentucky Burn Unit between 1975 and 1995. In the first decade (1975 to 1983), patients were managed conservatively, namely, with spontaneous eschar separation and late skin grafting. In the second half of the study period (1984 to 1994), elderly patients were managed by early operative excision (<7 days) and grafting. A total of 73 elderly patients were admitted to the unit, 6 of whom were not resuscitated and died shortly (<96 hours) after admission. Twenty-eight patients had early excision and grafting (average age 78.1 years, total body surface area 23.6 percent), and 39 were managed conservatively (average age 79.3 years, total body surface area 20.9 percent). The mortality rate was 57 percent in the first group and 41 percent in the second group (p = 0.22). In an effort to further define the two groups, the other patient variable that contributes to burn mortality besides age and total body surface area, inhalation injury, was subtracted and the mortality rates were recalculated. Excluding patients with inhalation injury, the mortality rate was 48 percent in the first group and 27 percent in the second group (p = 0.15). We conclude that, in our unit, the management of elderly patients by early excision and grafting was of no benefit and may have resulted in a higher mortality rate.

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.

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.

High-energy gunshot wounds to the face.

Thirty-three patients with high-energy gunshot wounds to the face were treated at the University of Kentucky Chandler Medical Center between 1976 and 1993. Wounds were classified according to the mass and velocity of the projectile, and the range from weapon to target. More than half the injuries involved multiple facial regions. Twenty patients underwent immediate definitive reconstructive procedures. Intervals between injury and initial nondefinitive reconstruction for the other patients ranged from 1 day to 1 month. Toward the end of the study period, reconstruction was undertaken earlier and more aggressively, and included more attention to primary bone grafting and free tissue transfer. These patients developed fewer problems with infection, long-term scarring, and contracture, and they required fewer operative procedures. There was no operative mortality and none of the patients with self-inflicted injuries reattempted suicide. We conclude that early aggressive treatment of these wounds can produce better structural, functional, and rehabilitative results.

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.