Engineering Flavin-Dependent Halogenases.

In the two decades since the discovery of the first flavin-dependent halogenase (FDH), great strides have been made in elucidating the diversity of enzymes in this class as well as their structures and functions. More recently, efforts to engineer these enzymes for synthetic applications have yielded their first successes. FDH variants with improved stability, expanded substrate scope, and altered regioselectivity have been developed. Here, we review these efforts and provide representative protocols that have been employed for FDH engineering. Random and structure-guided mutagenesis approaches and screening methods, including HPLC, mass spectrometry, and spectrophotometric methods, are discussed. The protocols described herein should be generalizable to many FDHs and a wide variety of engineering goals.

Treatment of canine hemangiopericytomas with photodynamic therapy.

BACKGROUND AND OBJECTIVES: Canine hemangiopericytomas are a commonly occurring neoplasm with a clinical course of recurrence after surgical removal. This study sought to evaluate Photochlor (HPPH) photodynamic therapy (HPPH-PDT) as an adjuvant therapy to prevent recurrence of tumor after surgical removal. STUDY DESIGN/MATERIALS AND METHODS: Sixteen dogs with naturally occurring hemangiopericytomas were treated with surgical removal of the tumor followed by PDT using Photochlor as the photosensitizer. Photochlor was injected intravenously at a dose of 0.3 mg/kg. Forty-eight hours later the treatment consisted of surgical removal of the tumor followed by HPPH-PDT. RESULTS: Nine dogs (56%) had recurrence of tumor from 2 to 29 (median 9) months after treatment. These results are comparable or not as good as other forms of therapy. CONCLUSIONS: Photochlor photodynamic therapy applied after surgery appears to have no advantage over other forms of therapy in regards to preventing recurrence. Delayed wound healing and infections are problematic and make HPPH-PDT an undesirable addition to surgery for the treatment of this tumor type.

Cortical bone healing following laser osteotomy using 6.1 microm wavelength.

BACKGROUND AND OBJECTIVE: Use of laser wavelengths in the 6.1 microm (amide I) to 6.45 microm (amide II) regions and a macropulse width of 4.0 microseconds delivered by a computer-controlled delivery system have produced clean, deep cortical bone ablations with minimal collateral thermal injury and no char formation. The purpose of this study was to evaluate the healing of cortical bone following 6.1 microm wavelength laser osteotomy using a 4.0 microsecond pulse, and compare that response to the response of similar osteotomies made with a standard pneumatic surgical bone saw. STUDY DESIGN/MATERIALS AND METHODS: Sixteen mature rabbits were divided equally into 2, 4, 6 and 8-week post-surgical survival groups. A nitrogen driven sagittal bone saw and an FEL generating 6.1 microm wavelength in 4.0 microsecond macropulses of 22.5 +/- 2.5 mJ/pulse directed into a 200 microm diameter spot were used to make 6.6 mm linear cuts into rabbit tibial cortex, and the healing response over time was monitored. Bone saw cuts were made halfway through the thickness of the cortex. Laser cuts were directed by a computer-controlled delivery system, and were either partial or full thickness cortical cuts. Location of the cortical bone cuts (saw or laser, partial or full thickness cut, proximal or distal, medial right or medial left tibia) were randomly assigned. At each predetermined post-surgical time point, rabbits of the appropriate group were euthanized, and the tibias of each subject collected, processed for histologic evaluation, and analyzed by light microscopy. RESULTS: At 2 weeks post-surgery, bone saw cuts showed no evidence of a healing response, while both the partial and full laser cut sites were filled with trabecular bone and primitive bone marrow. By 4 weeks post-surgery, the bone saw cuts showed filling of the defect with trabecular bone and primitive marrow, and an intense osteonal remodeling of the original cortex adjacent to the cuts was evident. All laser cut defects were filled, reactive periosteal bone was being converted to osteons and consolidating, and secondary osteons were appearing in the original cortex. At 6 weeks following surgery, the bone saw defects were filed with a mixture of woven and lamellar bone. All laser defects were filled with lamellar osteons and woven bone, the osteons were remodeling from primary to secondary osteons. By 8 weeks following the surgery, all bone saw and laser cut specimens revealed complete healing. CONCLUSIONS: Histologic evaluation of osteotomy sites made in skeletally mature rabbit tibia using the 6.1 microm wavelength, 4.0 microsecond macropulse FEL, delivered at 6 Hz at the osteotomy site, reveals a healing response which is at least as good as the healing of bone saw osteotomies, and appears to proceed at a faster rate during the first 2-4 weeks following surgery.

Treatment of canine oral squamous cell carcinomas with photodynamic therapy.

Eleven dogs with naturally occurring oral squamous cell carcinomas were treated with photodynamic therapy (PDT) using Photochlor (HPPH) as the photosensitizer. The largest length of the tumours measured in a two-dimensional plane ranged from 0.9 to 6.8 cm. Seven of the tumours invaded underlying bone as determined by radiograph appearance. Photochlor was injected intravenously at a dose of 0.3 mg kg(-1). Forty-eight hours later the tumours were treated. Tumours with a surface to base depth of greater than 1 cm were surgically reduced to less than 1 cm. Irradiation with 665 nm light with an energy density of 100 J cm(-2) was administered. Eight dogs were considered cured with no tumour recurrence for at least 17 months after treatment. Local treatment of oral squamous cell carcinomas with PDT appears to give results similar to those obtained with surgical removal of large portions of the mandible or maxilla. The cosmetic results with PDT are superior to those of radical surgical removal. The new sensitizer, Photochlor, appears effective for oral squamous carcinomas with results similar to those reported for other sensitizers.

Comparison of cortical bone ablations by using infrared laser wavelengths 2.9 to 9.2 microm.

BACKGROUND AND OBJECTIVE: The purpose of this study was to compare the ablation of cortical bone at wavelengths across the near and midinfrared region. STUDY DESIGN/MATERIALS AND METHODS: An free electron laser generating 4-micros macropulses at specific wavelengths between 2.9 and 9.2 microm was used to ablate cortical bone. The same pulse intensity, repetition rate, radiant exposure, number of pulses, and delivery was used for each wavelength. Tissue removal, collateral thermal injury, and morphologic characteristics of the ablation sites were measured by light and scanning electron microscopy, and compared with the infrared absorption characteristics of cortical bone. RESULTS: Within the parameters used, bone ablation was found to be wavelength dependent. Incisions were deepest where protein has strong absorption, and were most shallow where mineral is a strong absorber. No char was observed on ablation surfaces where 3.0, and 5.9-6.45 microm wavelengths were used. CONCLUSIONS: The use of wavelengths in the 6.1-microm amide I to 6.45-microm amide II region, with the pulse characteristics described, were the most efficient for cutting cortical bone and produced less collateral thermal injury than cutting with a surgical bone saw. This study confirms previous observations that the ablation mechanism below plasma threshold is consistent with an explosive process driven by internal vaporization of water in a confined space and demonstrates that ablation is enhanced by using wavelengths that target the protein matrix of cortical bone.

Closed reduction and lag screw fixation of sacroiliac luxations and fractures.

OBJECTIVE: To describe a technique for closed reduction and percutaneous insertion of a lag screw for fixation of sacroiliac fracture-luxations, and to report the success of this technique in stabilizing sacroiliac fracture-luxations. STUDY DESIGN: A retrospective clinical study. STUDY POPULATION: 13 consecutive client-owned dogs with sacroiliac fracture-luxations. METHODS: Sacroiliac fracture-luxations were stabilized by using a closed reduction and percutaneous lag screw fixation technique. Preoperative, postoperative, and last re-examination radiographs were used to assess the location and number of pelvic injuries, other orthopedic injuries, percent reduction of the sacroiliac joint, percent sacral width screw depth, position of the screw, pelvic canal diameter ratio, hemipelvic canal width ratio, and complications. Information on signalment, weight, weight-bearing status, neurologic status, and complications was obtained from the medical record. RESULTS: Mean percent reduction of the sacroiliac joint was 92.33%. All screws were placed within the sacral body with a mean screw depth/sacral width of 79.03%. No screw loosening occurred. Mean pelvic canal diameter ratios were 0.99, 1.20, and 1.14 preoperatively, immediately postoperatively, and at the last re-examination, respectively. Nine of 13 dogs were willing to walk on the ipsilateral rear leg the day after surgery. CONCLUSIONS: Closed reduction and percutaneous insertion of a lag screw for stabilization of fracture-luxation of the sacroiliac joint is an acceptable method of repair. CLINICAL RELEVANCE: Sacroiliac fracture-luxations can be successfully reduced and stabilized using a minimally invasive technique.

Biomechanical evaluation of a crimp clamp system for loop fixation of monofilament nylon leader material used for stabilization of the canine stifle joint.

OBJECTIVE: To test a crimp clamp system designed to secure monofilament nylon leader (MNL) material commonly used as lateral fabellotibial sutures (LFS) in extra-articular stabilization of the canine stifle joint. STUDY DESIGN: In vitro biomechanical tests of MNL loops secured with either the crimp clamp system or knots were performed. Suture loops (n = 94) were created from 27.3 kg tensile strength MNL and fastened with knots or crimp clamps. Tests were conducted on steam-sterilized, ethylene-oxide-sterilized, and nonsterilized MNL sutures. Loops were evaluated in single load tests and cycled tests. Values for load to failure, initial loop tension, loop elongation, mode of failure, and point of failure were determined. RESULTS: Crimp-clamped loops were superior to knotted loops in all parameters tested in both cycled and noncycled tests. Loop failure generally occurred by breaking within 3 mm of the fixation in both clamped and knotted tests. Loop elongation after cycling was greater in the knotted loops compared with clamped loops (P < .001). Load to failure was greater in clamped tests than in knotted tests (P < .001), regardless of sterilization technique used. Significantly higher initial loop tension could be achieved with the clamp system compared with knot fixation (P < .001). CONCLUSIONS: The crimp clamp system provides superior in vitro loop fixation characteristics compared with knot fixation in 27.3 kg test MNL. CLINICAL RELEVANCE: Based on the results of biomechanical testing and the known biocompatibility of the system's implant components, clinical trials using the crimp clamp system are warranted.

Pulp remains viable following vital amputation of the mesio-buccal root of the maxillary fourth premolar in dogs.

Vital amputation of the mesio-buccal root of the maxillary fourth premolar (P4) was performed bilaterally in 8 dogs. Histopathologic evaluation of the tooth structure revealed normal pulp in the remaining mesio-buccal, mesio-palatal, and distal roots and crown during a mean follow-up period of 3.5 +/- 1.9 months. Amputation site healing was characterized by a reparative dentin bridge produced by odontoid cells. Tunnel defects were observed in 13 of 16 (81%) dentin bridges. It is concluded that the pulp of a tri-rooted, periodontal disease-free tooth remains viable during a 6-month period following vital root amputation. These results point to the intriguing possibility that this may also occur in humans, however, this needs to be directly tested in man.

Scapular fractures in dogs: epidemiology, classification, and concurrent injuries in 105 cases (1988-1994).

A retrospective study of canine scapular fractures diagnosed and treated from 1988 through 1994 at four veterinary teaching hospitals was performed. Dogs (n = 105) with 109 scapular fractures were included. Most scapular fractures occurred in young (i.e., less than four years of age), male, medium- to large-breed (i.e., greater than 10 kg) dogs as the result of vehicular trauma. Concurrent injuries (primarily thoracic trauma) occurred in approximately 70% of cases. In-house follow-up evaluations were considered adequate in only 17% of the cases. A classification system that includes biomechanical principles for categorization is described to avoid discrepancies between various traditional classification systems.

Surgical treatment of osteoarthritis.

Surgical treatment of OA is appropriate when conservative therapy fails or is inadequate. The veterinary orthopedist's goals in treatment should be to alleviate pain, maintain function, and prevent or remove the potential for further degeneration of the joint. Currently, in veterinary surgery, THR and femoral head and neck excision are the primary treatments for OA of the coxofemoral joint. Other joints are treated primarily by arthrodesis or excision arthroplasty. Arthroscopy is proving to be a valuable tool in the diagnosis and treatment of OA, and total stifle and elbow replacement and cartilage resurfacing through chondrocyte grafting are on the horizon as potential treatment options.

Evaluation of percutaneous gastrostomy as a technique for permanent gastropexy.

OBJECTIVE: This study was designed to evaluate right-sided percutaneous endoscopic gastrostomy (PEG) as a method for creation of a permanent gastropexy. STUDY DESIGN: Percutaneous endoscopic gastrostomy adhesions were evaluated by gross examination and by mechanical testing and the results were compared with those obtained by conventional incisional gastropexy. ANIMALS OR SAMPLE POPULATION: Fourteen mixed-breed dogs. METHODS: Incisional gastropexies were performed on the dogs of group one (N = 7) and PEG tubes were placed in the dogs of group 2 (N = 7). All skin sutures (group 1) and PEG tubes (group 2) were removed on day 14. The animals were maintained for an additional 44 days before euthanasia and immediate necropsy. Gastropexy adhesions were evaluated and collected for biomechanical evaluation using a materials testing machine. RESULTS: The duration of the procedure for group 2 was less (32.86 min +/- 7.65) than for group 1 (56.29 min +/- 8.28). The number of complications was not significantly different between group 1 and group 2 (P = .103). Gastroperitoneal adhesions were present in 7 of 7 dogs in group 1 and 4 of 7 dogs in group 2. The adhesion lengths and widths were significantly lager in dogs in group 1 compared with those in group 2. The adhesions present in group 1 dogs sustained significantly greater tensile loads to failure (61.98 +/- 14.65 N), compared with the adhesions present in group 2 dogs (22.31 +/- 26.87 N). CONCLUSIONS: Right-sided PEG inconsistently formed a weak gastropexy and the procedure was associated with a trend toward greater morbidity than incisional gastropexy. CLINICAL RELEVANCE: Right-sided PEG is not recommended as a means of prophylactic gastropexy.

Three-dimensional culture of canine articular chondrocytes on multiple transplantable substrates.

OBJECTIVE: To determine the effects of transplantable substrates on canine chondrocytes grown in three-dimensional culture. ANIMALS: 3 canine cadavers. PROCEDURE: Articular cartilage harvested from canine cadavers was used to obtain chondrocytes for primary culture. Subcultured chondrocytes were grown in agarose alone (AG), or in agarose on canine cancellous bone (CB), polypropylene mesh, or oxidized regenerated cellulose substrate. Cell proliferation, proteoglycan and glycosaminoglycan (GAG) production, and collagen production were assessed on days 3, 6, 10, 15 and 20. RESULTS: Chondrocytes from groups AG and CB proliferated and produced matrix over the entire 20-day study period. Group-CB chondrocytes had significantly more GAG than did chondrocytes of all other groups on days 6 (P = 0.0297) and 15 (P = 0.00272). Those of groups AG and CB contained significantly (P = 0.0235) more GAG on day 20. Chondrocytes of the polypropylene mesh group proliferated and produced matrix through day 10 in culture, but were no longer viable and had no matrix production on days 15 and 20. Regenerated cellulose appeared to be toxic to canine chondrocytes during all stages of in vitro three-dimensional culture. CONCLUSIONS: Three-dimensional culture of canine chondrocytes in agarose appears to produce favorable results with respect to chondrocyte proliferation and matrix production. Canine CB appears to have beneficial effects with regard to early GAG synthesis. Polypropylene mesh and oxidized regenerated cellulose had detrimental effects on cellular proliferation and matrix production.

Bioavailability of lead to juvenile swine dosed with soil from the Smuggler Mountain NPL Site of Aspen, Colorado.

Bioavailability of lead (Pb) has become an issue in quantifying exposure of sensitive populations and, where necessary, establishing cleanup levels for contaminated soil. Immature swine were used as a model for young children to estimate the degree to which Pb from two fully characterized composite samples from the Smuggler Mountain Superfund Site in Aspen, Colorado may be bioavailable to resident children. The composite soils contained 14,200 and 3870 micrograms Pb/g of soil. Relative and absolute enteric bioavailabilities of Pb in soil (oral dose groups of 75,225, and 675 micrograms Pb/kg body wt/day) were estimated by comparison with an orally administered soluble Pb salt (lead acetate = PbAc2.3H2O) (dose groups of 0, 75, and 225 micrograms Pb/kg body wt/day) and an intravenously administered aqueous solution of Pb (100 micrograms Pb/kg/ day) from the same trihydrate salt administered daily for 15 days to 50 juvenile swine. The biological responses (area under the blood Pb concentration-time curve, and the terminal liver-, kidney-, and bone-lead concentrations) produced by Pb from PbAc2.3H2O and lead-contaminated soils were determined. This study revealed Pb from soil containing 14,200 micrograms Pb/g of soil had a bioavailability relative to Pb from PbAc (RBA), ranging from 56% based on the area under the blood lead concentration-time curve (AUC) versus dose, to 86% based on calculations from liver-Pb loading versus dose. Similarly, Pb from soil containing 3870 micrograms Pb/g of soil had an RBA ranging from 58% based on the AUC versus dose, to 74% based on calculations from liver- and kidney-Pb loading versus dose. Bioavailability of Pb in soils may be more or less than EPA's default RBA of 60%, therefore, measuring site-specific RBAs provides a basis for improved exposure and risk assessment.

Atraumatic rupture of the gastrocnemius muscle after corticosteroid administration in a dog.

A 6-year-old spayed female Shetland Sheepdog was referred for evaluation of lameness, muscle atrophy, and a partial plantigrade stance of the right hind limb of 5 weeks' duration. Without history of trauma, atraumatic rupture of the right gastrocnemius muscle was diagnosed. Surgical repair was unsuccessful. The dog then developed signs of hyperadrenocorticism. Results of ACTH stimulation and low-dose dexamethasone suppression tests were consistent with iatrogenic adrenal suppression. One deleterious effect of excessive use of corticosteroids on muscle and connective tissue is degenerative myopathy. Steroid-induced myopathy with subsequent rupture of the gastrocnemius muscle was suspected in this dog. Clinical signs of myopathy most often develop with use of triamcinolone acetonide; therefore, care should be taken when administering this and other corticosteroids.

Pharmacokinetics of pyropheophorbide-a-hexyl ether in the dog.

BACKGROUND AND OBJECTIVES: Pyropheophorbide-a-hexyl ether (HPPH) is a new compound being investigated for use as a photosensitizer for photodynamic therapy; however, the pharmacokinetics are not known for any of the target species likely to be treated with this drug. The objective of this study was to determine the pharmacokinetic parameters of this drug prior to institution of a clinical trial in canine patients with various cancers. STUDY DESIGN, MATERIALS AND METHODS: HPPH (0.3mg/kg i.v.) was administered to 12 dogs and blood samples were drawn at intervals for 24 hours and plasma HPPH concentrations were determined. Pharmacokinetic parameters were calculated for each dog. RESULTS: No evidence of toxicity was noted in any dog. The mean half-life was calculated to be 26.98 +/- 2.35 hrs. The mean clearance was 5.061 +/- 0.214 ml/hr/kg. The mean volume of distribution of the central compartment was 0.069 +/- 0.003 L/kg, and the mean steady state volume of distribution was 4.47 +/- 0.25 L/kg. CONCLUSION: The conclusion is that 0.3 mg/kg HPPH injected intravenously resulted in measurable plasma levels for 24 hrs, and resulted in no detectable adverse reactions.

Percutaneous iodine-125 seed implantation for carcinoma of the prostate.

This study was performed to assess the early results of treating stages T1-T3 adenocarcinoma prostate with either Iodine-125 (125I) implant alone (Group 1), for smaller more well differentiated cancers, or with low dose external beam radiation followed by a 125I boost (XRT + 125I) (Group 2) for larger less well differentiated tumours. Eighty-six patients were followed for between 11 and 60 months with a mean follow up of 26.1. All patients were followed by regular prostate specific antigen (PSA) evaluations, and digital rectal examinations (DRE). Eighty patients had a follow-up biopsy at 1 year. Prostate specific antigen progression-free survival (PSA-PFS) was determined and defined. Complications and potency were also assessed. Early results of 125I prostate seed implantation are very promising especially for selected cases of localized carcinoma.

General management considerations for the trauma patient.

Proper evaluation, nursing care, physical therapy, and attention to the patient's general comfort are essential to the successful management of animals with severe trauma. Evaluation is important because failure to identify all injuries early in the management of a trauma patient may have unfavorable effects on the eventual outcome of the case. Once therapy is underway, ensuring the patient's comfort, maintaining fluid and electrolyte balance, and providing a well-planned rehabilitation program are essential to speed an enhance the recovery of these patients.

Effects of temperature and storage time on pin pull-out testing in harvested canine femurs.

Effects of temperature and storage time on canine bone-transfixation pin specimens were tested by comparing pin pull-out forces. A total of 16 femurs from 8 mature dogs were tested. Five nonthreaded Steinmann pins were placed through both cortices in the diaphysis of each femur. The femurs were then sectioned transversely between each pin, with a bone-pin specimen placed evenly into each of 5 groups prior to biomechanical testing. Four bone-pin specimen groups were stored at -20 or -70 C for 14 or 28 days, while 1 specimen group was immediately tested. Pull-out forces for frozen groups were compared with pull-out forces for the fresh group. Using two-way ANOVA, there was no statistical difference in mean axial-extraction forces among bone-pin specimens in any of the tested groups. It is concluded that acute pin pull-out forces are not significantly affected by freezing temperature or time. However, specimens stored at -20 C for as few as 14 days had a trend for increased pull-out forces, compared with freshly harvested specimens. Therefore, the authors recommend storage of bone-pin specimens at -70 C when possible.

Detailed preliminary analysis of 125iodine implantation for localized prostate cancer using percutaneous approach.

Recent developments have permitted accurate seed placement and dosimetry for interstitial brachytherapy of selected patients with localized prostate cancer. We present our experience with 76 patients divided into 2 groups. Group 1 included 45 patients with smaller, more well differentiated tumors, usually less than 2 cm. in diameter on digital rectal examination or transrectal ultrasound and a Gleason score of less than 7 who were treated with 125iodine alone. Group 2 consisted of 31 patients with localized tumors greater than 2 cm. in diameter and/or a Gleason sum equal to or greater than 7 who were treated with low dose external beam radiation followed by 125iodine boost 4 weeks later. Complete clinical progression-free survival, including prostate specific antigen, digital rectal examination and biopsy, was 51% for group 1 and 63.3% for group 2, with a mean followup of 26.3 months. Prostate specific antigen progression-free survival was 97.7% for group 1 and 94.7% for group 2. These results appear to be superior to external beam radiation only although longer followup is needed to substantiate these favorable early results. The procedures were well tolerated with good potency sparing. They were performed on an outpatient or short stay basis and provided a good alternative to external beam radiation only or hormonal treatment for select patients with localized prostate cancer who may not be candidates for radical prostatectomy.

Articular fractures of the distal portion of the humerus in Vietnamese pot-bellied pigs: six cases (1988-1992)

Medical records of 6 Vietnamese pot-bellied pigs with articular fractures of the distal portion of the humerus were reviewed. Evaluation of the medical records did not reveal a sex predilection. All fractures were associated with minor traumatic episodes in young pigs. Of 6 fractures involving the humeral condyle, 4 involved the medial portion, 1 involved the lateral portion, and 1 was a Y-shaped fracture. Five of the pigs underwent surgical repair of the fracture, and all 5 did not have signs of lameness at follow-up evaluations (mean, 11 months). Of 4 pigs that had follow-up radiography, all had evidence of mild to moderate degenerative joint disease. Articular fractures of the distal portion of the humerus should be considered as a differential diagnosis in all Vietnamese pot-bellied pigs with forelimb lameness, even if the trauma sustained appeared mild. Surgical repair in Vietnamese pot-bellied pigs is straightforward, and excellent clinical results can be expected.