Standardization of Pneumococcal Biofilm Release to PncO Expression, a Predictive Measurement of Virulence.

A critical component in clinical trials for vaccines against pneumococcal disease is the establishment of robust preclinical models and clinical correlates of protection, which, in the case of the causative bacterial agent Streptococcus pneumoniae, include standard sepsis/pneumonia mouse models and opsonophagocytic activity (OPA), respectively. Despite broad usage, these gold-standard measures are ill equipped to evaluate nontraditional antigens that target virulence factors beyond capsular polysaccharides and/or proteins not associated with colonization or routine growth. These assays are further complicated by observed inconsistencies in the expression of target protein antigens and in the quantity of usable bacteria provided from respective growth processes. In an effort to overcome these issues, we performed an extensive optimization study of the critical steps in a bacterial biofilm dispersion model (termed "the biofilm model") to identify conditions that yield the greatest quantity of released pneumococci displaying a consistent virulence phenotype. Using this knowledge, we developed a secondary immune absorbance assay to provide immediate insight into the phenotypic state of bacteria conditioned using the biofilm model. Specifically, positive correlations between the expression of PncO (a key virulence-associated protein antigen) and immune absorbance (R2 = 0.96), capsule shedding, and OPA assay titers were translated into a predictive readout of virulence in sepsis and pneumonia challenge models. These results present a methodology for generating consistent lots of virulent bacteria to standardize inputs in preclinical and clinical models for testing vaccines against biofilm-associated bacteria.

Time-series lipidomic analysis of the oleaginous green microalga species Ettlia oleoabundans under nutrient stress.

BACKGROUND: Microalgae are uniquely advantageous organisms cultured and harvested for several value-added biochemicals. A majority of these compounds are lipid-based, such as triacylglycerols (TAGs), which can be used for biofuel production, and their accumulation is most affected under nutrient stress conditions. As such, the balance between cellular homeostasis and lipid metabolism becomes more intricate to achieve efficiency in bioproduct synthesis. Lipidomics studies in microalgae are of great importance as biochemical diversity also plays a major role in lipid regulation among oleaginous species. METHODS: The aim of this study was to analyze time-series changes in lipid families produced by microalga under different nutrient conditions and growth phases to gain comprehensive information at the cellular level. For this purpose, we worked with a highly adaptable, oleaginous, non-model green microalga species, Ettlia oleoabundans (a.k.a. Neochloris oleoabundans). Using a mass spectrometry-based untargeted and targeted metabolomics' approach, we analyzed the changes in major lipid families under both replete and deplete nitrogen and phosphorus conditions at four different time points covering exponential and stationary growth phases. RESULTS: Comprehensive analysis of the lipid metabolism highlighted the accumulation of TAGs, which can be utilized for the production of biodiesel via transesterification, and depletion of chlorophylls and certain structural lipids required for photosynthesis, under nutrient deprived conditions. We also found a correlation between the depletion of digalactosyldiacylglycerols (DGDGs) and sulfoquinovosyldiacylglycerols (SQDGs) under nutrient deprivation. CONCLUSIONS: High accumulation of TAGs under nutrient limitation as well as a depletion of other lipids of interest such as phosphatidylglycerols (PGs), DGDGs, SQDGs, and chlorophylls seem to be interconnected and related to the microalgal photosynthetic efficiency. Overall, our results provided key biochemical information on the lipid regulation and physiology of a non-model green microalga, along with optimization potential for biodiesel and other value-added product synthesis.

LC-MS/MS quantification of short-chain acyl-CoA's in Escherichia coli demonstrates versatile propionyl-CoA synthetase substrate specificity.

AIMS: This paper utilized quantitative LC-MS/MS to profile the short-chain acyl-CoA levels of several strains of Escherichia coli engineered for heterologous polyketide production. To further compare and potentially expand the levels of available acyl-CoA molecules, a propionyl-CoA synthetase gene from Ralstonia solanacearum (prpE-RS) was synthesized and expressed in the engineered strain BAP1. METHODS AND RESULTS: Upon feeding propionate, the engineered E. coli strains had increased the levels of both propionyl- and methylmalonyl-CoA of 6- to 30-fold and 3·7- to 6·8-fold, respectively. Expression of prpE-RS resulted in no significant increases in acetyl-, butyryl- and propionyl-CoA when fed the corresponding substrates (sodium acetate, butyrate or propionate). More interesting, however, were the results from strain BAP1 engineered for native prpE overexpression, which indicated increases in the same range of acyl-CoA formation. CONCLUSIONS: The increased acyl-CoA levels across the strains profiled in this study reflect the genetic modifications implemented for improved polyketide production and also indicate flexibility of the native PrpE. SIGNIFICANCE AND IMPACT OF THE STUDY: The results provide direct evidence of enhanced acyl-CoA levels correlating to those strains engineered for polyketide biosynthesis. This information and the inherent flexibility of the native PrpE enzyme support future efforts to characterize, engineer and extend acyl-CoA precursor supply for additional heterologous biosynthetic attempts.

Construction and performance of heterologous polyketide-producing K-12- and B-derived Escherichia coli.

AIMS: Escherichia coli has emerged as a viable heterologous host for the production of complex, polyketide natural compounds. In this study, polyketide biosynthesis was compared between different E. coli strains for the purpose of better understanding and improving heterologous production. METHODS AND RESULTS: Both B and K-12 E. coli strains were genetically modified to support heterologous polyketide biosynthesis [specifically, 6-deoxyerythronolide B (6dEB)]. Polyketide production was analysed using a helper plasmid designed to overcome rare codon usage within E. coli. Each strain was analysed for recombinant protein production, precursor consumption, by-product production, and 6dEB biosynthesis. Of the strains tested for biosynthesis, 6dEB production was greatest for E. coli B strains. When comparing biosynthetic improvements as a function of mRNA stability vs codon bias, increased 6dEB titres were observed when additional rare codon tRNA molecules were provided. CONCLUSIONS: Escherichia coli B strains and the use of tRNA supplementation led to improved 6dEB polyketide titres. SIGNIFICANCE AND IMPACT OF THE STUDY: Given the medicinal potential and growing field of polyketide heterologous biosynthesis, the current study provides insight into host-specific genetic backgrounds and gene expression parameters aiding polyketide production through E. coli.

Biosynthesis of complex polyketides in a metabolically engineered strain of E. coli.

The macrocyclic core of the antibiotic erythromycin, 6-deoxyerythronolide B (6dEB), is a complex natural product synthesized by the soil bacterium Saccharopolyspora erythraea through the action of a multifunctional polyketide synthase (PKS). The engineering potential of modular PKSs is hampered by the limited capabilities for molecular biological manipulation of organisms (principally actinomycetes) in which complex polyketides have thus far been produced. To address this problem, a derivative of Escherichia coli has been genetically engineered. The resulting cellular catalyst converts exogenous propionate into 6dEB with a specific productivity that compares well with a high-producing mutant of S. erythraea that has been incrementally enhanced over decades for the industrial production of erythromycin.

Biosynthesis of polyketides in heterologous hosts.

Polyketide natural products show great promise as medicinal agents. Typically the products of microbial secondary biosynthesis, polyketides are synthesized by an evolutionarily related but architecturally diverse family of multifunctional enzymes called polyketide synthases. A principal limitation for fundamental biochemical studies of these modular megasynthases, as well as for their applications in biotechnology, is the challenge associated with manipulating the natural microorganism that produces a polyketide of interest. To ameliorate this limitation, over the past decade several genetically amenable microbes have been developed as heterologous hosts for polyketide biosynthesis. Here we review the state of the art as well as the difficulties associated with heterologous polyketide production. In particular, we focus on two model hosts, Streptomyces coelicolor and Escherichia coli. Future directions for this relatively new but growing technological opportunity are also discussed.

Intraoperative computerized tomography scanning to assess the adequacy of decompression in anterior cervical spine surgery.

OBJECT: The purpose of this study was to improve the accuracy of bone removal during anterior spinal surgery. Intraoperative computerized tomography (CT) scanning was used to assess the success of bone resection and permit immediate correction in the event of inadequate bone removal. METHODS: The Phillips Tomoscan M was used to obtain preoperative cervical scans before and after cervical bone resection was complete. The completeness of bone removal was assessed by the operating neurosurgeon by reviewing the postresection CT scan. If the bone removal was deemed inadequate, additional bone was removed using a high-speed drill. A CT scan was obtained after each subsequent decompression until adequate bone removal was achieved. In 31 patients undergoing anterior cervical decompression intraoperative CT scanning was performed. Nineteen patients underwent corpectomy and 12 discectomy. Of the 31 patients, assessment of intraoperative CT scans obtained in 17 indicated further bone removal was required. CONCLUSIONS: Intraoperative CT scanning to monitor bone removal during anterior cervical surgery is a valuable tool to ensure the adequacy of surgery.

Single Price/Case Price Purchasing in orthopaedic surgery: experience at the Lahey Clinic.

BACKGROUND: Hospital revenues for orthopaedic operations are not keeping pace with inflation or with rising hospital expenses. In an attempt to reduce the hospital cost of orthopaedic operations by reducing the cost of operating-room supplies, we developed a Single Price/Case Price Purchasing Program for implants used in total hip arthroplasty, total knee arthroplasty, and total shoulder arthroplasty as well as for arthroscopic shavers and burrs, interference screws, and bone-suture anchors. METHODS: The Lahey Clinic asked orthopaedic vendors to supply all instruments, implants, and disposable items related to these selected products for one single price per unit or case. For example, a single price for total hip arthroplasty implants included instruments, acetabular cups, acetabular liners, acetabular screws, femoral stems, femoral heads, and stem centralizers, if required. The hospital implemented the Single Price/ Case Price Purchasing Program with a competitive-bid request for proposal. Surgeons evaluated the responses to the bidding process, and they made final decisions on product selection. RESULTS: The Single Price/Case Price Purchasing Program at the Lahey Clinic was successful in reducing the cost of orthopaedic implants and supplies. In the present article, we could not disclose the specific prices that we agreed to pay our vendors. The specific cost reductions were 32 percent for hip implants with a change of vendor, 23 percent for knee implants without a change of vendor, 25 percent for shoulder implants with a change of vendor, 45 percent for arthroscopic shavers and burrs without a change of vendor, 45 percent for interference screws without a change of vendor, and 23 percent for bone-suture anchors without a change of vendor. CONCLUSIONS: The Single Price/Case Price Purchasing Program at the Lahey Clinic allowed the hospital to reduce its cost of orthopaedic operations by lowering the cost of operating-room supplies. This cost reduction is important in a health-care economy in which hospital revenues per unit of service or care are decreasing.

Knee implant standardization: an implant selection and cost reduction program.

The largest single unit cost in the hospital cost for total knee arthroplasty (TKA) is the cost of knee implants. We developed a knee implant standardization program to provide guidelines for knee implant selection and to reduce the cost of knee implants for hospitals. Patients are assigned to demand categories based on five criteria: age, weight, expected activity, general health, and bone stock. Implants are assigned to demand categories based on an implant's projected capacity to handle the patient's projected demand. The program was applied retrospectively to 127 knee replacement operations performed on 93 patients during 1992. If this program had been in place, 8.4% of what was actually spent on knee implants for these 127 patients would have been saved. If the most expensive implants allowed in each demand category had actually been used, the program would have saved our hospital 12.8% of the cost of knee implants for these patients. Potential savings were noted in higher demand categories I and II by reducing the use of expensive cementless, porous-coated implants. The greatest potential savings were noted in lower demand categories III and IV: 11% savings could have been realized in demand category III, and 27% savings could have been achieved in the cost of knee implants in demand category IV. Potential savings would have been realized in these lower demand categories because of the recommended use of an all-polyethylene tibial component in 38 of 92 patients. This knee implant standardization program has the potential to assist surgeons in selecting knee implants and reduce the cost of knee implants without compromising outcome following TKA.

Impact of a clinical pathway and implant standardization on total hip arthroplasty: a clinical and economic study of short-term patient outcome.

This study evaluates the impact of a clinical pathway (CP) and a hip implant standardization program (HISP) on the quality and cost of total hip arthroplasty (THA). Two hundred six unilateral THA operations for osteoarthritis were evaluated: 89 operations were performed in 1991 without a CP or HISP (4-year follow-up period); 117 operations were performed in 1993 with a CP and HISP (2-year follow-up period). All patients had good clinical results and excellent outcomes with short-term follow-up evaluation. No differences were seen between groups in terms of patient ratings of outcome and satisfaction or in terms of complication rates in the hospital. Implementation of a CP and HISP did not adversely affect the short-term outcome of THA but did reduce hospital length of stay and hospital cost for THA.

Clinical outcome and survivorship analysis of core decompression for early osteonecrosis of the femoral head.

Thirty-three hips in 23 patients (followed for 5.3 years) were treated with core decompression for early osteonecrosis of the femoral head (Ficat and Arlet I, IIA, IIB). When the clinical endpoint of severe pain was used for survivorship, 76% of hips survived 1 year, 52% survived 2 years, and 44% survived 5 years. When the radiographic endpoint of progression to stage III disease was used, no progression was found in 72% of hips at 1 year, 61% at 2 years, and 37% at 5 years. When total hip arthroplasty was used as an endpoint, 90% of hips survived 1 year, 70% survived 2 years, and 61% survived 5 years. Lower radiographic stage was associated with a better result. Patients who weighed less than 79.4 kg (175 lb.) (P = .03) or whose bone stock was good (femoral index < 0.56, P < .001) had significantly improved survival. Outcome evaluation documented a 70% overall patient satisfaction rate in patients not undergoing total hip arthroplasty.

Advanced practice roles for nurses in tomorrow's healthcare systems.

Advanced practice nurses (APNs) have traditionally been a diverse group in terms of titles, education, credentials, and roles. The classification of APN usually encompasses the nurse practitioner (NP), certified nurse midwife (CNM), certified registered nurse anesthetist (CRNA), and clinical nurse specialist (CNS). NP, CRNA, and CNM roles have been more clearly delineated than the CNS roles. In light of healthcare reform, the CNS roles have been critically reviewed and analyzed. Attempts have been made to clarify responsibilities and outcomes, and to quantify the financial impact of this role. In this article, seven APN roles clearly defined in terms of minimal qualifications and competencies critical to accomplishment of the duties, responsibilities, and expected outcomes are presented. Comparison of competency requirements are also presented. The roles presented are designed to facilitate healthcare institutions in meeting the demands for serving patients with increasingly complex needs, as well as cost-containment goals.

Case report: radiation prevention of heterotopic ossification after bone and joint surgery in sites other than hips.

Five patients were given single dose irradiation in an attempt to prevent heterotopic ossification after bone and joint surgery in sites other than hips. All patients were at risk for the development of post-operative heterotopic ossification. Two patients were treated with 6 Gy and three patients were treated with 7 Gy the day after operation. No complications were encountered. Post-operative heterotopic ossification did not develop in patients who received 7 Gy, whereas treatment failed in the two patients who received 6 Gy. Because this is a case report study, no conclusion could be made. Further investigation is needed to assess the efficacy of post-operative single dose irradiation in heterotopic ossification prophylaxis in sites other than hips in high risk patients.

Implant standardization for total hip arthroplasty. An implant selection and a cost reduction program.

Total hip arthroplasty (THA) has been targeted by the United States federal government for cost control because of its high cost and rising incidence in the aging population. The hospital cost for THA during the 1980s was controlled by utilization review and a reduction in the volume of services delivered for each THA. The single largest increase in the cost of THA during the 1980s was the cost of hip implants. The Lahey Clinic Hip Implant Standardization Program was developed to provide objective guidelines for hip implant selection. These guidelines are based on the demands a patients is expected to place on his or her hip prosthesis. Because not every patient requires an expensive high-demand hip prosthesis, the standardization program also has the potential to reduce the hospital cost for hip implants without compromising patient care. Patients are assigned to four demand categories based on five objective criteria: age, weight, expected activity, general health, and bone stock. Selection of the prosthesis in each of the four demand categories is intended to match the implant's capacity with expected patient demand. The standardization program was retrospectively applied to 103 THAs performed during 1991. Analysis of variance demonstrated that patient variables and demand categories were statistically significant groupings. The cost of hip implants would have been reduced by 25.7% with the Lahey Clinic Hip Implant Standardization Program. A prospective outcome study is required to determine the long-term validity of this standardization program.

Single-dose irradiation for the prevention of heterotopic ossification after total hip arthroplasty. A comparison of doses of five hundred and fifty and seven hundred centigray.

One hundred and seven hips (ninety-four patients) that had risk factors associated with the development of heterotopic ossification after total hip arthroplasty were treated with a single dose of radiation after the operation in an attempt to prevent the formation of heterotopic bone. A study was conducted to compare the efficacy of a single dose of 550 centigray (nineteen hips) with that of a single dose of 700 centigray (eighty-eight hips). Heterotopic ossification developed in twelve (63 per cent) of the nineteen hips that were treated with 550 centigray; grades 1, 2, and 3, according to the classification of Brooker et al., developed in four hips each. Two of the patients who received 550 centigray were symptomatic. Heterotopic ossification developed in nine (10 per cent) of the eighty-eight hips that were treated with 700 centigray; the lesion was grade 1 in six, grade 2 in one, and grade 3 in two. None of the patients who received 700 centigray were symptomatic. We concluded that single-dose irradiation consisting of 550 centigray is inadequate for the prevention of heterotopic ossification in high-risk patients after total hip arthroplasty. We recommend a dose of 700 centigray as effective prophylaxis for these patients.

Repair of injured vertebral artery in anterior cervical procedures.

STUDY DESIGN: A case report of treatment of injury to the vertebral artery in anterior cervical procedures is presented with a review of the literature. OBJECTIVES: The feasibility of direct surgical repair is suggested. SUMMARY OF BACKGROUND DATA: Currently, ligation or tamponade is accepted as the treatment of injury to the vertebral artery in anterior cervical surgery. Significant morbidity can result from this method. Direct repair should eliminate this morbidity. METHODS: A report is presented of a patient who sustained injury to the vertebral artery, and the technique of repair is discussed. The literature was reviewed. RESULTS: The outcome was successful regarding both repair and the primary procedure. CONCLUSIONS: Although it is impossible to make a recommendation based on a single incident, this technique is believed to minimize postoperative sequelae that can be associated with occlusion of the vertebral artery.

Evaluation of autologous shed blood for autotransfusion after orthopaedic surgery.

Autologous shed blood for autotransfusion was evaluated at four medical centers in a prospective randomized study. One hundred twenty-eight patients were studied after hip replacement, knee replacement, or spine fusion. The efficacy of autologous shed blood in reducing homologous transfusion was evaluated. The relative risk of transfusion with homologous blood was 0.4 in patients who received shed blood compared with patients who did not receive shed blood. The reinfusion of shed blood reduced the requirement for homologous blood by 60%. Two filter systems were evaluated in reinfusing autologous shed blood. The Pall RC100 filter appeared to be more effective than the Pall 40-mu screen filter in removing fat particles and white blood cells. No significant clinical abnormalities were discovered after autotransfusion with autologous shed blood. Evaluation of clotting studies showed no significant differences between patients who received shed blood and patients who received liquid-preserved red blood cells. These data indicate that unwashed autologous shed blood from orthopaedic wound drainage is a safe and effective substitute for transfusion of autologous predonated blood or homologous liquid-preserved red blood cells.

Cold compressive dressing after total knee arthroplasty.

The efficacy of a cold compressive dressing after total knee arthroplasty (TKA) was prospectively studied in 105 knees in 76 patients. All components were cemented. All patients were placed in continuous passive motion machines after operation. A cold compressive Cryocuff dressing was applied to 50 knees after operation. An ACE wrap and ice pack were applied to the knees of 55 control patients after operation. Postoperative range of motion was recorded as maximum active flexion at two to four days (interval one), at seven to 14 days (interval two), and four to six weeks (interval three). Swelling was measured at the same time intervals by circumference at the midpatella and circumference at the distal thigh one inch proximal to the superior pole of the patella. Use of postoperative narcotics was calculated for postoperative days zero to three and for postoperative days four to seven. Wound drainage was recorded for all knees. The use of a cold compressive dressing after TKA was not associated with an increase in range of motion at any point after the operation. The Cryocuff dressing did not appreciably reduce swelling around the knee after TKA. No significant difference was found in the amount of postoperative wound drainage between the two groups of patients. In patients undergoing unilateral TKA, no significant difference existed between the narcotic requirements of control patients and patients wearing the cold compressive dressing.

Repair of failed transpedicle screw fixation. A biomechanical study comparing polymethylmethacrylate, milled bone, and matchstick bone reconstruction.

In a random, controlled laboratory study, pedicle screws placed in human cadaveric vertebrae were axially loaded to failure. Three repair methods were tested. Use of low pressure polymethylmethacrylate yielded 149% of the original pullout strength, milled bone yielded 70% of the initial pullout strength, and matchstick bone yielded 56% of the initial pullout strength. Two incidents of cortical penetration during reinsertion in the matchstick group occurred. The results were statistically analyzed.

Burst fractures of the fifth lumbar vertebra.

Eleven patients with burst fractures of the fifth lumbar vertebra were reviewed. The results of nonoperative treatment were compared with that of immediate surgery and stabilization with pedicle screw fixation. Five patients were treated nonoperatively and six patients underwent pedicle screw instrumentation and spinal fusion. Five patients had neurologic injury associated with their L5 burst fracture. Nonoperative treatment yielded excellent results in young patients with minimal canal compromise. Neurologic deficits responded more predictably to surgical decompression than to conservative treatment and internal fixation with pedicle screws restores spinal stability and allows early mobilization.