Robotic-Assisted Total Knee Arthroplasty: What are Patients' Perspectives, Understanding and Expectations?

BACKGROUND: The rate of using robotic-assisted total knee arthroplasty (RA-TKA) has increased markedly. Understanding how patients view the role of robotics during total knee arthroplasty (TKA) informs shared decision making and facilitate efforts to appropriately educate patients regarding the risks and benefits of robotic assistance. METHODS: A self-administered questionnaire was completed by 440 potential TKA patients at the time of their surgery scheduling. Participants answered 25 questions regarding RA-TKA, socioeconomic factors, and their willingness to pay (WTP) for RA-TKA. Logistic regressions were used to determine if population characteristics and surgeon preferences influenced the patients' perceptions of RA-TKA. RESULTS: There were 39.7% of respondents who said that they had no knowledge regarding RA-TKA. Only 40.7% of participants had expressed a desire for RA-TKA to be used. There were 8.7% who were WTP extra for the use of RA-TKA. Participants believed that the main 3 benefits of RA-TKA compared to conventional methods were: more accurate implant placement (56.2%); better results (49.0%); and faster recovery (32.1%). The main 3 patient concerns were harm from malfunction (55.2%), reduced surgeon role in the procedure (48.1%), and lack of supportive research (28.3%). Surgeon preference of RA-TKA was associated with patient's willingness to have RA-TKA (odds ratio 4.60, confidence interval 2.98-7.81, P < .001), and with WTP extra for RA-TKA (odds ratio 2.05, confidence interval: 1.01-4.26, P = .049). CONCLUSION: Patient knowledge regarding RA-TKA is limited. Nonpeer-reviewed online information may make prospective TKA candidates vulnerable to misinformation and aggressive advertising. The challenge for orthopaedic surgeons is to re-establish control and reliably educate patients about the proven advantages and disadvantages of this emerging technology.

Cis- and trans-acting variants contribute to survivorship in a naïve Drosophila melanogaster population exposed to ryanoid insecticides.

Insecticide resistance is a paradigm of microevolution, and insecticides are responsible for the strongest cases of recent selection in the genome of Drosophila melanogaster Here we use a naïve population and a novel insecticide class to examine the ab initio genetic architecture of a potential selective response. Genome-wide association studies (GWAS) of chlorantraniliprole susceptibility reveal variation in a gene of major effect, Stretchin Myosin light chain kinase (Strn-Mlck), which we validate with linkage mapping and transgenic manipulation of gene expression. We propose that allelic variation in Strn-Mlck alters sensitivity to the calcium depletion attributable to chlorantraniliprole's mode of action. GWAS also reveal a network of genes involved in neuromuscular biology. In contrast, phenotype to transcriptome associations identify differences in constitutive levels of multiple transcripts regulated by cnc, the homolog of mammalian Nrf2. This suggests that genetic variation acts in trans to regulate multiple metabolic enzymes in this pathway. The most outstanding association is with the transcription level of Cyp12d1 which is also affected in cis by copy number variation. Transgenic overexpression of Cyp12d1 reduces susceptibility to both chlorantraniliprole and the closely related insecticide cyantraniliprole. This systems genetics study reveals multiple allelic variants segregating at intermediate frequency in a population that is completely naïve to this new insecticide chemistry and it foreshadows a selective response among natural populations to these chemicals.

Minimizing Surgical Instrument Burden Increases Operating Room Efficiency and Reduces Perioperative Costs in Total Joint Arthroplasty.

BACKGROUND: Optimizing surgical trays to improve operating room efficiency and reduce costs in instrument processing is an under-appreciated strategy for cost containment. This study aimed to assess the economic impact of instrument tray optimization in total joint arthroplasty. METHODS: Thirty-five randomly selected elective primary total knee arthroplasty and total hip arthroplasty performed by 4 fellowship-trained surgeons were analyzed. Type and number of instruments used as well as timing of different steps in the sterilization process were recorded by an independent observer. Using Lean methodology, surgeons identified redundant or underutilized instruments and agreed upon the fewest number needed for each tray. Instrument utilization rates and processing time were analyzed before and after tray modifications. Annual cost savings were calculated based on a processing factor of $0.59-$11.52 per instrument. RESULTS: Only 45.5% of instruments opened for total knee arthroplasty were utilized. After optimization, 28 of 87 (32.2%) instruments were removed and the remainder could be stored in one tray. Mean set-up time decreased from 20.7 to 14.2 minutes, while 40-75 minutes were saved during the sterilization process. For total hip arthroplasty, only 36.0% of instruments were utilized. Using Lean methods, 46 of 112 (41.1%) instruments were removed and tray count was reduced to 2 trays. Mean set-up time decreased from 27.9 to 18.6 minutes, while 45-150 minutes were saved during processing. Average annual savings amounted to $281,298.05. CONCLUSION: Lean methodology can be used to eliminate redundant or underutilized instruments in total joint arthroplasty, improving surgical efficiency and generating substantial cost savings.

Prolonged Scar-in-a-Jar: an in vitro screening tool for anti-fibrotic therapies using biomarkers of extracellular matrix synthesis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a rapidly progressing disease with challenging management. To find novel effective therapies, better preclinical models are needed for the screening of anti-fibrotic compounds. Activated fibroblasts drive fibrogenesis and are the main cells responsible for the accumulation of extracellular matrix (ECM). Here, a prolonged Scar-in-a-Jar assay was combined with clinically validated biochemical markers of ECM synthesis to evaluate ECM synthesis over time. To validate the model as a drug screening tool for novel anti-fibrotic compounds, two approved compounds for IPF, nintedanib and pirfenidone, and a compound in development, omipalisib, were tested. METHODS: Primary human lung fibroblasts from healthy donors were cultured for 12 days in the presence of ficoll and were stimulated with TGF-ß1 with or without treatment with an ALK5/TGF-ß1 receptor kinase inhibitor (ALK5i), nintedanib, pirfenidone or the mTOR/PI3K inhibitor omipalisib (GSK2126458). Biomarkers of ECM synthesis were evaluated over time in cell supernatants using ELISAs to assess type I, III, IV, V and VI collagen formation (PRO-C1, PRO-C3, PRO-C4, PRO-C5, PRO-C6), fibronectin (FBN-C) deposition and α-smooth muscle actin (α-SMA) expression. RESULTS: TGF-ß1 induced synthesis of PRO-C1, PRO-C6 and FBN-C as compared with unstimulated fibroblasts at all timepoints, while PRO-C3 and α-SMA levels were not elevated until day 8. Elevated biomarkers were reduced by suppressing TGF-ß1 signalling with ALK5i. Nintedanib and omipalisib were able to reduce all biomarkers induced by TGF-ß1 in a concentration dependent manner, while pirfenidone had no effect on α-SMA. CONCLUSIONS: TGF-ß1 stimulated synthesis of type I, III and VI collagen, fibronectin and α-SMA but not type IV or V collagen. Synthesis was increased over time, although temporal profiles differed, and was modulated pharmacologically by ALK5i, nintedanib, pirfenidone and omipalisib. This prolonged 12-day Scar-in-a-Jar assay utilising biochemical markers of ECM synthesis provides a useful screening tool for novel anti-fibrotic compounds.

Intraoperative Surgeon Administered Adductor Canal Blockade Is Not Inferior to Anesthesiologist Administered Adductor Canal Blockade: A Prospective Randomized Trial.

BACKGROUND: Controlling postoperative pain and reducing opioid requirements after total knee arthroplasty remain a challenge, particularly in an era stressing rapid recovery protocols and early discharge. A single-shot adductor canal blockade (ACB) has been shown to be effective in decreasing postoperative pain. The purpose of the present study is to compare the efficacy of an anesthesiologist administered ACB and a surgeon administered intraoperative ACB. METHODS: Patients undergoing primary total knee arthroplasty were prospectively randomized to receive either an anesthesiologist administered (group 1) or surgeon administered (group 2) ACB using 15 mL of ropivacaine 0.5%. Primary outcomes were pain visual analog scale, range of motion, and opioid consumption. RESULTS: Thirty-four patients were randomized to group 1 and 29 to group 2. Opioid equivalents consumed were equal on postoperative day (POD) 0, 1, and 2. Patients in group 1 had statistically less pain on POD 0, but this did not reach clinical significance and there was no difference in pain on POD 1 or 2. Patients in group 1 had significantly increased active flexion POD 1, but there was no difference in active flexion on POD 0 or 6 weeks postop. There was no difference in patient satisfaction with pain control or short-term functional outcomes. CONCLUSION: Surgeon administered ACB is not inferior to anesthesiologist administered ACB with respect to pain, opioid consumption, range of motion, patient satisfaction, or short-term functional outcomes. Surgeon administered ACB is an effective alternative to anesthesiologist administered ACB.

Routine Patellar Resurfacing During Total Knee Arthroplasty Is Not Cost-Effective in Patients Without Patellar Arthritis.

BACKGROUND: Currently, the decision to resurface the patella is often made irrespective of the presence of patellar arthritis. The purpose of this study is to utilize the existing literature to assess cost-utility of routinely vs selectively resurfacing the patella. METHODS: Prospective randomized studies of patella resurfacing vs non-resurfacing in total knee arthroplasty (TKA) were identified through literature review. Data from these studies represented probabilities of varied outcomes following TKA dependent upon patella resurfacing. Using previously validated utility scores from the McKnee modified Health Utilities Index, endpoint utility values were provided for each potential outcome. RESULTS: Literature review yielded a total of 14 studies with 3,562 patients receiving 3,823 TKAs, of which 1,873 (49.0%) patellae were resurfaced. Persistent postoperative anterior knee pain occurred in 20.9% vs 13.2% (P < .001) and patella reoperation was performed in 3.7% vs 1.6% (P < .001) of unresurfaced and resurfaced patella, respectively. In studies excluding those with arthritic patellae, the incidence of anterior knee pain was equivalent between groups and reoperation decreased to 1.2% vs 0% (P = .06). Patella resurfacing provided marginally improved quality-adjusted life-years (QALY) for both selective and indiscriminate patella resurfacing. When including all studies, the incremental cost per QALY was $3,032. However, when analyzing only those studies with nonarthritic patellae, the incremental cost per QALY to resurface the patella increased to $183,584. CONCLUSION: Patellar resurfacing remains a controversial issue in TKA. Utilizing data from new prospective randomized studies, this analysis finds that routinely resurfacing arthritis-free patellae in TKA are not cost-effective.

Intraoperative Ketamine in Total Knee Arthroplasty Does Not Decrease Pain and Narcotic Consumption: A Prospective Randomized Controlled Trial.

BACKGROUND: Multiple studies have demonstrated that ketamine, a glutamate receptor blocker, may decrease postoperative pain in abdominal and orthopedic surgeries. However, its role with spinal anesthesia and total knee arthroplasty (TKA) remains unknown. The purpose of this study is to determine the efficacy of subanesthetic dosing of ketamine during TKA on postoperative pain and narcotic consumption. METHODS: In this prospective, randomized, double-blinded clinical trial, we enrolled 91 patients undergoing primary TKA with spinal anesthesia in a single institution from 2017 to 2018. Patients were randomized to receive intraoperative ketamine infusion at a rate of 6 mcg/kg/min for 75 minutes or a saline placebo. All patients received spinal anesthesia and otherwise identical surgical approaches, pain management, and rehabilitation protocols. Patient-reported visual analog pain scores were calculated preoperatively, postoperative days (POD) 0-7, and 2 weeks. Narcotic consumption was evaluated on POD 0 and 1. RESULTS: There was no difference in average pain between ketamine and placebo at all time points except for at PODs 1 (45 vs 56, P = .041) and 4 (39 vs 49, P = .040). For least pain experienced, patients administered with ketamine experienced a reduction in pain only at POD 4 (22 vs 35, P = .011). There was no difference in maximum pain cohorts at all time points of the study or in-hospital morphine equivalents between the 2 cohorts. CONCLUSION: As part of multimodal pain management protocol, intraoperative ketamine does not result in a clinically significant improvement in pain and narcotic consumption following TKA.

Endothelial to Mesenchymal Transition Contributes to Endothelial Dysfunction in Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is a progressive disease characterized by lung endothelial cell dysfunction and vascular remodeling. Normally, the endothelium forms an integral cellular barrier to regulate vascular homeostasis. During embryogenesis endothelial cells exhibit substantial plasticity that contribute to cardiac development by undergoing endothelial-to-mesenchymal transition (EndoMT). We determined the presence of EndoMT in the pulmonary vasculature in vivo and the functional effects on pulmonary artery endothelial cells (PAECs) undergoing EndoMT in vitro. Histologic assessment of patients with systemic sclerosis-associated PAH and the hypoxia/SU5416 mouse model identified the presence von Willebrand factor/α-smooth muscle actin-positive endothelial cells in up to 5% of pulmonary vessels. Induced EndoMT in PAECs by inflammatory cytokines IL-1ß, tumor necrosis factor α, and transforming growth factor ß led to actin cytoskeleton reorganization and the development of a mesenchymal morphology. Induced EndoMT cells exhibited up-regulation of mesenchymal markers, including collagen type I and α-smooth muscle actin, and a reduction in endothelial cell and junctional proteins, including von Willebrand factor, CD31, occludin, and vascular endothelial-cadherin. Induced EndoMT monolayers failed to form viable biological barriers and induced enhanced leak in co-culture with PAECs. Induced EndoMT cells secreted significantly elevated proinflammatory cytokines, including IL-6, IL-8, and tumor necrosis factor α, and supported higher immune transendothelial migration compared with PAECs. These findings suggest that EndoMT may contribute to the development of PAH.

Impaired bone morphogenetic protein receptor II signaling in a transforming growth factor-ß-dependent mouse model of pulmonary hypertension and in systemic sclerosis.

RATIONALE: Up to 10% of patients with systemic sclerosis (SSc) develop pulmonary arterial hypertension (PAH). This risk persists throughout the disease and is time dependent, suggesting that SSc is a susceptibility factor. Outcome for SSc-PAH is poor compared with heritable or idiopathic forms, despite clinical and pathological similarities. Although susceptibility in heritable PAH and idiopathic PAH is strongly associated with gene mutations leading to reduced expression of bone morphogenetic protein receptor (BMPR) II, these mutations have not been observed in SSc-PAH. OBJECTIVES: To explore BMPRII expression and function in a mouse model of SSc (TßRIIΔk-fib) that is susceptible to developing pulmonary hypertension and in SSc lung. METHODS: BMPRII and downstream signaling pathways were profiled in lung tissue and fibroblasts from the TßRIIΔk-fib model, which develops pulmonary vasculopathy with pulmonary hypertension that is exacerbated by SU5416. Complementary studies examined SSc or control lung tissue and fibroblasts. MEASUREMENTS AND MAIN RESULTS: Our study shows reduced BMPRII, impaired signaling, and altered receptor turnover activity in a transforming growth factor (TGF)-ß-dependent mouse model of SSc-PAH. Similarly, a significant reduction in BMPRII expression is observed in SSc lung tissue and fibroblasts. Increased proteasomal degradation of BMPRII appears to underlie this and may result from heightened TGF-ß activity. CONCLUSIONS: We found reduced BMPRII protein in patients with SSc-PAH and a relevant mouse model associated with increased proteasomal degradation of BMPRII. Collectively, these results suggest that impaired BMP signaling, resulting from TGF-ß-dependent increased receptor degradation, may promote PAH susceptibility in SSc and provide a unifying mechanism across different forms of PAH.

Gene duplication in the major insecticide target site, Rdl, in Drosophila melanogaster.

The Resistance to Dieldrin gene, Rdl, encodes a GABA-gated chloride channel subunit that is targeted by cyclodiene and phenylpyrazole insecticides. The gene was first characterized in Drosophila melanogaster by genetic mapping of resistance to the cyclodiene dieldrin. The 4,000-fold resistance observed was due to a single amino acid replacement, Ala(301) to Ser. The equivalent change was subsequently identified in Rdl orthologs of a large range of resistant insect species. Here, we report identification of a duplication at the Rdl locus in D. melanogaster. The 113-kb duplication contains one WT copy of Rdl and a second copy with two point mutations: an Ala(301) to Ser resistance mutation and Met(360) to Ile replacement. Individuals with this duplication exhibit intermediate dieldrin resistance compared with single copy Ser(301) homozygotes, reduced temperature sensitivity, and altered RNA editing associated with the resistant allele. Ectopic recombination between Roo transposable elements is involved in generating this genomic rearrangement. The duplication phenotypes were confirmed by construction of a transgenic, artificial duplication integrating the 55.7-kb Rdl locus with a Ser(301) change into an Ala(301) background. Gene duplications can contribute significantly to the evolution of insecticide resistance, most commonly by increasing the amount of gene product produced. Here however, duplication of the Rdl target site creates permanent heterozygosity, providing unique potential for adaptive mutations to accrue in one copy, without abolishing the endogenous role of an essential gene.

Vascular Complications After Total Knee Arthroplasty-A Single Institutional Experience.

BACKGROUND: Vascular complications during total knee arthroplasty (TKA) are uncommon but potentially devastating. We evaluated cases of vascular complication during TKA in our high-volume, community hospital system. METHODS: Patients were identified by cross-referencing billing codes for TKA with diagnosis codes for vascular complication during the same hospital stay between January 1, 2010 and December 31, 2014. Clinical and radiographic data, time to diagnosis, intervention, and outcomes were collected. RESULTS: We identified 13 vascular complications in 9951 TKAs (0.13%). Average age was 66.2 years (95% CI: 5.55; range: 54.1-87.9), 12 (92.3%) were female, average body mass index was 32.3 (5.17; 20-50), and average Charlson Comorbidity Index was 4.08 (1.03; 2-7). Black females (relative risk = 18.33, 95% CI: 6.20-54.22) were at particularly high risk. Preoperatively, 6 knees exhibited varus coronal malalignment and 2 valgus malalignment (only 1 >15°). None had flexion contracture >10°. Four knees exhibited vascular calcifications on preoperative radiographs. Twelve were diagnosed and treated the same day as index TKA and 1 on postoperative day 2. All underwent interventions: 9 stents, 2 endarterectomies, 1 thrombectomy, and 1 bypass. One patient sustained a peroneal nerve injury; 3 had persistent stiffness postoperatively that improved after manipulation. There were no revision surgeries, further vascular intervention, compartment syndrome, periprosthetic joint infection, amputation, or mortality. CONCLUSION: Incidence of vascular complications at our community-based hospital system is in line with previous reports. Black race and female gender were significant risk factors. Although outcomes were remarkable for a high rate of stiffness and one peroneal neuropathy, the devastating complications of amputation, compartment syndrome, periprosthetic joint infection, or early mortality were not observed.

Do Standard Surgical Guides Produce Accurate and Precise Femoral Bone Resections During Total Knee Arthroplasty?

PURPOSE: Accurate alignment and balanced flexion and extension gaps are critical elements in achieving a successful outcome following total knee arthroplasty (TKA). The ability to make accurate and precise bone cuts is essential in the creation of balanced gaps. We sought to determine if one type of modern-day standard surgical instrument using an intramedullary rod and posterior referencing produces accurate and precise distal and posterior femoral bone resections. MATERIALS AND METHODS: Seventy-five consecutive patients undergoing TKA were divided into three groups, with 25 patients in each group receiving one of three implant designs: 25 Stryker Triathlon® CR (Stryker, Mahwah, NJ), 25 Zimmer NexGen® Flex CR (Zimmer, Warsaw, IN), and 25 StelKast Proven Gen-FlexTM CR (StelKast, Pittsburgh, PA). Flexion-extension gap matching was determined using only the medial flexion and extension gaps. Accuracy was determined by comparing actual resection thickness to desired resection thickness. "Optimal" accuracy was considered to be within 1.0mm of desired, and "near-optimal" accuracy was considered to be within 2.0mm of the desired resection thickness. Precision was determined by the variability of resection thicknesses within each system. RESULTS: Data demonstrated a lack of accuracy and precision across all three tested systems, with each system resulting in certain unique tendencies. Only one out of 75 cases resulted in optimal resection accuracy with all three cuts (Zimmer). When lowering the threshold to include both optimal and near-optimal (within 2 mm of error) with all three cuts, only one third of Stryker and Zimmer cases and two thirds of StelKast cases achieved this threshold, representing 44% of cases (33/75). CONCLUSIONS: Improvements in instrumentation to increase accuracy and precision may be warranted. Errors in fixation may be due to the instrumentation itself, and altering instrumentation to include less modularity, provide more stable fixation, and more reliably seal the implant on the femur may be of benefit.

Imatinib attenuates hypoxia-induced pulmonary arterial hypertension pathology via reduction in 5-hydroxytryptamine through inhibition of tryptophan hydroxylase 1 expression.

RATIONALE: Whether idiopathic, familial, or secondary to another disease, pulmonary arterial hypertension (PAH) is characterized by increased vascular tone, neointimal hyperplasia, medial hypertrophy, and adventitial fibrosis. Imatinib, a potent receptor tyrosine kinase inhibitor, reverses pulmonary remodeling in animal models of PAH and improves hemodynamics and exercise capacity in selected patients with PAH. OBJECTIVES: Here we use both imatinib and knockout animals to determine the relationship between platelet-derived growth factor receptor (PDGFR) and serotonin signaling and investigate the PAH pathologies each mediates. METHODS: We investigated the effects of imatinib (100 mg/kg) on hemodynamics, vascular remodeling, and downstream molecular signatures in the chronic hypoxia/SU5416 murine model of PAH. MEASUREMENTS AND MAIN RESULTS: Treatment with imatinib reduced all measures of PAH pathology observed in hypoxia/SU5416 mice. In addition, 5-hydroxytryptamine (5-HT) and tryptophan hydroxylase 1 (Tph1) expression were reduced compared with the normoxia/SU5416 control group. Imatinib attenuated hypoxia-induced increases in Tph1 expression in pulmonary endothelial cells in vitro via inhibition of the PDGFR-ß pathway. To better understand the consequences of this novel mode of action for imatinib, we examined the development of PAH after hypoxic/SU5416 exposure in Tph1-deficient mice (Tph1(-/-)). The extensive changes in pulmonary vascular remodeling and hemodynamics in response to hypoxia/SU5416 were attenuated in Tph1(-/-) mice and further decreased after imatinib treatment. However, imatinib did not significantly further impact collagen deposition and collagen 3a1 expression in hypoxic Tph1(-/-) mice. Post hoc subgroup analysis suggests that patients with PAH with greater hemodynamic impairment showed significantly reduced 5-HT plasma levels after imatinib treatment compared with placebo. CONCLUSIONS: We report a novel mode of action for imatinib, demonstrating TPH1 down-regulation via inhibition of PDGFR-ß signaling. Our data reveal interplay between PDGF and 5-HT pathways within PAH, demonstrating TPH1-dependent imatinib efficacy in collagen-mediated mechanisms of fibrosis.

Performance characteristics of broth-only cultures after revision total joint arthroplasty.

BACKGROUND: Surgeons frequently obtain intraoperative cultures at the time of revision total joint arthroplasty. The use of broth or liquid medium before applying the sample to the agar medium may be associated with contamination and false-positive cultures; however, the degree to which this is the case is not known. QUESTIONS/PURPOSES: We (1) calculated the performance characteristics of broth-only cultures (sensitivity, specificity, positive predictive value, and negative predictive value) and (2) characterized the organisms identified in broth to determine whether a specific organism showed increased proclivity for true-positive periprosthetic joint infection (PJI). METHODS: A single-institution retrospective chart review was performed on 257 revision total joint arthroplasties from 2009 through 2010. One hundred ninety (74%) had cultures for review. All culture results, as well as treatment, if any, were documented and patients were followed for a minimum of 1 year for evidence of PJI. Cultures were measured as either positive from the broth only or broth negative. The true diagnosis of infection was determined by the Musculoskeletal Infection Society criteria during the preoperative workup or postoperatively at 1 year for purposes of calculating the performance characteristics of the broth-only culture. RESULTS: The sensitivity, specificity, positive predictive value, and negative predictive value were 19%, 88%, 13%, and 92%, respectively. The most common organism identified was coagulase-negative Staphylococcus (16 of 24 cases, 67%). Coagulase-negative Staphylococcus was present in all three true-positive cases; however, it was also found in 13 of the false-positive cases. CONCLUSIONS: The broth-only positive cultures showed poor sensitivity and positive predictive value but good specificity and negative predictive value. The good specificity indicates that it can help to rule in the presence of PJI; however, the poor sensitivity makes broth-only culture an unreliable screening test. We recommend that broth-only culture results be carefully scrutinized and decisions on the diagnosis and treatment of infection should be based specifically on the Musculoskeletal Infection Society criteria. LEVEL OF EVIDENCE: Level IV, diagnostic study. See Instructions for Authors for a complete description of levels of evidence.

Microsatellite loci and the complete mitochondrial DNA sequence characterized through next generation sequencing and de novo genome assembly for the critically endangered orange-bellied parrot, Neophema chrysogaster.

A suite of polymorphic microsatellite markers and the complete mitochondrial genome sequence was developed by next generation sequencing (NGS) for the critically endangered orange-bellied parrot, Neophema chrysogaster. A total of 14 polymorphic loci were identified and characterized using DNA extractions representing 40 individuals from Melaleuca, Tasmania, sampled in 2002. We observed moderate genetic variation across most loci (mean number of alleles per locus = 2.79; mean expected heterozygosity = 0.53) with no evidence of individual loci deviating significantly from Hardy-Weinberg equilibrium. Marker independence was confirmed with tests for linkage disequilibrium, and analyses indicated no evidence of null alleles across loci. De novo and reference-based genome assemblies performed using MIRA were used to assemble the N. chrysogaster mitochondrial genome sequence with mean coverage of 116-fold (range 89 to 142-fold). The mitochondrial genome consists of 18,034 base pairs, and a typical metazoan mitochondrial gene content consisting of 13 protein-coding genes, 2 ribosomal subunit genes, 22 transfer RNAs, and a single large non-coding region (control region). The arrangement of mitochondrial genes is also typical of Avian taxa. The annotation of the mitochondrial genome and the characterization of 14 microsatellite markers provide a valuable resource for future genetic monitoring of wild and captive N. chrysogaster populations. As found previously, NGS provides a rapid, low cost and reliable method for polymorphic nuclear genetic marker development and determining complete mitochondrial genome sequences when only a fraction of a genome is sequenced.

Copy number variation and transposable elements feature in recent, ongoing adaptation at the Cyp6g1 locus.

The increased transcription of the Cyp6g1 gene of Drosophila melanogaster, and consequent resistance to insecticides such as DDT, is a widely cited example of adaptation mediated by cis-regulatory change. A fragment of an Accord transposable element inserted upstream of the Cyp6g1 gene is causally associated with resistance and has spread to high frequencies in populations around the world since the 1940s. Here we report the existence of a natural allelic series at this locus of D. melanogaster, involving copy number variation of Cyp6g1, and two additional transposable element insertions (a P and an HMS-Beagle). We provide evidence that this genetic variation underpins phenotypic variation, as the more derived the allele, the greater the level of DDT resistance. Tracking the spatial and temporal patterns of allele frequency changes indicates that the multiple steps of the allelic series are adaptive. Further, a DDT association study shows that the most resistant allele, Cyp6g1-[BP], is greatly enriched in the top 5% of the phenotypic distribution and accounts for approximately 16% of the underlying phenotypic variation in resistance to DDT. In contrast, copy number variation for another candidate resistance gene, Cyp12d1, is not associated with resistance. Thus the Cyp6g1 locus is a major contributor to DDT resistance in field populations, and evolution at this locus features multiple adaptive steps occurring in rapid succession.

Molecular basis of adaptive shift in body size in Drosophila melanogaster: functional and sequence analyses of the Dca gene.

Latitudinal body size clines in animals conforming to Bergmann's rule occur on many continents but isolating their underlying genetic basis remains a challenge. In Drosophila melanogaster, the gene Dca accounts for approximately 5-10% of the natural wing size variation (McKechnie SW, Blacket MJ, Song SV, Rako L, Carroll X, Johnson TK, Jensen LT, Lee SF, Wee CW, Hoffmann AA. 2010. A clinally varying promoter polymorphism associated with adaptive variation in wing size in Drosophila. Mol Ecol. 19:775-784). We present here functional evidence that Dca is a negative regulator of wing size. A significant negative latitudinal cline of Dca gene expression was detected in synchronized third instar larvae. In addition, we clarified the evolutionary history of the three most common Dca promoter alleles (Dca237-1, Dca237-2, and Dca247) and showed that the insertion allele (Dca247), whose frequency increases with latitude, is associated with larger wing centroid size and higher average cell number in male flies. Finally, we showed that the overall linkage disequilibrium (LD) was low in the Dca promoter and that the insertion/deletion polymorphism that defines the Dca alleles was in strong LD with two other upstream sites. Our results provide strong support that Dca is a candidate for climatic adaptation in D. melanogaster.

A novel murine model of severe pulmonary arterial hypertension.

RATIONALE: The complex pathologies associated with severe pulmonary arterial hypertension (PAH) in humans have been a challenge to reproduce in mice due to the subtle phenotype displayed to PAH stimuli. OBJECTIVES: Here we aim to develop a novel murine model of PAH that recapitulates more of the pathologic processes, such as complex vascular remodeling and cardiac indices, that are not characteristic of alternative mouse models. METHODS: Inhibition of vascular endothelial growth factor receptor (VEGFR) with SU5416 combined with 3 weeks of chronic hypoxia was investigated. Hemodynamics, cardiac function, histological assessment of pulmonary vasculature, and molecular pathway analysis gauged the extent of PAH pathology development. MEASUREMENTS AND MAIN RESULTS: The combination of VEGFR inhibition with chronic hypoxia profoundly exacerbated all measures of PAH-like pathology when compared with hypoxia alone (> 45 mm Hg right ventricular pressure, > 0.35 right ventricular hypertrophy). The changes in pulmonary vascular remodeling in response to hypoxia were further enhanced on SU5416 treatment. Furthermore, hypoxia/SU5416 treatment steadily decreased cardiac output, indicating incipient heart failure. Molecular analysis showed a dysregulated transforming growth factor-ß/bone morphogenetic protein/Smad axis in SU5416- and/or hypoxia-treated mice as well as augmented induction of IL-6 and Hif-1α levels. These changes were observed in accordance with up-regulation of Tph1 and Pdgfr gene transcripts as well as a rise in platelet-rich serotonin. Biomarker analysis in response to VEGFR inhibition and/or hypoxia revealed distinct signatures that correlate with cytokine profiles of patients with idiopathic PAH. CONCLUSIONS: These data describe a novel murine model of PAH, which displays many of the hallmarks of the human disease, thus opening new avenues of investigation to better understand PAH pathophysiology.

Platelet-rich plasma does not reduce blood loss or pain or improve range of motion after TKA.

BACKGROUND: Numerous reports suggest the application of platelet-rich plasma (PRP) during TKA may decrease postoperative bleeding. Because excessive bleeding can increase postoperative pain and inflammation, use of PRP also reportedly decreases the need for narcotics and increases speed of recovery after TKA. Because previous investigations of PRP and TKA reflect a weak level of medical evidence, we sought to confirm these findings. QUESTIONS/PURPOSES: We asked whether an intraoperative application of PRP gel to the deep wound reduced postoperative bleeding after TKA. METHODS: We retrospectively reviewed the charts of all 134 patients who received an intraoperative application of PRP during TKA from November 2009 to April 2010 and all 139 patients undergoing TKA who did not receive PRP between September 2009 to November 2009. Patients' charts were reviewed to identify detailed data, including hemoglobin level, ROM, postoperative narcotic use, and length of hospital stay. Blood loss was determined by the hemoglobin drop on postoperative Day 2. RESULTS: The blood loss between study groups was similar (3.6 g/dL [study] versus 3.8 g/dL [controls]). Differences in passive ROM (88° versus 88°), narcotic requirement (27 versus 32 morphine equivalent), and length of stay (2.4 versus 2.6 days) were also similar. CONCLUSION: We found no clinically important differences in patients who received an intraoperative application of PRP compared with patients who did not receive PRP and therefore could not confirm the findings of previous studies.

Is it time to include vancomycin for routine perioperative antibiotic prophylaxis in total joint arthroplasty patients?

In an effort to reduce methicillin-resistant Staphylococcus aureus (MRSA) and overall periprosthetic joint infections (PJI), we switched the perioperative prophylactic antibiotic during total knee arthroplasty and total hip arthroplasty from cefazolin to vancomycin in June 2008. We retrospectively reviewed the total and MRSA PJI in 5036 primary total joint arthroplasties, as well as the cure rate of PJI from January 2006 to June 2008 (Ancef Period) and June 2008 to December 2010 (Vanco Period). With vancomycin, total PJI was significantly reduced (1.0%-0.5%) and MRSA PJI (0.23%-0.07%). Periprosthetic joint infections that occurred were more successfully treated with irrigation and debridement only, not requiring spacer (76.9% vs 22.2%). The use of vancomycin as the perioperative prophylactic antibiotic for primary total joint arthroplasties appeared to be effective in decreasing the rate of PJI and may result, when they occur, in infections with less virulent organisms.