Anterior vertebral body tethering shows clinically comparable shoulder balance outcomes to posterior spinal fusion.

PURPOSE: Posterior spinal fusion (PSF) is the current gold standard in surgical treatment for adolescent idiopathic scoliosis. Vertebral body tethering (VBT) is a fusionless alternative. Shoulder balance is an important metric for outcomes and patient satisfaction. Here we compare shoulder balance outcomes between PSF and VBT. METHODS: In this retrospective review, the pre-operative and post-operative absolute radiographic shoulder height (|RSH|) of 45 PSF patients were compared to 46 VBT patients. Mean values were compared and then collapsed into discrete groups (|RSH| GROUP) and compared. Patients were propensity score matched. Regression models based on pretest-posttest designs were used to compare procedure type on post-operative outcomes. RESULTS: Pre-operatively there were no differences in |RSH| between PSF and VBT, however, at latest post-operative follow-up PSF maintained a larger |RSH| imbalance compared to VBT (0.91 cm vs 0.63 cm, p = 0.021). In an ANCOVA regression, PSF was associated with a larger |RSH| imbalance compared to VBT, F(1, 88) = 5.76, p = 0.019. An ordinal logistic regression found that the odds ratio of being in a worse |RSH| GROUP for PSF vs VBT is 2.788 (95% CI = 1.099 to 7.075), a statistically significant effect χ2(1) = 4.658, p = 0.031. Results were similar in subgroup analyses of Lenke 1 and Lenke 2 patients, though to less statistical significance. CONCLUSION: While PSF was found to be associated with worse |RSH| outcomes, the actual numbers (2-3 mm) are unlikely to be clinically meaningful. Thus, in this analysis, VBT can be said to show comparable shoulder balance outcomes to PSF.

Does Medicare Insurance Mitigate Racial/Ethnic Disparities in Access to Lumbar Spinal Surgery When Compared to Commercial Insurance?

STUDY DESIGN: Retrospective cohort study. OBJECTIVE: Compare disparities in lumbar surgical care utilization in Commercially insured versus Medicare patients. SUMMARY OF BACKGROUND DATA: While disparities in spinal surgery have been previously described, less evidence exists on effective strategies to mitigate them. Theoretically, universal health care coverage under Medicare should improve health care access. MATERIALS AND METHODS: Utilizing National Inpatient Sample data (2003-2018), we included inpatient lumbar discectomy or laminectomy/fusion cases in black, white, or Hispanic patients aged 18-74 years, with Commercial or Medicare insurance. A multivariable Poisson distribution model determined race/ethnicity subgroup-specific rate ratios (RRs) of patients undergoing lumbar surgery compared to their respective population distribution (using US Census data) based on race/ethnicity, region, gender, primary payor, and age (Commercially insured age subgroups: 18-39, 40-54, and 55-64 y; Medicare age subgroup: 65-74 y). RESULTS: Of the 2,310,956 lumbar spine procedures included, 88.9%, 6.1%, and 5.0% represented white, black, and Hispanic patients, respectively. Among Commercially insured patients, black and Hispanic (compared to white) patients had lower rates of surgical care utilization; however, these disparities decreased with increasing age: black (RR=0.37, 95% CI: 0.37-0.38) and Hispanic patients (RR=0.53, 95% CI: 0.52-0.54) aged 18-39 years versus black (RR=0.72, 95% CI: 0.71-0.73) and Hispanic patients (RR=0.64, 95% CI: 0.63-0.65) aged 55-64 years. Racial/ethnic disparities persisted in Medicare patients, especially when compared to the neighboring age subgroup that was Commercially insured: black (RR=0.61, 95% CI: 0.60-0.62) and Hispanic patients (RR=0.61, 95% CI: 0.60-0.61) under Medicare. CONCLUSIONS: Disparities in surgical care utilization among black and Hispanic patients persist regardless of health care coverage, and an expansion of Medicare eligibility alone may not comprehensively address health care disparities. LEVEL OF EVIDENCE: Level III.

Assessment of Risk Factors and Rate of Conversion to Total Hip Arthroplasty Within 2 Years After Hip Arthroscopy Utilizing a Large Database of Commercially Insured Patients in the United States.

Background: The conversion rate of hip arthroscopy (HA) to total hip arthroplasty (THA) has been reported to be as high as 10%. Despite identifying factors that increase the risk of conversion, current studies do not stratify patients by type of arthroscopic procedure. Purpose/Hypothesis: To analyze the rate and predictors of conversion to THA within 2 years after HA. It was hypothesized that osteoarthritis (OA) and increased patient age would negatively affect the survivorship of HA. Study Design: Cohort study; Evidence level, 3. Methods: The IBM MarketScan database was utilized to identify patients who underwent HA and converted to THA within 2 years at inpatient and outpatient facilities between 2013 and 2017. Patients were split into 3 procedure cohorts as follows: (1) femoroacetabular osteoplasty (FAO), which included treatment for femoroacetabular impingement; (2) isolated debridement; and (3) isolated labral repair. Cohort characteristics were compared using standardized differences. Conversion rates between the 3 cohorts were compared using chi-square tests. The relationship between age and conversion was assessed using linear regression. Predictors of conversion were analyzed using multivariable logistic regression. The median time to conversion was estimated using Kaplan-Meier tests. Results: A total of 5048 patients were identified, and the rates of conversion to THA were 12.86% for isolated debridement, 8.67% for isolated labral repair, and 6.76% for FAO (standardized difference, 0.138). The isolated labral repair cohort had the shortest median time to conversion (isolated labral repair, 10.88 months; isolated debridement, 10.98 months; and FAO, 11.9 months [P = .034). For patients >50 years, isolated debridement had the highest rate of conversion at 18.8%. The conversion rate increased linearly with age. Factors that increased the odds of conversion to THA were OA, having an isolated debridement procedure, and older patient age (P < .05). Conclusion: Older patients and those with preexisting OA of the hip were at a significantly increased risk of failing HA and requiring a total hip replacement within 2 years of the index procedure. Younger patients were at low risk of requiring a conversion procedure no matter which arthroscopic procedure was performed.

There Is Wide Variation in Platelet-rich Plasma Injection Pricing: A United States Nationwide Study of Top Orthopaedic Hospitals.

BACKGROUND: Demand for platelet-rich plasma (PRP) injections for osteoarthritis has dramatically increased in recent years despite conflicting evidence regarding its efficacy and highly variable pricing in the top orthopaedic centers in the United States, because PRP is typically not covered by insurance. A previous study investigating the mean price of PRP injections obtained information only from centers advertising online the availability of PRP injections. Thus, there is a need for further clarification of the overall availability and variability in cost of PRP injections in the orthopaedic community as well as an analysis of relevant regional demographic and hospital characteristics that could be associated with PRP pricing. QUESTIONS/PURPOSES: Our study purposes were to (1) report the availability and price variation of knee PRP injections at top-ranked United States orthopaedic centers, (2) characterize the availability of pricing information for a PRP injection over the telephone, (3) determine whether hospital characteristics (Orthopaedic Score [ U . S. News & World Report measure of hospital orthopaedic department performance], size, teaching status, and rural-urban status) were associated with PRP injection availability and pricing, and (4) characterize the price variation, if it exists, of PRP injections in three metropolitan areas and individual institutions. METHODS: In this prospective study, a scripted telephone call to publicly listed clinic telephone numbers was used to determine the availability and price estimate (amount to be paid by the patient) of a PRP injection for knee osteoarthritis from the top 25 hospitals from each United States Census region selected from the U.S. News & World Report ranking of best hospitals for orthopaedics. Univariable analyses examined factors associated with PRP injection availability and willingness to disclose pricing, differences across regions, and the association between hospital characteristics (Orthopaedic Score, size, teaching status, and rural-urban status) and pricing. The Orthopaedic Score is a score assigned to each hospital by U . S. News & World Report as a measure of hospital performance based partly on patient outcomes, with higher scores indicating better outcomes. RESULTS: Overall, 87% (87 of 100) of respondents stated they offered PRP injections. Pricing ranged from USD 350 to USD 2815 (median USD 800) per injection, with the highest prices in the Northeast. The largest price range was in the Midwest, where more than two-thirds of PRP injections given at hospitals that disclosed pricing cost USD 500 to USD 1000. Of the hospitals that offered PRP injections, 68% (59 of 87) were willing to disclose price information over the telephone. PRP injection pricing was inversely correlated with hospital Orthopaedic Score (-3% price change [95% CI -5% to -1%]; p = 0.01) and not associated with any of the other hospital characteristics that were studied, such as patient population median income and total hospital expenses. An intracity analysis revealed wide variations in PRP pricing in all metropolitan areas that were analyzed, ranging from a minimum of USD 300 within 10 miles of metropolitan area B to a maximum of USD 1269 within 20 miles of metropolitan area C. CONCLUSION: We found that although PRP injections are widely available, pricing continues to be a substantial financial burden on patients, with large price variability among institutions. We also found that if patients are willing to shop around in a metropolitan area, there is potential to save a meaningful amount of money. CLINICAL RELEVANCE: As public interest in biologics in orthopaedic surgery increases, knowledge of its pricing should be clarified to consumers. The debated efficacy of PRP injections, combined with our findings that it is an expensive out-of-pocket procedure, suggests that PRP has limited cost-effectiveness, with variable, discrete pricing. As such, the price of PRP injections should be clearly disclosed to patients so they can make informed healthcare decisions.

Synthesis of 'joint class' curricula at high volume joint replacement centres and a preliminary model for development and evaluation.

RATIONALE: Preoperative patient education through 'joint class' has potential to improve quality of care for total joint replacement (TJR). However, no formal guidance exists regarding curriculum content, potentially resulting in inter-institutional variation. OBJECTIVE: We aimed to (a) synthesize curriculum components of 'joint classes' across high-volume institutions and (b) develop a preliminary theory of change model for development and evaluation guided by the existing curricula and related literature. METHODS: We reviewed 'joint class' curricula from the websites of the 10 highest-volume TJR centres (by average annual 2017-2019 volume) that publicly disclosed this information. Two reviewers qualitatively compared available content and noted common categories, which were synthesized into key domains across institutions. We then reviewed the PubMed database for literature on pre-TJR patient education and education needs in the past 10 years. Drawing on our curriculum synthesis and related literature, we proposed a theory of change model: hypothesized mechanisms through which 'joint class' confers benefits to patients and health systems. RESULTS: We identified 30 categories in our review of existing class content, which we synthesized into seven key domains: (I) Practical Elements, (II) Logistics, (III) Medical Information, (IV) Modifiable Risk Factors, (V) Expected Outcomes, (VI) Patient Role in Recovery and (VII) Enhanced Education. Variation across institutions was noted. Our preliminary model based on the curriculum synthesis and related literature on the impact of 'joint class' includes three levels: (1) Practical Elements ('joint class' accessibility and information quality), (2) Class Goals (increased health literacy, increased adherence, risk mitigation, realistic expectations, and reduced anxiety) and (3) Target Outcomes (improved clinical outcomes, positive patient experience and increased patient satisfaction). CONCLUSION: Our synthesis identified core common topics included in pre-TJR education but also highlighted variation across institutions, supporting opportunities for standardization. Clinicians and researchers can use our preliminary model to systematically develop and evaluate 'joint classes,' with the goal of establishing a standard of care for TJR preoperative education.

Understanding Prescribing Practices and Patient Experiences with Renin Angiotensin System Inhibitors Use in Chronic Kidney Disease: A Qualitative Study.

INTRODUCTION: Angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin receptor blockers (ARBs) improve outcomes but are underutilized in patients with chronic kidney disease (CKD). Little is known about reasons for discontinuation and lack of reinitiating these medications. We aimed to explore clinicians' and patients' experiences and perceptions of ACEI/ARB use in CKD. METHODS: A multi-profession sample of health care clinicians and patients with documented ACEI/ARB-associated side effects in the past 6 months. Participants were recruited from 2 Veterans Affairs healthcare systems in Texas and Tennessee. A total of 15 clinicians and 10 patients completed interviews. We used inductive and deductive qualitative data analysis approaches to identify themes related to clinician and patient experiences with ACEI/ARB. Thematic analysis focused on prescribing decisions and practices, clinical guidelines, and perception of side effects. Data were analyzed as they amassed, and recruitment was stopped at the point of thematic saturation. RESULTS: Clinicians prescribe ACEI/ARB for blood pressure control and kidney protection and underscored the importance of these medications in patients with diabetes. While clinicians described providing comprehensive patient education about ACEI/ARB in CKD, patient interviews revealed significant knowledge gaps about CKD and ACEI/ARB use. Many patients were unaware of their CKD status, and some did not know why they were prescribed ACEI/ARB. Clinicians' drug management strategies varied widely, as did their understanding of prescribing guidelines. They identified structural and patient-level barriers to prescribing and many endorsed the development of a decision support tool to facilitate ACEI/ARB prescribing and management. DISCUSSION/CONCLUSION: Our qualitative study of clinicians and providers identified key target areas for improvement to increase ACEI/ARB utilization in patients with CKD with the goal to improve long-term outcomes in high-risk patients. These findings will also inform the development of a decision support tool to assist with prescribing ACEI/ARBs for patients with CKD.

Sleep Disturbance Trends in the Short-Term Postoperative Period for Patients Undergoing Total Joint Arthroplasty.

BACKGROUND: Patients undergoing total joint arthroplasty (TJA) often experience preoperative/postoperative sleep disturbances. Although sleep quality generally improves > 6 months after surgery, patterns of sleep in the short-term postoperative period are poorly understood. This study sought to (1) characterize sleep disturbance patterns over the 3-month postoperative period and (2) investigate clinical and sociodemographic factors associated with 3-month changes in sleep. METHODS: This retrospective analysis of prospectively collected data included 104 primary elective TJA patients. Patients were administered the PROMIS Sleep Disturbance questionnaire preoperatively and at 2 weeks, 6 weeks, and 3 months postoperatively. Median sleep scores were compared between time points using Wilcoxon signed-rank tests, stratified by preoperative sleep impairment. A multivariable logistic regression model identified factors associated with 3-month clinically improved sleep. RESULTS: The percentage of patients reporting sleep within normal limits increased over time: 54.8% preoperatively and 58.0%, 62.5%, and 71.8% at 2 weeks, 6 weeks, and 3 months post-TJA, respectively. Patients with normal preoperative sleep experienced a transient 4.7-point worsening of sleep at 2 weeks ( P = 0.003). For patients with moderate/severe preoperative sleep impairment, sleep significantly improved by 5.4 points at 2 weeks ( P = 0.002), with improvement sustained at 3 months. In multivariable analysis, patients undergoing total hip arthroplasty (versus knee; OR: 3.47, 95% CI: 1.06 to 11.32, P = 0.039) and those with worse preoperative sleep scores (OR: 1.13, 95% CI: 1.04 to 1.23, P = 0.003) were more likely to achieve clinically improved sleep from preoperatively to 3 months postoperatively. DISCUSSION: Patients experience differing patterns in postoperative sleep changes based on preoperative sleep disturbance. Hip arthroplasty patients are also more likely to experience clinically improved sleep by 3 months compared with knee arthroplasty patients. These results may be used to counsel patients on postoperative expectations and identify patients at greater risk of impaired postoperative sleep. STUDY DESIGN: Retrospective analysis of prospectively collected data.

Documented Adverse Drug Reactions and Discontinuation of Angiotensin-Converting Enzyme Inhibitors and Angiotensin Receptor Blockers in Chronic Kidney Disease.

INTRODUCTION: Angiotensin-converting enzyme inhibitors (ACEis) and angiotensin receptor blockers (ARBs) are frequently discontinued in patients with chronic kidney disease (CKD). Documented adverse drug reactions (ADRs) in medical records may provide insight into the reasons for treatment discontinuation. METHODS: In this retrospective cohort of US veterans from 2005 to 2019, we identified individuals with CKD and a current prescription for an ACEi or ARB (current user group) or a discontinued prescription within the preceding 5 years (discontinued group). Documented ADRs in structured datasets associated with an ACEi or ARB were categorized into 17 pre-specified groups. Logistic regression assessed associations of documented ADRs with treatment discontinuation. RESULTS: There were 882,441 (73.0%) individuals in the current user group and 326,794 (27.0%) in the discontinued group. There were 26,434 documented ADRs, with at least one documented ADR in 7,520 (0.9%) current users and 9,569 (2.9%) of the discontinued group. ADR presence was associated with treatment discontinuation, aOR 4.16 (95% CI: 4.03, 4.29). The most common documented ADRs were cough (37.3%), angioedema (14.2%), and allergic reaction (10.4%). ADRs related to angioedema (aOR 3.81, 95% CI: 3.47, 4.17), hyperkalemia (aOR 2.03, 95% CI: 1.84, 2.24), peripheral edema (aOR 1.53, 95% CI: 1.33, 1.77), or acute kidney injury (aOR 1.32, 95% CI: 1.15, 1.51) were associated with treatment discontinuation. CONCLUSION: ADRs leading to drug discontinuation were infrequently documented. ADR types were differentially associated with treatment discontinuation. An understanding of which ADRs lead to treatment discontinuation provides an opportunity to address them at a healthcare system level.

Hip Arthroscopy Trends: Increasing Patient Out-of-Pocket Costs, Lower Surgeon Reimbursement, and Cost Reduction With Utilization of Ambulatory Surgery Centers.

PURPOSE: To (1) report on trends in immediate procedure reimbursement, patient out-of-pocket expenditures, and surgeon reimbursement in hip arthroscopy (2) compare trends in ambulatory surgery centers (ASC) versus outpatient hospitals (OH) utilization; (3) quantify the cost differences (if any) associated with ASC versus OH settings; and 4) determine the factors that predict ASC utilization for hip arthroscopy. METHODS: The cohort for this descriptive epidemiology study was any patient over 18 years identified in the IBM MarketScan Commercial Claims Encounter database who underwent an outpatient hip arthroscopy, identified by Current Procedural Terminology codes, in the United States from 2013 to 2017. Immediate procedure reimbursement, patient out-of-pocket expenditure, and surgeon reimbursement were calculated, and a multivariable model was used to determine the influence of specific factors on these outcome variables. Statistically significant P values were less than .05, and significant standardized differences were more than 0.1. RESULTS: The cohort included 20,335 patients. An increasing trend in ASC utilization was observed (P = .001), and ASC utilization for hip arthroscopy was 32.4% in 2017. Patient out-of-pocket expenditures for femoroacetabular impingement surgery increased 24.3% over the study period (P = .003), which was higher than the rate for immediate procedure reimbursement (4.2%; P = .007). ASCs were associated with $3,310 (28.8%; P = .001) reduction in immediate procedure reimbursement and $47 (6.2%; P = .001) reduction in patient out-of-pocket expenditure per hip arthroscopy. CONCLUSIONS: ASCs provide a significant cost difference for hip arthroscopy. Although there is an increasing trend toward ASC utilization, it remains relatively low at 32.4% in 2017. Thus, there are opportunities for expanded ASC utilization, which is associated with significant immediate procedure reimbursement difference of $3,310 and patient out-of-pocket expenditure difference of $47 per hip arthroscopy case, ultimately benefiting healthcare systems, surgeons, and patients alike. LEVEL OF EVIDENCE: Level III, retrospective comparative trial.

Ambulatory Surgery Centers Significantly Decrease Total Health Care Expenditures in Primary Anterior Cruciate Ligament Reconstruction.

BACKGROUND: Anterior cruciate ligament reconstruction (ACLR) is a commonly performed orthopaedic procedure. The volume and cost of ACLR procedures are increasing annually, but the drivers of these cost increases are not well described. PURPOSE: To analyze the modifiable drivers of total health care utilization (THU), immediate procedure reimbursement, and surgeon reimbursement for patients undergoing ACLR using a large national commercial insurance database from 2013 to 2017. STUDY DESIGN: Descriptive epidemiology study. METHODS: For this study, the cohort consisted of patients identified in the MarketScan Commercial Claims and Encounters database who underwent outpatient arthroscopic ACLR in the United States from 2013 to 2017. Patients with Current Procedural Terminology code 29888 were included. THU was defined as the sum of any payment related to the ACLR procedure from 90 days preoperatively to 180 days postoperatively. A multivariable model was utilized to describe the patient- and procedure-related drivers of THU, immediate procedure reimbursement, and surgeon reimbursement. RESULTS: There were 34,862 patients identified. On multivariable analysis, the main driver of THU and immediate procedure reimbursement was an outpatient hospital as the surgical setting (US$6789 increase in THU). The main driver of surgeon reimbursement was an out-of-network surgeon (US$1337 increase). Health maintenance organization as the insurance plan type decreased THU, immediate procedure reimbursement, and surgeon reimbursement (US$955, US$108, and US$38 decrease, respectively, compared with preferred provider organization; P < .05 for all). CONCLUSION: Performing procedures in more cost-efficient ambulatory surgery centers had the largest effect on decreasing health care expenditures for ACLR. Health maintenance organizations aided in cost-optimization efforts as well, but had a minor effect on surgeon reimbursement. Overall, this study increases transparency into what drives reimbursement and serves as a foundation for how to decrease health care expenditures related to ACLR.

Ethical challenges in genetic research among Philippine Indigenous Peoples: Insights from fieldwork in Zamboanga and the Sulu Archipelago.

The Philippines, with the recent discovery of an archaic hominin in Luzon and an extensive ethnolinguistic diversity of more than 100 Indigenous peoples, is crucial to understanding human evolution and population history in Island Southeast Asia. Advances in DNA sequencing technologies enable the rapid generation of genomic data to robustly address questions about origins, relatedness, and population movements. With the increased genetic sampling in the country, especially by international scientists, it is vital to revisit ethical rules and guidelines relevant to conducting research among Indigenous peoples. Our team led fieldwork expeditions between 2019 and February 2020 in Zamboanga and the Sulu Archipelago, a chain of islands connecting the Mindanao and Borneo landmasses. The trips concluded with a collection of 2,149 DNA samples from 104 field sites. We present our fieldwork experience among the mostly sea-oriented Sama-Bajaw and Tausug-speaking communities and propose recommendations to address the ethical challenges of conducting such research. This work contributes toward building an enabling research environment in the Philippines that respects the rights and autonomy of Indigenous peoples, who are the rightful owners of their DNA and all genetic information contained therein.

Interprofessional Collaboration Between a Clinical Pharmacist and Specialty Physicians to Treat Hepatitis C in an Interdisciplinary Medical Practice Setting.

Objective: Describes the activities of a clinical pharmacist in a gastroenterology (GI) clinic providing services to hepatitis C virus (HCV) patients, with a focus on practice management activities and tools. Practice Description: Located inside a GI specialty clinic in Fort Worth, Texas, the pharmacist provides comprehensive medication management under a collaborative practice agreement (CPA). Once referred by the GI physician, the pharmacist has face-to-face patient visits, develops the care plan, orders medications, and follows patients through sustained virologic response and the development of a hepatocellular carcinoma surveillance plan. Practice Innovation: The role of pharmacists in the management of HCV is important to understand. This article details a pharmacist-led clinic in an open GI medical practice. Evaluation: A retrospective chart review study was conducted to assess outcomes related to the integration of the clinical pharmacist. Methods: Completed by the study team, this study included manual chart reviews of patients with the ambulatory care pharmacist-driven HCV practice to pull data and information that were then tabulated using Qualtrics. Results: A total of 95 charts were surveyed, 78 records were created, and 49 patients were started on direct-acting antiviral (DAA) treatment by the pharmacist. Patients required multiple pharmacist communication actions. The minimum duration of the pharmacist service was 6 months and could extend more than 9 months depending on the time it took to get the patient started on medication. Pharmacist integration into the practice resulted in improved intake for the GI clinic, improved interprofessional interaction, and increased utilization of newer treatment modalities for HCV which feature cure rates up to 99% with limited side effects. Conclusion: Clinical pharmacists are well positioned to help navigate patients through the complexities of the medication use system, medication access, drug interactions and adverse effects, promote medication adherence, and allow patients to start and complete therapy.

Coupled Natural Fusion Enzymes in a Novel Biocatalytic Cascade Convert Fatty Acids to Amines.

Tambjamine YP1 is a pyrrole-containing natural product. Analysis of the enzymes encoded in the Pseudoalteromonas tunicata "tam" biosynthetic gene cluster (BGC) identified a unique di-domain biocatalyst (PtTamH). Sequence and bioinformatic analysis predicts that PtTamH comprises an N-terminal, pyridoxal 5'-phosphate (PLP)-dependent transaminase (TA) domain fused to a NADH-dependent C-terminal thioester reductase (TR) domain. Spectroscopic and chemical analysis revealed that the TA domain binds PLP, utilizes l-Glu as an amine donor, accepts a range of fatty aldehydes (C7-C14 with a preference for C12), and produces the corresponding amines. The previously characterized PtTamA from the "tam" BGC is an ATP-dependent, di-domain enzyme comprising a class I adenylation domain fused to an acyl carrier protein (ACP). Since recombinant PtTamA catalyzes the activation and thioesterification of C12 acid to the holo-ACP domain, we hypothesized that C12 ACP is the natural substrate for PtTamH. PtTamA and PtTamH were successfully coupled together in a biocatalytic cascade that converts fatty acids (FAs) to amines in one pot. Moreover, a structural model of PtTamH provides insights into how the TA and TR domains are organized. This work not only characterizes the formation of the tambjamine YP1 tail but also suggests that PtTamA and PtTamH could be useful biocatalysts for FA to amine functional group conversion.

BioWF: A Naturally-Fused, Di-Domain Biocatalyst from Biotin Biosynthesis Displays an Unexpectedly Broad Substrate Scope.

The carbon backbone of biotin is constructed from the C7 di-acid pimelate, which is converted to an acyl-CoA thioester by an ATP-dependent, pimeloyl-CoA synthetase (PCAS, encoded by BioW). The acyl-thioester is condensed with ʟ-alanine in a decarboxylative, Claisen-like reaction to form an aminoketone (8-amino-7-oxononanoic acid, AON). This step is catalysed by the pyridoxal 5'-phosphate (PLP)-dependent enzyme (AON synthase, AONS, encoded by BioF). Distinct versions of Bacillus subtilis BioW (BsBioW) and E. coli BioF (EcBioF) display strict substrate specificity. In contrast, a BioW-BioF fusion from Corynebacterium amycolatum (CaBioWF) accepts a wider range of mono- and di-fatty acids. Analysis of the active site of the BsBioW : pimeloyl-adenylate complex suggested a key role for a Phe (F192) residue in the CaBioW domain; a F192Y mutant restored the substrate specificity to pimelate. This surprising substrate flexibility also extends to the CaBioF domain, which accepts ʟ-alanine, ʟ-serine and glycine. Structural models of the CaBioWF fusion provide insight into how both domains interact with each other and suggest the presence of an intra-domain tunnel. The CaBioWF fusion catalyses conversion of various fatty acids and amino acids to a range of AON derivatives. Such unexpected, natural broad substrate scope suggests that the CaBioWF fusion is a versatile biocatalyst that can be used to prepare a number of aminoketone analogues.

A Laboratory Exercise Simulating Antibody and Antigen Reactions of the Ouchterlony Double Immunodiffusion Assay Using Inorganic Salts.

The Ouchterlony double immunodiffusion assay is a serological technique used in the detection of antibodies and antigens for diagnostic purposes and also used in immunology laboratory courses as a common teaching assay where students observe the geometrical precipitation line patterns that form in the agarose, elucidating degrees of homology between antigens. In this classical technique, students must wait several hours to days to obtain results when protein antigens and antibodies are used. Furthermore, these proteins degrade over time if not frozen or stored in the refrigerator and are the most expensive consumables of the laboratory exercise. In this study, inexpensive and commonly used inorganic ionic salt solutions that are stable and can be stored at room temperature for several years were used to mimic antigens and antibodies. The precipitation lines started to form in the agarose plates after 15 min and fully developed within an hour, showing different geometrical precipitation patterns and spur formations that could be identified by students as full identity, partial identity, and nonidentity between the simulated (inorganic) antigens. Students conducting this exercise in a combined lecture and laboratory immunology course were able to finish the exercise as well as record and discuss results within class time, and tvhey showed increased interest in the laboratory exercise and had a better understanding of antibody-antigen reactions. Thus, this simulated laboratory experiment is an inexpensive, safe, and fast exercise that allows students to observe precipitations reactions of the Ouchterlony assay within the class session time.

Genetic origins and diversity of bushpigs from Madagascar (Potamochoerus larvatus, family Suidae).

The island of Madagascar, situated off the southeast coast of Africa, shows the first evidence of human presence ~ 10,000 years ago; however, other archaeological data indicates a settlement of the modern peoples of the island distinctly more recent, perhaps > 1500 years ago. Bushpigs of the genus Potamochoerus (family Suidae), are today widely distributed in Madagascar and presumed to have been introduced from Africa at some stage by human immigrants to the island. However, disparities about their origins in Madagascar have been presented in the literature, including the possibility of endemic subspecies, and few empirical data are available. Furthermore, the separation of bushpigs in Madagascar from their mainland relatives may have favoured the evolution of a different repertoire of immune genes first due to a founder effect and then as a response to distinct pathogens compared to their ancestors. Molecular analysis confirmed the species status of the bushpig in Madagascar as P. larvatus, likely introduced from the central region of southern Africa, with no genetic evidence for the recognition of eastern and western subspecies as suggested from previous cranial morphology examination. Investigation of the immunologically important SLA-DQB1 peptide-binding region showed a different immune repertoire of bushpigs in Madagascar compared to those on the African mainland, with seventeen exon-2 haplotypes unique to bushpigs in Madagascar (2/28 haplotypes shared). This suggests that the MHC diversity of the Madagascar populations may have enabled Malagasy bushpigs to adapt to new environments.

European and Asian contribution to the genetic diversity of mainland South American chickens.

Chickens (Gallus gallus domesticus) from the Americas have long been recognized as descendants of European chickens, transported by early Europeans since the fifteenth century. However, in recent years, a possible pre-Columbian introduction of chickens to South America by Polynesian seafarers has also been suggested. Here, we characterize the mitochondrial control region genetic diversity of modern chicken populations from South America and compare this to a worldwide dataset in order to investigate the potential maternal genetic origin of modern-day chicken populations in South America. The genetic analysis of newly generated chicken mitochondrial control region sequences from South America showed that the majority of chickens from the continent belong to mitochondrial haplogroup E. The rest belongs to haplogroups A, B and C, albeit at very low levels. Haplogroup D, a ubiquitous mitochondrial lineage in Island Southeast Asia and on Pacific Islands is not observed in continental South America. Modern-day mainland South American chickens are, therefore, closely allied with European and Asian chickens. Furthermore, we find high levels of genetic contributions from South Asian chickens to those in Europe and South America. Our findings demonstrate that modern-day genetic diversity of mainland South American chickens appear to have clear European and Asian contributions, and less so from Island Southeast Asia and the Pacific Islands. Furthermore, there is also some indication that South Asia has more genetic contribution to European chickens than any other Asian chicken populations.

East African origins for Madagascan chickens as indicated by mitochondrial DNA.

The colonization of Madagascar by Austronesian-speaking people during AD 50-500 represents the most westerly point of the greatest diaspora in prehistory. A range of economically important plants and animals may have accompanied the Austronesians. Domestic chickens (Gallus gallus) are found in Madagascar, but it is unclear how they arrived there. Did they accompany the initial Austronesian-speaking populations that reached Madagascar via the Indian Ocean or were they late arrivals with Arabian and African sea-farers? To address this question, we investigated the mitochondrial DNA control region diversity of modern chickens sampled from around the Indian Ocean rim (Southeast Asia, South Asia, the Arabian Peninsula, East Africa and Madagascar). In contrast to the linguistic and human genetic evidence indicating dual African and Southeast Asian ancestry of the Malagasy people, we find that chickens in Madagascar only share a common ancestor with East Africa, which together are genetically closer to South Asian chickens than to those in Southeast Asia. This suggests that the earliest expansion of Austronesian-speaking people across the Indian Ocean did not successfully introduce chickens to Madagascar. Our results further demonstrate the complexity of the translocation history of introduced domesticates in Madagascar.

Origin and timing of New Zealand's earliest domestic chickens: Polynesian commensals or European introductions?

Human settlers transported chickens (Gallus gallus domesticus) to most East Polynesian archipelagos between AD 1000 and 1300; however, it has long been assumed that New Zealand was an exception. Despite the fact that chicken bones have been recovered from localities of early archaeological middens in New Zealand, their age and genetic relationships have never been critically assessed. Here, we test the assumption that chickens were not introduced to New Zealand during prehistory through ancient DNA and radiocarbon analyses of chicken bones from sites of Maori middens containing prehistoric material. The chickens belong to the widespread mitochondrial control region haplogroup E. Radiocarbon dating reveals that the bones are not prehistoric, but are still the earliest chicken remains known from New Zealand. Two of the bones pre-date permanent European settlement (ca 1803s onwards) but overlap with the arrival of James Cook's second voyage (1773-1774), and, therefore, they are likely to be chickens, or progeny thereof, liberated during that voyage. Our results support the idea that chickens were first introduced to New Zealand by Europeans, and provide new insights into Maori uptake and integration of resources introduced during the early post-European period.

Quantitative determination of tilmicosin in canine serum by high performance liquid chromatography-tandem mass spectrometry.

A highly sensitive and quantitative LC/MS/MS assay for the determination of tilmicosin in serum has been developed and validated. For sample preparation, 0.2 mL of canine serum was extracted with 3 mL of methyl tert-butyl ether. The organic layer was transferred to a new vessel and dried under nitrogen. The sample was then reconstituted for analysis by high performance liquid chromatography-tandem mass spectrometry. A Phenomenex Luna C8(2) analytical column was used for the chromatographic separation. The eluent was subsequently introduced to the mass spectrometer by electrospray ionization. A single range was validated for 50-5000 ng/mL for support of toxicokinetic studies. The inter-day relative error (inaccuracy) for the LLOQ samples ranged from -5.5% to 0.3%. The inter-day relative standard deviations (imprecision) at the respective LLOQ levels were < or =10.1%.