Extra-articular Osteotomy for the Management of Intra-articular Tibial Plateau Valgus Malunion.

OBJECTIVES: To report a case series of extra-articular osteotomies for the management of intra-articular tibial plateau malunions and to assess the ability to correct deformity and improve knee range of motion (ROM). METHODS: . DESIGN: Retrospective case series. SETTING: Academic, tertiary, referral center. PATIENT SELECTION CRITERIA: Adult patients with tibial plateau fracture malunion treated with extra-articular osteotomy of the femur and/or tibia between 2014 and 2023. OUTCOME MEASURES AND COMPARISON: Mechanical axis deviation (MAD), medial proximal tibia angle (MPTA), lateral distal femoral angle (LDFA), and posterior proximal tibia angle (PPTA) correction; knee ROM; and time to weight bearing. RESULTS: There were 7 patients included, 6 (85.7%) were female and 1 (14.3%) were male. The median age was 43.5 years (IQR 38.5-51, range 32-62). Four (57.1%) patients were treated with a high tibial osteotomy (HTO), and 3 (42.9%) patients were treated with an HTO and distal femoral osteotomy. One patient had concomitant supramalleolar osteotomy with HTO to address distal tibia procurvatum and valgus. Four were treated with hexapod frames, and 3 were treated with plates and screws. Median follow-up was 22.5 months (IQR 10.5-107 months, range 7-148 months). Surgical intervention corrected median radiographic measures of valgus malalignment preoperatively relative to postoperative values. This included MAD (42.5 mm-0 mm), valgus angle (12.5 degrees-1.5 degrees), MPTA (95 degrees-88.0°), and LDFA (86.0°-87.3 degrees). Surgical intervention increased maximal knee range of motion preoperatively to postoperatively. Median time to full weight bearing was 81.5 days (IQR 46-57 days, range 41-184 days). Two patients were converted to total knee arthroplasty after 5 and 10 years following HTO with hexapod frame. CONCLUSIONS: Extra-articular osteotomy is an effective treatment for addressing intra-articular malunion after tibia plateau fractures. It is effective in correcting the MAD, valgus deformity, MPTA, LDFA, PPTA, and improving knee ROM (measured through knee extension and flexion). LEVEL OF EVIDENCE: Therapeutic Level IV. See Instructions for Authors for a complete description of levels of evidence.

Percutaneous removal of hardware after orthopedic surgery: Is it safe? and What are patients saying?

OBJECTIVES: To analyze our patient's complication profile and rate after removal of hardware (ROH) surgery, and survey our patients to ask their overall status and improvement in symptomatology post-operatively. DESIGN: Retrospective chart review and survey. SETTING: Academic, tertiary referral center. PATIENTS/PARTICIPANTS: 173 patients with 314 pieces of hardware. Seventy-six patients (43.9%) responded to our survey. INTERVENTION: ROH surgery. MAIN OUTCOME MEASUREMENTS: Patient demographics and complications were recorded. All patients were sent a brief 3-question survey which asked: (1) Why did you get your hardware removed? (2) How did your overall status change after ROH? (3) How did the ROH affect your stiffness, pain, swelling, and mobility? RESULTS: There were 10 complications (5.5%): 5 infections, 2 with unresolved pain, 1 hematoma, 1 chronic regional pain syndrome exacerbation, and 1 recurrent deformity. All infections were treated with oral antibiotics and improved. All other complications resolved with treatment except for the patient who developed recurrent deformity. Patients underwent ROH surgery because their doctor suggested it (76.3%) and to improve mobility (39.5%). 86.9% reported their overall status improved after ROH. They improved regarding stiffness (73.7%), pain (73.6%), swelling (61.8%), and mobility (76.3%). Similar results were seen among different implants removed. CONCLUSIONS: The majority of patients who underwent percutaneous ROH were satisfied. They reported improvement in stiffness, pain, swelling and mobility (greatest improvement). The complication rate was low (5.5%). ROH can be a meaningful operation to patients allowing them to improve their quality of life with a low complication rate. LEVEL OF EVIDENCE: Level IV.

Distal Femoral Osteotomy for the Management of Coronal Deformity Following Total Knee Arthroplasty: A Report of 2 Cases.

CASE: We present 2 cases demonstrating the management of coronal malalignment with an existing total knee arthroplasty using opening wedge distal femur osteotomy. The mechanical axis was corrected, and the primary total knee implants were maintained. Patients resumed full activity 3 to 4 months after surgery. CONCLUSION: In certain cases, opening wedge distal femoral osteotomy is an option to correct coronal malalignment with an existing total knee arthroplasty without revising the implant.

Signficant Femoral Version Abnormalities and Patient-Reported Quality of Life.

INTRODUCTION: The purpose of this study was to determine how the Limb Deformity-Modified Scoliosis Research Society (LD-SRS) and Patient-Reported Outcomes Measurement Information System (PROMIS) questionnaire scores differ between patients with femoral version abnormalities and healthy control subjects. METHODS: A retrospective database review identified patients with femoral version abnormalities between December 2018 and September 2022. A total of 21 adult patients scheduled for femoral derotational osteotomy and 33 control subjects were included. All individuals completed the LD-SRS and PROMIS questionnaires. RESULTS: Patients with femoral version abnormalities reported significantly worse scores than control subjects on all LD-SRS and PROMIS domains: LD-SRS (Total [3.46 ± 0.66 vs. 4.58 ± 0.37, P < 0.001]; Function/Activity [3.48 ± 0.83 vs. 4.44 ± 0.4, P < 0.001]; Mental Health [3.41 ± 0.96 vs. 4.3 ± 0.73, P < 0.001]; Pain [3.55 ± 0.9 vs. 4.81 ± 0.31, P < 0.001]; and Self-Image/Appearance [3.37 ± 0.79 vs. 4.75 ± 0.43, P < 0.001]) and PROMIS (Function [41.6 ± 7.58 vs. 60.0 ± 7.28, P < 0.001]; Pain Intensity [45.85 ± 8.04 vs. 33.7 ± 4.89, P < 0.001]; Pain Interference [56.78 ± 9.63 vs. 42.8 ± 6.6, P < 0.001]; Global Mental Health [47.97 ± 9.68 vs. 55.3 ± 7.81, P = 0.004]; and Global Physical Health [45.23 ± 7.49 vs. 58.2 ± 7.07, P < 0.001]). DISCUSSION: Patients with femoral version abnormalities reported markedly worse quality of life as measured on the LD-SRS and PROMIS scores compared with healthy control subjects. The combination of these two surveys effectively captures the multifaceted quality-of-life-deficit individuals with excessive femoral version may experience.

Staged Extra-Articular Deformity Correction in the Setting of Total Knee Arthroplasty.

Background: Extra-articular lower-leg deformities mandate unique considerations when planning total knee arthroplasty (TKA). Poor limb alignment may increase perioperative complications and cause early implant failure. This study reports on the safety and efficacy of staged, extra-articular deformity correction about the knee in the setting of osteoarthritis and TKA. Methods: A retrospective review was conducted from December 2007 to December 2019 identifying 30 deformities in 27 patients (average age: 52.7 years; range 31-74) who underwent staged surgical correction of extra-articular deformity in preparation for TKA. Patient demographics, surgical details, clinical and radiographic measurements, severity of knee arthritis, and complications were collected. Results: There were 17 femur and 12 tibia deformities. There was an average improvement of 14.7° of deformity measured in the coronal plane and 12.7° of deformity in the sagittal plane in the femur and 13.5° in the coronal plane and 10.3° in the sagittal plane in the tibia. Leg length discrepancies improved by 26 mm on average (1-100 mm). After an average 3.1-year follow-up, 12 out of 27 patients proceeded with primary or revision TKA. There were no cases of blood transfusion, nerve palsy, or compartment syndrome, and all patients achieved bony union. Conclusions: Staged, extra-articular deformity correction is a safe and effective approach to improve limb alignment in the setting of knee osteoarthritis and TKA.

The Management of Tibial Bone Defects: A Multicenter Experience of Hexapod and Ilizarov Frames.

INTRODUCTION: Bone defects may be managed with bone transport or acute shortening and lengthening using circular external fixation devices. We performed a multicenter retrospective cohort study to compare the outcomes between the Ilizarov frames and hexapod frames for the management of bone defects. METHODS: Patients treated for bone defects using either Ilizarov or hexapod frames were included for analysis in two specialist institutions. Primary outcomes were time to consolidation, bone healing index (BHI), and external fixator index (EFI). Radiographic parameters included the medial proximal tibial angle, lateral distal tibial angle, posterior proximal tibial angle, and anterior distal tibial angle. RESULTS: There were 137 hexapods and 90 Ilizarov frames in total. The mean time to follow-up was 3.7 years in the hexapod group and 4.0 years in the Ilizarov group. Hexapods had a significantly lower time to consolidation (253 days versus 449 days) (P < 0.0001) and BHI (59.1 days/cm versus 87.5 days/cm) (P < 0.0001). Hexapods had a significantly better EFI (72.3 days/cm versus 96.1 days/cm) (P = 0.0009). CONCLUSION: Hexapods may confer a significant advantage over Ilizarov frames in the management of bone defects. Time to consolidation, radiographic parameters, BHI, and EFI are all superior in hexapods.

Comparative fixation devices for preventing migration of the proximal tibiofibular joint during tibial lengthening: a tether versus screw fixation.

BACKGROUND: When lengthening the tibia segment using motorized internal lengthening nails (MILN), undesired distal migration of the proximal fibula segment is prevented by tibiofibular stabilization, traditionally using a screw. A tightened cortical suspensory fixation rope (tether) is an alternative option, but its appropriateness has never been studied. The primary outcome was comparing the amount of proximal fibular migration between patients who were stabilized with either a tether or a screw. The secondary outcome was to evaluate the effect of fibular migration on the clinical outcome between both groups. METHODS: A retrospective study was conducted on patients who underwent tibial lengthening with MILN between April 2016 and June 2022. Two cohorts were compared: 18 limbs with tether fixation versus 29 limbs with screw fixation. Data on the patient's age, sex, etiologies, and clinical outcomes were collected. Radiographic measurements included the lengthening distance and the amount of proximal fibular migration. RESULTS: In total, 47 limbs from 41 patients, with average age 35.01 ± 13.72 years old. There were 28 males (68.29%) and 13 females (31.71%). The tether group demonstrated a statistically significant greater distance of migration than the screw group (p < 0.001), with an average migration distance of 8.39 ± 5.09 mm and 2.59 ± 3.06 mm, respectively. No correlation was found between the amount of tibial lengthening and the distance of proximal fibular migration in both the tether group (p = 0.96) and the screw group (p = 0.32). There was no significant difference in the change of knee extension between both groups (p = 0.3), and no patients reported knee pain or tightness. CONCLUSION: A screw provides better resistance to proximal tibiofibular joint migration during MILN lengthening, but the difference appears clinically inconsequential. Either option appears suitable.

Outcomes of percutaneous femoral derotational osteotomy in pediatric patients.

Background: Percutaneous femoral derotational osteotomies are performed in both adult and pediatric patients. There is little published on the outcomes after femoral derotational osteotomy in pediatric patients. Methods: A retrospective cohort study of pediatric patients treated with percutaneous femoral derotational osteotomy by one of two surgeons between 2016 and 2022 was performed. Data collected included patient demographics; surgical indications; femoral version; tibial torsion; magnitude of rotational correction; complications; time to hardware removal; pre-operative and post-operative patient-reported outcome scores, including Limb Deformity-Scoliosis Research Society and Patient-Reported Outcomes Measurement Information System; and time to consolidation. Descriptive statistics were used to summarize the data and t tests used to compare means. Results: Thirty-one femoral derotational osteotomies in 19 patients were included with an average age of 14.7 (9-17) years. The average rotational correction was 21.5° ± 6.4° (10°-40°). The average length of follow-up was 17.9 ± 6.7 months. There were no instances of nonunion, joint stiffness, or nerve injury. No patients returned to the operating room for additional surgeries other than routine hardware removal. There were no cases of avascular necrosis of the femoral head. Of the 19 patients, 8 completed both a pre-operative and post-operative survey set. There were significant improvements in the Limb Deformity-Scoliosis Research Society Self-Image/Appearance sub-category and the Patient-Reported Outcomes Measurement Information System Physical Function sub-category. Conclusion: Femoral derotational osteotomy using a percutaneous drill hole technique with antegrade trochanteric entry femoral nail is safe in the pediatric population and improves self-image in patients with symptomatic femoral version abnormalities.

Modern Internet Search Analytics and Osseointegration: What are Patients Asking and Reading Online?

Background: Osseointegration (OI) limb has been performed for over 30 years and is an example of an advance in technology and surgical technique which has led to improvements in patient mobility and quality of life. An increasing number of patients seek information about osseointegration. The aim of this study was to categorise the most frequently asked questions by patients using the Google search engine and the most frequently accessed websites with the highest return on answers. The secondary aims of this study were to assess the quality of the information provided on those websites and to stratify, by category, which websites provide the best quality information. Materials and methods: Ten permutations and conjugations of the word 'osseointegration' were entered into Google. The first fifty 'People also ask' and associated websites by Google's machine learning and natural language processing engine were collected for each search term. The Rothwell classification system of questions by topic (Fact, Value, Policy) and websites by category was used (Commercial, Academic, Medical Practice, Single Surgeon Personal, Government, Social Media). Website quality was assessed using the Journal of the American Medical Association (JAMA) benchmark criteria (Likert-style rating 0-4). Pearson's Chi-squared and Student's t-tests were performed for statistical analysis as appropriate (significance, p < 0.05). Results: The 10 search terms generated 454 questions and referenced 408 websites. Of the 454 questions generated, the most common question categories were fact (70.8%), value (19.2%), and policy (10%). The most common website type was social media (37.4%). The most common question types were technical details (30.4%), specific activity (20.6%), and cost (14.1%). Only 1.6% of questions related to risks and complications. Generally, website quality was poor with 64.1% having a JAMA score of 0 or 1. Websites that were categorised as 'Government' had the highest overall JAMA scores: 71.4% had a score of 4. Conclusion: Based on Google search engine's results, the most commonly asked questions about osteointegration related to technical details, specific activities and cost; only 1.6% related to risks and complications. Interestingly, social media websites represented the highest volume of search result referrals. Overall, the quality of websites was poor with the most factual information coming from governmental websites. How to cite this article: Murphy EP, Sheridan GA, Page BJ, et al. Modern Internet Search Analytics and Osseointegration: What Are Patients Asking and Reading Online? Strategies Trauma Limb Reconstr 2023;18(3):163-168.

Anger Increases Susceptibility to Misinformation.

The effect of anger on acceptance of false details was examined using a three-phase misinformation paradigm. Participants viewed an event, were presented with schema-consistent and schema-irrelevant misinformation about it, and were given a surprise source monitoring test to examine the acceptance of the suggested material. Between each phase of the experiment, they performed a task that either induced anger or maintained a neutral mood. Participants showed greater susceptibility to schema-consistent than schema-irrelevant misinformation. Anger did not affect either recognition or source accuracy for true details about the initial event, but suggestibility for false details increased with anger. In spite of this increase in source errors (i.e., misinformation acceptance), both confidence in the accuracy of source attributions and decision speed for incorrect judgments also increased with anger. Implications are discussed with respect to both the general effects of anger and real-world applications such as eyewitness memory.

Development of a biodosimeter for radiation triage using novel blood protein biomarker panels in humans and non-human primates.

Purpose: In a significant nuclear event, hundreds of thousands of individuals will require rapid triage for absorbed radiation to ensure effective medical treatment and efficient use of medical resources. We are developing a rapid screening method to assess whether an individual received an absorbed dose of ≥2 Gy based on the analysis of a specific panel of blood proteins in a fingerstick blood sample.Materials and methods: We studied a data set of 1051 human blood samples obtained from radiotherapy patients, normal healthy individuals, and several special population groups. We compared the findings in humans with those from irradiation studies in non-human primates (NHPs).Results: We identified a panel of three protein biomarkers, salivary alpha amylase (AMY1), Flt3 ligand (FLT3L), and monocyte chemotactic protein 1 (MCP1), which are upregulated in human patients receiving fractionated doses of total body irradiation (TBI) therapy as a treatment for cancer. These proteins exhibited a similar radiation response in NHPs after single acute or fractionated doses of ionizing radiation.Conclusion: Our work provides confidence in this biomarker panel for biodosimetry triage using fingerstick blood samples and in the use of NHPs as a model for irradiated humans.

Not all Anchors Weigh Equally.

The anchoring bias is a reliable effect wherein a person's judgments are affected by initially presented information, but it is unknown specifically why this effect occurs. Research examining this bias suggests that elements of both numeric and semantic priming may be involved. To examine this, the present research used a phenomenon wherein people treat numeric information presented differently in Arabic numeral or verbal formats. We presented participants with one of many forms of an anchor that represented the same value (e.g., twelve hundred or 1,200). Thus, we could examine how a concept's meaning and its absolute numeric value affect anchoring. Experiments 1 and 2 showed that people respond to Arabic and verbal anchors differently. Experiment 3 showed that these differences occurred largely because people tend to think of numbers in digit format. This suggests that one's conceptual understanding of the anchored information matters more than its strict numeric value.

In-Group Versus Out-Group Source Memory.

A common finding in the source monitoring literature is that greater similarity impairs source discriminability. Experiments traditionally manipulate similarity overtly by describing or showing sources with explicitly differentiable features. However, people may also infer source characteristics themselves, which should also affect discriminability. Two studies examined inferred source characteristics by capitalizing on the out-group homogeneity effect, whereby in-group members are conceptualized as more diverse than out-group members. Participants learned about two sources who were described only as members of an in-group or an out-group and whose actions did not have higher a priori association with either group. Source memory was superior when participants believed the sources to be in-group members. This demonstrates that people spontaneously include inferred features with source representations and can capitalize on these features during source monitoring. Interestingly, information suggesting membership in one's in-group improved performance even for sources who had previously been considered out-group members (Experiment 2).

When anticipation beats accuracy: Threat alters memory for dynamic scenes.

Threat frequently leads to the prioritization of survival-relevant processes. Much of the work examining threat-related processing advantages has focused on the detection of static threats or long-term memory for details. In the present study, we examined immediate memory for dynamic threatening situations. We presented participants with visually neutral, dynamic stimuli using a representational momentum (RM) paradigm, and manipulated threat conceptually. Although the participants in both the threatening and nonthreatening conditions produced classic RM effects, RM was stronger for scenarios involving threat (Exps. 1 and 2). Experiments 2 and 3 showed that this effect does not generalize to the nonthreatening objects within a threatening scene, and that it does not extend to arousing happy situations. Although the increased RM effect for threatening objects by definition reflects reduced accuracy, we argue that this reduced accuracy may be offset by a superior ability to predict, and thereby evade, a moving threat.

On-chip quantitative PCR using integrated real-time detection by capillary electrophoresis.

Quantitative PCR (qPCR) has been widely used for the detection and monitoring of a variety of infectious diseases. PCR and CE were integrated into a microfluidic chip that was designed to achieve rapid real-time amplicon sampling, separation, and quantitation without requiring various probes. A novel chip design allows the overlapped execution of PCR and CE, minimizing the time required for CE analysis after each PCR cycle. The performance of the on-chip qPCR method was demonstrated using a 45-minutes model assay protocol for the phiX174 bacteriophage, and the multiplexing capability of the method was demonstrated by adding a second target, E. coli genomic DNA, to the model assay. The results indicate good sensitivity, reproducibility, and linearity over the tested assay range, 50 to 2 × 10(4) copies/25 µL reaction. Based on this performance, the on-chip qPCR method should be applicable to a wide variety of infectious disease detection and monitoring assays with the addition of suitable sample preparation protocols.

Production of fungal antibiotics using polymeric solid supports in solid-state and liquid fermentation.

The use of inert absorbent polymeric supports for cellular attachment in solid-state fungal fermentation influenced growth, morphology, and production of bioactive secondary metabolites. Two filamentous fungi exemplified the utility of this approach to facilitate the discovery of new antimicrobial compounds. Cylindrocarpon sp. LL-Cyan426 produced pyrrocidines A and B and Acremonium sp. LL-Cyan416 produced acremonidins A-E when grown on agar bearing moist polyester-cellulose paper and generated distinctly different metabolite profiles than the conventional shaken or stationary liquid fermentations. Differences were also apparent when tenfold concentrated methanol extracts from these fermentations were tested against antibiotic-susceptible and antibiotic-resistant Gram-positive bacteria, and zones of inhibition were compared. Shaken broth cultures of Acremonium sp. or Cylindrocarpon sp. showed complex HPLC patterns, lower levels of target compounds, and high levels of unwanted compounds and medium components, while agar/solid support cultures showed significantly increased yields of pyrrocidines A and B and acremonidins A-E, respectively. This method, mixed-phase fermentation (fermentation with an inert solid support bearing liquid medium), exploited the increase in surface area available for fungal growth on the supports and the tendency of some microorganisms to adhere to solid surfaces, possibly mimicking their natural growth habits. The production of dimeric anthraquinones by Penicillium sp. LL-WF159 was investigated in liquid fermentation using various inert polymeric immobilization supports composed of polypropylene, polypropylene cellulose, polyester-cellulose, or polyurethane. This culture produced rugulosin, skyrin, flavomannin, and a new bisanthracene, WF159-A, after fermentation in the presence and absence of polymeric supports for mycelial attachment. The physical nature of the different support systems influenced culture morphology and relative metabolite yields, as determined by HPLC analysis and measurement of antimicrobial activity. The application of such immobilized-cell fermentation methods under solid and liquid conditions facilitated the discovery of new antibiotic compounds, and offers new approaches to fungal fermentation for natural product discovery.

Biosynthetic pathway for mannopeptimycins, lipoglycopeptide antibiotics active against drug-resistant gram-positive pathogens.

The mannopeptimycins are a novel class of lipoglycopeptide antibiotics active against multidrug-resistant pathogens with potential as clinically useful antibacterials. This report is the first to describe the biosynthesis of this novel class of mannosylated lipoglycopeptides. Included here are the cloning, sequencing, annotation, and manipulation of the mannopeptimycin biosynthetic gene cluster from Streptomyces hygroscopicus NRRL 30439. Encoded by genes within the mannopeptimycin biosynthetic gene cluster are enzymes responsible for the generation of the hexapeptide core (nonribosomal peptide synthetases [NRPS]) and tailoring reactions (mannosylation, isovalerylation, hydroxylation, and methylation). The NRPS system is noncanonical in that it has six modules utilizing only five amino acid-specific adenylation domains and it lacks a prototypical NRPS macrocyclizing thioesterase domain. Analysis of the mannopeptimycin gene cluster and its engineering has elucidated the mannopeptimycin biosynthetic pathway and provides the framework to make new and improved mannopeptimycins biosynthetically.

A simple, rapid, sensitive method detecting homoserine lactone (HSL)-related compounds in microbial extracts.

A simple, rapid, sensitive microtiter plate method detecting N-acyl homoserine lactone (HSL)-related compounds was established using an Agrobacterium tumefaciens strain harboring a traG::lacZ/traR reporter gene responsive to HSLs. This strain did not produce its own HSL, but the traG::lacZ reporter gene was induced only when its transcription activator TraR detected a cognate exogenous HSL. Therefore, the assay was expected to be highly specific for HSL-related compounds. Induction of the reporter gene, leading to production of beta-galactosidase enzyme, was measured by using two different beta-galactosidase substrates, X-gal and Galacton-Star, for colorimetric and chemiluminometric detection, respectively. The screen was validated in both the 96-well and 384-well plate formats, and extracts derived from 696 different microbial isolates, mostly unidentified actinomycetes isolated from diverse locations, were tested. Crude extracts of 81 (11.64%) cultures tested positive for HSL-related compounds, and an additional 34 (4.8%) crude extracts showed a moderate to weak signal for HSLs. Data from the fractionated samples, however, suggested a much higher prevalence of HSL signals in these extracts. Of 144 crude extracts fractionated into 10 individual samples at a 10x concentration, 72 (50%) cultures tested positive for HSLs. Six cultures were active only in the crude extract, 18 were active both in crude and one or more of their fractions, and an additional 48 were active in just one or more of their fractions. This finding may be the first to suggest such a high prevalence of HSL-signals found in nature, and a large number of actinomycetes in our collection appeared to produce HSL-related compounds.

Investigating beta-hydroxyenduracididine formation in the biosynthesis of the mannopeptimycins.

The mannopeptimycins (MPPs) are potent glycopeptide antibiotics that contain both D and L forms of the unique, arginine-derived amino acid beta-hydroxyenduracididine (betahEnd). The product of the mppO gene in the MPP biosynthetic cluster resembles several non-heme iron, alpha-ketoglutarate-dependent oxygenases, such as VioC and clavaminate synthase. The role of MppO in betahEnd biosynthesis was confirmed through inactivation of mppO, which yielded a strain that produced dideoxy-MPPs, indicating that mppO is essential for generating the beta-hydroxy functionality for both betahEnd residues. Characterization in vitro of recombinant His6-MppO expressed in E. coli revealed that MppO selectively hydroxylates the beta carbon of free L-enduracididine.

Production of novel rapamycin analogs by precursor-directed biosynthesis.

The natural product rapamycin, produced during fermentation by Streptomyces hygroscopicus, is known for its potent antifungal, immunosuppressive, and anticancer activities. During rapamycin biosynthesis, the amino acid l-pipecolate is incorporated into the rapamycin molecule. We investigated the use of precursor-directed biosynthesis to create new rapamycin analogs by substitution of unusual l-pipecolate analogs in place of the normal amino acid. Our results suggest that the l-pipecolate analog (+/-)-nipecotic acid inhibits the biosynthesis of l-pipecolate, thereby limiting the availability of this molecule for rapamycin biosynthesis. We used (+/-)-nipecotic acid in our precursor-directed biosynthesis studies to reduce l-pipecolate availability and thereby enhance the incorporation of other pipecolate analogs into the rapamycin molecule. We describe here the use of this method for production of two new sulfur-containing rapamycin analogs, 20-thiarapamycin and 15-deoxo-19-sulfoxylrapamycin, and report measurement of their binding to FKBP12.