Metabolic engineering of oleaginous yeast Rhodotorula toruloides for overproduction of triacetic acid lactone.

The plant-sourced polyketide triacetic acid lactone (TAL) has been recognized as a promising platform chemical for the biorefinery industry. However, its practical application was rather limited due to low natural abundance and inefficient cell factories for biosynthesis. Here, we report the metabolic engineering of oleaginous yeast Rhodotorula toruloides for TAL overproduction. We first introduced a 2-pyrone synthase gene from Gerbera hybrida (GhPS) into R. toruloides and investigated the effects of different carbon sources on TAL production. We then systematically employed a variety of metabolic engineering strategies to increase the flux of acetyl-CoA by enhancing its biosynthetic pathways and disrupting its competing pathways. We found that overexpression of ATP-citrate lyase (ACL1) improved TAL production by 45% compared to the GhPS overexpressing strain, and additional overexpression of acetyl-CoA carboxylase (ACC1) further increased TAL production by 29%. Finally, we characterized the resulting strain I12-ACL1-ACC1 using fed-batch bioreactor fermentation in glucose or oilcane juice medium with acetate supplementation and achieved a titer of 28 or 23 g/L TAL, respectively. This study demonstrates that R. toruloides is a promising host for the production of TAL and other acetyl-CoA-derived polyketides from low-cost carbon sources.

Toll-like receptor 7 and 8 imidazoquinoline-based agonist/antagonist pairs.

Toll-like receptors (TLRs) 7 and 8 are key targets in the development of immunomodulatory drugs for treating infectious disease, cancer, and autoimmune disorders. These receptors can adopt both agonist and antagonist binding conformations that switch the receptor signal on or off to the downstream production of cytokines. In this study, we examined the effect of simple isomeric substitutions to the C2-butyl group of two imidazoquinoline agonists and evaluated the activity of these analogs using both TLR7 and TLR8 reporter cells and cytokine induction assays. Results are presented showing the C2-isobutyl and C2-cyclopropylmethyl isomers are both mixed TLR7/8 competitive antagonists of the parent agonist [4-Amino-1-(4-(aminomethyl)benzyl)-2-butyl-7-methoxycarbonyl-1H-imidazo[4,5-c]quinoline], indicating the conformation of the dimeric receptor complex is highly sensitive to steric perturbations to the ligand binding pocket. This observation is consistent with prior work demonstrating TLR7 and TLR8 activity is directly correlated to C2-alkyl substitutions that project into a hydrophobic pocket at the dimer interface of the receptor. The close structural relationship of the agonist/antagonist pairs identified here highlights the importance of this pocket in tipping the balance between the agonist and antagonist binding states of the receptor which may have significant ramifications to the design of imidazoquinoline-based immunomodulatory agents.

Stable carbon and nitrogen isotope inter- and intra-individual dietary reconstruction from the late 14th to early 18th century site of Alytus, Lithuania.

OBJECTIVES: The Baltic region, particularly Lithuania, was a politically vibrant area of Eastern Europe during the medieval and early modern period. To better understand the diet of Lithuanians during the late 14th to early 18th century, we examine stable carbon and nitrogen isotope ratios from bone and dentin samples from the site of Alytus. We investigate possible dietary differences based on sex, age, and religious practice, as well as dietary changes throughout an individual's lifetime, within the broader European milieu. MATERIALS AND METHODS: Stable carbon and nitrogen isotope analysis was conducted on bone (n = 35) and dentin (n = 38) collagen samples from a total of 39 individuals buried in the cemetery at Alytus (late 14th to early 18th centuries). RESULTS: Results indicate individuals at Alytus consumed a C3 terrestrial based diet. The δ13 C and δ15 N values are not significantly different between bone and dentin, and did not vary by sex. DISCUSSION: The diet at Alytus was homogeneous between males and females and between tissue types. The lack of evidence indicating substantial consumption of fish is unexpected given widespread Catholic fasting practices and marine resource trade throughout Europe. Comparisons with other populations indicate that individuals from Alytus differ in diet from contemporaneous Polish-Lithuanian Commonwealth elites. Comparison of the diets of non-elite individuals in the Eastern Baltic region also reveals dietary variability.

Reconstructing diet in Napoleon's Grand Army using stable carbon and nitrogen isotope analysis.

OBJECTIVES: Historical evidence has provided information regarding disease and mortality in Napoleon Bonaparte's Grand Army, but dietary information beyond individual soldier accounts remains scarce. The purpose of this research is to reconstruct the diets of Napoleon's multiethnic army who were associated with the Russian Campaign of 1812. MATERIALS AND METHODS: We conducted stable carbon and nitrogen isotope ratio analysis on femoral bone collagen of 78 individuals recovered from a salvage excavation at the mass gravesite of Siaures miestelis in Vilnius, Lithuania. These individuals were later discovered to be Napoleonic soldiers and camp followers who participated in the 1812 Russian Campaign. RESULTS: Stable carbon isotope ratios range from -19.2‰ to -11.8‰, with a mean of -17.8‰ ± 1.5‰ (1 σ). Stable nitrogen isotope ratios range from 7.1‰ to 13.6‰, with a mean of 10.5‰ ± 1.4‰ (1 σ). Both δ13 C and δ15 N values show a wide range of variation. DISCUSSION: Stable isotope data indicate considerable dietary variation in this population associated with a multiethnic and socially stratified military population. Diets ranged from predominantly C3 -based to predominantly C4 -based, with varying inputs of terrestrial, freshwater, and marine animal protein. Comparison with other European populations further denotes the exceptional range of dietary variation of soldiers and camp followers in Napoleon's army.

Intra-discal injection of autologous, hypoxic cultured bone marrow-derived mesenchymal stem cells in five patients with chronic lower back pain: a long-term safety and feasibility study.

BACKGROUND: Chronic low back pain due to disc degeneration represents a major social and economic burden worldwide. The current standard of care is limited to symptomatic relief and no current approved therapy promotes disc regeneration. Bone marrow-derived mesenchymal stem cells (MSCs) are easily accessible and well characterized. These MSCs are multipotent and exhibit great tissue regenerative potential including bone, cartilage, and fibrous tissue regeneration. The use of this cell-based biologic for treating protruding disc herniation and/or intervertebral disc degeneration is a promising therapeutic strategy, due to their known regenerative, immuno-modulatory and anti-inflammatory properties. METHODS: Five patients diagnosed with degenerative disc disease received an intra-discal injection of autologous, hypoxic cultured, bone marrow-derived mesenchymal stem cells (15.1-51.6 million cells) as part of a previous study. These patients were re-consented to participate in this study in order to assess long-term safety and feasibility of intra-discal injection of autologous, hypoxic cultured, bone marrow-derived mesenchymal stem cells 4-6 years post mesenchymal stem cell infusion. The follow-up study consisted of a physical examination, a low back MRI, and a quality of life questionnaire. RESULTS: Patients' lower back MRI showed absence of neoplasms or abnormalities surrounding the treated region. Based on the physical examination and the quality of life questionnaire, no adverse events were reported due to the procedure or to the stem cell treatment 4-6 years post autologous, hypoxic cultured mesenchymal stem cell infusion. All patients self-reported overall improvement, as well as improvement in strength, post stem cell treatment, and four out of five patients reported improvement in mobility. CONCLUSION: This early human clinical data suggests the safety and feasibility of the clinical use of hypoxic cultured bone marrow-derived mesenchymal stem cells for the treatment of lower back pain due to degenerative disc disorders and support further studies utilizing hypoxic cultured bone marrow-derived stem cells. The overall improvements reported are encouraging, but a larger double-blind, controlled, randomized clinical study with significant number of patients and implementation of validated endpoint measurements are next steps in order to demonstrate efficacy of this cell-based biologic.

Chemical Differentiation of Osseous, Dental, and Non-skeletal Materials in Forensic Anthropology using Elemental Analysis.

Forensic anthropologists are generally able to identify skeletal materials (bone and tooth) using gross anatomical features; however, highly fragmented or taphonomically altered materials may be problematic to identify. Several chemical analysis techniques have been shown to be reliable laboratory methods that can be used to determine if questionable fragments are osseous, dental, or non-skeletal in nature. The purpose of this review is to provide a detailed background of chemical analysis techniques focusing on elemental compositions that have been assessed for use in differentiating osseous, dental, and non-skeletal materials. More recently, chemical analysis studies have also focused on using the elemental composition of osseous/dental materials to evaluate species and provide individual discrimination, but have generally been successful only in small, closed groups, limiting their use forensically. Despite significant advances incorporating a variety of instruments, including handheld devices, further research is necessary to address issues in standardization, error rates, and sample size/diversity.

Sex estimation research trends in forensic anthropology between 2000 and 2022 in five prominent journals.

In forensic anthropology, osteological sex estimation methods are continuously reevaluated and updated to improve classification accuracies. Therefore, to gain a comprehensive understanding of recent trends in sex estimation research in forensic anthropology, a content analysis of articles published between 2000 and 2022 in Forensic Science International, the Journal of Forensic Sciences, the International Journal of Legal Medicine, the American Journal of Biological Anthropology, and Forensic Anthropology, was performed. The main goals of this content analysis were to (1) examine trends in metric versus morphological research, (2) examine which areas of the skeleton have been explored, (3) examine which skeletal collections and population affinities have been most frequently utilized, and (4) determine which statistical methods were commonly implemented. A total of 440 articles were coded utilizing MAXQDA and the resulting codes were exported for analysis. Statistical analyses were conducted utilizing the Cochran-Armitage and Jonckheere-Terpstra tests for trends, as well as Fisher-Freeman-Halton tests. The results demonstrate that sex estimation research published in these journals has prioritized metric over morphological methods. Further, the most utilized skeletal regions continue to be the skull and pelvis, while the most popular classification statistics continue to be discriminant function analysis and logistic regression. This study also demonstrates that a substantial portion of research has been conducted utilizing U.S. and Europe-based collections and limited populations. Based on these results, future sex estimation research must continue exploring the use of long bones and other postcranial elements, testing newer methods of analysis, as well as developing population-inclusive methods.

Cardiac evaluation of hospitalized children with 2019 coronavirus (COVID-19) infection at a single large quaternary center.

Background: Cardiac complications of serious SARS-CoV-2 infections, especially Multisystem Inflammatory Syndrome of Children (MIS-C) are well described, however current studies have not considered pediatric patients hospitalized with no cardiac concerns. We established a protocol for cardiac evaluation of all admitted COVID-19 patients three weeks post-discharge, irrespective of cardiac concerns. We assessed cardiovascular outcomes and hypothesized that patients with absent cardiac concerns are at lower risk for cardiac abnormalities. Methods: This was a retrospective study of 160 patients admitted for COVID-19 (excluding MIS-C) between March 2020 and September 2021 with subsequent echocardiogram(s) performed at our center. Patients were divided into 4 subgroups: Group 1 included patients with absent cardiac concerns, admitted to acute care (1a) and intensive care unit (ICU) (1 b). Group 2 included patients with cardiac concerns, admitted to acute care (2a) and ICU (2 b). Groups were compared based on clinical endpoints and echocardiographic measurements, including tissue Doppler imaging (TDI) assessment of diastolic function (z-score of septal Mitral E/TDI E' and lateral E/TDI E'). Chi-squared, Fisher's exact, and Kruskal-Wallis tests were used. Results: Traditional cardiac abnormalities varied significantly between the groups; with Group 2 b having the most (n = 8, 21%), but still found in Group 1a (n = 2, 3%) and Group 1 b (n = 1, 5%). No patients in Group 1 demonstrated abnormal systolic function, compared to Group 2a (n = 1, 3%) and Group 2 b (n = 3, 9%, p = 0.07). When including TDI assessment of diastolic function, the total incidence of abnormalities found on echocardiogram was increased in all groups. Conclusion: Cardiac abnormalities were found in pediatric patients admitted with COVID-19, even those without apparent cardiovascular concerns. The risk was greatest in ICU-admitted patients with cardiac concerns. The clinical significance of diastolic function assessment in these patients remains unknown. Further studies are needed to assess long-term cardiovascular sequelae of children with COVID-19, irrespective of cardiac concerns.

Controlling an effector with eye movements: The effect of entangled sensory and motor responsibilities.

Restoring arm and hand function has been indicated by individuals with tetraplegia as one of the most important factors for regaining independence. The overall goal of our research is to develop assistive technologies that allow individuals with tetraplegia to control functional reaching movements. This study served as an initial step toward our overall goal by assessing the feasibility of using eye movements to control the motion of an effector in an experimental environment. We aimed to understand how additional motor requirements placed on the eyes affected eye-hand coordination during functional reaching. We were particularly interested in how eye fixation error was affected when the sensory and motor functions of the eyes were entangled due to the additional motor responsibility. We recorded participants' eye and hand movements while they reached for targets on a monitor. We presented a cursor at the participant's point of gaze position which can be thought of as being similar to the control of an assistive robot arm. To measure eye fixation error, we used an offline filter to extract eye fixations from the raw eye movement data. We compared the fixations to the locations of the targets presented on the monitor. The results show that not only are humans able to use eye movements to direct the cursor to a desired location (1.04 ± 0.15 cm), but they can do so with error similar to that of the hand (0.84 ± 0.05 cm). In other words, despite the additional motor responsibility placed on the eyes during direct eye-movement control of an effector, the ability to coordinate functional reaching movements was unaffected. The outcomes of this study support the efficacy of using the eyes as a direct command input for controlling movement.

Proof-of-Concept: A Hands-Free Interface for Robot-Assisted Self-Feeding.

Eating and drinking is an essential part of every-day life. And yet, there are many people in the world today who rely on others to feed them. In this work, we present a prototype robot-assisted self-feeding system for individuals with movement disorders. The system is capable of perceiving, localizing, grasping, and delivering non-compliant food items to an individual. We trained an object recognition network to detect specific food items, and we compute the grasp pose for each item. Human input is obtained through an interface consisting of an eye-tracker and a display screen. The human selects options on the monitor with their eye and head movements and triggers responses with mouth movements. We performed a pilot study with four able-bodied participants and one participant with a spinal cord injury (SCI) to evaluate the performance of our prototype system. Participants selected food items with their eye movements, which were then delivered by the robot. We observed an average overall feeding success rate of 89.1% and an average overall task time of $31.4 \pm 2.4$ seconds per food item. The SCI participant gave scores of 90.0 and 8.3 on the System Usability Scale and NASA Task Load Index, respectively. We also conducted a custom, post-study interview to gather participant feedback to drive future design decisions. The quantitative results and qualitative user feedback demonstrate the feasibility of robot-assisted self-feeding and justify continued research into mealtime-related assistive devices.

Identification of 5-(Aryl/Heteroaryl)amino-4-quinolones as Potent Membrane-Disrupting Agents to Combat Antibiotic-Resistant Gram-Positive Bacteria.

Nosocomial infections caused by resistant Gram-positive organisms are on the rise, presumably due to a combination of factors including prolonged hospital exposure, increased use of invasive procedures, and pervasive antibiotic therapy. Although antibiotic stewardship and infection control measures are helpful, newer agents against multidrug-resistant (MDR) Gram-positive bacteria are urgently needed. Here, we describe our efforts that led to the identification of 5-amino-4-quinolone 111 with exceptionally potent Gram-positive activity with minimum inhibitory concentrations (MICs) ≤0.06 µg/mL against numerous clinical isolates. Preliminary mechanism of action and resistance studies demonstrate that the 5-amino-4-quinolones are bacteriostatic, do not select for resistance, and selectively disrupt bacterial membranes. While the precise molecular mechanism has not been elucidated, the lead compound is nontoxic displaying a therapeutic index greater than 500, is devoid of hemolytic activity, and has attractive physicochemical properties (clog P = 3.8, molecular weight (MW) = 441) that warrant further investigation of this promising antibacterial scaffold for the treatment of Gram-positive infections.

The effects of a positional feedback device on rollator walker use: a validation study.

BACKGROUND: According to clinical guidelines, rolling walker users should walk with their feet between the posterior wheels of the walker; however approximately 50% of users do not. OBJECTIVE: To describe the development and effects of a custom device designed to attach to a walker and provide visual feedback to encourage improved user position. METHODS: Fourteen older adults participated in this study to validate the effects of this device when a 10% decrease in the users' habitual distance away from the walker was encouraged via feedback. Users' relative distances were recorded using a non-contact distance sensor within the device, while kinematics were measured using commercial wearable wireless inertial sensors. RESULTS: Individuals were able to ambulate on average 20% closer or more to their walker when prescribed the visual feedback. This was primarily achieved through a reduction in shoulder flexion. Trunk and cervical postures were less generalizable as only small and variable changes were observed. CONCLUSIONS: These findings suggest that the device has promise, as individuals attended to the device and walked in a position closer to that recommended by clinical guidelines. The device did not appear to improve posture. Future work is needed to determine long-term effects.

Children's Hospital Ambulatory Response to the 2019 Novel Coronavirus Disease (COVID-19) Pandemic.

The 2019 novel coronavirus disease (COVID-19) pandemic produced an abrupt and near shutdown of nonemergent patient care. Children's National Hospital (CNH) mounted a multidisciplinary, coordinated ambulatory response that included supply chain management, human resources, risk management, infection control, and information technology. To ensure patient access, CNH expanded telemedicine and instituted operational innovations for outpatient procedures. While monthly in-person ambulatory subspecialty visits decreased from 25 889 pre-COVID-19 to 4484 at nadir of the COVID-19 pandemic, telemedicine visits increased from 70 to 13 539. Further studies are needed to assess the impact of innovations in health care delivery and operations that the crisis prompted.

Massive carbon dioxide gas embolism: a near catastrophic situation averted by use of cardiopulmonary bypass.

Endovascular vein harvesting is used as a less invasive method when compared to the standard open surgical method of dissecting and procurement of the greater saphenous vein. The benefits include smaller incision, decreased blood loss, less pain, decreased transfusion, decreased risk of infection and enhanced epidermal cosmetics. However, endovascular vein harvesting does have a learning curve to master the technique and although endovascular vein harvesting is the preferred standard of practice over the open surgical technique, it does have inherent potential complications. During endovascular vein harvesting, there is a potential for the patient's circulatory system to collapse which can be identified initially through hemodynamic monitoring, blood gas results, and vigilance through clinical observation. The suspected source of the crisis was confirmed when the surgeon cannulated the right atrium, where a massive gas source that (appeared pressurized) escaped upon incision of the right atrial appendage. Cardiopulmonary bypass (CPB) was utilized to support the patient and rectify the impending catastrophic event. Once full CPB was attained, we achieved hemodynamic stability and eventually all blood gases were normalized. Massive CO2 embolism is a life threatening emergency which must be identified and corrected instantaneously. CPB was the modality used to salvage this situation. Attention to the set-up of the EVH equipment, use of the transesophageal echocardiography, cerebral cximetry, vigilance and cooperation of all disciplines in the OR are definite recommendations to prevent such an occurrence. Experience gained by the perfusion team with a previous case was applied and helped to solve the immediate problems presented in this case.

Ethnicity and the distance to the epidural space in parturients.

BACKGROUND AND OBJECTIVES: In a pilot study, we previously demonstrated a higher average skin to lumbar epidural space distance (STLESD) in our obstetric population compared with the published literature. Furthermore, we demonstrated differences in STLESD based on ethnicity. The aim of this study was to perform a comprehensive analysis of the STLESD in our patient population by expanding the number of patients and ethnic groups included. METHODS: Data from 3,305 patients were obtained from our electronic database from September 2003 through November 2005. Self-declared ethnicity included 1,177 Caucasians (36%), 1,162 African Americans (35%), 760 Hispanics (23%), 135 Asians (4%), and 71 Indian/Pakistani/Bangladeshi/Sri Lankans (2%). The influences of body mass index (BMI), ethnicity, and their interaction on the STLESD were tested with a multiple linear regression model. RESULTS: The mean +/- SD STLESD differed among the ethnic groups ranging from 4.8 +/- 0.9 cm in Asian patients to 6.3 +/- 1.6 cm in African American parturients. When all ethnic groups were compared, BMI had a significant influence on STLESD (P < .0001), but so did ethnicity (P = .0004). The Hispanic group demonstrated STLESDs that were significantly lower than the African American and Caucasian groups at high BMI (P < .0001). In a subanalysis performed without the Hispanic group, the influence of BMI on STLESD was found to be similar for each group. In this subanalysis, the African American group had STLESDs that were deeper compared with the other 3 ethnic groups (P < .0001), regardless of BMI. CONCLUSIONS: In this study we found that the STLESD was deeper than what was previously reported in the literature. Furthermore, ethnicity, in addition to BMI, influenced the STLESD.

Sequential monitoring of burials containing small pig cadavers using ground penetrating radar.

Ground-penetrating radar (GPR) was used to monitor 12 pig burials in Florida, each of which contained a small pig cadaver. Six of the cadavers were buried in sand at a depth of 0.50-0.60 m, and the other six were buried in sand at a depth of 1.00-1.10 m to represent deep and shallow burials that are generally encountered in forensic scenarios. Four control excavations with no pig interment were also constructed as blank graves and monitored with GPR. The burials were monitored for durations of either 13 or 21 months, and were then excavated to correlate the decomposition state of the cadaver with the GPR imagery. Overall, this study demonstrated that it may be difficult to detect small cadavers buried in sand soon after they are skeletonized because the area surrounding the body, or the grave, may not provide a strong enough contrasting area to be detected by GPR when compared to that of the surrounding undisturbed soil. Also, depth of burial appears to influence grave detection because bodies that are buried at deeper depths may be detected for a longer period of time due to reduced decomposition rates.

Sclerotherapy of Baker's cyst with imaging confirmation of resolution.

BACKGROUND: Baker's cysts are commonly encountered in pain management practices. OBJECTIVE: To ascertain if sclerotherapy treatment of a Baker's cyst could produce objectively verifiable MRI imaging changes. DESIGN: Case report. METHODS: A 52-year-old white male with a posterior horn of the medial meniscus tear and a large Baker's cyst who had failed conservative care and drainage was imaged before treatment with sclerosing. Three injections of 12.5% dextrose and anesthetic with sodium morrhuate were injected intraarticular into the right knee after drainage. RESULTS: The Baker's cyst resolved on both postoperative imaging after the completion of care as well as on physical examination. CONCLUSIONS: Prolotherapy in this case study seemed to be an effective treatment for Baker's cyst in this patient.

Avian Scavenging of Small-Sized Pig Carcasses in Central Florida: Utilizing GIS to Analyze Site Variables Affecting Skeletal Dispersal.

Scavengers can significantly alter a forensic scene and consume, modify, disarticulate, and disperse bodies on the ground surface. The research purpose was to examine vulture scavenging in central Florida, USA. Four small-sized pig (Sus scrofa) carcasses were left on the ground surface of two microenvironments (shaded and open) at a secure site with game cameras. Dispersal data were mapped and analyzed using geographical information systems spatial analysis digital mapping tools. The primary avian scavengers recorded included black vultures (Coragyps atratus) and turkey vultures (Cathartes aura), as well as bald eagles (Haliaeetus leucocephalus). Carcass dispersal patterns were impacted by foliage density (grass height and concentrations of bushes and trees) and proximity to the perimeter fence. While the majority of skeletal elements were dispersed within 6 m of the carcass deposition locations, dispersion occurred over a greater distance in the wooded microenvironment. Overall, vulture behaviors deleteriously destroyed and changed the context of the scene, with black vultures having the greatest impact.

Skeletal weathering in central Florida: A preliminary approach for developing a scoring protocol to estimate time since death.

Regardless of region, skeletal remains deposited in subaerial contexts are subjected to a progression of taphonomic alterations. However, there are limited postmortem interval (PMI) standards developed, particularly in environments where skeletonization can occur quickly. The research purpose was to evaluate the timing and progression of early weathering alterations in two microenvironments (shaded and open) using pig carcasses (Sus scrofa) in the subtropical humid environment of central Florida. Over nine months, sun bleaching was assessed using Munsell® Soil Color Charts (2009) and scored based on percentage of the bone sun bleached, while bone weathering was further evaluated using Behrensmeyer's (1978) stages. Results indicate that microenvironment has a significant influence, with the onset and progression of sun bleaching and Behrensmeyer's (1978) Stages 2 and 3 occurring earlier in the open microenvironment. Sun bleaching stages and Behrensmeyer's (1978) weathering stages can be utilized in conjunction for developing regionally specific PMI taphonomic models for different microenvironments.

Structural Basis of Polyketide Synthase O-Methylation.

Modular type I polyketide synthases (PKSs) produce some of the most chemically complex metabolites in nature through a series of multienzyme modules. Each module contains a variety of catalytic domains to selectively tailor the growing molecule. PKS O-methyltransferases ( O-MTs) are predicted to methylate ß-hydroxyl or ß-keto groups, but their activity and structure have not been reported. We determined the domain boundaries and characterized the catalytic activity and structure of the StiD and StiE O-MTs, which methylate opposite ß-hydroxyl stereocenters in the myxobacterial stigmatellin biosynthetic pathway. Substrate stereospecificity was demonstrated for the StiD O-MT. Key catalytic residues were identified in the crystal structures and investigated in StiE O-MT via site-directed mutagenesis and further validated with the cyanobacterial CurL O-MT from the curacin biosynthetic pathway. Initial structural and biochemical analysis of PKS O-MTs supplies a new chemoenzymatic tool, with the unique ability to selectively modify hydroxyl groups during polyketide biosynthesis.