Terpene cyclases (TCs) catalyze the generation of complex polycyclic terpenoids through elaborate mechanistic cascades. Very little is known about the unusual type II TCs responsible for generation of a large diverse family of meroterpenoids known as indole diterpenoids (IDTs). The radarins are insecticidal and cytotoxic IDTs that possess an unusual core architecture for which the catalytic machinery is unknown. Here, we interrogate the genome of Aspergillus fresenii to reveal the biosynthetic machinery responsible for delivering the specific radarin scaffold. We demonstrate that a bespoke regioselective IDT epoxidase (RadM) acts at the terminal olefin of precursor 3 '-geranylgeranylindole, providing access to this distinct class of IDT architectures. Coexpression of this epoxidase with a noncanonical type II TC RadB led to the biosynthesis of radarin C, in a complex skeletal rearrangement that proceeds via rare alcoholic deprotonation and ketone formation. Our modeling of RadB reveals that catalysis by RadB is enabled by a unique His residue that acts as both the proton donor and acceptor. Intriguingly, coexpression of RadM with an alternative A. fresenii IDT cyclase gene, which does not share this catalytic His, revealed an alternate partial cyclization of 14,15-epoxy-3 '-GGI, producing a new decalin IDT. This work solves the final piece of the biosynthetic puzzle to access the full suite of known IDT architectures, informs type II cyclase engineering efforts, and expands the repertoire of impressive chemistry catalyzed by noncanonical TCs.

The enzyme MetX is a homoserine O-acetyltransferase that catalyzes the first step in methionine biosynthesis and is essential for survival and virulence of various pathogens. It is an attractive target for antifungal and antibacterial drug development. MetX catalyzes the acetyl transfer from acetyl-CoA (AcCoA) to homoserine via a ping-pong mechanism involving an acyl-enzyme intermediate. The active site contains a Ser-His-Asp catalytic triad, which constitutes its core catalytic machinery. Here we investigated the mechanistic details of MetX from Mycobacterium tuberculosis (MtbMetX) using a combination of quantum mechanics/molecular mechanics (QM/MM) calculations, mutagenesis, and mass spectrometry. QM/MM calculations suggest that D320 of the catalytic triad participates in the proton transfer during homoserine acetylation, but not during acyl-enzyme formation. Experiments showed that a D320N substitution, which removes the proton-accepting capability of D320 as well as the pKa modulation of H350 by D320, still allowed acyl-enzyme formation at a markedly reduced rate, but significantly impaired the production of acetyl-homoserine. To isolate the effect of D320's participation in proton transfer from its pKa modulation role, we used QM/MM calculations to simulate a system where D320 could modulate H350 pKa but not accept a proton. These calculations suggest that while D320's proton-accepting role is not required for the AcCoA reaction, it contributes thermodynamically in the homoserine reaction by lowering the energy of the forward pathway. Elucidating the mechanistic details of MtbMetX reactions offers valuable insights that will facilitate the development of mechanism-based inhibitors, contributing to future therapeutic strategies.

Indole diterpenes (IDTs) are a large class of highly complex fungal natural products that possess a wide array of intriguing bioactivities. While IDTs are structurally diverse, the first four steps of IDT biosynthesis are highly conserved and result typically in the formation of a tetrahydropyran (THP)-ring containing structure, most commonly paspaline. The biosynthetic genes responsible for these steps are the most extensively studied of all IDT genes and collectively define the core biosynthetic pathway. Here we show that the fourth fundamental step, formation of the THP ring, is catalysed by a terpene cyclase encoded by an overlooked and uncharacterised fifth gene, idtA. All previously delineated biosynthetic routes have incorrectly attributed this step to the terpene cyclase IdtB, leading to imprecise pathway reconstructions and ignoring the fully evolved biosynthetic solution for core IDT generation. Moreover, while IdtA terpene cyclases are found in Eurotiomycetes fungi, in Sordariomycetes fungi this step is catalysed by the unrelated protein IdtS, demonstrating that two distinct solutions to this chemistry exist. All biosynthetic gene clusters known to specify production of THP-containing IDTs include an idtA or idtS gene. These findings reset the paradigm for core IDT biosynthesis and support accurate heterologous biosynthesis of these complex natural products.

BACKGROUND:Liver cirrhosis is a chronic disease that is known as a "silent killer" and its true prevalence is difficult to describe. It is imperative to accurately characterize the prevalence of cirrhosis because of its increasing healthcare burden.METHODS:In this retrospective cohort study, trends in cirrhosis prevalence were evaluated using administrative data from one of the largest national health insurance providers in the US. (2011-2018). Enrolled adult (≥18-years-old) patients with cirrhosis defined by ICD-9 and ICD-10 were included in the study. The primary outcome measured in the study was the prevalence of cirrhosis 2011-2018.RESULTS:Among the 371,482 patients with cirrhosis, the mean age was 62.2 (±13.7) years; 53.3% had commercial insurance and 46.4% had Medicare Advantage. The most frequent cirrhosis etiologies were alcohol-related (26.0%), NASH (20.9%) and HCV (20.0%). Mean time of follow-up was 725 (±732.3) days. The observed cirrhosis prevalence was 0.71% in 2018, a 2-fold increase from 2012 (0.34%). The highest prevalence observed was among patients with Medicare Advantage insurance (1.67%) in 2018. Prevalence increased in each US. state, with Southern states having the most rapid rise (2.3-fold). The most significant increases were observed in patients with NASH (3.9-fold) and alcohol-related (2-fold) cirrhosis.CONCLUSION:Between 2012-2018, the prevalence of liver cirrhosis doubled among insured patients. Alcohol-related and NASH cirrhosis were the most significant contributors to this increase. Patients living in the South, and those insured by Medicare Advantage also have disproportionately higher prevalence of cirrhosis. Public health interventions are important to mitigate this concerning trajectory of strain to the health system.

OBJECTIVE This study updates previous estimates of the economic burden of diagnosed diabetes and calculates the health resource use and indirect costs attributable to diabetes in 2022. RESEARCH DESIGN AND METHODS We combine the demographics of the US population in 2022 with diabetes prevalence from national survey data, epidemiological data, healthcare cost, and economic data into a Cost of Diabetes Economic Model to estimate the economic burden at the population and per capita levels. Health resource use and associated medical costs are analyzed by age, sex, race/ethnicity, comorbid condition, and health service category. Data sources include national surveys (2015-2020, or most recent available), Medicare standard analytic files (2020), and administrative claims data from 2018-2021 for a large commercially insured population in the US. RESULTS The total estimated cost of diagnosed diabetes in 2022 is $412.9 billion, including $306.6 billion in direct medical costs and $106.3 billion in indirect costs attributable to diabetes. For cost categories analyzed, care for people diagnosed with diabetes accounts for 1 in 4 healthcare dollars in the US, 61% of which is attributable to diabetes. People with diabetes incur average annual medical expenditures of $19,736, of which approximately $12,022 is attributable to diabetes. People diagnosed with diabetes, on average, have medical expenditures 2.6-times higher than what would be expected without diabetes. Glucose lowering medications and diabetic supplies account for about 17% of the total direct medical costs attributable to diabetes. Major contributors to indirect costs are reduced employment due to disability ($28.3 billion), presenteeism ($35.8 billion), and lost productivity due to 338,526 premature deaths ($32.4 billion). CONCLUSIONS The inflation-adjusted economic costs of diabetes are estimated to rise slightly (7%) compared to the 2017 calculations (stated in 2022 dollars). Following decades of steadily increasing prevalence of diabetes, the overall estimated prevalence in 2022 remains relatively stable. However, the absolute number of people with diabetes has grown and contributes to increased healthcare expenditures, particularly per capita spending on inpatient hospital stays and prescription medications. The enormous economic toll of diabetes continues to burden society through direct medical and indirect costs.

BACKGROUND:Musculoskeletal consultations constitute a growing portion of primary care physician (PCP) referrals. Optimizing communication between PCPs and orthopaedists can potentially reduce time spent in the electronic medical record (EMR) as well as physician burnout. Little is known about the preferences of PCPs regarding communication from orthopaedic surgeons. Hence, the present study investigated, across a large health network, the preferences of PCPs regarding communication from orthopaedists. METHODS:A total of 175 PCPs across 15 practices within our health network were surveyed. These providers universally utilized Epic as their EMR platform. Five-point, labeled Likert scales were utilized to assess the PCP-perceived importance of communication from orthopaedists in specific clinical scenarios. PCPs were further asked to report their preferred method of communication in each scenario and their overall interest in communication from orthopaedists. Logistic regression analyses were performed to determine whether any PCP characteristics were associated with the preferred method of communication and the overall PCP interest in communication from orthopaedists. RESULTS:A total of 107 PCPs (61.1%) responded to the survey. PCPs most commonly rated communication from orthopaedists as highly important in the scenario of an orthopaedist needing information from the PCP. In this scenario, PCPs preferred to receive an Epic Staff Message. Scenarios involving a recommendation for surgery, hospitalization, or a major clinical change were also rated as highly important. In these scenarios, an Epic CC'd Chart rather than a Staff Message was preferred. Increased after-hours EMR use was associated with diminished odds of having a high interest in communication from orthopaedists (odds ratio, 0.65; 95% confidence interval, 0.48 to 0.88; p = 0.005). Ninety-three PCPs (86.9%) reported spending 1 to 1.5 hours or more per day in Epic after normal clinical hours, and 27 (25.2%) spent >3 hours per day. Forty-six PCPs (43.0%) reported experiencing ≥1 symptom of burnout. CONCLUSIONS:There were distinct preferences among PCPs regarding clinical communication from orthopaedic surgeons. There was also evidence of substantial burnout and after-hours work effort by PCPs. These results may help to optimize communication between PCPs and orthopaedists while reducing the amount of time that PCPs spend in the EMR.

Anthranilate phosphoribosyltransferase catalyses the second reaction in the biosynthesis of tryptophan from chorismate in microorganisms and plants. The enzyme is homodimeric with the active site located in the hinge region between two domains. A range of structures in complex with the substrates, substrate analogues and inhibitors have been determined, and these have provided insights into the catalytic mechanism of this enzyme. Substrate 5-phospho- d -ribose 1-diphosphate (PRPP) binds to the C-terminal domain and coordinates to Mg 2+ , in a site completed by two flexible loops. Binding of the second substrate anthranilate is more complex, featuring multiple binding sites along an anthranilate channel. This multi-modal binding is consistent with the substrate inhibition observed at high concentrations of anthranilate. A series of structures predict a dissociative mechanism for the reaction, similar to the reaction mechanisms elucidated for other phosphoribosyltransferases. As this enzyme is essential for some pathogens, efforts have been made to develop inhibitors for this enzyme. To date, the best inhibitors exploit the multiple binding sites for anthranilate. This article is part of the theme issue ‘Reactivity and mechanism in chemical and synthetic biology’.

BACKGROUND:Although the medial clear space (MCS) is commonly used to assess talar alignment and ankle stability, its measurement is variable with multiple reported normal values. We have observed that the lateral tibial shaft is a reliable landmark to assess talar alignment. The objective of the current investigation was to determine the normal relationship of the lateral tibia to the superolateral talus using a tangent drawn inferiorly from the lateral tibial shaft, which we refer to as the "lateral tibial line" (LTL). METHODS:The relationship of the LTL to the superolateral talus was assessed by three reviewers on 99 standing ankle mortise radiographs in uninjured patients. This relationship was quantified by measuring the distance (in millimeters) between the LTL and the superolateral talus. In addition, the interobserver reliability of the LTL measurement was recorded and compared with that of the MCS. RESULTS:The median value for the distance between the superolateral talus and LTL was -0.50 mm with an interquartile range of -1.4 to 0.0 mm. The LTL was within 1 mm of the lateral talus in 176 of 297 reviewer measurements (59.3%). Moreover, it was either lateral to or at most 1 mm medial to the lateral talus in 90.9% of cases. The LTL measurement also demonstrated good interobserver reliability (0.764, 95% confidence interval, 0.670 to 0.834), similar to the measurement of MCS (0.742, 95% confidence interval, 0.539 to 0.846). CONCLUSIONS:The relationship between the LTL and superolateral talus is easily measured with good reliability for assessing the anatomic relationship of the tibia and talus. The LTL uncommonly fell more than 1 mm medial to the superolateral talus, as might be seen with displaced ankle fractures. These findings will hopefully serve as a basis for future studies evaluating its role in assessing lateral displacement and stability of isolated fibula fractures. LEVEL OF EVIDENCE:Level III, retrospective review.

α-Isopropylmalate synthase (IPMS) catalyzes the first step in leucine (Leu) biosynthesis and is allosterically regulated by the pathway end product, Leu. IPMS is a dimeric enzyme with each chain consisting of catalytic, accessory, and regulatory domains, with the accessory and regulatory domains of each chain sitting adjacent to the catalytic domain of the other chain. The IPMS crystal structure shows significant asymmetry because of different relative domain conformations in each chain. Owing to the challenges posed by the dynamic and asymmetric structures of IPMS enzymes, the molecular details of their catalytic and allosteric mechanisms are not fully understood. In this study, we have investigated the allosteric feedback mechanism of the IPMS enzyme from the bacterium that causes meningitis, Neisseria meningitidis (NmeIPMS). By combining molecular dynamics simulations with small-angle X-ray scattering, mutagenesis, and heterodimer generation, we demonstrate that Leu-bound NmeIPMS is in a rigid conformational state stabilized by asymmetric interdomain polar interactions. Furthermore, we found removing these polar interactions by mutagenesis impaired the allosteric response without compromising Leu binding. Our results suggest that the allosteric inhibition of NmeIPMS is achieved by restricting the flexibility of the accessory and regulatory domains, demonstrating that significant conformational flexibility is required for catalysis.

Emerging SARS-CoV-2 variants pose a threat to human health worldwide. SARS-CoV-2 receptor binding domain (RBD)-based vaccines are suitable candidates for booster vaccines, eliciting a focused antibody response enriched for virus neutralizing activity. Although RBD proteins are manufactured easily, and have excellent stability and safety properties, they are poorly immunogenic compared to the full-length spike protein. We have overcome this limitation by engineering a sub-unit vaccine composed of an RBD tandem dimer fused to the N-terminal domain (NTD) of the spike protein. We found that inclusion of the NTD (1) improved the magnitude and breadth of the T cell and anti-RBD response, and (2) enhanced T follicular helper cell and memory B cell generation, antibody potency, and cross-reactive neutralization activity against multiple SARS-CoV-2 variants, including B.1.1.529 (Omicron BA.1). In summary, our uniquely engineered RBD-NTD-subunit protein vaccine provides a promising booster vaccination strategy capable of protecting against known SARS-CoV-2 variants of concern.

Physical organic chemistry and mechanistic thinking provide a strong intellectual framework for understanding the chemical logic of evolvable informational macromolecules and metabolic transformations in living organisms. These concepts have also led to numerous successes in designing and applying tools to delineate biological function in health and disease, chemical ecology and possible alternative chemistries employed by extraterrestrial life. A symposium at the 2020 Pacifichem meeting was scheduled in December 2020 to discuss designing and exploiting expanded genetic alphabets, methods to understand the biosynthesis of natural products and re-engineering primary metabolism in bacteria. The COVID-19 pandemic led to postponement of in-person discussions, with the symposium eventually being held on 20-21 December 2021 as an online event. This issue is a written record of work presented on biosynthetic pathways and enzyme catalysis, engineering microorganisms with new metabolic capabilities, and the synthesis of non-canonical, nucleobases for medical applications and for studies of alternate chemistries for living organisms. The variety of opinion pieces, reviews and original research articles provide a starting point for innovations that clarify how complex biological systems emerge from the rules of chemical reactivity and mechanism.This article is part of the themed issue 'Reactivity and mechanism in chemical and synthetic biology'.

There is limited research using real‐world data to evaluate protective cardiovascular effects of glucagon‐like peptide‐1 (GLP‐1) agonists among adults with type 2 diabetes (T2D) early in treatment.

The significant structural diversity and potent bioactivity of the fungal indole diterpenes (IDTs) has attracted considerable interest in their biosynthesis. Although substantial skeletal diversity is generated by the action of noncanonical terpene cyclases, comparatively little is known about these enzymes, particularly those involved in the generation of the subgroup containing emindole SA and DA, which show alternate terpenoid skeletons. Here, we describe the IDT biosynthetic machinery generating these unusual IDT architectures from Aspergillus striatus and Aspergillus desertorum. The function of four putative cyclases was interrogated via heterologous expression. Two specific cyclases were identified that catalyze the formation of epimers emindole SA and DA from A. striatus and A. desertorum, respectively. These cyclases are both clustered along with all the elements required for basic IDT biosynthesis yet catalyze an unusual Markovnikov-like cyclization cascade with alternate stereochemical control. Their identification reveals that these alternate architectures are not generated by mechanistically sloppy or promiscuous enzymes, but by cyclases capable of delivering precise regio- and stereospecificities.

Indole terpenoids make up a large group of secondary metabolites that display an enticing array of bioactivities. While indole diterpene (IDT) and rarely indole sesquiterpene (IST) pathways have been found individually in filamentous fungi, here we show that both cluster types are encoded within the genome of Tolypocladium album. Through heterologous reconstruction, we demonstrate the SES cluster encodes for IST biosynthesis and can tailor IDT substrates produced by the TER cluster.

Category: Other Introduction/Purpose: Patient Reported Outcome Measures (PROMs) are used to inform treatment, payment, and health policy decisions. As such, it is crucial to develop a robust understanding of extrinsic factors influencing PROM scores. Prior studies suggest sociodemographic factors are associated with variation in outcome scores not only in response to treatment, but also at initial presentation. The purpose of this study was twofold: first, to deepen our understanding of sociodemographic factors affecting baseline PROMs in a foot and ankle population; and second, to assess the extent to which modifiable sociodemographic factors may explain perceived racial and ethnic disparities in outcomes. Increased understanding of the complex relationships between health and social factors may facilitate conscientious use of PROMs and help identify points of intervention for reducing healthcare disparities. Methods: We retrospectively reviewed baseline Foot and Ankle Ability Measure (FAAM) and PROMIS Global-Mental scores of 26,409 foot and ankle patients within our institutional database from 2015 to 2021. Primary predictors included age, sex, race, ethnicity, primary language, education level, median household income, and Charlson Comorbidity Index. All continuous predictors were transformed into categorical variables for analyses. Descriptive analyses, two-tailed T-tests, and Bonferroni post hoc analyses were utilized to assess variation in unadjusted baseline PROM scores stratified by sociodemographic factors. Multivariable modeling was utilized to investigate the independent correlations with and relative importance of the predictors in accounting for variability in outcome scores while adjusting for confounding by all. To examine if the effects of race and ethnicity were modulated by related predictors, we used a series of sequential regression models and evaluated the change in race and ethnicity parameter estimates and R2 values upon including additional predictors in the models. Results: On unadjusted analysis, our data revealed differences in FAAM and PROMIS Global-Mental scores that exceeded the minimal clinically important differences (MCIDs) when stratified by educational level and primary language spoken, but not age, sex, race, ethnicity, median household income, or Charlson Comorbidity Index. Our models accounted for less than 20% of the observed variation in PROM scores. In adjusted models, education level was most prominently associated with baseline scores. Black vs White race had a minor effect in unadjusted models, well below the MCID, and appears to be influenced by differences in education level and household income, which do importantly impact outcomes. Hispanic ethnicity had a larger effect than race in unadjusted analyses and appears to be explained by primary language spoken, education level, and median household income. Conclusion: FAAM and PROMIS Global-Mental baseline scores differed by amounts exceeding the MCID when stratified by educational level and primary language spoken. In adjusted models, race and ethnicity did not independently correlate with clinically significant variations in PROMs scores. Interventions to eliminate disparities among racial and ethnic groups should address modifiable factors such as improving health literacy and removing language and financial barriers to care. To avoid widening health disparities, differences in education level and primary language spoken should be acknowledged when using PROMs as a metric in research, clinical care, or health policy.

Enzyme-catalyzed hydrolysis is a fundamental chemical transformation involved in many essential metabolic processes. The enzyme 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) catalyzes the hydrolysis of adenosine-containing metabolites in cysteine and methionine metabolism. Although MTAN enzymes contain highly similar active site architecture and generally follow a dissociative (DN*AN) reaction mechanism, substantial differences in reaction rates and chemical transition state structures have been reported. To understand how subtle changes in sequence and structure give rise to differences in chemistry between homologous enzymes, we have probed the reaction coordinates of two MTAN enzymes using quantum mechanical/molecular mechanical and molecular dynamics simulations combined with experimental methods. We show that the transition state structure and energy are significantly affected by the recruitment and positioning of the catalytic water molecule and that subtle differences in the noncatalytic active site residues alter the environment of the catalytic water, leading to changes in the reaction coordinate and observed reaction rate.

Introduction: Progressive collapsing foot deformity (PCFD) is frequently associated with a gastrocnemius contracture. Surgical treatment of PCFD often includes a gastrocnemius recession in addition to other corrective procedures, which typically requires a period of restricted weight bearing postoperatively. Isolated gastrocnemius recession may allow passive correction of the deformity, improve orthotic fit, and obviate the need for full reconstruction and restricted weight bearing. The goal of this study was to evaluate patient-reported outcomes after an isolated gastrocnemius recession for flexible PCFD in patients anticipated to have difficulty with postoperative restricted weight bearing. Methods: A total of 47 patients met the inclusion criteria: isolated gastrocnemius recession for flexible PCFD, no previous ipsilateral surgery, and more than 6 months of follow-up. Of 47 eligible patients, 29 (31 feet) participated. Available preoperative and postoperative patient-reported outcomes were gathered, including the Foot and Ankle Ability Measure Activities of Daily Living, visual analog scale, and the Patient-Reported Outcome Measurement Information System Physical Function Short Form 10a. In addition, patients were asked about satisfaction, willingness to undergo the procedure again, and whether orthotics provided better relief. Results: At a mean of 5.1 (range, 0.6 to 9.0) years postoperatively, median Foot and Ankle Ability Measure Activities of Daily Living was 82.1, mean Patient-Reported Outcome Measurement Information System Physical Function Short Form 10a was 44.2, and median visual analog scale was 10 (of 100). Sixty-nine percent of patients were either satisfied or very satisfied, 69% would undergo the procedure again, and 62% reported improved relief with use of orthotics postoperatively. Among the 47 eligible patients, there were 5 (11%) subsequent flatfoot reconstructions. Conclusions: Isolated gastrocnemius recession for the management of flexible PCFD can be effective as this procedure demonstrated good outcomes scores with high procedural satisfaction and 11% of patients proceeding to subsequent flatfoot reconstruction. This alternative approach may be of particular value for patients anticipated to have difficulty with postoperative weight-bearing restrictions. Level of Evidence :IV

Nodulisporic acids (NAs) are structurally complex potent antiinsectan indole diterpenes. We previously reported the biosynthetic gene cluster for these metabolites in Hypoxylon pulicicidum and functionally characterised the first five steps of the biosynthetic pathway. Here we reveal a highly complex biosynthetic array, furnishing multiple end products through expression of cluster components in Penicillium paxilli. We show that seven additional cluster-encoded gene products comprise the biosynthetic machinery that elaborate precursor NAF in this highly branched pathway. The combined action of these enzymes delivers 37 NA congeners including four major end products, NAA, NAA(1), NAA(2) and NAA(4). The plethora of intermediates arises due to modification of the carboxylated prenyl tail by a single promiscuous P450 monooxygenase, NodJ, a pivotal branchpoint enzyme which produces four distinct biosynthetic products giving rise to the complex metabolic grid that characterises NA biosynthesis.

Decoration of the core scaffolds of indole diterpene (IDT) natural products is key to generating structural and bioactivity diversity. Aminoacylation as a tailoring step is rarely linked to terpene biosynthesis and is extremely rare in IDT biosynthesis. Through heterologous pathway reconstruction, we have illuminated the genetic and biochemical basis for the only reported examples of aminoacylation in IDT biosynthesis, demonstrating the unusual involvement of monomodular nonribosomal peptide synthetase (NRPS)-like enzymes in IDT decoration.