Preparation of fluorescent polyurethane microspheres and their applications as reusable sensor for 4-nitrophenol detection and as microplastics model for visualizing polyurethane in cells and zebrafish.

Fluorescent microspheres are of significant interests due to their wide applications in biotechnology fields. However, their preparation presents several challenges, such as the need for dye labeling, the complexity of materials and often sophisticated preparation conditions. Here a simple process for hydrophilic and crosslinked polyurethane (CPU) microspheres, with carboxyl groups on the surface via one-step precipitation polymerization in 40 min, is presented. The microsphere size is easily adjusted by varying experimental conditions. CPU microspheres exhibit high thermal and pH stability with good redispersibility in water, and emit fluorescence without any modification or dye labeling. The emission mechanism is discussed. CPU microspheres are used as fluorescent probe to detect 4-nitrophenol (4-NP) based on their emission in UV light region, with excellent selectivity and sensitivity. In addition, they are reusable with detection limit unchanged after 7 cycles of reuses, a significant feature of this work. The mechanism of fluorescence detection is thoroughly explored and ascribed to the internal filtration effect. Based on the emission in visible light region, CPU microspheres are used as a model of PU microplastics (MPs) to visualize their biodistribution in HeLa and macrophage cells, as well as in zebrafish larvae, providing a reliable tracer for the visualization and tracking of PU MPs in organisms.

Acetyl-CoA Synthetase Mutations S868G and V949I Do Not Confer Resistance to Antimalarial Drugs in vitro in Plasmodium falciparum.

Plasmodium falciparum acetyl-CoA synthetase (PfACAS) protein is an important source of acetyl-CoA. We detected the mutations S868G and V949I in PfACAS by whole-genome sequencing analysis in some recrudescent parasites after antimalarial treatment with artesunate and dihydroartemisinin-piperaquine, suggesting that they may confer drug resistance. Using CRISPR/Cas9 technology, we engineered parasite lines carrying the PfACAS S868G and V949I mutations in two genetic backgrounds and evaluated their susceptibility to antimalarial drugs in vitro. The results demonstrated that PfACAS S868G and V949I mutations alone or in combination were not enough to provide resistance to antimalarial drugs.

Analysis of risk factors for the sigmoid stoma complications in patients after abdominoperineal resection surgery: An observational study.

To analyze the risk factors for intraperitoneal sigmoid stoma complications after abdominoperineal resection (APR) surgery to guide clinical practice. Patients who were diagnosed with rectal cancer and underwent APR surgery from June 2013 to June 2021 were retrospectively enrolled. The characteristics of the stoma complication group and the no stoma complication group were compared, and univariate and multivariate logistic analyses were employed to identify risk factors for sigmoid stoma-related complications. A total of 379 patients who were diagnosed with rectal cancer and underwent APR surgery were enrolled in this study. The average age of the patients was 61.7 ±â€…12.1 years, and 226 (59.6%) patients were males. Patients in the short-term stoma complication group were younger (55.7 vs 62.0, P < .05) and had a more advanced tumor stage (P < .05). However, there was no significant difference between the long-term stoma complication group and the no stoma complication group. Multivariate logistic regression analysis revealed that operation time was an independent risk factor (P < .05, OR = 1.005, 95% CI = 1.000-1.010) for short-term stoma complications. Both the short-term and long-term stoma complication rates in our institution were low. A longer operation time was an independent risk factor for short-term stoma complications after APR surgery.

A Dual-Passband Frequency Selective Surface with High Angular Stability and Polarization Insensitivity.

In this paper, a dual-passband frequency selective surface (FSS) with high angular stability and polarization insensitivity is proposed. The unit structure consists of a circular aperture, two annular apertures and four cross apertures. The designed FSS can achieve a double-passband at the interested frequencies of 8.45 GHz and 12.76 GHz with an insertion loss of less than 1 dB, and it can retain a stable transmission characteristic with the incident angle ranging from 0° to 86° for TE mode and from 0° to 83° for TM mode. Good agreement between the experimental results and the simulated response verifies the feasibility of the proposed FSS.

Patrinia villosa (Thunb.) Juss alleviates CCL4-induced acute liver injury by restoring bile acid levels and inhibiting apoptosis/autophagy.

Background: Patrinia villosa (Thunb.) Juss is one of the plant resources of the famous traditional Chinese medicine "Bai jiang cao (herba patriniae)," and it is considered to function at the liver meridian, thereby treating diseases of the liver as demonstrated by the traditional theory of TCM. Unfortunately, the therapeutic mechanism of the whole plant of PV is so far unknown. Method: UPLC QTOF-MS/MS was used to analyze the profile of PV. Male Sprague-Dawley rats were categorized into five groups, and PV groups (125 and 375 mg/kg) were administered by oral gavage for seven consecutive days. The model of liver injury was induced by intraperitoneal injection of 40% CCl4 oil solution. H&E staining was performed for histological evaluation. The ELISA method was used to assess the serum level of ALT, AST, and T-BIL. Serum and liver bile acid (BA) profiling was analyzed by LC-MS/MS. TUNEL-stained liver sections were used to monitor apoptosis caused by CCl4. HepG2 cells were used to detect autophagy caused by CCl4. Results: A total of 16 compounds were identified from the 70% methanol extract of PV. PV (125 and 375 mg/kg) could reverse the ectopic overexpression of AST, ALT, and T-BIL caused by CCl4 administration. H&E staining indicated that PV (125 and 375 mg/kg) could reduce the infiltration of inflammatory cells and restore liver tissue and hepatocyte structures. Six bile acids, including DCA, HDCA, GCA, TCA, TCDCA, and TUDCA, were significantly altered both in the serum and liver tissue after CCl4 administration, and the level of all these six bile acids was restored by PV treatment. Moreover, PV inhibited apoptosis caused by CCl4 stimulation in liver tissue and suppressed autophagy in HepG2 cells treated with CCl4. Conclusion: The results in this paper for the first time reveal the alteration of the bile acid profile in CCl4-induced liver injury and demonstrate that inhibiting apoptosis and autophagy was involved in P. villosa-elicited liver protection, providing a scientific basis for the clinical utilization of P. villosa as a natural hepatic protective agent.

Promising roles of combined therapy based on immune response and iron metabolism in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an intricate autoimmune disease with diverse manifestations. Immunometabolism reprogramming contributes to the progression of SLE by regulating the phenotype and function of immune cells. Dysregulated iron metabolism is implicated in SLE pathogenesis, affecting both systemic and immune cell-specific iron homeostasis. This review explores the systemic and cellular iron handling and regulation. Additionally, the advancements regarding iron metabolism in SLE with a focus on the distinct subsets of immune cells are highlighted. By gaining insight into the interplay between iron dysregulation and immune dysfunction, the potential therapeutic avenues may be unveiled. However, challenges remain in elucidating cell-specific iron metabolic reprogramming and its contribution to SLE pathogenesis needs further research for personalized therapeutic interventions and biomarker discovery. This review provides an in-depth understanding of immune cell-specific regulatory mechanisms of iron metabolism and new insights in current challenges as well as possible clinical applications.

Predictors of complications after prophylactic ileostomy reversal for rectal cancer: A retrospective study.

BACKGROUND: Previous studies have analyzed the risk factors for complications after ileostomy reversal for rectal cancer (RC), but there were significant differences in the reported risk factors for complications after stoma reversal. No studies have analyzed the risk factors for stoma-related complications and overall postoperative complications separately. AIM: To analyze the risk factors for overall complications and stoma-related complications after ileostomy reversal for patients with RC. METHODS: This was a retrospective study of 439 patients who underwent ileostomy reversal at a clinical center and were followed up between September 2012 and September 2022. Continuous variables are expressed as the mean ± SD and were analyzed with independent-sample t tests, while frequency variables are expressed as n (%), and the χ2 test or Fisher's exact test was used. Univariate and multivariate logistic regression analyses were used to identify predictors of overall complications and stoma-related complications. RESULTS: The overall complication rate after ileostomy reversal was 11.4%. Patients with lower preoperative albumin concentration (P < 0.01), greater blood loss (P = 0.017), and longer operative times (P < 0.01) were more likely to experience postoperative complications. The incidence of stoma-related complications was 6.4%. Analysis of the study showed that a higher body mass index (BMI) (P < 0.01), preoperative comorbid hypertension (P = 0.049), time from primary surgery to ileostomy reversal (P < 0.01) and longer operation time (P = 0.010) were more likely to result in stoma-related complications postoperatively. Multivariate logistic regression analysis revealed that a lower preoperative albumin level (P < 0.01, OR = 0.888, 95%CI: 0.828-0.958) was an independent risk factor for overall complications. Moreover, multivariate analysis revealed that BMI (P < 0.01, OR = 1.176, 95%CI: 1.041-1.330) and time from primary surgery to ileostomy reversal (P < 0.01, OR = 1.140, 95%CI: 1.038-1.252) were independent risk factors for stoma-related complications after stoma reversal. CONCLUSION: The preoperative albumin level was a predictor of overall complications. Preoperative BMI and the time from primary surgery to ileostomy reversal were predictors of stoma-related complications.

Correlation between omega-3 intake and the incidence of diabetic retinopathy based on NHANES from 2005 to 2008.

AIMS: To identify correlations between omega-3 intake and incidence of diabetic retinopathy (DR). METHODS: This was a cross-sectional study using data from participants over age 40 in the National Health and Nutrition Examination Survey (NHANES) 2005-2008. Metrics included participants' intake of omega-3 fatty acids, specifically three types of representative polyunsaturated fatty acids, DR prevalence, and demographic characteristics. Multiple logistic regression models were used to assess the relationship between omega-3 intake and DR. RESULTS: Of the 1243 participants included in this study, omega-3 intake was lower in patients with DR relative to those without DR. Of the three polyunsaturated fatty acids within the omega-3 fatty acid family that we focused on, participants without DR consumed more docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA) than those with DR. In contrast, there was no significant difference in the intake of eicosapentaenoic acid (EPA). Higher omega-3 intake was associated with a decreased risk of DR. In a crude model, the odds ratio (OR) was 0.548 (95% CI 0.315, 0.951; p = 0.033). In the fully adjusted model of omega-3 (model II), the adjusted OR was 0.525 (95% CI 0.306, 0.901; p = 0.021). DPA and DHA were also associated with a decreased risk of DR. In the full adjustment model (model II) of DPA and DHA, the adjusted ORs were 0.0002 (95% CI 0.000, 0.166; p = 0.014) and 0.293 (95% CI 0.105, 0.819; p = 0.020). Subgroup analysis showed that the protective effect of omega-3 against DR was more significant in younger patients (p value = 0.015). CONCLUSIONS: In this cross-sectional study of the U.S. general population, we found that increased intake of omega-3 and its components, specifically DPA and DHA were negatively associated with DR incidence. This suggests that omega-3 may be a potential protective factor for DR and may help to prevent or delay the onset and progression of DR.

Development and validation of a nomogram to predict the risk factors of major complications after radical rectal cancer surgery.

Purpose: The aim of this study was to establish a validated nomogram to predict risk factors for major post-operative complications in patients with rectal cancer (RC) by analyzing the factors contributing to major post-operative complications in RC patients. Methods: We retrospectively collected baseline and surgical information on patients who underwent RC surgery between December 2012 and December 2022 at a single-center teaching hospital. The entire cohort was randomly divided into two subsets (60% of the data for development, 40% for validation). Independent risk factors for major post-operative complications were identified using multivariate logistic regression analyses, and predictive models were developed. Area under the curve (AUC) was calculated using receiver operating characteristic curve (ROC) to assess predictive probability, calibration curves were plotted to compare the predicted probability of the nomogram with the actual probability, and the clinical efficacy of the nomogram was assessed using decision curve analysis (DCA). Results: Our study included 3151 patients who underwent radical surgery for RC, including 1892 in the development set and 1259 in the validation set. Forty (2.1%) patients in the development set and 26 (2.1%) patients in the validation set experienced major post-operative complications. Through multivariate logistic regression analysis, age (p<0.01, OR=1.044, 95% CI=1.016-1.074), pre-operative albumin (p<0.01, OR=0.913, 95% CI=0.866-0.964), and open surgery (p<0.01, OR=2.461, 95% CI=1.284-4.761) were identified as independent risk factors for major post-operative complications in RC, and a nomogram prediction model was established. The AUC of the ROC plot for the development set was 0.7161 (95% Cl=0.6397-0.7924), and the AUC of the ROC plot for the validation set was 0.7191 (95% CI=0.6182-0.8199). The predicted probabilities in the calibration curves were highly consistent with the actual probabilities, which indicated that the prediction model had good predictive ability. The DCA also confirmed the good clinical performance of the nomogram. Conclusion: In this study, a validated nomogram containing three predictors was created to identify risk factors for major complications after radical RC surgery. Due to its accuracy and convenience, it could contribute to personalized management of patients in the perioperative period.

FEN1-assisted DNA logic amplifier circuit for fast and compact DNA computing.

This work developed DNA amplifier logic gates (AND-OR, OR-AND, FAN-IN, FAN-OUT, and 4-bit square-root circuits) using a flap endonuclease 1 (FEN1)-catalyzed signal amplification reaction, for the fastest and compact DNA computing. Moreover, the logic circuit can use input strands with concentrations of less than 1 nM, which is more than 100 times lower than the input concentration of other DNA logic circuits, providing a promising methodology for constructing fast and compact DNA computations.

Comprehensive Analysis of Immune Signatures in Primary Biliary Cholangitis and Autoimmune Hepatitis.

Primary Biliary Cholangitis (PBC) and Autoimmune Hepatitis (AIH) are autoimmune diseases that target hepatocytes and bile duct cells, respectively. Despite their shared autoimmune nature, the differences in immunologic characteristics between them remain largely unexplored. This study seeks to elucidate the unique immunological profiles of PBC and AIH, and to identify key differences. We comprehensively analyzed various T-cell subsets and their receptor expression in a cohort of 45 patients, including 27 PBC and 18 AIH cases. Both diseases exhibited T cell exhaustion and senescence along with a surge in inflammatory cytokines. Significantly increased CD38+HLA-DR+CD8+T cell populations were observed in both diseases. AIH was characterized by an upregulation of CD8+TEMRA, CD4+TEM, and CD4+TEMRA cells, and a concurrent reduction in Treg cells. In contrast, PBC displayed a pronounced presence of Tfh cells and a contraction of CD4-CD8-T cell populations. Correlation analysis revealed that NKP46+NK frequency was closely tied to ALT and AST levels, and TIGIT expression on T cells was associated with GLB level in AIH. In PBC, there is a significant correlation between Tfh cells and ALP levels. Moreover, the identified immune landscapes in both diseases strongly related to disease severity. Through logistic regression analysis, γδ T, TIGIT+Vδ2 T, and Tfh1 cell frequencies emerged as distinct markers capable of differentiating PBC from AIH. In conclusion, our analyses reveal that PBC and AIH share similarities and differences regarding to immune profiles. And γδ T, TIGIT+Vδ2 T, and Tfh1 cell frequencies are potential noninvasive immunological markers that can differentiate PBC from AIH.

An Artificial Enzyme for Asymmetric Nitrocyclopropanation of α,ß-Unsaturated Aldehydes-Design and Evolution.

The introduction of an abiological catalytic group into the binding pocket of a protein host allows for the expansion of enzyme chemistries. Here, we report the generation of an artificial enzyme by genetic encoding of a non-canonical amino acid that contains a secondary amine side chain. The non-canonical amino acid and the binding pocket function synergistically to catalyze the asymmetric nitrocyclopropanation of α,ß-unsaturated aldehydes by the iminium activation mechanism. The designer enzyme was evolved to an optimal variant that catalyzes the reaction at high conversions with high diastereo- and enantioselectivity. This work demonstrates the application of genetic code expansion in enzyme design and expands the scope of enzyme-catalyzed abiological reactions.

More potential uses of specific perforator flaps in the calf - A cadaveric study on the subdermal vascular structure of the lower leg.

BACKGROUND: The perforator flap has garnered significant interest since its inception due to its advantage of not needing a vascular network at the deep fascial level. Perforator flaps are commonly utilized in different flap transplant surgeries, and the thigh flap is presently the most widely used perforator flap. Is it possible for the calf to replace the thigh as a more suitable site for harvesting materials? Currently, there is a lack of relevant anatomical research. This study aims to address this question from an anatomical and imaging perspective. METHODS: This study used cadavers to observe the branches and courses of perforators on the calf and the distribution of skin branches using microdissection techniques, digital X-ray photography, and micro-computed tomography techniques. RESULTS: The perforators had three main branches: the vertical cutaneous branch, the oblique cutaneous branch, and the superficial fascial branch. The superficial fascial branch traveled in the superficial fascia and connected with the nearby perforators. The vertical and oblique cutaneous branches entered the subdermal layer and connected with each other to create the subdermal vascular network. CONCLUSIONS: We observed an intact calf cutaneous branch chain between the cutaneous nerve and the perforator of the infrapopliteal main artery at the superficial vein site. Utilizing this anatomical structure, the calfskin branch has the potential to serve as a substitute for thigh skin flap transplantation and may be applied to perforator flap transplantation in more locations.

Comparison of the Efficacy of Different Radiofrequency Techniques for the Treatment of Lumbar Facet Joint Pain: Combined with Anatomy.

PURPOSE OF REVIEW: Lumbar facet pain is generally considered to be one of the major causes of chronic low back pain. Each lumbar facet joint is innervated by the medial branch of the posterior spinal nerve from its own level and above. Radiofrequency (RF) of the medial branch of the posterior branch of the spinal nerve is an effective method for the treatment of lumbar facet pain. RF technology is diverse, including traditional radiofrequency (TRF), pulsed radiofrequency (PRF), cooled radiofrequency (CRF), low-temperature plasma radiofrequency ablation (CA), and other treatment methods. The purpose of this paper is to compare the efficacy of different radiofrequency techniques and to analyze the reasons for this in the context of anatomy. RECENT FINDINGS: There have been studies confirming the differences in efficacy of different RF techniques. However, most of the studies only compared two RF techniques, not four techniques, TRF, CRF, PRF, and CA, and did not analyze the reasons for the differences in efficacy. This article reviews the differences in the efficacy of the above four RF techniques, clarifies that the differences are mainly due to the inability to precisely localize the medial branch of the posterior branch of the spinal nerve, analyzes the reasons for the inability to precisely localize the posterior branch of the spinal nerve in conjunction with anatomy, and proposes that the development of RF technology for lumbar facet pain requires more in-depth anatomical, imaging, and clinical studies.

Absence of association between Pfnfs1 mutation and in vitro susceptibility to lumefantrine in Plasmodium falciparum.

Artemether-lumefantrine (AL) is the most widely used antimalarial drug for treating uncomplicated falciparum malaria. This study evaluated whether the K65Q mutation in the Plasmodium falciparum cysteine desulfurase IscS (Pfnfs1) gene was associated with alternated susceptibility to lumefantrine using clinical parasite samples from Ghana and the China-Myanmar border area. Parasite isolates from the China-Myanmar border had significantly higher IC50 values to lumefantrine than parasites from Ghana. In addition, the K65 allele was significantly more prevalent in the Ghanaian parasites (34.5%) than in the China-Myanmar border samples (6.8%). However, no difference was observed in the lumefantrine IC50 value between the Pfnfs1 reference K65 allele and the non reference 65Q allele in parasites from the two regions. These data suggest that the Pfnfs1 K65Q mutation may not be a reliable marker for reduced susceptibility to lumefantrine.

PDGFBB facilitates tumorigenesis and malignancy of lung adenocarcinoma associated with PI3K-AKT/MAPK signaling.

Lung adenocarcinoma (LUAD) remains one of the most aggressive tumors and the efficacy of conventional treatment has been bleak. Nowadays, gene-targeted therapy has become a new favorite in tumor therapy. Herein, we investigated the effect of platelet derived growth factor BB (PDGFBB) on LUAD. Firstly, PDGFBB was upregulated in LUAD patients and closely linked with poor survival. Furthermore, the expression of PDGFBB and PDGFRα/ß in LUAD cells was higher than that in normal lung cells. By loss-of-function with herpes simplex virus (HSV)-PDGFi-shRNA, we found that PDGFBB knockdown caused a significant decrease in proliferation and migration, but evoked apoptosis of LUAD cells in vitro. Conversely, exogenous PDGFBB held adverse effect. Additionally, A549 cells with PDGFBB knockdown had a low probability of tumorigenesis in vivo. Moreover, PDGFBB knockdown restrained the growth of xenografts derived from normal A549 cells. Mechanistically, PDGFBB knockdown suppressed PI3K/AKT and Ras/MAPK signaling, while PDGFBB was the opposite. Therefore, we concluded that PDGFBB might facilitate the tumorigenesis and malignancy of LUAD through its functional downstream nodes-PI3K/AKT and Ras/MAPK signaling, which supported that PDGFBB could serve as a rational therapeutic target for LUAD.

The Efficacy and Safety of Nirmatrelvir/Ritonavir Against COVID-19 in Elderly Patients.

Objective: To assess the key factors influencing the effectiveness of nirmatrelvir/ritonavir in treating elderly patients with COVID-19. Methods: This study was conducted on patients aged ≥60 who were admitted to the Second Affiliated Hospital of Soochow University for COVID-19 infection and were treated with nirmatrelvir/ritonavir. Clinical information was collected from patients and steady-state blood concentrations of nirmatrelvir and ritonavir were measured. Factors associated with treatment effects were searched by univariate and multivariate analysis. Results: A total of 68 (51 males and 17 females) patients had a median age of 80 (73.0-84.8) years were enrolled in this study. The blood concentration measurements (trough concentrations) of nirmatrelvir and ritonavir were 5.1 (2.6-7.1) and 0.4 (0.2-0.9) µg/mL, respectively. Adverse drug reaction was reported in 4 (5.9%) patients. Univariate analysis showed that age, clinical classification, APACHE II score, total bilirubin (TBil), aspartate transaminase (AST), lactate dehydrogenase (LDH), and total cholesterol (TC) were significantly associated with the effectiveness of treatment (P value <0.05). Concentration of nirmatrelvir was also associated with treatment outcome (P value <0.1). Based on the results of univariate analysis, the above factors were introduced into the multiple linear regression equation as independent variables, and the results showed that clinical classification was included in the regression equation model and was the most important factor affecting the treatment outcome. By receiver operating characteristic curve analysis, the area under curve of age + biochemical indicators + APACHE II score + clinical classification was 0.968 (95% CI = 0.919-1.000; P <0.0001). Among the 68 patients included in the study, 4 cases experienced adverse drug reactions. Conclusion: Age, clinical classification, APACHE II score, TBil, AST, LDH, and TC were significantly associated with the effectiveness of treatment in elderly patients with COVID-19.

Molecular and cellular pruritus mechanisms in the host skin.

Pruritus, also known as itching, is a complex sensation that involves the activation of specific physiological and cellular receptors. The skin is innervated with sensory nerves as well as some receptors for various sensations, and its immune system has prominent neurological connections. Sensory neurons have a considerable impact on the sensation of itching. However, immune cells also play a role in this process, as they release pruritogens. Disruption of the dermal barrier activates an immune response, initiating a series of chemical, physical, and cellular reactions. These reactions involve various cell types, including keratinocytes, as well as immune cells involved in innate and adaptive immunity. Collective activation of these immune responses confers protection against potential pathogens. Thus, understanding the molecular and cellular mechanisms that contribute to pruritus in host skin is crucial for the advancement of effective treatment approaches. This review provides a comprehensive analysis of the present knowledge concerning the molecular and cellular mechanisms underlying itching signaling in the skin. Additionally, this review explored the integration of these mechanisms with the broader context of itch mediators and the expression of their receptors in the skin.

The Diagnostic Value of Carnett's Test with Chronic Abdominal Pain: A Narrative Review.

PURPOSE OF REVIEW: Chronic abdominal wall pain is a poorly recognized cause of chronic abdominal pain, and patients frequently go misdiagnosed despite a battery of medical tests. The Carnett's test is a diagnostic tool used to distinguish between abdominal wall pain and visceral pain. This review synthesizes the current literature on the Carnett's test, merges the viewpoints of diverse writers, and evaluates and reports on the Carnett's test's applicability. RECENT FINDINGS: Several clinical investigations have established the usefulness of the Carnett's test in the diagnosis of chronic abdominal wall pain. Furthermore, the Carnett's test is quite useful in determining the depth of the mass and detecting psychogenic abdominal pain. However, its diagnostic use for acute abdominal pain is limited. The Carnett's test is a simple and safe point-of-care diagnostic technique, with several studies supporting its usefulness. Early detection of abdominal wall pain is critical for chronic abdominal wall pain therapy. Carnett's test is very useful in patients with chronic, unexplained local abdominal discomfort who are compliant and do not have a clear rationale for surgery.

Conformational Control in Diterpene Synthase TlnA: Elucidating the Mechanism of 5-8-6 Skeleton Formation through Ring Expansion.

TlnA produces a distinct cyclohexane-fused 5-8-6 ring system, different from the prevalent 5-8-5 scaffold synthesized by well-established enzymes. This study identifies two conformations of a carbocation intermediate, revealing how the enzyme environment prohibits one conformation due to steric hindrance, thereby directing the formation of the 5-8-6 system over the 5-8-5 scaffold. This investigation enhances our understanding of diterpene biosynthesis and the impact of enzyme environments on chemical reactions, providing valuable insights into the formation of complex cyclic structures.