A Unifying Mechanism of DNA Translocation Underlying Chromatin Remodeling.

Chromatin remodelers alter the position and composition of nucleosomes, and play key roles in the regulation of chromatin structure and various chromatin-based transactions. Recent cryo-electron microscopy (cryo-EM) and single-molecule fluorescence resonance energy transfer (smFRET) studies have shed mechanistic light on the fundamental question of how the remodeling enzymes couple with ATP hydrolysis to slide nucleosomes. Structures of the chromatin remodeler Snf2 bound to the nucleosome reveal the conformational cycle of the enzyme and the induced DNA distortion. Investigations on ISWI, Chd1, and INO80 support a unifying fundamental mechanism of DNA translocation. Finally, studies of the SWR1 complex suggest that the enzyme distorts the DNA abnormally to achieve histone exchange without net DNA translocation.

Bioinspired FeN5 Sites with Enhanced Peroxidase-like Activity Enable Colorimetric Sensing of Uranyl Ions in Seawater.

Nanozymes with peroxidase (POD)-like activity have garnered significant attention due to their exceptional performance in colorimetric assays. However, nanozymes often possess oxidase (OD) and POD-like activity simultaneously, which affects the accuracy and sensitivity of the detection results. To address this issue, inspired by the catalytic pocket of natural POD, a single-atom nanozyme with FeN5 configuration is designed, exhibiting enhanced POD-like activity in comparison with a single-atom nanozyme with FeN4 configuration. The axial N atom in FeN5 highly mimics the amino acid residues in natural POD to optimize the electronic structure of the metal active center Fe, realizing the efficient activation of H2O2. In addition, in the presence of both H2O2 and O2, FeN5 enhances the activation of H2O2, effectively avoiding the interference of dissolved oxygen in colorimetric sensing. As a proof-of-concept application, a colorimetric detection platform for uranyl ions (UO22+) in seawater is successfully constructed, demonstrating satisfactory sensitivity and specificity.

Survival benefits of postoperative radiotherapy in patients with cT1 - 2N1M0 breast cancer after neoadjuvant chemotherapy: a SEER-based population study.

BACKGROUND: Whether patients with cT1 - 2N1M0 breast cancer can benefit from postoperative radiotherapy (RT) after receiving neoadjuvant chemotherapy (NAC) has been controversial. Therefore, the purpose of this study was to explore whether postoperative RT can benefit this group of patients in terms of survival. METHODS: We used Surveillance, Epidemiology, and End Results (SEER) data to conduct a retrospective review of women with cT1 - 2N1M0 breast cancer diagnosed between 20 and 80 years of age who received NAC between 2010 and 2015. Our study compared the impact of postoperative RT on overall survival (OS) and cancer-specific survival (CSS) in breast cancer patients using propensity score matching (PSM) and performed subgroup analysis. RESULTS: This study finally included 1092 cT1 - 2N1M0 breast cancer patients. Regardless of the patient's PSM status, postoperative RT was significantly associated with OS of cT1-2N1M0 breast cancer patients who received NAC. Specifically, the 10-year OS rate was 78.7% before PSM matching, compared with 71.1% in patients who did not receive postoperative RT, and the difference was more significant after PSM matching, which was 83.1% and 71.1% respectively. However, postoperative RT did not significantly benefit CSS in patients with cT1 - 2N1M0 breast cancer who received NAC. The 10-year CSS rate was 81.4% VS 76.2% (P = 0.085) before PSM matching and 85.8% VS 76.2%(P = 0.076) after matching. Due to the intersection of OS and CSS curves, this restricted mean survival time (RMST) method was chosen as a supplement. After 60 months, the OS difference in RMST between the postoperative RT group and the non-radiotherapy (noRT) group was 7.37 months (95%CI: 0.54-14.21; P = 0.034), and the CSS difference was 5.18 months (95%CI: -1.31-11.68; P = 0.118). Subgroup analysis found that in patients with right-sided breast cancer, postoperative RT improved the patient's OS (HR = 0.45, 95%CI: 0.21-0.95, P = 0.037) and CSS (HR = 0.42, 95%CI: 0.18-0.98, P = 0.045). CONCLUSIONS: Our results showed that additional postoperative RT improved the OS of cT1 - 2N1M0 breast cancer patients who received NAC, but failed to improve their CSS. It is worth noting that in the subgroup analysis of patients with right-sided breast cancer, we observed significant improvements in OS and CSS. And further prospective studies are still needed to verify the effect of postoperative RT in different subgroups.

Habitual salt preference worsens blood pressure in hospitalized hypertensive patients with omicron infection under epidemic-related stress.

BACKGROUND: We investigated the synergistic effect of stress and habitual salt preference (SP) on blood pressure (BP) in the hospitalized Omicron-infected patients. METHODS: From 15,185 hospitalized Omicron-infected patients who reported having high BP or hypertension, we recruited 662 patients. All patients completed an electronic questionnaire on diet and stress, and were required to complete morning BP monitoring at least three times. RESULTS: The hypertensive group (n = 309) had higher habitual SP (P = 0.015) and COVID-19 related stress (P < 0.001), and had longer hospital stays (7.4 ± 1.5 days vs. 7.2 ± 0.5 days, P = 0.019) compared with controls (n = 353). After adjusting for a wide range of covariates including Omicron epidemic-related stress, habitual SP was found to increase both systolic (4.9 [95% confidence interval (CI), 2.3-7.4] mmHg, P < 0.001) and diastolic (2.1 [95%CI, 0.6-3.6] mmHg, P = 0.006) BP in hypertensive patients, and increase diastolic BP (2.0 [95%CI, 0.2-3.7] mmHg, P = 0.026) in the control group. 31 (8.8%) patients without a history of hypertension were discovered to have elevated BP during hospitalization, and stress was shown to be different in those patients (P < 0.001). In contrast, habitual SP was more common in hypertensive patients with uncontrolled BP, compared with patients with controlled BP (P = 0.002). CONCLUSIONS: Habitual SP and psychosocial stress were associated with higher BP in Omicron-infected patients both with and without hypertension. Nonpharmaceutical intervention including dietary guidance and psychiatric therapy are crucial for BP control during the long COVID-19 period.

Peripheral Blood Non-Coding RNA as Biomarker for Schizophrenia: A Review.

Schizophrenia (SCZ) is a complex and heterogeneous neuropsychiatric disorder that lacks objective diagnostic indicators and the pathogenesis remain unclear. Genetic factors may exert a significant impact on the development of the condition. While obtaining brain tissue for biopsy in the course of adjuvant diagnosis of SCZ patients may not be possible, the collection of peripheral blood is more accessible and easier to implement. In recent years, the development and application of RNA sequencing technology has made seeking biomarkers of SCZ becomes more feasible. There is emerging evidence suggesting that certain non-coding RNAs (ncRNA) are distinctly different in the peripheral blood of SCZ patients and healthy controls. Although the mechanisms remain unclear, these aberrantly expressed ncRNAs may be intimately associated with the onset and development of SCZ and may be of great significance for the diagnosis and treatment of SCZ. Therefore, we reviewed the expression of distinct types of ncRNAs that have been found in the peripheral blood of SCZ patients and explored their potential application as diagnostic biomarkers of SCZ. Differentially expressed ncRNAs in the peripheral blood of SCZ patients could not only serve as potential diagnostic biomarkers and therapeutic targets for SCZ but may also have implications for advancing understanding of the molecular mechanisms underlying the development of SCZ and elucidating the complex etiology of SCZ. Early diagnostic biomarkers obtained directly from peripheral blood are of great significance for the timely diagnosis and treatment of SCZ. Our review will enhance the comprehension of molecular mechanisms of SCZ and contribute to the identification of promising ncRNAs in peripheral blood for both diagnosis and therapy of SCZ.

Increased thoracic fluid content is associated with higher risk for pneumonia in patients undergoing maintenance hemodialysis.

BACKGROUND: Pneumonia is the most common infectious disease in patients undergoing maintenance hemodialysis (MHD). The aim of this study is to determine the possible predictive value of thoracic fluid content (TFC) for pneumonia in this population. METHOD: Clinical data were recorded for 1412 MHD patients who were hospitalized for certain comorbidities or complications. Each patient underwent an impedance cardiography (ICG) examination before next dialysis session after admission. Patients were divided into Having-, Will-have-, and Non-pneumonia groups based on whether they had pneumonia at the time of ICG examination after the admission and within five months after the examination. Hemodynamic parameters and other clinical data were compared and analyzed. RESULTS: Patients who were going to develop pneumonia were older, and had a higher proportion of diabetes, poorer nutritional status, a higher level of inflammatory, poorer cardiac function, and more fluid volume load than those who did not develop pneumonia. Multivariate binary logistic analysis revealed that for each 1/KΩ increase in TFC and 1 increase in neutrophil-to-lymphocyte ratio (NLR), the risk of the development of pneumonia increased by 3.1% (p ˂ 0.01) and 7.2% (p = 0.035), respectively, whereas for each 1 g/L increase in hemoglobin and 1 g/L increase in serum albumin, the risk of the development of pneumonia decreased by 1.3% (p = 0.034) and 5% (p = 0.048), respectively. CONCLUSIONS: TFC, NLR, hemoglobin, and serum albumin were independent risk factors for the development of pneumonia in MHD patients. Given the advantages of ICG, TFC can be used clinically as a helpful predictor of pneumonia in MHD patients.

Large B7 Triangles in Hollow Spherical Trihedral Metallo-borospherenes and Their Endohedral Complexes of B20TMn (TM = Sc, Y; n = 3, 4): a Theoretical Characterization.

The spherical trihedral metallo-borospherene D3h B18Ln3- (Ln = La, Tb), in which both Ln and B atoms are integral parts of the cage surface, represents a brand new geometric structure. On the basis of particle swarm optimization searches and density functional theory calculations, a large spherical trihedral metallo-borospherene D3h B20TM3 (TM = Sc, Y) is predicted herein, of which the B20 framework can be viewed as two equivalent B7 triangles, rather than two routine equivalent B6 triangles in B18Ln3-, connected by three B2 units. More interestingly, an additional TM can be embedded into the center of surface cage, first designing the co-existence of empty and endohedral spherical trihedral metallo-borospherene, of which the stability is further enhanced and the energy gaps of 1.25 and 1.20 eV are enlarged to 2.86 and 2.61 eV at the PBE0 level for Sc and Y as dopants, respectively. Detailed orbital and bonding analyses show that B20TM4 resembles B18Ln3-, except for the six missing 2c-2e B-B σ bonds, the 10 more 3c-2e B-B σ bonds, and the lack of one delocalized π bonds for the B20 skeleton, where the absence of two totally delocalized Sc-B bonds lead to the relative instability of B20Sc3. The novel hollow geometric structure can be expanded to their charged states with one or three negative charges and to the systems doped by three Ti, Zr, or Hf atoms. Their infrared and Raman spectra are theoretically simulated to facilitate the experimental characterizations in future. Our findings enrich the family of trihedral metallo-borospherene and provide a B7 triangular motif for the B-framework.

Mechanistic Insights into the Anaerobic Degradation of Globally Abundant Dihydroxypropanesulfonate Catalyzed by the DHPS-Sulfolyase (HpsG).

2(S)-Dihydroxypropanesulfonate (DHPS) is the main abundant organosulfonate in the biosphere generated by the microbial degradation of the abundant organosulfur species 6-deoxy-6-sulfo-d-glucopyranose (sulfoquinovose, SQ). Massive amounts of DHPS can also be produced by the highly abundant oceanic diatoms. The quantity of degradation DHPS is so large that it has become an important part of the earth's sulfur. The recently characterized O2-sensitive glycyl radical enzyme DHPS-sulfolyase HpsG in anaerobic bacteria was found to be capable of cleaving the C-S bond of DHPS under anaerobic conditions. However, the detailed degradation mechanism is still unclear. Here, on the basis of the crystal structure of HpsG, we constructed the computational model and performed QM/MM calculations to illuminate the anaerobic degradation mechanism of DHPS. Our calculations revealed that the degradation reaction follows an unusual radical-dependent mechanism that does not require a conserved Glu464 to deprotonate the C2 hydroxyl of substrate to promote the C-S cleavage; instead, after the first hydrogen abstraction triggered by the thiyl radical (Cys462), the C-S bond in 2(S)-dihydroxypropanesulfonate can directly collapse. Thus, conserved Glu464 mainly plays a role in stabilizing the substrate and reaction intermediate by forming a hydrogen bond. After the release of the sulfonic acid group from the protein environment, the deprotonated Glu464 spontaneously accepts a proton from the C2 hydroxyl of the substrate radical. Our findings clarified an unusual C-S cleavage mechanism involved in the DHPS degradation reaction catalyzed by GREs.

Structure and regulation of the chromatin remodeller ISWI.

ISWI is a member of the SWI2/SNF2 family of chromatin remodellers, which also includes Snf2, Chd1, and Ino80. ISWI is the catalytic subunit of several chromatin remodelling complexes, which mobilize nucleosomes along genomic DNA, promoting replication progression, transcription repression, heterochromatin formation, and many other nuclear processes. The ATPase motor of ISWI is an autonomous remodelling machine, whereas its carboxy (C)-terminal HAND-SAND-SLIDE (HSS) domain functions in binding extranucleosomal linker DNA. The activity of the catalytic core of ISWI is inhibited by the regulatory AutoN and NegC domains, which are in turn antagonized by the H4 tail and extranucleosomal DNA, respectively, to ensure the appropriate chromatin landscape in cells. How AutoN and NegC inhibit ISWI and regulate its nucleosome-centring activity remains elusive. Here we report the crystal structures of ISWI from the thermophilic yeast Myceliophthora thermophila and its complex with a histone H4 peptide. Our data show the amino (N)-terminal AutoN domain contains two inhibitory elements, which collectively bind the second RecA-like domain (core2), holding the enzyme in an inactive conformation. The H4 peptide binds to the core2 domain coincident with one of the AutoN-binding sites, explaining the ISWI activation by H4. The H4-binding surface is conserved in Snf2 and functions beyond AutoN regulation. The C-terminal NegC domain is involved in binding to the core2 domain and functions as an allosteric element for ISWI to respond to the extranucleosomal DNA length.

Serum Fat-Soluble Vitamin Levels of 6,082 Minors in Zhuzhou City.

Objective: To explore the nutritional status of serum fat-soluble vitamins such as vitamin A, 25-hydroxyvitamin D, and vitamin E of minors in the Zhuzhou area to provide a scientific basis for clinical guidance to supplement fat-soluble vitamins reasonably. Method: A total of 6,082 minors who underwent physical examination from January 2017 to February 2019 in the Children's Health Department of Zhuzhou Hospital affiliated with XiangYa School of Medicine of Central South University were selected as the subjects to measure the levels of serum fat-soluble vitamins A, D, and E. Results: (1) Their average levels of serum vitamin A, 25-hydroxyvitamin D, and vitamin E were (0.34 ± 0.08) mg/mL, (34.65 ± 10.24) ng/mL, and (10.11 ± 2.65) mg/mL, respectively. (2) Serum vitamin E showed a gender difference (P < 0.001). (3) The average levels of serum 25-hydroxyvitamin D and vitamin E in infancy, early childhood, preschool age, school age, and adolescence decreased gradually (P < 0.05). In contrast, the average level of serum vitamin A ranged between 0.32 mg/mL and 0.37 mg/mL. (4) The age was negatively correlated with serum 25-hydroxyvitamin D (r = -0.517, P < 0.001) and weakly negatively correlated with vitamin E (r = -0.366, P < 0.001), but weakly positively correlated with vitamin A (r = 0.269, P < 0.001). Conclusion: Minors from infancy to adolescence in Zhuzhou should strengthen their supplementation of fat-soluble vitamins.

Superwetting Electrospun PDMS/PMMA Membrane for PM2.5 Capture and Microdroplet Transfer.

Efficient acquiring and removal of a hazardous particulate matter (PM) have significant effects on human health. Here, we illustrate the fabrication of a superwetting electrospun polydimethylsiloxane/polymethyl methacrylate (PDMS/PMMA) membrane (EPPM) with multifunctional performance for PM2.5 capture and microdroplet transfer, where PMMA was added as a carrier polymer to the superhydrophobic PDMS, which has very low cohesive energy density. The obtained EPPM, which is composed of special bead-on-string fibers with a mean fiber diameter of 350 nm, shows a porous structure with an aperture of 7.87 µm (calculated by the bubble pressure method) and superb thermostability (up to 325 °C). The EPPM possesses an excellent PM2.5 purification efficiency of nearly up to 100% at a very low pressure drop (70 Pa, <0.07% of the atmospheric pressure) under the condition of high humidity (96 ± 3%), which is greatly advantageous over those hydrophilic filters frequently suffering the drawbacks of low efficiency or total invalidation in humid environments. In addition, benefitting from the superhydrophobic and strong adhesive properties of the membrane surface, the EPPM could complete the trace aqueous sample analysis such as "robotic hand" from superhydrophobic to hydrophilic surfaces without any contamination or loss and hold a high contact angle of 161.6° for water. Altogether, the EPPM may have technological advantages as a kind of novel fibrous filter in diverse environmental applications, including PM2.5 capture, separation, microdroplet transfer, and so on.

Computational Study of the C5-Hydroxylation Mechanism Catalyzed by the Diiron Monooxygenase PtmU3 as Part of the Platensimycin Biosynthesis.

PtmU3 is a newly identified nonheme diiron monooxygenase, which installs a C-5 ß-hydroxyl group into the C-19 CoA-ester intermediate involved in the biosynthesis of unique diterpene-derived scaffolds of platensimycin and platencin. PtmU3 possesses a noncanonical diiron active site architecture of a saturated six-coordinate iron center and lacks the µ-oxo bridge. Although the hydroxylation process is a simple reaction for nonheme mononuclear iron-dependent enzymes, how PtmU3 employs the diiron center to catalyze the H-abstraction and OH-rebound is still unknown. In particular, the electronic characteristic of diiron is also unclear. To understand the catalytic mechanism of PtmU3, we constructed two reactant models in which both the Fe1II-Fe2III-superoxo and Fe1II-Fe2IV═O are considered to trigger the H-abstraction and performed a series of quantum mechanics/molecular mechanics calculations. Our calculation results reveal that PtmU3 is a special monooxygenase, that is, both atoms of the dioxygen molecule can be incorporated into two molecules of the substrate by the successive reactions. In the first-round reaction, PtmU3 uses the Fe1II-Fe2III-superoxo to install a hydroxyl group into the substrate, generating the high-reactive Fe1II-Fe2IV═O complex. In the second-round reaction, the Fe1II-Fe2IV═O species is responsible for the hydroxylation of another molecule of the substrate. In the diiron center, Fe2 adopts the high spin state (S = 5/2) during the catalysis, whereas for Fe1, in addition to its structural role, it may also play an assistant role for Fe1 catalysis. In the two successive OH-installing steps, the H-abstraction is always the rate-liming step. E241 and D308 not only act as bridging ligands to connect two Fe ions but also take part in the electron reorganization. Owing to the high reactivity of Fe1II-Fe2IV═O compared to Fe1II-Fe2III-superoxo, besides the C5-hydroxylation, the C3- or C18-hydroxylation was also calculated to be feasible.

Formation Mechanism of Cofactor Cys-Tyr in the Cysteine Dioxygenases (CDO and F2-CDO) and Its Influence on Catalysis: A QM/MM Study.

Cysteine dioxygenase (CDO) is a nonheme mononuclear iron enzyme, which catalyzes the oxidation of cysteine to cysteine sulfinic acid. Crystal structure studies of mammalian CDO showed that there is a cross-linked cysteine-tyrosine (Cys-Tyr) cofactor in its active site. Moreover, the formation of the Cys-Tyr cofactor requires the metal cofactor (Fe2+) and O2, and it was previously considered to substantially enhance the catalytic efficiency and half-life of CDO. Recently, a pure human CDO (F2-CDO) without including the Cys-Tyr cofactor was crystalized by the site-directed mutagenesis approach in the anaerobic condition. In this work, to gain insights into the formation mechanism of the Cys-Tyr cofactor and whether it can really promote the catalytic reactivity of CDO, a series of computational models have been constructed, and quantum mechanical/molecular mechanical (QM/MM) calculations have been performed. Our calculation results reveal that WT-CDO and F2-CDO follow different mechanisms for the formation of the Cys-Tyr cofactor. In F2-CDO, the cofactor formation contains the H-abstraction, C-S bond formation, intramolecular F migration, and aromatization of the residue F2Y157, in which the Fe-coordinate dioxygen can be recovered after the formation cofactor; however, in the WT-CDO, the cofactor formation shows some differences. During the reaction, hydrogen peroxide is generated, and the final aromatization requires the assistance of one water molecule. Furthermore, the overall barriers of cofactor formation are always higher than l-cysteine oxidation for both WT-CDO and F2-CDO irrespective of the absence or presence of the cofactor. Thus, we can theoretically confirm that the Cys-Tyr cofactor is not essential for the oxidation activity of CDO, and cofactor formation is just an accompanying reaction but not a prerequisite for the oxidation reaction. These results may provide useful information for understanding the catalysis of CDO.

Inactivation Mechanism of Neuronal Nitric Oxide Synthase by (S)-2-Amino-5-(2-(methylthio)acetimidamido)pentanoic Acid: Chemical Conversion of the Inactivator in the Active Site.

Neuronal nitric oxide synthase (nNOS) is one of the three isoforms of nitric oxide synthase (NOS). The other two isoforms include inducible NOS (iNOS) and endothelial NOS (eNOS). These three isoforms of NOS are widely present in both human and other mammals and are responsible for the biosynthesis of NO. As an essential biological molecule, NO plays an essential role in neurotransmission, immune response, and vasodilation; however, the overproduction of NO can cause a series of diseases. Thus, the selective inhibition of three isoforms of NOS has been considered to be important in treating related diseases. The active sites of the three enzymes are highly conserved, causing the selective inhibition of the three enzymes to be a great challenge. (S)-2-Amino-5-(2-(methylthio)acetimidamido)pentanoic acid (1) has been experimentally proved to be a selective and time-dependent irreversible inhibitor of nNOS, and three pathways, including sulfide oxidation, oxidative dethiolation, and oxidative demethylation, have been suggested. In this work, we performed quantum mechanics/molecular mechanics calculations to verify the chemical conversion of inactivator 1. Although we agree with the previously suggested chemical transformation process, our calculations demonstrated that there are lower energy pathways to accomplish both oxidative dethiolation and oxidative demethylation. These three branching reactions are competitive, but only dethiolation and demethylation reactions can generate inhibitory intermediates. As a powerful time-dependent irreversible inhibitor of nNOS, the key sulfur atom and middle imine are all necessary. Our calculation results not only verified the chemical reaction of inhibitor 1 occurring in the enzymatic active site but also explained the inactivation mechanism of inhibitor 1. This is also the first verified example of the heme-enzyme-catalyzed S-demethylation mechanism.

Characterization of PmDGM Conferring Powdery Mildew Resistance in Chinese Wheat Landrace Duanganmang.

Wheat powdery mildew, caused by Blumeria graminis f. sp. tritici, is a devastating disease that threatens yield and quality. Host resistance is considered the most effective and preferred means to control this disease. Wheat landrace Duanganmang (DGM) showed high resistance or near immunity to Blumeria graminis f. sp. tritici mixture from Henan Province, China. DGM was crossed with highly susceptible Chinese wheat landrace Huixianhong (HXH) and cultivar 'Shimai 15' (SM15) to produce genetic populations. The resistance of DGM to Blumeria graminis f. sp. tritici isolate E09 was shown to be controlled by a single dominant Mendelian factor, tentatively designated PmDGM. Marker analysis and 55K single nucleotide polymorphism (SNP) array scanning showed that this gene was positioned in the Pm5 interval (2.4 cM or 1.61 Mb) flanked by Xhenu099 and Xmp1158 in the Chinese Spring reference genome. Homology-based cloning and sequence analysis demonstrated that DGM has the identical NLR gene (Pm5e) and RXL gene reported in Fuzhuang 30 (FZ30), conferring and modifying powdery mildew resistance, respectively. However, based on the different reaction patterns to the Blumeria graminis f. sp. tritici isolate B15 between DGM and FZ30, the authors speculate that DGM may have two tightly linked genes that could not be separated in the current mapping population, one of which is PmDGM and the other being Pm5e. Hence, this study provides a valuable resistance resource for improvement of powdery mildew resistance.

The Retaining Mechanism of Xylose Transfer Catalyzed by Xyloside α-1,3-Xylosyltransferase (XXYLT1): a Quantum Mechanics/Molecular Mechanics Study.

Glycosyltransferases (GTs) are a ubiquitous group of enzymes that catalyze the synthesis of glycosidic bonds. In this work, we focused on the retained reaction catalyzed by xyloside α-1,3-xylosyltransferase (XXYLT1) from Mus musculus. Our calculations revealed that the xylose transfer reaction follows the SNi-like mechanism, which involves a short-lived oxocarbenium-phosphate ion-pair intermediate (IP). The previously obtained crystal structure of the UDP-Xyl ternary Michaelis reaction complex was found to be an inactive form. Accordingly, the ß-phosphate oxygen O3B of the donor should first undergo a conformational change to reach an active state where the donor forms a strong hydrogen bond with the acceptor, facilitating the departure of the phosphate group. Our calculations also revealed that two predicated transition states for the sugar-phosphate bond cleavage and glycosidic bond formation are structurally similar to the short-lived intermediate, which contains a three-member ring formed by the ß-phosphate oxygen, the hydroxyl oxygen in the acceptor, and the anomeric carbon. It can be considered as a typical characteristic of the SNi-like mechanism. In addition, a nearby polar residue, Q330, is responsible for stabilizing the short-lived intermediate by electrostatic interactions. Thus, the Q330A mutant can abolish the activity of XXYLT1. In addition, using UDP-glucose as the donor, our calculations revealed that glucose transfer would correspond to a higher energy barrier owing to the steric repulsion between the glucosyl moiety and the nearby residue L327, indicating the requirement of active site architecture for glucose transfer. These findings not only explain the experimental observations but also are meaningful for clarifying the mechanism of GTs.

Rapid exacerbation featuring acute leukemoid reaction after retrolaparoscopic nephrectomy: a rare case report of renal cell carcinoma with postoperative comprehensive genomic profiling.

BACKGROUND: Rapid lethal exacerbation and recurrence featuring acute leukemoid reaction (ALR) after retrolaparoscopic radical nephrectomy (RN) is a relatively rare clinical incident. Performing the reoperation for the patient and analyzing the tissue-based genetic mutation information postoperatively are a skill-demanding and meaningful task, which have been even more rarely reported. CASE PRESENTATION: We present a case with a large right renal mass (13.0 × 10.0 × 8.0 cm). This 71-year-old male patient underwent the retrolaparoscopic RN in our department. The operation was technically precise and successful with final pathological diagnosis of hybrid (clear cell and papillary type) renal cell carcinoma (RCC). However, 10 days after the patient was discharged, he was readmitted with the chief complaint of high fever with severe right flank pain. CT scanning revealed that right retroperitoneal hematoma and the blood routine showed the dramatic elevation of white blood cell count (WBC). Even though the immediate broad-spectrum antibiotics were administered without delay and subsequent percutaneous puncturing and drainage was performed, the patient's condition still exacerbated rapidly. In spite of the reoperation of hematoma evacuation, the patient died of multiple organ failure 10 days after the reoperation. The pathological result of reoperation showed the necrotic and hematoma tissue blended with RCC tumor cells (nuclear grading III), and both of the postoperative tissue-originated comprehensive genomic profiling by using the specimens from the RN and reoperation respectively indicated significant mutations of some oncogenes which might have potential relevance with ALR. Besides, both of the immunohistochemical (IHC) staining results from primary surgical renal mass and reoperative resected tissue revealed the positive expressions of granulocyte colony-stimulating factor (G-CSF). CONCLUSIONS: ALR may be a predictor of poor prognosis in patients with RCC, and comprehensive genomic profiling as well as the alterative expression of G-CSF can help to provide potential valuable genetic etiological information and evidence for guiding the potential effective molecular-targeting therapy.

Insights into the Mechanism and Enantioselectivity in the Biosynthesis of Ergot Alkaloid Cycloclavine Catalyzed by Aj_EasH from Aspergillus japonicus.

Cycloclavine is a complex ergot alkaloid containing an unusual cyclopropyl moiety, which has a wide range of biological activities and pharmaceutical applications. The biosynthesis of cycloclavine requires a series of enzymes, one of which is a nonheme FeII/α-ketoglutarate-dependent (aKG) oxidase (Aj_EasH). According to the previous proposal, the cyclopropyl ring formation catalyzed by Aj_EasH follows an unprecedented oxidative mechanism; however, the reaction details are unknown. In this article, on the basis of the recently obtained crystal structure of Aj_EasH (EasH from Aspergillus japonicas), the reactant models were built, and the reaction details were investigated by performing QM-only and combined QM and MM calculations. Our calculation results reveal that the biosynthesis of cyclopropyl moiety involves a radical intermediate rather than a carbocationic or carbanionic intermediate as in the biosynthesis of terpenoid family. The iron(IV)-oxo first abstracts a hydrogen atom from the substrate to trigger the reaction, and then the generated radical intermediate undergoes ring rearrangement to form the fused 5-3 ring system of cycloclavine. On the basis of our calculations, the absolute configuration of the cycloclavine catalyzed by Aj_EasH from Aspergillus japonicus should be (5R,8R,10R), which is different from the product isolated from Ipomoea hildebrandtii (5R,8S,10S). Residues at the active site play an important role in substrate binding, ring rearrangement, and enantioselectivity.

Catalytic mechanism of the PrhA (V150L/A232S) double mutant involved in the fungal meroterpenoid biosynthetic pathway: a QM/MM study.

PrhA from Penicillium brasilianum and AusE from Aspergillus nidulans are nonheme Fe(ii)/α-ketoglutarate-dependent oxygenases, which are involved in the fungal meroterpenoid biosynthetic pathways. Both enzymes use preaustinoid A1 as a common substrate to form divergent products through dynamic skeletal rearrangement. Importantly, structure-guided mutagenesis results in the successful interconversion of AusE and PrhA functions, for example, the PrhA(V150L/A232S) double mutant carried out the same catalysis as AusE. Here, on the basis of the crystal structure of the PrhA (V150L/A232S) double mutant in complex with Fe(ii), αKG and the substrate preaustinoid A1, computational models were constructed, and combined quantum mechanics/molecular mechanics (QM/MM) calculations were performed to illuminate the reaction mechanism at the atomistic level. According to our calculation results, the whole reaction occurs on the quintet state surface. All three steps, including desaturation, ring rearrangement and hydroxylation, require three hydrogen abstractions by FeIV[double bond, length as m-dash]O to trigger the reaction. Owing to the relative position of FeIV[double bond, length as m-dash]O to the hydrogen atoms in the substrate to be extracted, the three H-abstractions correspond to different energy barriers, which are 17.9, 23.6 and 21.8 kcal mol-1, respectively. For the ring rearrangement, as soon as the H5 is extracted, the skeletal rearrangement is very easy. However, in the hydroxylation of intermediate preaustinoid A3, the final O-rebound corresponds to a high barrier, which is mainly caused by the long distance between the Fe-OH and -CH2 radical. It is the relative orientation of the substrate to the highly reactive FeIV[double bond, length as m-dash]O that controls the catalytic chemistry of these enzymes. The reaction barriers are sensitive to the geometry of FeIV[double bond, length as m-dash]OHx (Hx is the hydrogen atom to be extracted). These results may provide useful information for understanding the mechanisms of AusE and PrhA as well as other nonheme Fe(ii)/α-ketoglutarate-dependent oxygenases.

Conversion mechanism of enoyl thioesters into acyl thioesters catalyzed by 2-enoyl-thioester reductases from Candida Tropicalis.

Enoyl thioester reductase from Candida tropicalis (Etr1p) catalyzes the NADPH-dependent conversion of enoyl thioesters into acyl thioesters, which are essential in fatty acid and second metabolite biosynthesis. In this paper, we explored the detailed catalytic mechanism of Etr1p by performing QM/MM calculations. Here, we focused on the formation of the covalent ene adduct intermediate and the proton transfer from Tyr79 to the substrate. Our calculation results reveal that the formation of the stable covalent ene adduct follows the Michael addition mechanism rather than the electrocyclic ene reaction. In addition, the ene adduct intermediate can reversibly decompose into the carbanion, and the proton of Tyr79 undertakes a direct electrophilic attack on the substrate to yield the product. In addition, three crystal water molecules do not participate in the catalytic reaction, but they play a crucial role in the hydride transfer and the proton transfer processes by forming a hydrogen bond network. These findings presented here would benefit our understanding of the catalytic mechanism of the NADPH-dependent enzyme.