Computer-directed rational design enhanced the thermostability of carbonyl reductase LsCR for the synthesis of ticagrelor precursor.

Carbonyl reductases are useful for producing optically active alcohols from their corresponding prochiral ketones. Herein, we applied a computer-assisted strategy to increase the thermostability of a previously constructed carbonyl reductase, LsCRM4 (N101D/A117G/F147L/E145A), which showed an outstanding activity in the synthesis of the ticagrelor precursor (1S)-2-chloro-1-(3,4-difluorophenyl)ethanol. The stability changes introduced by mutations at the flexible sites were predicted using the computational tools FoldX, I-Mutant 3.0, and DeepDDG, which demonstrated that 12 virtually screened mutants could be thermally stable; 11 of these mutants exhibited increased thermostability. Then a superior mutant LsCRM4-V99L/D150F was screened out from the library that was constructed by iteratively combining the beneficial sites, which showed a 78% increase in activity and a 17.4°C increase in melting temperature compared to LsCRM4. Our computer-assisted design and combinatorial strategy dramatically increased the efficiency of thermostable enzyme production.

Surgical outcomes of robotic-assisted percutaneous fixation for thoracolumbar fractures in patients with ankylosing spondylitis.

BACKGROUND: Spinal fractures in patients with ankylosing spondylitis (AS) mainly present as instability, involving all three columns of the spine, and surgical intervention is often considered necessary. However, in AS patients, the significant alterations in bony structure and anatomy result in a lack of identifiable landmarks, which increases the difficulty of pedicle screw implantation. Therefore, we present the clinical outcomes of robotic-assisted percutaneous fixation for thoracolumbar fractures in patients with AS. METHODS: A retrospective review was conducted on a series of 12 patients diagnosed with AS. All patients sustained thoracolumbar fractures between October 2018 and October 2022 and underwent posterior robotic-assisted percutaneous fixation procedures. Outcomes of interest included operative time, intra-operative blood loss, complications, duration of hospital stay and fracture union. The clinical outcomes were assessed using the visual analogue scale (VAS) and Oswestry Disability Index (ODI). To investigate the achieved operative correction, pre- and postoperative radiographs in the lateral plane were analyzed by measuring the Cobb angle. RESULTS: The 12 patients had a mean age of 62.8 ± 13.0 years and a mean follow-up duration of 32.7 ± 18.9 months. Mean hospital stay duration was 15 ± 8.0 days. The mean operative time was 119.6 ± 32.2 min, and the median blood loss was 50 (50, 250) ml. The VAS value improved from 6.8 ± 0.9 preoperatively to 1.3 ± 1.0 at the final follow-up (P < 0.05). The ODI value improved from 83.6 ± 6.1% preoperatively to 11.8 ± 6.6% at the latest follow-up (P < 0.05). The average Cobb angle changed from 15.2 ± 11.0 pre-operatively to 8.3 ± 7.1 at final follow-up (P < 0.05). Bone healing was consistently achieved, with an average healing time of 6 (5.3, 7.0) months. Of the 108 screws implanted, 2 (1.9%) were improperly positioned. One patient experienced delayed nerve injury after the operation, but the nerve function returned to normal upon discharge. CONCLUSION: Posterior robotic-assisted percutaneous internal fixation can be used as an ideal surgical treatment for thoracolumbar fractures in AS patients. However, while robot-assisted pedicle screw placement can enhance the accuracy of pedicle screw insertion, it should not be relied upon solely.

Ivonescimab Plus Chemotherapy in Non-Small Cell Lung Cancer With EGFR Variant: A Randomized Clinical Trial.

Importance: For patients with non-small cell lung cancer whose disease progressed while receiving EGFR tyrosine kinase inhibitor (EGFR-TKI) therapy, particularly third-generation TKIs, optimal treatment options remain limited. Objective: To compare the efficacy of ivonescimab plus chemotherapy with chemotherapy alone for patients with relapsed advanced or metastatic non-small cell lung cancer with the epidermal growth factor receptor (EGFR) variant. Design, Setting, and Participants: Double-blind, placebo-controlled, randomized, phase 3 trial at 55 sites in China enrolled participants from January 2022 to November 2022; a total of 322 eligible patients were enrolled. Interventions: Participants received ivonescimab (n = 161) or placebo (n = 161) plus pemetrexed and carboplatin once every 3 weeks for 4 cycles, followed by maintenance therapy of ivonescimab plus pemetrexed or placebo plus pemetrexed. Main Outcomes and Measures: The primary end point was progression-free survival in the intention-to-treat population assessed by an independent radiographic review committee (IRRC) per Response Evaluation Criteria in Solid Tumors version 1.1. The results of the first planned interim analysis are reported. Results: Among 322 enrolled patients in the ivonescimab and placebo groups, the median age was 59.6 vs 59.4 years and 52.2% vs 50.9% of patients were female. As of March 10, 2023, median follow-up time was 7.89 months. Median progression-free survival was 7.1 (95% CI, 5.9-8.7) months in the ivonescimab group vs 4.8 (95% CI, 4.2-5.6) months for placebo (difference, 2.3 months; hazard ratio [HR], 0.46 [95% CI, 0.34-0.62]; P < .001). The prespecified subgroup analysis showed progression-free survival benefit favoring patients receiving ivonescimab over placebo across almost all subgroups, including patients whose disease progressed while receiving third-generation EGFR-TKI therapy (HR, 0.48 [95% CI 0.35-0.66]) and those with brain metastases (HR, 0.40 [95% CI, 0.22-0.73]). The objective response rate was 50.6% (95% CI, 42.6%-58.6%) with ivonescimab and 35.4% (95% CI, 28.0%-43.3%) with placebo (difference, 15.6% [95% CI, 5.3%-26.0%]; P = .006). The median overall survival data were not mature; at data cutoff, 69 patients (21.4%) had died. Grade 3 or higher treatment-emergent adverse events occurred in 99 patients (61.5%) in the ivonescimab group vs 79 patients (49.1%) in the placebo group, the most common of which were chemotherapy-related. Grade 3 or higher immune-related adverse events occurred in 10 patients (6.2%) in the ivonescimab group vs 4 (2.5%) in the placebo group. Grade 3 or higher vascular endothelial growth factor-related adverse events occurred in 5 patients (3.1%) in the ivonescimab group vs 4 (2.5%) in the placebo group. Conclusions: Ivonescimab plus chemotherapy significantly improved progression-free survival with tolerable safety profile in TKI-treated non-small cell lung cancer. Trial Registration: ClinicalTrials.gov Identifier: NCT05184712.

Identification of Crucial Photosensitizing Factors to Promote CO2 -to-CO Conversion.

The sensitizing ability of a catalytic system is closely related to the visible-light absorption ability, excited-state lifetime, redox potential, and electron-transfer rate of photosensitizers (PSs), however it remains a great challenge to concurrently mediate these factors to boost CO2 photoreduction. Herein, a series of Ir(III)-based PSs (Ir-1-Ir-6) were prepared as molecular platforms to understand the interplay of these factors and identify the primary factors for efficient CO2 photoreduction. Among them, less efficient visible-light absorption capacity results in lower CO yields of Ir-1, Ir-2 or Ir-4. Ir-3 shows the most efficient photocatalytic activity among these mononuclear PSs due to some comprehensive parameters. Although the Kobs of Ir-3 is ≈10 times higher than that of Ir-5, the CO yield of Ir-3 is slightly higher than that of Ir-5 due to the compensation of Ir-5's strong visible-light-absorbing ability. Ir-6 exhibits excellent photocatalytic performance due to the strong visible-light absorption ability, comparable thermodynamic driving force, and electron transfer rate among these PSs. Remarkably, the CO2 photoreduction to CO with Ir-6 can achieve 91.5 µmol, over 54 times higher than Ir-1, and the optimized TONC-1 can reach up to 28160. Various photophysical properties of the PSs were concurrently adjusted by fine ligand modification to promote CO2 photoreduction.

Single-cluster Functionalized TiO2 Nanotube Array for Boosting Water Oxidation and CO2 Photoreduction to CH3OH.

Solar-driven CO2 reduction and water oxidation to liquid fuels represents a promising solution to alleviate energy crisis and climate issue, but it remains a great challenge for generating CH3OH and CH3CH2OH dominated by multi-electron transfer. Single-cluster catalysts with super electron acceptance, accurate molecular structure, customizable electronic structure and multiple adsorption sites, have led to greater potential in catalyzing various challenging reactions. However, accurately controlling the number and arrangement of clusters on functional supports still faces great challenge. Herein, we develop a facile electrosynthesis method to uniformly disperse Wells-Dawson- and Keggin-type polyoxometalates on TiO2 nanotube arrays, resulting in a series of single-cluster functionalized catalysts P2M18O62@TiO2 and PM12O40@TiO2 (M=Mo or W). The single polyoxometalate cluster can be distinctly identified and serves as electronic sponge to accept electrons from excited TiO2 for enhancing surface-hole concentration and promote water oxidation. Among these samples, P2Mo18O62@TiO2-1 exhibits the highest electron consumption rate of 1260 µmol g-1 for CO2-to-CH3OH conversion with H2O as the electron source, which is 11 times higher than that of isolated TiO2 nanotube arrays. This work supplied a simple synthesis method to realize the single-dispersion of molecular cluster to enrich surface-reaching holes on TiO2, thereby facilitating water oxidation and CO2 reduction.

Recent advances in structure-based enzyme engineering for functional reconstruction.

Structural information can help engineer enzymes. Usually, specific amino acids in particular regions are targeted for functional reconstruction to enhance the catalytic performance, including activity, stereoselectivity, and thermostability. Appropriate selection of target sites is the key to structure-based design, which requires elucidation of the structure-function relationships. Here, we summarize the mutations of residues in different specific regions, including active center, access tunnels, and flexible loops, on fine-tuning the catalytic performance of enzymes, and discuss the effects of altering the local structural environment on the functions. In addition, we keep up with the recent progress of structure-based approaches for enzyme engineering, aiming to provide some guidance on how to take advantage of the structural information.

Structural-guided design to improve the catalytic performance of aldo-keto reductase KdAKR.

Aldo-keto reductases (AKRs) are important biocatalysts that can be used to synthesize chiral pharmaceutical alcohols. In this study, the catalytic activity and stereoselectivity of a NADPH-dependent AKR from Kluyveromyces dobzhanskii (KdAKR) toward t-butyl 6-chloro (5S)-hydroxy-3-oxohexanoate ((5S)-CHOH) were improved by mutating its residues in the loop regions around the substrate-binding pocket. And the thermostability of KdAKR was improved by a consensus sequence method targeted on the flexible regions. The best mutant M6 (Y28A/L58I/I63L/G223P/Y296W/W297H) exhibited a 67-fold higher catalytic efficiency compared to the wild-type (WT) KdAKR, and improved R-selectivity toward (5S)-CHOH (dep value from 47.6% to >99.5%). Moreover, M6 exhibited a 6.3-fold increase in half-life (t1/2 ) at 40°C compared to WT. Under the optimal conditions, M6 completely converted 200 g/L (5S)-CHOH to diastereomeric pure t-butyl 6-chloro-(3R, 5S)-dihydroxyhexanoate ((3R, 5S)-CDHH) within 8.0 h, with a space-time yield of 300.7 g/L/day. Our results deepen the understandings of the structure-function relationship of AKRs, providing a certain guidance for the modification of other AKRs.

Molecular dynamics study on thermal conductance between a nanotip and a substrate under vertical forces and horizontal sliding.

The heat transfer between a nanotip and its substrate is extremely complex but is a key factor in determining the measurement accuracy in tip-assisted nanomanufacturing and thermometry. In this work, the heat transfer from the nanotip to the substrate during sliding is investigated using molecular dynamics simulations. Interfacial interaction and bond formation are analyzed during the sliding process. The results show that the increase of vertical force would greatly improve the interface thermal conductance between the nanotip and the substrate. It is found that more bonds are formed and there are larger contact areas at the interface. In addition, we found that the thermal conductivity of the nanotip is another obstacle for heat transfer between the tip and substrate and it is greatly limited by the nanotip diameter near contact which is close to or even smaller than the phonon mean free path. Meanwhile, the dynamic formation and breakage of the covalent bonds during the sliding could gradually smoothen the tip apex and enhance the thermal transport at the interface. This work provides guidance for the thermal design of a nanotip-substrate system for nanoscale thermal transport measurements.

Human nails as a valuable noninvasive alternative for estimating exposure to parabens.

Exposure of human to parabens (commonly used preservatives) is inevitable due to their extensively applied in numerous consumer products. Thus, a reliable noninvasive matrix reflecting long-term exposure to parabens is essential for human biomonitoring study. Human nails are potentially a valuable alternative for measuring intergrated exposure to parabens. In this work, we collected 100 paired nail and urine samples from university students in Nanjing, China, and measured simultaneously for six parent parabens and four metabolites. Methylparaben (MeP), ethylparaben (EtP), and propylparaben (PrP) were three predominant paraben analogue in both matrices, with the median concentrations being 12.9, 0.753, and 3.42 ng/mL in urine, and 1540, 154, and 961 ng/g in nail, respectively, while 4-hydroxybenzoic acid (4-HB) and 3,4-dihydroxybenzoic acid (3,4-DHB) were the most abundant metabolites (median values of 143 and 35.9 ng/mL, respectively) in urine. Gender-related analysis suggested that females exposed to more higher parabens than males. Significantly positive correlations were found between levels of MeP, PrP, EtP, and OH-MeP (r = 0.54-0.62, p < 0.01) in paired urine and nail samples. Our result here suggests that human nails, as an emerging biospecimen, are a potentially valuable biological matrix to evaluate human long-term exposure to parabens.

Comparison of in vitro strategies for predicting Dichlorodiphenyltrichloroethane (DDT) and its metabolites bioavailability from soils.

In vitro strategies have widely been used to assess bioaccessibility of organic pollutants in soils. However, studies for comparing in vitro models with in vivo data are still limited. In this study, Dichlorodiphenyltrichloroethane (DDT) and its metabolites (called as DDTr) bioaccessibility in nine contaminated soils were measured using physiologically based extraction test (PBET), in vitro digestion model (IVD), and Deutsches Institut für Normung (DIN) with/without Tenax as an absorptive sink, and DDTr bioavailability was assessed using an in vivo mouse model. Whether or not Tenax was added, DDTr bioaccessibility significantly varied among three methods, suggesting that DDTr bioaccessibility depended on the in vitro method employed. Multiple linear regression analysis indicated that sink, intestinal incubation time and bile content are identified to be the dominant factors in controlling DDTr bioaccessibility. Comparison of in vitro and in vivo results demonstrated that DIN assay with Tenax (TI-DIN) provided the best prediction for DDTr bioavailability (r2 = 0.66, slope=0.78). After extending intestinal incubation time to 6 h or increasing bile content to 4.5 g/L (same to DIN assay) of the TI-PBET and TI-IVD assays, the in vivo-in vitro correlation will improved significantly, with r2 = 0.76 and slope= 1.4 for TI-PBET and r2 = 0.84 and slope= 1.9 for TI-IVD under 6 h intestinal incubation, and r2 = 0.59 and slope= 0.96 for TI-PBET and r2 = 0.51 and slope= 1.0 for TI-IVD under 4.5 g/L of bile content. The results suggest that it is essential to understand these key factors influencing bioaccessibility for the development of standardized in vitro methods, which helps to refine the risk assessment of human exposure to contaminants via soil ingestion.

Accelerating Ethanol Complete Electrooxidation via Introducing Ethylene as the Precursor for the C-C Bond Splitting.

The crucial issue restricting the application of direct ethanol fuel cells (DEFCs) is the incomplete and sluggish electrooxidation of ethanol due to the chemically stable C-C bond thereof. Herein, a unique ethylene-mediated pathway with a 100 % C1-selectivity for ethanol oxidation reaction (EOR) is proposed for the first time based on a well-structured Pt/Al2 O3 @TiAl catalyst with cascade active sites. The electrochemical in situ Fourier transform infrared spectroscopy (FTIR) and differential electrochemical mass spectrometry (DEMS) analysis disclose that ethanol is primarily dehydrated on the surface of Al2 O3 @TiAl and the derived ethylene is further oxidized completely on nanostructured Pt. X-ray absorption and density functional theory (DFT) studies disclose the Al component doped in Pt nanocrystals can promote the EOR kinetics by lowering the reaction energy barriers and eliminating the poisonous species. Strikingly, Pt/Al2 O3 @TiAl exhibits a specific activity of 3.83 mA cm-2 Pt , 7.4 times higher than that of commercial Pt/C and superior long-term durability.

Nomogram to predict 3-month unfavorable outcome after thrombectomy for stroke.

BACKGROUND: Mechanical thrombectomy (MT) is an effective treatment for large-vessel occlusion in acute ischemic stroke, however, only some revascularized patients have a good prognosis. For stroke patients undergoing MT, predicting the risk of unfavorable outcomes and adjusting the treatment strategies accordingly can greatly improve prognosis. Therefore, we aimed to develop and validate a nomogram that can predict 3-month unfavorable outcomes for individual stroke patient treated with MT. METHODS: We analyzed 258 patients with acute ischemic stroke who underwent MT from January 2018 to February 2021. The primary outcome was a 3-month unfavorable outcome, assessed using the modified Rankin Scale (mRS), 3-6. A nomogram was generated based on a multivariable logistic model. We used the area under the receiver-operating characteristic curve to evaluate the discriminative performance and used the calibration curve and Spiegelhalter's Z-test to assess the calibration performance of the risk prediction model. RESULTS: In our visual nomogram, gender (odds ratio [OR], 3.40; 95%CI, 1.54-7.54), collateral circulation (OR, 0.46; 95%CI, 0.28-0.76), postoperative mTICI (OR, 0.06; 95%CI, 0.01-0.50), stroke-associated pneumonia (OR, 5.76; 95%CI, 2.79-11.87), preoperative Na (OR, 0.82; 95%CI, 0.72-0.92) and creatinine (OR, 1.02; 95%CI, 1.01-1.03) remained independent predictors of 3-month unfavorable outcomes in stroke patients treated with MT. The area under the nomogram curve was 0.8791 with good calibration performance (P = 0.873 for the Spiegelhalter's Z-test). CONCLUSIONS: A novel nomogram consisting of gender, collateral circulation, postoperative mTICI, stroke-associated pneumonia, preoperative Na and creatinine can predict the 3-month unfavorable outcomes in stroke patients treated with MT.

Critical problems faced in Raman-based energy transport characterization of nanomaterials.

In the last two decades, tremendous research has been conducted on the discovery, design and synthesis, characterization, and applications of two-dimensional (2D) materials. Thermal conductivity and interface thermal conductance/resistance of 2D materials are two critical properties in their applications. Raman spectroscopy, which measures the inelastic scattering of photons by optical phonons, can distinct a 2D material's temperature from its surrounding materials', featuring unprecedented spatial resolution (down to the atomic level). Raman-based thermometry has been used tremendously for characterizing the thermal conductivity of 2D materials (suspended or supported) and interface thermal conductance/resistance. Very large data deviations have been observed in literature, partly due to physical phenomena and factors not considered in measurements. Here, we provide a critical review, analysis, and perspectives about a broad spectrum of physical problems faced in Raman-based thermal characterization of 2D materials, namely interface separation, localized stress due to thermal expansion mismatch, optical interference, conjugated phonon, and hot carrier transport, optical-acoustic phonon thermal nonequilibrium, and radiative electron-hole recombination in monolayer 2D materials. Neglect of these problems will lead to a physically unreasonable understanding of phonon transport and interface energy coupling. In-depth discussions are also provided on the energy transport state-resolved Raman (ET-Raman) technique to overcome these problems and on future research challenges and needs.

Directed evolution of a carbonyl reductase LsCR for the enantioselective synthesis of (1S)-2-chloro-1-(3,4-difluorophenyl) ethanol.

Traditional screening methods of enzyme engineering often require building large mutant libraries to screen for potentially beneficial sites, which are often time-consuming and labor-intensive with low mining efficiency. In this study, a novel enzyme engineering strategy was established to modify carbonyl reductase LsCR for the synthesis of (1S)-2-chloro-1-(3,4-difluorophenyl) ethanol ((S)-CFPL), which is a key intermediate of anticoagulant drug ticagrelor. The strategy was developed by combining HotSpot, FireProt and multiple sequence alignment, resulting in the construction of a "small and smart" mutant library including 10 mutations. Among them, 5 mutations were positive, resulting in a 50% mining accuracy of beneficial sites. Finally, a highly active mutant LsCRM3 (N101D/A117G/F147L) was obtained by further screening through saturation mutation and iterative mutation. Compared with wild type (WT) LsCR, the catalytic activity of LsCRM3 was increased by 4.7 times, the catalytic efficiency kcat/KM value was increased by 2.9 times, and the half-life t1/2 at 40 °C was increased by 1.3 times. Due to the low aqueous solubility of the substrate 2-chloro-1-(3,4-difluorophenyl) ethanone (CFPO), isopropanol was used as not only the co-substrate but also co-solvent. In the presence of 40% (v/v) isopropanol, LsCRM3 completely reduced 400 g/L CFPO to enantiomerically pure CFPL (99.9%, e.e.) in 11 h with a space-time yield (STY) as high as 809 g/L∙d.

A Pilot Study: Nails as a Non-invasive Biospecimen of Human Exposure to Phthalate Esters.

Human biomonitoring provides a scientific approach that systematically reveals exposure to phthalates through all possible routes. In this pilot study, fingernail was chosen as a non-destructive biospecimen to assess human exposure to nine phthalates. Concentrations of total phthalates ranged from 17.8 to 176 µg/g (median: 65.4 µg/g). Di(2-ethylhexyl) phthalate, dibutyl phthalate (DBP), and di-isobutyl phthalate were the major compounds found in fingernails, accounting for 64.3%, 19.4%, and 12.9% of the total phthalates, respectively. No significant difference in phthalates concentrations was found among genders and age-related distribution (p > 0.05). The concentration of DBP was positively correlated with participant's body mass index (r = 0.83). Our results suggested that fingernail can be used as a non-invasive biospecimen for the biomonitoring of human exposure to phthalates. Further studies are needed to investigate the relationship between phthalates or their metabolites in fingernail and other biological samples, such as urine and blood.

Assessment of DDT and its Metabolites Bioaccessibility in Historically Contaminated Soils Using Unfed and Fed in Vitro Methods.

Bioaccessibility of hydrophobic organic contaminants (HOCs) from unintentional ingestion of soil is increasingly assessed with in vitro gastrointestinal models incorporating a sorption sink. In this study, the bioaccessibility of DDTs in contaminated soils (n = 11) was determined using "unfed" unified bioaccessibility method (UBM) and fed organic estimation human simulation test (FOREhST) with/without Tenax as an absorbent. By adding Tenax, the bioaccessibility of DDTs determined using UBM was significantly increased from 4.9-30.6% to 31.6-86.0%. In contrast, the bioaccessibility of DDTs determined using FOREhST without/with Tenax were similar with values of 20.0-60.9% vs 31.5-47.6%, implying that the influence of food components on the absorption efficiency of the sink should not be overlooked. Much high fraction of DDTs (bioaccessibility: 11.7-24.8%) remained in FOREhST supernatant after Tenax collection, suggesting that prediction of bioavailability through bioaccessibility obtained by absorbent needs to be treated with caution when bioaccessibility is determined using a "fed state" in vitro method.

Semirational engineering of an aldo-keto reductase KmAKR for overcoming trade-offs between catalytic activity and thermostability.

Enzyme engineering usually generates trade-offs between activity, stability, and selectivity. Herein, we report semirational engineering of an aldo-keto reductase (AKR) KmAKR for simultaneously enhancing its thermostability and catalytic activity. Previously, we constructed KmAKRM9 (W297H/Y296W/K29H/Y28A/T63M/A30P/T302S/N109K/S196C), which showed outstanding activity towards t-butyl 6-chloro-(3R,5S)-dihydroxyhexanoate ((3R,5S)-CDHH), and t-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate, the key chiral building blocks of rosuvastatin and atorvastatin. Under the guidance of computer-aided design including consensus residues analysis and molecular dynamics (MD) simulations, K164, S182, S232, and Q266 were dug out for their thermostability conferring roles, generating the "best" mutant KmAKRM13 (W297H/Y296W/K29H/Y28A/T63M/A30P/T302S/N109K/S196C/K164E/S232A/S182H/Q266D). The Tm and T5015 values of KmAKRM13 were 10.4 and 6.1°C higher than that of KmAKRM9 , respectively. Moreover, it displayed a significantly elevated organic solvent tolerance over KmAKRM9 . Structural analysis indicated that stabilization of the α-helixes mainly contributed to thermostability enhancement. Under the optimized conditions, KmAKRM13 completely asymmetrically reduced 400 g/l t-butyl 6-chloro-(5S)-hydroxy-3-oxohexanoate ((5S)-CHOH) in 8.0 h at a high substrate to catalyst ratio (S/C) of 106.7 g/g, giving diastereomerically pure (3R,5S)-CDHH (>99.5% d.e.P ) with a space-time yield (STY) of 449.2 g/l·d.

Different fuel regulation in two types of myofiber results in different antioxidant strategies in Daurian ground squirrels (Spermophilus dauricus) during hibernation.

We previously showed that different skeletal muscles in Daurian ground squirrels (Spermophilus dauricus) possess different antioxidant strategies during hibernation; however, the reason for these varied strategies remains unclear. To clarify this issue, we studied REDD1, FOXO4, PGC-1α, FOXO1 and atrogin-1 proteins to determine the potential cause of the different antioxidant strategies in Daurian ground squirrels during hibernation, and to clarify whether different strategies affect atrophy-related signals. Results showed that the soleus (SOL) muscle experienced intracellular hypoxia during interbout arousal, but no oxidative stress. This may be due to increased PGC-1α expression enhancing antioxidant capacity in the SOL under hypoxic conditions. Extensor digitorum longus (EDL) muscle showed no change in oxidative stress, hypoxia or antioxidant capacity during hibernation. The FOXO1 and PGC-1α results strongly suggested differentially regulated fuel metabolism in the SOL and EDL muscles during hibernation, i.e. enhanced lipid oxidation and maintained anaerobic glycolysis, respectively. Atrogin-1 expression did not increase during hibernation in either the SOL or EDL, indicating that protein synthesis was not inhibited by atrogin-1. Thus, our results suggest that different fuel regulation may be one mechanism related to antioxidant defense strategy formation in different kinds of skeletal muscle fibers of Daurian ground squirrels during hibernation.

Long-term vitamin D deficiency promotes renal fibrosis and functional impairment in middle-aged male mice.

Renal fibrosis is common especially in the elderly population. Recently, we found that vitamin D deficiency caused prostatic hyperplasia. This study aimed to investigate whether vitamin D deficiency promotes renal fibrosis and functional impairment. All mice except controls were fed with vitamin D-deficient (VDD) diets, beginning from their early life. The absolute and relative kidney weights on postnatal week 20 were decreased in VDD diet-fed male pups but not in female pups. A mild pathological damage was observed in VDD diet-fed male pups but not in females. Further analysis showed that VDD-induced pathological damage was aggravated, accompanied by renal dysfunction in 40-week-old male pups. An obvious collagen deposition was observed in VDD diet-fed 40-week-old male pups. Moreover, renal α-smooth muscle actin (α-SMA), a marker of epithelial-mesenchymal transition (EMT), and Tgf-ß mRNA were up-regulated. The in vitro experiment showed that 1,25-dihydroxyvitamin D3 alleviated transforming growth factor-ß1 (TGF-ß1)-mediated down-regulation of E-cadherin and inhibited TGF-ß1-evoked up-regulation of N-cadherin, vimentin and α-SMA in renal epithelial HK-2 cells. Moreover, 1,25-dihydroxyvitamin D3 suppressed TGF-ß1-evoked Smad2/3 phosphorylation in HK-2 cells. These results provide experimental evidence that long-term vitamin D deficiency promotes renal fibrosis and functional impairment, at least partially, through aggravating TGF-ß/Smad2/3-mediated EMT in middle-aged male mice.

Lipopolysaccharide Downregulates 11ß-Hydroxysteroid Dehydrogenase 2 Expression through Inhibiting Peroxisome Proliferator-Activated Receptor-γ in Placental Trophoblasts.

It is increasingly recognized that excessive glucocorticoids induce fetal intrauterine growth restriction (IUGR). Placental 11ß-hydroxysteroid dehydrogenase 2 (11ß-HSD2), a glucocorticoid-catalyzing enzyme, prevents active glucocorticoids from maternal circulation into the fetus, thus protecting against IUGR. Previous studies demonstrated gestational LPS exposure caused fetal IUGR. The aim of the current study was to investigate the effects of LPS on 11ß-HSD2 in mice placentas and human placental trophoblasts. Pregnant ICR(CD-1) mice were i.p. injected with LPS (200 µg/kg) on gestational day 16. As expected, gestational LPS exposure downregulated 11ß-HSD2 in mice placentas. In vitro, LPS downregulated 11ß-HSD2 in human placental trophoblasts. Additional experiment showed that LPS, which activated NF-κB, suppressed rosiglitazone-induced activation of peroxisome proliferator-activated receptor-γ (PPARγ) in mice placentas and human placental trophoblasts. Moreover, NF-κB p65 knockdown and specific NF-κB inhibitor attenuated LPS-induced suppression of PPARγ nuclear translocation in human placental trophoblasts. In addition, NF-κB p65 knockdown attenuated LPS-induced downregulation of 11ß-HSD2 in human placental trophoblasts. Mechanically, LPS promoted physical interaction between NF-κB p65 and PPARγ in the cytoplasm and nucleus of placental trophoblasts. Finally, pretreatment with rosiglitazone, a PPARγ agonist, partially alleviated LPS-induced reduction of fetal weight and crown-rump length. Taken together, these results suggest that LPS downregulates 11ß-HSD2 through suppressing PPARγ in placental trophoblasts. Placental 11ß-HSD2 downregulation may contribute partially to LPS-induced fetal IUGR.