Cytochrome P450 Enzyme Design by Constraining the Catalytic Pocket in a Diffusion Model.

Although cytochrome P450 enzymes are the most versatile biocatalysts in nature, there is insufficient comprehension of the molecular mechanism underlying their functional innovation process. Here, by combining ancestral sequence reconstruction, reverse mutation assay, and progressive forward accumulation, we identified 5 founder residues in the catalytic pocket of flavone 6-hydroxylase (F6H) and proposed a "3-point fixation" model to elucidate the functional innovation mechanisms of P450s in nature. According to this design principle of catalytic pocket, we further developed a de novo diffusion model (P450Diffusion) to generate artificial P450s. Ultimately, among the 17 non-natural P450s we generated, 10 designs exhibited significant F6H activity and 6 exhibited a 1.3- to 3.5-fold increase in catalytic capacity compared to the natural CYP706X1. This work not only explores the design principle of catalytic pockets of P450s, but also provides an insight into the artificial design of P450 enzymes with desired functions.

Lateral Quadratus Lumborum Block Versus Acupuncture for Postcaesarean Analgesia: A Randomized Clinical Trial.

BACKGROUND: Improved pain control after caesarean section remains a challenging objective. Although both the lateral quadratus lumborum block and acupuncture have been reported to provide superior postoperative analgesia after caesarean section when compared to placebo, the efficacy of these techniques has never been compared head-to-head. OBJECTIVE: This study was conducted to investigate the comparative analgesic efficacy of lateral quadratus lumborum block and acupuncture following elective caesarean section. STUDY DESIGN: In this prospective, randomized, controlled clinical trial, a total of 190 patients with singleton term pregnancies scheduled for caesarean section under spinal-epidural anesthesia were enrolled. Patients were randomized 1:1 to acupuncture group or lateral quadratus lumborum block group. Lateral quadratus lumborum block group received bilateral lateral quadratus lumborum block with 0.33% ropivacaine and sham acupuncture, acupuncture group received transcutaneous electrical acupoint stimulation and press needle therapy and sham lateral quadratus lumborum block. All patients received the standard postoperative pain treatment. The primary outcome was pain scores on movement at 24 h. Secondary endpoints included pain scores in the first 48 h postoperatively, patient-controlled intravenous analgesia demands, analgesia-related adverse effects, postoperative complications, QoR-15, the time to mobilization, and gastrointestinal function. RESULTS: Median (IQR [range]) pain scores at 24 h on movement was similar in patients receiving acupuncture or lateral quadratus lumborum block (3 (2-4) vs. 3 (2-4), respectively; P = 0.40). Patient-controlled intravenous analgesia consumption and pain scores within 48 h postoperatively also showed no difference between the two groups. The acupuncture improved QoR-15 scores at 24 and 48 h postoperatively (P<0.001), as well as shortened the time to first flatus (P=0.03) and first drinking (P<0.001) compared to lateral quadratus lumborum block. In addition, the median time to mobilization in the lateral quadratus lumborum block group was markedly prolonged compare with acupuncture group (17.0 (15.0-19.0) h vs. 15.3 (13.3-17.0) h, estimated median difference, 1.5; 95%CI, 1-2; P<0.001;). CONCLUSIONS: As a component of multimodal analgesia regimen after caesarean section, acupuncture did not lower postoperative pain scores or reduce analgesic medication consumption compared to lateral quadratus lumborum block.

LUZP1 regulates the maturation of contractile actomyosin bundles.

Contractile actomyosin bundles play crucial roles in various physiological processes, including cell migration, morphogenesis, and muscle contraction. The intricate assembly of actomyosin bundles involves the precise alignment and fusion of myosin II filaments, yet the underlying mechanisms and factors involved in these processes remain elusive. Our study reveals that LUZP1 plays a central role in orchestrating the maturation of thick actomyosin bundles. Loss of LUZP1 caused abnormal cell morphogenesis, migration, and the ability to exert forces on the environment. Importantly, knockout of LUZP1 results in significant defects in the concatenation and persistent association of myosin II filaments, severely impairing the assembly of myosin II stacks. The disruption of these processes in LUZP1 knockout cells provides mechanistic insights into the defective assembly of thick ventral stress fibers and the associated cellular contractility abnormalities. Overall, these results significantly contribute to our understanding of the molecular mechanism involved in actomyosin bundle formation and highlight the essential role of LUZP1 in this process.

A mild deprotection of isopropylidene ketals from 2-deoxyglycosides using AcOH/H2O/DME: An efficient regioselective deprotection of terminal isopropylidene ketals.

This paper describes a mild and efficient catalytic deprotection method for isopropylidene ketals and benzylidene acetals using AcOH/H2O/DME(1,2-Dimethoxyethane). The method effectively removes ketal and acetal protecting groups from 2-deoxyglycosides which are prone to hydrolysis under acidic conditions. Moreover, it enables the selective removal of the terminal ketal over an internal one.

Dialdehyde cellulose films covalently crosslinked with porphyrin-based covalent organic polymers for photodynamic sterilization.

Foodborne pathogens result in a great harm to human, which is an urgent problem to be addressed. Herein, a novel cellulose-based packaging films with excellent anti-bacterial properties under visible light were prepared. A porphyrin-based covalent organic polymer (Por-COPs) was constructed, then covalently grafted onto dialdehyde cellulose (DAC). The addition of Por-COPs enhanced the mechanical, hydrophobicity, and water resistance of the DAC-based composite films. DAC/Por-COP-2.5 film exhibited outstanding properties for the photodynamic inactivation of bacteria under visible light irradiation, delivering inactivation efficiencies of 99.90 % and 99.45 % towards Staphylococcus aureus and Escherichia coli within 20 min. The DAC/Por-COPs films efficiently generated •O2- and 1O2 under visible light, thereby causing oxidative stress to cell membranes for bacterial inactivation. The prepared composite film forms a protective barrier against bacterial contamination. Results guide the development of high performance and more sustainable packaging films for the food sector.

DMAP-promoted oxidative functionalization of α-amino ketones via oxygen delivery from water/alcohols.

This paper shows a novel oxidative functionalization of α-amino ketones to yield the corresponding α-ketoamides and α-acylimidates. The reaction proceeds via oxygen delivery from water/alcohols in conjunction with an electron acceptor and 4-dimethylaminopyridine (DMAP). Mechanistic study indicates that DMAP exhibits a dual function of nucleophilic catalysis and proton abstraction.

Biochemical synthesis of taxanes from mevalonate.

Taxanes are kinds of diterpenoids with important bioactivities, such as paclitaxel (taxol®) is an excellent natural broad-spectrum anticancer drug. Attempts to biosynthesize taxanes have made with limited success, mainly due to the bottleneck of the low efficiency catalytic elements. In this study, we developed an artificial synthetic system to produce taxanes from mevalonate (MVA) by coupling biological and chemical methods, which comprises in vitro multi-enzyme catalytic module, chemical catalytic module and yeast cell catalytic module. Through optimizing in vitro multienzyme catalytic system, the yield of taxadiene was increased to 946.7 mg/L from MVA within 8 h and the productivity was 14.2-fold higher than microbial fermentation. By incorporating palladium catalysis, the conversion rate of Taxa-4(20),11(12)-dien-5α-yl acetate (T5α-AC) reached 48 %, effectively addressing the product promiscuity and the low yield rate of T5αOH. Finally, we optimized the expression of T10ßOH in yeast resulting in the biosynthesis of Taxa-4(20),11(12)-dien-5α-acetoxy-10ß-ol(T5α-AC-10ß-ol) at a production of 15.8 mg/L, which displayed more than 2000-fold higher than that produced by co-culture fermentation strategy. These technologies offered a promising new approach for efficient synthesis of taxanes.

Mapping protein-protein interactions by mass spectrometry.

Protein-protein interactions (PPIs) are essential for numerous biological activities, including signal transduction, transcription control, and metabolism. They play a pivotal role in the organization and function of the proteome, and their perturbation is associated with various diseases, such as cancer, neurodegeneration, and infectious diseases. Recent advances in mass spectrometry (MS)-based protein interactomics have significantly expanded our understanding of the PPIs in cells, with techniques that continue to improve in terms of sensitivity, and specificity providing new opportunities for the study of PPIs in diverse biological systems. These techniques differ depending on the type of interaction being studied, with each approach having its set of advantages, disadvantages, and applicability. This review highlights recent advances in enrichment methodologies for interactomes before MS analysis and compares their unique features and specifications. It emphasizes prospects for further improvement and their potential applications in advancing our knowledge of PPIs in various biological contexts.

A splice site variant in MADD affects hormone expression in pancreatic ß cells and pituitary gonadotropes.

MAPK activating death domain (MADD) is a multifunctional protein regulating small GTPases RAB3 and RAB27, MAPK signaling, and cell survival. Polymorphisms in the MADD locus are associated with glycemic traits, but patients with biallelic variants in MADD manifest a complex syndrome affecting nervous, endocrine, exocrine, and hematological systems. We identified a homozygous splice site variant in MADD in 2 siblings with developmental delay, diabetes, congenital hypogonadotropic hypogonadism, and growth hormone deficiency. This variant led to skipping of exon 30 and in-frame deletion of 36 amino acids. To elucidate how this mutation causes pleiotropic endocrine phenotypes, we generated relevant cellular models with deletion of MADD exon 30 (dex30). We observed reduced numbers of ß cells, decreased insulin content, and increased proinsulin-to-insulin ratio in dex30 human embryonic stem cell-derived pancreatic islets. Concordantly, dex30 led to decreased insulin expression in human ß cell line EndoC-ßH1. Furthermore, dex30 resulted in decreased luteinizing hormone expression in mouse pituitary gonadotrope cell line LßT2 but did not affect ontogeny of stem cell-derived GnRH neurons. Protein-protein interactions of wild-type and dex30 MADD revealed changes affecting multiple signaling pathways, while the GDP/GTP exchange activity of dex30 MADD remained intact. Our results suggest MADD-specific processes regulate hormone expression in pancreatic ß cells and pituitary gonadotropes.

Assessing the Value of Incorporating a Polygenic Risk Score with Nongenetic Factors for Predicting Breast Cancer Diagnosis in the UK Biobank.

BACKGROUND: Previous studies have demonstrated that incorporating a polygenic risk score (PRS) to existing risk prediction models for breast cancer improves model fit, but to determine its clinical utility the impact on risk categorization needs to be established. We add a PRS to two well-established models and quantify the difference in classification using the net reclassification improvement (NRI). METHODS: We analyzed data from 126,490 post-menopausal women of "White British" ancestry, aged 40 to 69 years at baseline from the UK Biobank prospective cohort. The breast cancer outcome was derived from linked registry data and hospital records. We combined a PRS for breast cancer with 10-year risk scores from the Tyrer-Cuzick and Gail models, and compared these to the risk scores from the models using phenotypic variables alone. We report metrics of discrimination and classification, and consider the importance of the risk threshold selected. RESULTS: The Harrell's C statistic of the 10-year risk from the Tyrer-Cuzick and Gail models was 0.57 and 0.54, respectively, increasing to 0.67 when the PRS was included. Inclusion of the PRS gave a positive NRI for cases in both models [0.080 (95% confidence interval (CI), 0.053-0.104) and 0.051 (95% CI, 0.030-0.073), respectively], with negligible impact on controls. CONCLUSIONS: The addition of a PRS for breast cancer to the well-established Tyrer-Cuzick and Gail models provides a substantial improvement in the prediction accuracy and risk stratification. IMPACT: These findings could have important implications for the ongoing discussion about the value of PRS in risk prediction models and screening.

Disruption of KLHL6 Fuels Oncogenic Antigen Receptor Signaling in B-cell Lymphoma.

Pathomechanisms that activate oncogenic B-cell receptor (BCR) signaling in diffuse large B-cell lymphoma (DLBCL), are largely unknown. Kelch-like family member 6 (KLHL6) encoding a substrate-adapter for Cullin-3-RING E3 ubiquitin-ligase (CRL) with poorly established targets is recurrently mutated in DLBCL. By applying high-throughput protein interactome screens and functional characterization, we discovered that KLHL6 regulates BCR by targeting its signaling subunits CD79A and CD79B. Loss of physiological KLHL6 expression pattern was frequent among the MCD/C5-like activated B-cell DLBCLs and was associated with higher CD79B levels and dismal outcome. Mutations in the BTB domain of KLHL6 disrupted its localization and heterodimerization, and increased surface BCR levels and signaling, whereas Kelch domain mutants had the opposite effect. Malfunctions of KLHL6 mutants extended beyond proximal BCR signaling with distinct phenotypes from KLHL6 silencing. Collectively, our findings uncover how recurrent mutations in KLHL6 alter BCR signaling and induce actionable phenotypic characteristics in DLBCL.

Diamond-Like Carbon Depositing on the Surface of Polylactide Membrane for Prevention of Adhesion Formation During Tendon Repair.

Post-traumatic peritendinous adhesion presents a significant challenge in clinical medicine. This study proposes the use of diamond-like carbon (DLC) deposited on polylactic acid (PLA) membranes as a biophysical mechanism for anti-adhesion barrier to encase ruptured tendons in tendon-injured rats. The results indicate that PLA/DLC composite membrane exhibits more efficient anti-adhesion effect than PLA membrane, with histological score decreasing from 3.12 ± 0.27 to 2.20 ± 0.22 and anti-adhesion effectiveness increasing from 21.61% to 44.72%. Mechanistically, the abundant C=O bond functional groups on the surface of DLC can reduce reactive oxygen species level effectively; thus, the phosphorylation of NF-κB and M1 polarization of macrophages are inhibited. Consequently, excessive inflammatory response augmented by M1 macrophage-originated cytokines including interleukin-6 (IL-6), interleukin-1ß (IL-1ß), and tumor necrosis factor-α (TNF-α) is largely reduced. For biocompatibility evaluation, PLA/DLC membrane is slowly absorbed within tissue and displays prolonged barrier effects compared to traditional PLA membranes. Further studies show the DLC depositing decelerates the release of degradation product lactic acid and its induction of macrophage M2 polarization by interfering esterase and PLA ester bonds, which further delays the fibrosis process. It was found that the PLA/DLC membrane possess an efficient biophysical mechanism for treatment of peritendinous adhesion.

GTPBP8 modulates mitochondrial fission through a Drp1-dependent process.

Mitochondrial fission is a tightly regulated process involving multiple proteins and cell signaling. Despite extensive studies on mitochondrial fission factors, our understanding of the regulatory mechanisms remains limited. This study shows the critical role of a mitochondrial GTPase, GTPBP8, in orchestrating mitochondrial fission in mammalian cells. Depletion of GTPBP8 resulted in drastic elongation and interconnectedness of mitochondria. Conversely, overexpression of GTPBP8 shifted mitochondrial morphology from tubular to fragmented. Notably, the induced mitochondrial fragmentation from GTPBP8 overexpression was inhibited in cells either depleted of the mitochondrial fission protein Drp1 (also known as DNM1L) or carrying mutated forms of Drp1. Importantly, downregulation of GTPBP8 caused an increase in oxidative stress, modulating cell signaling involved in the increased phosphorylation of Drp1 at Ser637. This phosphorylation hindered the recruitment of Drp1 to mitochondria, leading to mitochondrial fission defects. By contrast, GTPBP8 overexpression triggered enhanced recruitment and assembly of Drp1 at mitochondria. In summary, our study illuminates the cellular function of GTPBP8 as a pivotal modulator of the mitochondrial division apparatus, inherently reliant on its influence on Drp1.

Incorporating polygenic risk into the Leicester Risk Assessment score for 10-year risk prediction of type 2 diabetes.

AIMS: We evaluated whether incorporating information on ethnic background and polygenic risk enhanced the Leicester Risk Assessment (LRA) score for predicting 10-year risk of type 2 diabetes. METHODS: The sample included 202,529 UK Biobank participants aged 40-69 years. We computed the LRA score, and developed two new risk scores using training data (80% sample): LRArev, which incorporated additional information on ethnic background, and LRAprs, which incorporated polygenic risk for type 2 diabetes. We assessed discriminative and reclassification performance in a test set (20% sample). Type 2 diabetes was ascertained using primary care, hospital inpatient and death registry records. RESULTS: Over 10 years, 7,476 participants developed type 2 diabetes. The Harrell's C indexes were 0.796 (95% Confidence Interval [CI] 0.785, 0.806), 0.802 (95% CI 0.792, 0.813), and 0.829 (95% CI 0.820, 0.839) for the LRA, LRArev and LRAprs scores, respectively. The LRAprs score significantly improved the overall reclassification compared to the LRA (net reclassification index [NRI] = 0.033, 95% CI 0.015, 0.049) and LRArev (NRI = 0.040, 95% CI 0.024, 0.055) scores. CONCLUSIONS: Polygenic risk moderately improved the performance of the existing LRA score for 10-year risk prediction of type 2 diabetes.

Visual identification for species and sex derived from bloodstain based on phosphate-mediated isothermal amplification colorimetric system.

Species and sex confirmation of the biological specimen play a crucial role in crime investigation. However, the specimen found in the scene is always trace quantity, which is hard to be analyzed by current methods. Moreover, the time-consuming DNA extraction, sophisticated apparatus, and complex data processing make it difficult to satisfy the demand of speediness and convenience for point-of-care tests. In this study, we first exhibit a phosphate-based visual system for field-based species and sex identification derived from trace bloodstain. By introducing phosphate ion-based colorimetry into loop-mediated isothermal amplification (LAMP) for result interpretation, not only the bloodstain can be directly submitted to mitochondrial variant amplification owing to the enhanced amplification efficiency by pyrophosphate ion hydrolyzation, but also the colorimetric signal can be recognized by the naked eye for result output within 30 min through molybdophosphate generation. Aerosol contamination, the major conflict of LAMP, has been solved once and for all by integrating uracil-DNA glycosylase into this system that still holds on a constant temperature. As a demonstration, cytochrome b and Y-chromosomal amelogenin are employed to identify species and sex respectively, which has achieved a highly sensitive and specific distinguishability under a strong interferential background. Accurate results can be obtained from both the simulative degraded and dated specimen, which indicates that this novel system may serve as a promising tool in forensic practice.

Swimming trapper decreases the proportion of chromosomally unbalanced spermatozoa in human Robertsonian translocation carriers.

BACKGROUND: Carriers of reciprocal translocations often have more unbalanced spermatozoa and higher DNA fragmentation rates, elevating reproductive risk. The simple swim-up method (SSUM) can decrease the amount of spermatozoa with abnormal chromatin structure and fragmented DNA, however, it has limited efficacy in eliminating chromosomally unbalanced sperm. METHODS: The spermatozoa of eight Robertsonian translocation (Rob) carriers were split into three groups: original raw semen group (control group); SSUM and swimming trapper method group (STM) processed semen samples. After different semen preparation procedures, semen qualities, sperm chromosomal aneuploidy, and sperm fragmented DNA were evaluated. RESULTS: Although spermatozoa with higher motility was obtained by both SSUM and STM, the population of faster forward moving sperm was greater with STM as compared to SSUM. While the rates of DNA fragmentation were statistically much lower in both groups than ejaculated semen sample, our data showed better effect on the decrease of DNA fragmentation index (DFI) after selection by STM for patients who have high DFI (>20%) in neat semen. For all patients, significant decrease in the frequency of chromosomally unbalanced spermatozoa was observed after selection using STM. Although similar trends can be seen in the SSUM group, a significant difference was identified in one patient only. CONCLUSIONS: Use of swimming trapper (STM) is superior for enriching high-motile and genetically competent sperm in comparison with SSUM.

A complementary learning systems model of how sleep moderates retrieval practice effects.

While many theories assume that sleep is critical in stabilizing and strengthening memories, our recent behavioral study (Liu & Ranganath, 2021, Psychonomic Bulletin & Review, 28[6], 2035-2044) suggests that sleep does not simply stabilize memories. Instead, it plays a more complex role, integrating information across two temporally distinct learning episodes. In the current study, we simulated the results of Liu and Ranganath (2021) using our biologically plausible computational model, TEACH, developed based on the complementary learning systems (CLS) framework. Our model suggests that when memories are activated during sleep, the reduced influence of temporal context establishes connections across temporally separated events through mutual training between the hippocampus and neocortex. In addition to providing a compelling mechanistic explanation for the selective effect of sleep, this model offers new examples of the diverse ways in which the cortex and hippocampus can interact during learning.

Antiadhesion Biomaterials in Tendon Repair: Application Status and Future Prospect.

The healing process after tendon injury is often accompanied by the formation of peritendinous adhesion, contributing to limb dysfunction and exerting detrimental effects on the individuals, as well as the development of society and economy. With the continuous development of material science, as well as the augmented understanding of tendon healing and the mechanism of peritendinous adhesion formation, materials used for the fabrication of barrier membranes against peritendinous adhesion emerge endlessly. In this article, based on the analysis of the mechanism of adhesion formation, we first review the commonly used natural and synthetic materials, along with their corresponding fabrication strategies, in order to furnish valuable insights for the future optimization and development of antiperitendinous adhesion barrier membranes. This article also discusses the interaction between antiadhesion materials and cells for ameliorating peritendinous adhesion.

One-year efficacy of myopia control by the defocus distributed multipoint lens: a multicentric randomised controlled trial.

AIMS: To report the 1-year results of the efficacy of a defocus distributed multipoint (DDM) lens in controlling myopia progression in a multicentre, randomised controlled trial. METHODS: Overall, 168 children aged 6-13 years were recruited and randomly assigned to wear a DDM lens (n=84) or single-vision (SV) lens (n=84) in three centres. Cycloplegic autorefraction (spherical equivalent refraction (SER)) and axial length (AL) were measured. Linear mixed model analysis was performed to compare between-group SER and AL changes. Logistic regression analysis was used to analyse the between-group difference in rapid myopia progression (SER increase≥0.75 D per year or AL growth≥0.40 mm per year). RESULTS: After 1 year, mean changes in SER were significantly lower in the DDM group (-0.47±0.37 D) than in the SV group (-0.71±0.42 D) (p<0.001). Similarly, mean changes in AL were significantly lower in the DDM group (0.21±0.17 mm) than in the SV group (0.34±0.16 mm) (p<0.001). After adjusting for age, sex, daily wearing time and parental myopia, rapid myopia progression risk was higher in the SV group than in the DDM group (OR=3.51, 95% CI: 1.77 to 6.99), especially for children who wore a lens for >12 hours per day, boys and younger children (6-9 years) with ORs (95% CIs) of 10.82 (3.22 to 36.37), 5.34 (1.93 to 14.78) and 8.73 (2.6 to 29.33), respectively. CONCLUSIONS: After 1 year, DDM lenses effectively retarded myopia progression in children. Longer daily wearing time of DDM lens improved the efficacy of myopia control. Future long-term studies are needed for validation. TRIAL REGISTRATION NUMBER: NCT05340699.

PABPC1L Induces IDO1 to Promote Tryptophan Metabolism and Immune Suppression in Renal Cell Carcinoma.

The tumor microenvironment (TME) in renal cell carcinomas (RCC) is marked by substantial immunosuppression and immune resistance despite having extensive T-cell infiltration. Elucidation of the mechanisms underlying immune evasion could help identify therapeutic strategies to boost the efficacy of immune checkpoint blockade (ICB) in RCC. This study uncovered a mechanism wherein the polyadenylate-binding protein PABPC1L modulates indoleamine 2,3-dioxygenase 1 (IDO1), a prospective target for immunotherapy. PABPC1L was markedly upregulated in RCC, and high PABPC1L expression correlated with unfavorable prognosis and resistance to ICB. PABPC1L bolstered tryptophan metabolism by upregulating IDO1, inducing T-cell dysfunction and Treg infiltration. PABPC1L enhanced the stability of JAK2 mRNA, leading to increased JAK2-STAT1 signaling that induced IDO1 expression. Additionally, PABPC1L-induced activation of the JAK2-STAT1 axis created a positive feedback loop to promote PABPC1L transcription. Conversely, loss of PABPC1L diminished IDO1 expression, mitigated cytotoxic T-cell suppression, and enhanced responsiveness to anti-PD-1 therapy in patient-derived xenograft models. These findings reveal the crucial role of PABPC1L in facilitating immune evasion in RCC and indicate that inhibiting PABPC1L could be a potential immunotherapeutic approach in combination with ICB to improve patient outcomes. SIGNIFICANCE: PABPC1L functions as a key factor in renal cell carcinoma immune evasion, enhancing IDO1 and impeding T-cell function, and represents a potential target to enhance the efficacy of immune checkpoint blockade therapy.