Charting the solid-state NMR signals of polysaccharides: A database-driven roadmap.

Solid-state nuclear magnetic resonance (ssNMR) measurements of intact cell walls and cellular samples often generate spectra that are difficult to interpret due to the presence of many coexisting glycans and the structural polymorphism observed in native conditions. To overcome this analytical challenge, we present a statistical approach for analyzing carbohydrate signals using high-resolution ssNMR data indexed in a carbohydrate database. We generate simulated spectra to demonstrate the chemical shift dispersion and compare this with experimental data to facilitate the identification of important fungal and plant polysaccharides, such as chitin and glucans in fungi and cellulose, hemicellulose, and pectic polymers in plants. We also demonstrate that chemically distinct carbohydrates from different organisms may produce almost identical signals, highlighting the need for high-resolution spectra and validation of resonance assignments. Our study provides a means to differentiate the characteristic signals of major carbohydrates and allows us to summarize currently undetected polysaccharides in plants and fungi, which may inspire future investigations.

Heterotypic Interactions between the 40- and 42-Residue Isoforms of ß-Amyloid Peptides on Lipid Bilayer Surfaces.

Co-aggregation involving different amyloidogenic sequences has been emphasized recently in the modified amyloid cascade hypothesis. Yet, molecular-level interactions between two predominant ß-amyloid peptide sequences, Aß40 and Aß42, in the fibrillation process in membrane-mimicked environments remain unclear. Here, we report biophysical evidence that demonstrates the molecular-level interactions between Aß40 and Aß42 at the membrane-associated conucleation stage using dynamic nuclear polarization-enhanced solid-state NMR spectroscopy. These residue-specific contacts are distinguished from those reported in mature fibrils formed by either Aß40 or Aß42. Meanwhile, site-specific interactions between Aß and lipid molecules and modulation of microsecond-time-scale lipid dynamics are observed, which may be responsible for the more rapid and significant membrane content leakage compared to that with Aß40 alone.

Polysaccharide assemblies in fungal and plant cell walls explored by solid-state NMR.

Structural analysis of macromolecular complexes within their natural cellular environment presents a significant challenge. Recent applications of solid-state NMR (ssNMR) techniques on living fungal cells and intact plant tissues have greatly enhanced our understanding of the structure of extracellular matrices. Here, we selectively highlight the most recent progress in this field. Specifically, we discuss how ssNMR can provide detailed insights into the chemical composition and conformational structure of pectin, and the consequential impact on polysaccharide interactions and cell wall organization. We elaborate on the use of ssNMR data to uncover the arrangement of the lignin-polysaccharide interface and the macrofibrillar structure in native plant stems or during degradation processes. We also comprehend the dynamic structure of fungal cell walls under various morphotypes and stress conditions. Finally, we assess how the combination of NMR with other techniques can enhance our capacity to address unresolved structural questions concerning these complex macromolecular assemblies.

The efficacy and safety of angiogenesis inhibitors for recurrent ovarian cancer: a meta­analysis.

OBJECTIVE: To investigate the efficacy and safety of angiogenesis inhibitors in the treatment of recurrent ovarian cancer (OC). METHODS: Electronic databases including PubMed, Web of Science, and the Cochrane Library were searched to find eligible studies until August 10, 2021. The data on overall survival (OS), progression-free survival (PFS), and objective response rate (ORR) were pooled. Furthermore, grade ≥ 3 adverse events (AEs) were investigated. RESULTS: A total of 13 studies with 3953 patients were included. Compared with control group, angiogenesis inhibitors resulted in significant improvement in PFS (hazard ratio (HR) = 0.61, 95%CI, 0.54-0.69), OS (HR = 0.88, 95%CI, 0.81-0.95), and ORR (odds ratio (OR) = 2.15, 95% CI, 1.74-2.65). However, angiogenesis inhibitors were associated with a higher risk of grade ≥ 3 AEs (relative risk (RR), 1.20, 95% CI, 1.04-1.38). CONCLUSION: Angiogenesis inhibitors can improve ORR, PFS, and OS in patients with recurrent OC, but they can increase the incidence of AEs ≥ 3.

Current limitations of solid-state NMR in carbohydrate and cell wall research.

High-resolution investigation of cell wall materials has emerged as an important application of biomolecular solid-state NMR (ssNMR). Multidimensional correlation experiments have become a standard method for obtaining sufficient spectral resolution to determine the polymorphic structure of carbohydrates and address biochemical questions regarding the supramolecular organization of cell walls. Using plant cellulose and matrix polysaccharides as examples, we will review how the multifaceted complexity of polysaccharide structure is impeding the resonance assignment process and assess the available biochemical and spectroscopic approaches that could circumvent this barrier. We will emphasize the ineffectiveness of the current methods in reconciling the ever-growing dataset and deriving structural information. We will evaluate the protocols for achieving efficient and homogeneous hyperpolarization across the cell wall material using magic-angle spinning dynamic nuclear polarization (MAS-DNP). Critical questions regarding the line-broadening effects of cell wall molecules at cryogenic temperature and by paramagnetic biradicals will be considered. Finally, the MAS-DNP method will be placed into a broader context with other structural characterization techniques, such as cryo-electron microscopy, to advance ssNMR research in carbohydrate and cell wall biomaterials.

Significance of TP53 Mutational Status-Associated Signature in the Progression and Prognosis of Endometrial Carcinoma.

Background: TP53 mutations are associated with poor outcome for patients with endometrial carcinoma (EC). However, to date, there have been no studies focused on the construction of TP53 mutational status-associated signature in EC. In this study, we aim to conduct a TP53 mutation-associated prognostic gene signature for EC. Methods: Hence, we explored the mutational landscape of TP53 in patients with EC based on the simple nucleotide variation data downloaded from The Cancer Genome Atlas (TCGA) database. Differential expression analysis and least absolute shrinkage and selection operator (LASSO)-Cox analysis was used to establish TP53 mutation-associated prognostic gene signature. The overall survival rate between the high-risk and low-risk groups was compared by the Kaplan-Meier (K-M) method. Results: We found that the TP53 mutation was associated with poor outcome, older age, lower BMI, and higher grade and stage of EC in patients. A TP53 mutational status-associated signature was established based on transcriptome profiling data. Moreover, the patients in TCGA database were categorized into high- and low-risk groups. Kaplan-Meier (K-M) analysis indicated that the patients in the high-risk group have poor survival outcome. Furthermore, receiver operating characteristic (ROC) curves confirmed the robust prognostic prediction efficiency of the TP53 mutational status-associated signature. Finally, the prognostic ability was successfully verified in the other two datasets from cBioPortal database as well as in 60 clinical specimens. Univariate (hazard ratio (HR) = 1.041, 95%CI = 1.031-1.051, p < 0.001) and multivariate (hazard ratio (HR) = 1.029, 95%CI = 1.018-1.040, p < 0.001) Cox regression analyses indicated that the TP53 mutational status-associated signature could be used as an independent prognostic factor for EC patients. Conclusion: In summary, our research constructed a powerful TP53 mutational status-associated signature that could be a potential novel prognostic biomarker and therapeutic target for EC.

A 13C three-dimensional DQ-SQ-SQ correlation experiment for high-resolution analysis of complex carbohydrates using solid-state NMR.

Complex carbohydrates are the key components of the protective cell walls of microbial pathogens and the bioenergy reservoir in plants and algae. Structural characterization of these polymorphic molecules requires assistance from multidimensional 13C correlation approaches. To facilitate the analysis of carbohydrate structure using solid-state NMR, we present a three-dimensional (3D) 13C-13C-13C experiment that includes a double-quantum (DQ) dimension and is thus free of the cube's body diagonal. The enhanced resolution supports the unambiguous resonance assignment of many polysaccharides in plant and fungal cell walls using uniformly 13C-labeled cells of spruce and Aspergillus fumigatus. Long-range structural restraints were effectively obtained to revisit our understanding of the spatial organization of plant cellulose microfibrils. The method is widely applicable to the investigations of cellular carbohydrates and carbon-based biomaterials.

Effect of cross polarization radiofrequency phases on signal phase.

Utilizing phases of radio frequency (RF) pulses to manipulate spin dynamics is routine in NMR and MRI, leading to spectacular techniques like phase cycling. In a very different area, cross polarization (CP) also has a long history as part of a vast number of solid-state NMR pulse sequences. However, a detailed study devoted to the effect of CP RF phases on NMR signal, seems not to be readily available. From first principles, we arrive at a simple dependence of NMR signal on arbitrary CP RF phases, for static and MAS conditions, accompanied by experimental verification. In the process, the CP propagator emerges as a product of RF "pulses" and a period of "free precession", conforming to coherence transfer pathway theory. The theoretical expressions may lend confidence for dealing with CP blocks with tunable phases in pulse sequences.

Carbohydrate-aromatic interface and molecular architecture of lignocellulose.

Plant cell walls constitute the majority of lignocellulosic biomass and serve as a renewable resource of biomaterials and biofuel. Extensive interactions between polysaccharides and the aromatic polymer lignin make lignocellulose recalcitrant to enzymatic hydrolysis, but this polymer network remains poorly understood. Here we interrogate the nanoscale assembly of lignocellulosic components in plant stems using solid-state nuclear magnetic resonance and dynamic nuclear polarization approaches. We show that the extent of glycan-aromatic association increases sequentially across grasses, hardwoods, and softwoods. Lignin principally packs with the xylan in a non-flat conformation via non-covalent interactions and partially binds the junction of flat-ribbon xylan and cellulose surface as a secondary site. All molecules are homogeneously mixed in softwoods; this unique feature enables water retention even around the hydrophobic aromatics. These findings unveil the principles of polymer interactions underlying the heterogeneous architecture of lignocellulose, which may guide the rational design of more digestible plants and more efficient biomass-conversion pathways.

Exploring the value of cesarean section diverticulum area to predict the safety of hysteroscopic management for cesarean scar pregnancy patients.

OBJECTIVE: To study indicators predicting the safety of hysteroscopic management for cesarean scar pregnancy (CSP) patients. METHODS: This was a retrospective study, starting from June 1, 2020. The study included 141 CSP patients who underwent hysteroscopic surgery and met the requirements of gestational age ≤12 weeks, stable vital signs, and preoperative magnetic resonance imaging. Patients were divided into control group and testing group according to surgical outcomes. Preoperative indicators were compared between the two groups, including a novel indicator, cesarean section diverticulum (CSD) area. RESULTS: Univariate analysis identified five statistically significant (P < 0.05) factors associated with hysteroscopy failure including a large CSD area. Multifactor logistic regression analysis showed that the only statistically significant indicator of all five factors was the CSD area. The area under the receiver operating characteristics curve of CSD area was 0.848. Next, we determined three cut-off values for CSD area that can be used to predict the outcome of surgery: 138, 189, and 300 mm2 . CONCLUSION: For the first time, we found that CSD area could predict the safety of hysteroscopic management for CSP patients and might be helpful for clinical decision making. The findings need to be verified by further research.

A pectin methyltransferase modulates polysaccharide dynamics and interactions in Arabidopsis primary cell walls: Evidence from solid-state NMR.

Plant cell walls contain cellulose embedded in matrix polysaccharides. Understanding carbohydrate structures and interactions is critical to the production of biofuel and biomaterials using these natural resources. Here we present a solid-state NMR study of cellulose and pectin in 13C-labeled cell walls of Arabidopsis wild-type and mutant plants. Using 1D 13C and 2D 13C-13C correlation experiments, we detected a highly branched arabinan structure in qua2 and tsd2 samples, two allelic mutants for a pectin methyltransferase. Both mutants show close physical association between cellulose and the backbones of pectic homogalacturonan and rhamnogalacturonan-I. Relaxation and dipolar order parameters revealed enhanced microsecond dynamics due to polymer disorder in the mutants, but restricted motional amplitudes due to tighter pectin-cellulose associations. These molecular data shed light on polymer structure and packing in these two pectin mutants, helping to elucidate how pectin could influence cell wall architecture at the nanoscale, cell wall mechanics, and plant growth.

Survival Outcomes in Patients With 2018 FIGO Stage IA2-IIA2 Cervical Cancer Treated With Laparoscopic Versus Open Radical Hysterectomy: A Propensity Score-Weighting Analysis.

OBJECTIVE: To compare the survival and recurrence outcomes between open and laparoscopic radically hysterectomy (RH) for stage IA2-IIA2 cervical cancer based on Federation International of Gynecology and Obstetrics (FIGO) 2018. METHODS: Data of 1,373 early cervical cancer patients undergoing open or laparoscopic radically hysterectomy at ShengJing Hospital of China Medical University between January 1, 2013, and December 31, 2016, were retrospectively reviewed. Propensity score-based inverse probability of treatment weighting (PS-IPTW) was used to balance the covariates between the two groups. RESULTS: A total of 705 cervical cancer patients of FIGO 2009 stage IA2-IIA2 were finally enrolled in this study. After IPTW adjustment, the OS (HR = 2.095, 95% CI: 1.233-3.562, P = 0.006) and PFS (HR = 1.950, 95%CI: 1.194-3.184, P = 0.008) rates were significantly higher in the open RH (ORH) group compared with the laparoscopic RH (LRH) group. Then after re-staging according to the FIGO 2018 staging system, 561 patients still belonged to stage IA2-IIA2, 144 patients were upgraded to stage IIIC1p-IIIC2p. The ORH group had a significantly superior OS (HR = 1.977, 95%CI: 1.077-3.626, P = 0.028) and PFS (HR = 1.811, 95%CI: 1.046-3.134, P = 0.034) compared with the LRH group after PS-IPTW analysis. Furthermore, in patients with no high and intermediate risks, difference of the OS (HR = 1.386, 95%CI: 0.287-6.69, P = 0.684) and PFS (HR = 1.524, 95%CI: 0.363-6.396, P = 0.565) rates between the two groups were with no statistical meaning. CONCLUSIONS: Outcomes of this retrospective cohort study were in compliance with indications for ORH recommended by the National Comprehensive Cancer Network guidelines Version 1, 2021. However, LRH showed non-inferiority for patients with no prognostic risk factors compared with ORH.

Imaging-Guided Chemo-Photothermal Polydopamine Carbon Dots for EpCAM-Targeted Delivery toward Liver Tumor.

We demonstrate a versatile nanoparticle with imaging-guided chemo-photothermal synergistic therapy and EpCAM-targeted delivery of liver tumor cells. EpCAM antibody (anti-EpCAM) and Pt(IV) were grafted onto the polydopamine carbon dots (PDA-CDs) by the amidation reaction. The EpCAM antibody of particles enables the targeted interaction with liver progenitor cells due to their overexpressed EpCAM protein. The tetravalent platinum prodrug [Pt(IV)] induces apoptosis with minimum toxic side effects through the interaction between cisplatin and tumor cell DNA. The nanoparticles displayed stable photothermal property and considerable anti-tumor therapeutic effect in vivo. Coupling with cellular imaging due to their fluorescence property, anti-EpCAM@PDA-CDs@Pt(IV) offers a convenient and effective platform for imaging-guided chemo-photothermal synergistic therapy toward liver cancers in the near future.

Identification and validation of significant gene mutations to predict clinical benefit of immune checkpoint inhibitors in lung adenocarcinoma.

OBJECTIVE: Immune checkpoint inhibitors (ICI) has achieved remarkable clinical benefit in advanced lung adenocarcinoma (LUAD). However, effective clinical use of ICI agents is encumbered by the high rate of innate resistance. The aim of our research is to identify significant gene mutations which can predict clinical benefit of immune checkpoint inhibitors in LUAD. METHODS: The "mafComapre" function of "MafTools" package was used to screen the differentially mutated genes between durable clinical benefit (DCB) group and no durable clinical benefit (NDB) group based on the somatic mutation data from NSCLc_PD1_mSK_2018. Machine learning was performed to select significantly mutated genes to accurately classify patients into DCB group and NDB group. A nomogram model was constructed based on the significantly mutated genes to predict the susceptibility of patients to ICI. Finally, we explored the correlation between two classifications of immune cell infiltration, PD-1 and PD-L1 expression, tumor mutational burden (TMB) and prognosis. RESULTS: Through utilize machine learning, 6 significantly mutated genes were obtained from 8 differentially mutated genes and used to accurately classify patients into DCB group and NDB group. The DCA curve and clinical impact curve revealed that the patients can benefit from the decisions made based on the nomogram model. Patients highly sensitive to ICI have elevated immune activity, higher expression of PD-1 and PD-L1, increased TMB, and well prognosis if they accept ICI treatment. CONCLUSIONS: Our research selected 6 significantly mutated genes that can predict clinical benefit of ICI in LUAD patients.

Lymph-Vascular Space Invasion in Patients with Stages IA2-IIA2 Cervical Cancer Treated with Laparoscopic versus Open Radical Hysterectomy.

OBJECTIVE: To explore the relationship between laparoscopic radical hysterectomy (LRH) and cervical cancer lymph-vascular space invasion (LVSI) by comparing the prevalence of LVSI in cervical cancer patients who underwent LRH versus open radical hysterectomy (ORH). METHODS: The study participants were 1087 cervical cancer patients (FIGO 2009 stages IA2-IIA2) with pathologically confirmed with or without LVSI who underwent radical hysterectomy at Shengjing Hospital of China Medical University from 2013 through 2018. The patients were divided according to the type of surgical procedure into an LRH group (n=148) and an ORH group (n=939). RESULTS: In the LRH group, 31.76% of patients (47/148) had LVSI-positive tumors compared to 33.23% of patients (312/939) in the ORH group; the difference was not significant (p=0.724). No between-group differences in LVSI prevalence according to lymph node metastasis, interstitial infiltration depth, differentiation degree, and parametrial infiltration were found. However, the number of LVSI-positive patients whose cervical cancer lesions >4 cm (stage I B2 and II A2) was significantly higher in the LRH group than in the ORH group (Odds Ratio [OR] 0.333, 95% confidence interval [CI] 0.157-0.706, p=0.005). The 3-Year disease-free survival (DFS) in the LRH group is lower than that in the ORH group (94.75% vs 97.27%), but there was no significance (P=0.187). Furthermore, the percentage of LVSI-positive tumors in patients with lymph node metastases was significantly higher than those without lymph node metastases (OR 2.897, 95% CI 2.129-3.942, p=0.000). The 3-Year DFS were 98.22% in the LVSI negative patients and 93.78% in the LVSI positive patients, the difference was significant (P=0.002). CONCLUSION: A higher risk of lymph node metastasis and a lower 3-Year DFS was found in the LVSI-positive patients. In case of LVSI, it would be dangerous to treat patient in laparoscopy, especially in case of cervical cancer lesions >4cm.

Solid-state NMR of unlabeled plant cell walls: high-resolution structural analysis without isotopic enrichment.

BACKGROUND: Multidimensional solid-state nuclear magnetic resonance (ssNMR) spectroscopy has emerged as an indispensable technique for resolving polymer structure and intermolecular packing in primary and secondary plant cell walls. Isotope (13C) enrichment provides feasible sensitivity for measuring 2D/3D correlation spectra, but this time-consuming procedure and its associated expenses have restricted the application of ssNMR in lignocellulose analysis. RESULTS: Here, we present a method that relies on the sensitivity-enhancing technique Dynamic Nuclear Polarization (DNP) to eliminate the need for 13C-labeling. With a 26-fold sensitivity enhancement, a series of 2D 13C-13C correlation spectra were successfully collected using the unlabeled stems of wild-type Oryza sativa (rice). The atomic resolution allows us to observe a large number of intramolecular cross peaks for fully revealing the polymorphic structure of cellulose and xylan. NMR relaxation and dipolar order parameters further suggest a sophisticated change of molecular motions in a ctl1 ctl2 double mutant: both cellulose and xylan have become more dynamic on the nanosecond and microsecond timescale, but the motional amplitudes are uniformly small for both polysaccharides. CONCLUSIONS: By skipping isotopic labeling, the DNP strategy demonstrated here is universally extendable to all lignocellulose materials. This time-efficient method has landed the technical foundation for understanding polysaccharide structure and cell wall assembly in a large variety of plant tissues and species.

N-doped carbon dots modified with the epithelial cell adhesion molecule antibody as an imaging agent for HepG2 cells using their ultra-sensitive response to Al3.

Carbon dots (CDs) are emerging as an ideal multifunctional materials due to their ease of preparation and excellent properties in medical imaging technology, environmental monitoring, chemical analysis and other fields. N-doped CDs modified with the epithelial cell adhesion molecule antibody (anti-EpCAM-NCDs) were synthesized in an ingenious and high-output approach. Due to the fluorescence enhancement effect of the introduced N atoms, the obtained anti-EpCAM-NCDs exhibited a strong green emission with an absolute quantum yield of up to 32.5%. Anti-EpCAM-NCDs have immunofluorescent properties and an active targeting function. The fluorescence effect and fluorescence quenching of anti-EpCAM-NCDs are used to image cells and detect Al3+, respectively. Experimental results show that this probe exhibited a wide linear response to Al3+over a concentration range of 0-100µM with a detection limit and quantification limit of 3 nM and 6 nM, respectively. Significantly, anti-EpCAM-NCDs, which have negligible cytotoxicity, excellent biocompatibility and high photostability, could be used for the intracellular imaging of HepG2 cells and the detection of Al3+in environmental and biological samples. As an efficient multifunctional material, anti-EpCAM-NCDs hold great promise for a number of applications in biological systems.

Combinatorial discovery of Mo-based polyoxometalate clusters for tumor photothermal therapy and normal cell protection.

Nanomaterials with multiple functions such as precision diagnosis, therapeutic efficacy and biosafety are attractive for tumor treatment but remain a technical challenge. In this study, molybdenum (Mo)-based polyoxometalate clusters (Mo-POM) with considerable photothermal conversion efficiency (∼56.6%) and high stability (>30 days) were prepared through a modification of the Folin-Ciocalteu method. These synthetic particles accumulated at the target site, and induced thermal ablation of the tumor following near infrared (NIR) absorption. Furthermore, the Mo-POM effectively scavenged reactive oxygen species (ROS) through charge transfer between Mo(vi) and Mo(v) states, thereby avoiding off-target effects on normal cells and improving the therapeutic efficiency both in vitro and in vivo. Therefore, for the first time, we prepared Mo-POM having two key functions, i.e., photothermal therapy (PTT) for cancer cells and protection of normal cells. These exceptional features may open up the exploration of Mo-POM as new tools for PTT against tumors in clinical applications.

Solid-state NMR of plant and fungal cell walls: A critical review.

The cell walls of plants and microbes are a central source for bio-renewable energy and the major targets of antibiotics and antifungal agents. It is highly challenging to determine the molecular structure of complex carbohydrates, protein and lignin, and their supramolecular assembly in intact cell walls. This article selectively highlights the recent breakthroughs that employ 13C/15N solid-state NMR techniques to elucidate the architecture of fungal cell walls in Aspergillus fumigatus and the primary and secondary cell walls in a large variety of plant species such as Arabidopsis, Brachypodium, maize, and spruce. Built upon these pioneering studies, we further summarize the underexplored aspects of fungal and plant cell walls. The new research opportunities introduced by innovative methods, such as the detection of proton and quadrupolar nuclei on ultrahigh-field magnets and under fast magic-angle spinning, paramagnetic probes, natural-abundance DNP, and software development, are also critically discussed.