Integrated multimodality microscope for accurate and efficient target-guided cryo-lamellae preparation.

Cryo-electron tomography (cryo-ET) is a revolutionary technique for resolving the structure of subcellular organelles and macromolecular complexes in their cellular context. However, the application of the cryo-ET is hampered by the sample preparation step. Performing cryo-focused ion beam milling at an arbitrary position on the sample is inefficient, and the target of interest is not guaranteed to be preserved when thinning the cell from several micrometers to less than 300 nm thick. Here, we report a cryogenic correlated light, ion and electron microscopy (cryo-CLIEM) technique that is capable of preparing cryo-lamellae under the guidance of three-dimensional confocal imaging. Moreover, we demonstrate a workflow to preselect and preserve nanoscale target regions inside the finished cryo-lamellae. By successfully preparing cryo-lamellae that contain a single centriole or contact sites between subcellular organelles, we show that this approach is generally applicable, and shall help in innovating more applications of cryo-ET.

Unveiling the Role of Water on π-π Stacking Through Microwave Spectroscopy of (Thiophene)2-(Water)1-2 Clusters.

There has been an ongoing debate about whether water enhances or hinders π-π stacking, a phenomenon crucial in various biological and chemical systems. In this study, the influence of water on π-π stacking is investigated by microwave spectroscopic observation of gas-phase hydrated clusters of thiophene dimers. Two isomers of (C4H4S)2-H2O and two isomers of (C4H4S)2-(H2O)2 have been unambiguously identified. These identifications are supported by quantum chemistry calculations and isotopic measurements. In each of these conformations, water molecules are situated between aromatic pairs, forming distinctive interactions. Water molecules engage with thiophene molecules either as hydrogen bond donors through OH···π interactions or as hydrogen bond acceptors through CH···O interactions. The energy decomposition analysis indicates that the bonding pattern of water molecules significantly affects the π···π interactions between aromatic rings. These findings offer valuable structural insights into the role of water in shaping π-π stacking.

Molecular-scale axial localization by repetitive optical selective exposure.

We introduce an axial localization with repetitive optical selective exposure (ROSE-Z) method for super-resolution imaging. By using an asymmetric optical scheme to generate interference fringes, a <2 nm axial localization precision was achieved with only ~3,000 photons, which is an approximately sixfold improvement compared to previous astigmatism methods. Nanoscale three-dimensional and two-color imaging was demonstrated, illustrating how this method achieves superior performance and facilitates the investigation of cellular nanostructures.

Hydrogen-Bonded Complex of the Parent Phosphinidene.

The diatomic molecule PH is very reactive, and it serves as the parent compound for phosphinidenes featuring a monovalent phosphorus atom. Herein, we report the characterization and reactivity of a rare hydrogen-bonded complex of PH. Specifically, the molecular complex between PH and HCl has been generated by photolysis of chlorophosphine (H2PCl) at 254 nm in a solid Ar-matrix at 10 K. The IR spectrum of the complex HP⋅⋅⋅HCl and quantum chemical calculations at the UCCSD(T)-F12a/haTZ level consistently prove that the phosphorus atom acts as a hydrogen bond acceptor with a binding energy (D0) of -0.6 kcal mol-1. In line with the observed absorption at 341 nm for the binary complex, the triplet phosphinidene PH undergoes prototype H-Cl bond insertion by reformation of H2PCl upon photoexcitation at 365 nm. However, this hydrogen-bonded complex is unstable in the presence of N2 and HCl, as both molecules prefers stronger interactions with HCl than PH in the observed complexes HP⋅⋅⋅HCl⋅⋅⋅N2 and HP⋅⋅⋅2HCl.

Glycine-Modified Co-MOF Pervaporation Membrane to Enhance Water Transporting.

Cobalt-based metal-organic frameworks (Co-MOFs) with a two-dimensional layered morphology have received increasing attention for pervaporation due to their stability and hydrophilic properties. Using amino glycine (Gly) as a cross-linking agent, the Co-MOF ultrathin two-dimensional membrane doped with organic filler sodium alginate (SA) with the "brick-mixed-sand" structure was proposed. Polyacrylonitrile (PAN) was selected as the support layer of the hybrid membrane. The introduction of Gly efficiently solved the nanomaterial stacking problem and controllably adjusted the interlayer spacing between the nanosheets, which demonstrated good performance for ethanol dehydration. The results of this experimental research showed that the total flux of alcohol/water (9:1) separation by Gly-Co-MOF-SA/PAN hybrid membranes reached 1902 g m-2 h-1, which was 67% higher than that of the pure SA membranes. The "brick-mixed-sand" lamellar dense morphology of Gly-Co-MOF not only enhances membrane hydrophilicity but also provides effective channels for the rapid transport of water, which is expected to be used for the dehydration of organic solvents.

A rotational spectroscopy study of microsolvation effects on intramolecular proton transfer in trifluoroacetylacetone-(H2O)1-3.

Trifluoroacetylacetone (TFAA) has two enol forms, which can switch to each other via proton transfer. While much attention has been paid to their conformational preferences, the influence of microsolvation on regulating the proton position remains unexplored. Herein, we report the rotational spectra of trifluoroacetylacetone-(water)n (n = 1-3) investigated by chirped pulse Fourier transform microwave spectroscopy in the 2-8 GHz frequency range. Two conformers were identified for both TFAA-H2O and TFAA-(H2O)2, while only one conformer was characterized for TFAA-(H2O)3. The results indicate that water binding on the CH3 side stabilizes the enolF form, whereas water binding on the CF3 side stabilizes the enolH form. The enolF form predominates over the enolH form in these hydrated complexes, which contrasts with the fact that only enolH exists in isolated TFAA. EnolH becomes preferred only when water inserts itself into the intramolecular hydrogen bond. Instanton theory calculations reveal that the proton transfer reaction is dominated by quantum tunneling at low temperatures, leading to the stable existence of only one enol form in each configuration of the hydrated clusters.

Tunable Construction of Chiral Nematic Cellulose Nanocrystals/ZnO Films for Ultra-Sensitive, Recyclable Sensing of Humidity and Ethanol.

The investigation of functional materials derived from sustainable and eco-friendly bioresources has generated significant attention. Herein, nanocomposite films based on chiral nematic cellulose crystals (CNCs) were developed by incorporating xylose and biocompatible ZnO nanoparticles (NPs) via evaporation-induced self-assembly (EISA). The nanocomposite films exhibited iridescent color changes that corresponded to the birefringence phenomenon under polarized light, which was attributed to the formation of cholesteric structures. ZnO nanoparticles were proved to successfully adjust the helical pitches of the chiral arrangements of the CNCs, resulting in tunable optical light with shifted wavelength bands. Furthermore, the nanocomposite films showed fast humidity and ethanol stimuli response properties, exhibiting the potential of stimuli sensors of the CNC-based sustainable materials.

Evolution of Solute-Water Interactions in the Benzaldehyde-(H2O)1-6 Clusters by Rotational Spectroscopy.

The investigation on the preferred arrangement and intermolecular interactions of gas phase solute-water clusters gives insights into the intermolecular potentials that govern the structure and dynamics of the aqueous solutions. Here, we report the investigation of hydrated coordination networks of benzaldehyde-(water)n (n = 1-6) clusters in a pulsed supersonic expansion using broadband rotational spectroscopy. Benzaldehyde (PhCHO) is the simplest aromatic aldehyde that involves both hydrophilic (CHO) and hydrophobic (phenyl ring) functional groups, which can mimic molecules of biological significance. For the n = 1-3 clusters, the water molecules are connected around the hydrophilic CHO moiety of benzaldehyde through a strong CO···HO hydrogen bond and weak CH···OH hydrogen bond(s). For the larger clusters, the spectra are consistent with the structures in which the water clusters are coordinated on the surface of PhCHO with both the hydrophilic CHO and hydrophobic phenyl ring groups being involved in the bonding interactions. The presence of benzaldehyde does not strongly interfere with the cyclic water tetramer and pentamer, which retain the same structure as in the pure water cluster. The book isomer instead of cage or prism isomers of the water hexamer is incorporated into the microsolvated cluster. The PhCHO molecule deviates from the planar structure upon sequential addition of water molecules. The PhCHO-(H2O)1-6 clusters may serve as a simple model system in understanding the solute-water interactions of biologically relevant molecules in an aqueous environment.

Denoising brain networks using a fixed mathematical phase change in independent component analysis of magnitude-only fMRI data.

Brain networks extracted by independent component analysis (ICA) from magnitude-only fMRI data are usually denoised using various amplitude-based thresholds. By contrast, spatial source phase (SSP) or the phase information of ICA brain networks extracted from complex-valued fMRI data, has provided a simple yet effective way to perform the denoising using a fixed phase change. In this work, we extend the approach to magnitude-only fMRI data to avoid testing various amplitude thresholds for denoising magnitude maps extracted by ICA, as most studies do not save the complex-valued data. The main idea is to generate a mathematical SSP map for a magnitude map using a mapping framework, and the mapping framework is built using complex-valued fMRI data with a known SSP map. Here we leverage the fact that the phase map derived from phase fMRI data has similar phase information to the SSP map. After verifying the use of the magnitude data of complex-valued fMRI, this framework is generalized to work with magnitude-only data, allowing use of our approach even without the availability of the corresponding phase fMRI datasets. We test the proposed method using both simulated and experimental fMRI data including complex-valued data from University of New Mexico and magnitude-only data from Human Connectome Project. The results provide evidence that the mathematical SSP denoising with a fixed phase change is effective for denoising spatial maps from magnitude-only fMRI data in terms of retaining more BOLD-related activity and fewer unwanted voxels, compared with amplitude-based thresholding. The proposed method provides a unified and efficient SSP approach to denoise ICA brain networks in fMRI data.

Multi-omics study identifies that PICK1 deficiency causes male infertility by inhibiting vesicle trafficking in Sertoli cells.

BACKGROUND: Infertility affects approximately 10-15% of reproductive-age men worldwide, and genetic causes play a role in one-third of cases. As a Bin-Amphiphysin-Rvs (BAR) domain protein, protein interacting with C-kinase 1 (PICK1) deficiency could lead to impairment of acrosome maturation. However, its effects on auxiliary germ cells such as Sertoli cells are unknown. PURPOSE: The present work was aimed to use multi-omics analysis to research the effects of PICK1 deficiency on Sertoli cells and to identify effective biomarkers to distinguish fertile males from infertile males caused by PICK1 deficiency. METHODS: Whole-exome sequencing (WES) was performed on 20 infertility patients with oligozoospermia to identify pathogenic PICK1 mutations. Multi-omics analysis of a PICK1 knockout (KO) mouse model was utilized to identify pathogenic mechanism. Animal and cell function experiments of Sertoli cell-specific PICK1 KO mouse were performed to verify the functional impairment of Sertoli cells. RESULTS: Two loss-of-function deletion mutations c.358delA and c.364delA in PICK1 resulting in transcription loss of BAR functional domain were identified in infertility patients with a specific decrease in serum inhibin B, indicating functional impairment of Sertoli cells. Multi-omics analysis of PICK1 KO mouse illustrated that targeted genes of differentially expressed microRNAs and mRNAs are significantly enriched in the negative regulatory role in the vesicle trafficking pathway, while metabolomics analysis showed that the metabolism of amino acids, lipids, cofactors, vitamins, and endocrine factors changed. The phenotype of PICK1 KO mouse showed a reduction in testis volume, a decreased number of mature spermatozoa and impaired secretory function of Sertoli cells. In vitro experiments confirmed that the expression of growth factors secreted by Sertoli cells in PICK1 conditional KO mouse such as Bone morphogenetic protein 4 (BMP4) and Fibroblast growth factor 2 (FGF2) were decreased. CONCLUSIONS: Our study attributed male infertility caused by PICK1 deficiency to impaired vesicle-related secretory function of Sertoli cells and identified a variety of significant candidate biomarkers for male infertility induced by PICK1 deficiency.

Rapid Construction of Caffeic Acid/p-Phenylenediamine Antifouling Hydrophilic Coating on a PVDF Membrane for Emulsion Separation.

The current methods of constructing modification strategies for hydrophilic membranes are time-consuming, complex in operation, and poor in universality, which limit their application on membranes. In this work, inspired by the adhesion properties and versatility of caffeic acid (CA) and p-phenylenediamine (PPDA), a simple, rapid, and universal method was designed for the separation of oil-in-water emulsion by preparing a stable hydrophilic coating separation membrane. The preparation time of the membrane was shortened to 40 min. The developed PVDF-PCA/PPDA membrane showed superhydrophilic and underwater superoleophobic properties. When applied to petroleum ether-in-water emulsion, isooctane-in-water emulsion, and dodecane-in-water emulsion separation, the oil rejection was more than 99.0%. In the circulating separation of 10 g/L soybean oil-in-water emulsion, the oil rejection was more than 99.3%, and the highest flux was 1036 L·m-2·h-1. The prepared PVDF-PCA/PPDA membrane performed well in the separation test of oily wastewater. The proposed strategy is simple and rapid; it may become a universal method for preparing membranes with super strong antifouling properties against viscous oil and accelerate the research progress of membrane separation of oil-in-water emulsions.

Multifeature analyses of vascular cambial cells reveal longevity mechanisms in old Ginkgo biloba trees.

Aging is a universal property of multicellular organisms. Although some tree species can live for centuries or millennia, the molecular and metabolic mechanisms underlying their longevity are unclear. To address this, we investigated age-related changes in the vascular cambium from 15- to 667-y-old Ginkgo biloba trees. The ring width decreased sharply during the first 100 to 200 y, with only a slight change after 200 y of age, accompanied by decreasing numbers of cambial cell layers. In contrast, average basal area increment (BAI) continuously increased with aging, showing that the lateral meristem can retain indeterminacy in old trees. The indole-3-acetic acid (IAA) concentration in cambial cells decreased with age, whereas the content of abscisic acid (ABA) increased significantly. In addition, cell division-, cell expansion-, and differentiation-related genes exhibited significantly lower expression in old trees, especially miR166 and HD-ZIP III interaction networks involved in cambial activity. Disease resistance-associated genes retained high expression in old trees, along with genes associated with synthesis of preformed protective secondary metabolites. Comprehensive evaluation of the expression of genes related to autophagy, senescence, and age-related miRNAs, together with analysis of leaf photosynthetic efficiencies and seed germination rates, demonstrated that the old trees are still in a healthy, mature state, and senescence is not manifested at the whole-plant level. Taken together, our results reveal that long-lived trees have evolved compensatory mechanisms to maintain a balance between growth and aging processes. This involves continued cambial divisions, high expression of resistance-associated genes, and continued synthetic capacity of preformed protective secondary metabolites.

Interaction of Phytohormones and External Environmental Factors in the Regulation of the Bud Dormancy in Woody Plants.

Bud dormancy and release are essential phenomena that greatly assist in adapting to adverse growing conditions and promoting the holistic growth and development of perennial plants. The dormancy and release process of buds in temperate perennial trees involves complex interactions between physiological and biochemical processes influenced by various environmental factors, representing a meticulously orchestrated life cycle. In this review, we summarize the role of phytohormones and their crosstalk in the establishment and release of bud dormancy. External environmental factors, such as light and temperature, play a crucial role in regulating bud germination. We also highlight the mechanisms of how light and temperature are involved in the regulation of bud dormancy by modulating phytohormones. Moreover, the role of nutrient factors, including sugar, in regulating bud dormancy is also discussed. This review provides a foundation for enhancing our understanding of plant growth and development patterns, fostering agricultural production, and exploring plant adaptive responses to adversity.

Author Correction: Molecular resolution imaging by repetitive optical selective exposure.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Molecular resolution imaging by repetitive optical selective exposure.

We introduce an interferometric single-molecule localization method for super-resolution fluorescence microscopy. Fluorescence molecules are located by the intensities of multiple excitation patterns of an interference fringe, providing around a twofold improvement in the localization precision compared with the conventional imaging with the same photon budget. We demonstrate this technique by resolving nanostructures down to 5 nm in size over a large 25 × 25 µm2 field of view.

Porous PVA-g-SPA/PVA-SA Catalytic Composite Membrane via Lyophilization for Esterification Enhancement.

A catalytic composite membrane was developed for the enhancement of esterification by lyophilization for the first time. The catalytic composite membrane was composed of a poly(vinyl alcohol) (PVA)-sodium alginate (SA) separation layer and a spongy porous catalytic layer cross-linked by PVA and 4-sulfophthalic acid (SPA). Fourier transform infrared (FTIR) spectroscopy and X-ray photoelectron spectroscopy (XPS) results indicated the successful synthesis of the catalytic composite membrane. The membrane properties were evaluated by ethanol dehydration and esterification. The conversion rate of acetic acid reached 95.9% after 12 h. Compared with batch reactions, the conversion rate increased by 24.4%. After five cycles, the membrane still maintained outstanding catalytic activity. The resistance of mass transfer was analyzed, and the results showed that the porous structure reduced the catalytic layer resistance to total resistance from 70.27 to 32.88%. The composite membrane with a spongy porous catalytic layer exhibited superior dehydration and catalytic performance.

Rapid Approach to Synthesizing a Tannic Acid (TA)-3-Aminopropyltrietoxysilane (APTES) Coating for Efficient Oil-Water Emulsion Separation.

Plant polyphenol-inspired surface modification of membranes is helpful for oil-water separation. However, the preparation of this coating is time-consuming. Herein, we introduce a rapid synthesis of the TA-APTES coating by the addition of sodium periodate (SP). The surface chemical composition and morphology of the resultant TA-APTES hybrid coatings were characterized using SEM, ATR-FTIR, and XPS. The hydrophilicity and membrane performance were investigated by the water contact angle, pure water permeability, and oil rejection for an isooctane-in-water emulsion. The experimental findings revealed that the optimal microfiltration (MF) membrane (MF-TA-APTES-SP-0.05) displayed exceptional hydrophilicity and water permeability (9558 L m-2 h-1 bar-1). The membrane realized highly efficient separation with a permeability (4117 L m-2 h-1 bar-1) and rejection of oils (>99%). Furthermore, it possessed outstanding chemical stability and maintained underwater superoleophobicity even after exposure to harsh conditions. This simple and rapid strategy of developing hydrophilic coatings as a modifier for the poly(vinylidene fluoride) membranes has potential applications in oil-water separation and wastewater treatment.

Application of biological variation and sigma metrics to evaluate the performance of HbA1c in external quality assessment.

BACKGROUND: It is crucial to improve the accuracy of HbA1c measurement as its essential role in diabetes diagnosis and treatment. We aimed to establish the biological variation (BV) and sigma metrics (SM) models and apply the models to evaluate the analytical performance of HbA1c in external quality assessment (EQA) program. METHODS: Data of HbA1c EQA (2021) and internal quality control (IQC) (March-August 2021) were collected. The group-specific bias and coefficient of variance (CV) were computed for measuring systems with laboratory number >9 in EQA program. The analytical bias and CV for individual laboratory were estimated from EQA and IQC data. The CV% and bias% were plotted in the BV-SM models for performance evaluation of measuring system and individual laboratory. RESULTS: Totally, 380 laboratories participated in EQA program. The overall inter-laboratory CV of five EQA samples ranged from 3.02% to 3.63%. There were five measuring systems that met the minimum performance for 5/5 samples: Arkary, Primus, Roche, Mindray and Tosoh, but none of them achieved the optimum performance. Half of the 196 laboratories that reported IQC and EQA results simultaneously achieved 3σ and minimum performance limits. Further analysis indicated that 88.8%, and 31.6% of the laboratories met the minimum performance for bias and CV, respectively. CONCLUSIONS: The biological variation and sigma metrics are appropriate quality management models for evaluating the performance of HbA1c in EQA program. The intra-laboratory and inter-laboratory imprecision need to be improved in order to achieve the required analytical goals for diabetes diagnosis.

Improved skill for tracheal intubation using optical stylets through remote training model: a before and after interventional study.

BACKGROUND: Conducting on-site, hands-on training during the Coronavirus disease 2019 (COVID-19) pandemic has been challenging. We conducted a before and after interventional study to estimate the efficacy of a new remote hands-on training model for improving the trainees' tracheal intubation competency using optical stylets. METHODS: Residents or physicians in anesthesiology apartment who have not received the nominated training in tracheal intubation using optical stylets were enrolled. The 4-week training course contains theoretical knowledge along with preclinical and clinical training of optical stylets techniques. Competency of intubation using optical stylets on patients with normal airways was evaluated according to an assessment tool with a maximum score of 29 points based on video recording pre-post training performance. Pre-post questionnaires measured theoretical knowledge and self-efficacy. RESULTS: Twenty-two participants were included (8 females, 14 men, mean age of 33.5 years). The total score of intubation competency was significantly improved after training from 14.6±3.7 to 25.3±2.6 (P < 0.0001). The scores of three subitems (anatomical identification, hand-eye coordination, and optimized intubation condition) were all significantly increased after training (P < 0.0001). The total percentage of correct answers in the multiple-choice questionnaire increased from 58.2%±8.2% before training to 85.2%±7.2% shortly after training (P < 0.0001). In addition, the self-efficacy score was significantly increased from 2.5±1.2 to 4.4±0.6 (P < 0.0001). CONCLUSIONS: The new remote and progressively advanced hands-on training model improved the competency of intubation using optical stylets under the COVID-19 pandemic.

Intravenous versus Volatile Anesthetic Effects on Postoperative Cognition in Elderly Patients Undergoing Laparoscopic Abdominal Surgery.

BACKGROUND: Delayed neurocognitive recovery after surgery is associated with poor outcome. Most surgeries require general anesthesia, of which sevoflurane and propofol are the most commonly used inhalational and intravenous anesthetics. The authors tested the primary hypothesis that patients with laparoscopic abdominal surgery under propofol-based anesthesia have a lower incidence of delayed neurocognitive recovery than patients under sevoflurane-based anesthesia. A second hypothesis is that there were blood biomarkers for predicting delayed neurocognitive recovery to occur. METHODS: A randomized, double-blind, parallel, controlled study was performed at four hospitals in China. Elderly patients (60 yr and older) undergoing laparoscopic abdominal surgery that was likely longer than 2 h were randomized to a propofol- or sevoflurane-based regimen to maintain general anesthesia. A minimum of 221 patients was planned for each group to detect a one-third decrease in delayed neurocognitive recovery incidence in propofol group compared with sevoflurane group. The primary outcome was delayed neurocognitive recovery incidence 5 to 7 days after surgery. RESULTS: A total of 544 patients were enrolled, with 272 patients in each group. Of these patients, 226 in the propofol group and 221 in the sevoflurane group completed the needed neuropsychological tests for diagnosing delayed neurocognitive recovery, and 46 (20.8%) in the sevoflurane group and 38 (16.8%) in the propofol group met the criteria for delayed neurocognitive recovery (odds ratio, 0.77; 95% CI, 0.48 to 1.24; P = 0.279). A high blood interleukin-6 concentration at 1 h after skin incision was associated with an increased likelihood of delayed neurocognitive recovery (odds ratio, 1.04; 95% CI, 1.01 to 1.07; P = 0.007). Adverse event incidences were similar in both groups. CONCLUSIONS: Anesthetic choice between propofol and sevoflurane did not appear to affect the incidence of delayed neurocognitive recovery 5 to 7 days after laparoscopic abdominal surgery. A high blood interleukin-6 concentration after surgical incision may be an independent risk factor for delayed neurocognitive recovery.