Electroacupuncture ameliorates depression-like behaviors in rats with post-stroke depression by inhibiting ferroptosis in the prefrontal cortex.

Introduction: Post-stroke depression (PSD) is the most common complication following a stroke, significantly hindering recovery and rehabilitation in affected patients. Despite its prevalence, the pathogenesis of PSD remains poorly understood. Electroacupuncture (EA) has shown antidepressant effects, yet its neuroprotective properties are not well defined. Ferroptosis, a recently identified form of cell death, is implicated in the pathological processes of stroke and is associated with the development of depression-like behaviors. So we aimed to investigate whether PSD induces ferroptosis, identify potential therapeutic targets within these pathways, and elucidate the underlying mechanisms in this study. Methods: Male Sprague-Dawley rats were subjected to middle carotid artery occlusion and chronic unpredictable mild stress to model PSD. To explore the role of ferroptosis in the effects of EA, the ferroptosis inducer erastin was administered into the rats' lateral ventricles, followed by 14 days of EA treatment, with sessions lasting 30 minutes per day. The Zea-Longa score was used to assess neurological deficits, while the sucrose preference test, elevated plus maze test, and open-field test were employed to evaluate depression-like behaviors in the rats. Hematoxylin-eosin, Nissl, and Perl's staining were used to observe the morphological changes and iron deposition in the prefrontal neurons. Transmission electron microscopy provided detailed observations of mitochondrial morphological changes in neurons. We utilized activity assay kits, enzyme-linked immunosorbent assay (ELISA), and Western blotting to explore potential molecular mechanisms underlying the effects of EA. Results: EA can reduce neurological deficits and enhance the spontaneous activity and exploration behavior of rats. In addition, EA could inhibit prefrontal cortex neuronal ferroptosis by reducing iron deposition, decreasing lipid peroxidation, and enhancing antioxidation. Discussion: EA improved depression-like behaviors, mitigated mitochondrial damage, and inhibited ferroptosis in prefrontal cortex neurons. Notably, the administration of erastin further enhanced these effects. In conclusion, EA appears to improve PSD by inhibiting ferroptosis in the prefrontal cortex.

Effects of Hydrogen Bond Networks on Viscosity in Aqueous Solutions.

In aqueous solutions, the impact of ions on hydrogen bond networks plays a crucial role in transport properties. We used molecular dynamics simulations to explain how ions affect viscosity through structural changes. We developed a quantitative model to describe the effect of ions on viscosity. The model comprises two parts: the addition of ions alters hydrogen bond networks, and changes in hydrogen bond networks exponentially lead to changes in viscosity. The influence of ions on hydrogen bond networks involves the following mechanisms: first, ions can disrupt the tetrahedral structures within the first solvation shell into three-coordinated structures through substitution; second, structural changes within the first shells affect the global hydrogen bond network through electrostatic forces and the hindrance of ionic volumes. By analyzing the mechanisms of how hydrogen bond networks determine viscosity through the decomposition of viscosity, we found that the proportion of potential viscosity in aqueous solutions primarily increases due to the enhancement of non-hydrogen bonding interactions, and the proportion of hydrogen bonding viscosity decreases accordingly. Our results demonstrate that hydrogen bond networks are crucial for describing the changes in transport phenomena affected by external factors.

Exploring the efficacy and safety of acupuncture versus sham acupuncture for diminished ovarian reserve: study protocol for a multicentre randomised controlled trial.

INTRODUCTION: The therapeutic needs of women with diminished ovarian reserve (DOR), coupled with the increasing application of acupuncture in improving ovarian function, have highlighted the need to verify the efficacy and safety of acupuncture for DOR. This study aims to provide high-quality evidence by evaluating both ovarian reserve and in vitro fertilisation (IVF) outcomes. METHODS AND ANALYSIS: A large-scale, multicentre, randomised controlled trial will be carried out across seven hospitals in China. 400 women with DOR will be randomised in a 1:1 ratio to an acupuncture group or a sham acupuncture group. Acupuncture or sham acupuncture will consist of 36 sessions per participant over 12 weeks. The primary outcome will be the change in antral follicle count (AFC) at week 12 from baseline. Secondary outcomes are AFC at week 24, the serum levels of basal follicle-stimulating hormone and anti-Mullerian hormone at weeks 12 and 24, the scores of the Self-Rating Anxiety Scale at weeks 12 and 24, clinical pregnancy rate, and IVF embryo transfer related outcomes. Any adverse events during treatment will be documented. ETHICS AND DISSEMINATION: The study protocol has been approved by all the participating institutions. Written informed consent will be obtained prior to participant enrolment. The results of this study will be published in peer-reviewed publications. TRIAL REGISTRATION NUMBER: ChiCTR2200062295 PROTOCOL VERSION: V2.0-20220317.

Whole-genome landscape of histone H3K4me3 modification during sperm cell lineage development in tomato.

BACKGROUND: During male gametogenesis of flowering plants, sperm cell lineage (microspores, generative cells, and sperm cells) differentiated from somatic cells and acquired different cell fates. Trimethylation of histone H3 on lysine 4 (H3K4me3) epigenetically contributes to this process, however, it remained unclear how H3K4me3 influences the gene expression in each cell type. Here, we conducted chromatin immunoprecipitation sequencing (ChIP-seq) to obtain a genome-wide landscape of H3K4me3 during sperm cell lineage development in tomato (Solanum lycopersicum). RESULTS: We show that H3K4me3 peaks were mainly enriched in the promoter regions, and intergenic H3K4me3 peaks expanded as sperm cell lineage differentiated from somatic cells. H3K4me3 was generally positively associated with transcript abundance and served as a better indicator of gene expression in somatic and vegetative cells, compared to sperm cell lineage. H3K4me3 was mutually exclusive with DNA methylation at 3' proximal of the transcription start sites. The microspore maintained the H3K4me3 features of somatic cells, while generative cells and sperm cells shared an almost identical H3K4me3 pattern which differed from that of the vegetative cell. After microspore division, significant loss of H3K4me3 in genes related to brassinosteroid and cytokinin signaling was observed in generative cells and vegetative cells, respectively. CONCLUSIONS: Our results suggest the asymmetric division of the microspore significantly reshapes the genome-wide distribution of H3K4me3. Selective loss of H3K4me3 in genes related to hormone signaling may contribute to functional differentiation of sperm cell lineage. This work provides new resource data for the epigenetic studies of gametogenesis in plants.

Utility of real-time 3D visualization system in the early stage of phacoemulsification training.

AIM: To determine the teaching effects of a real-time three dimensional (3D) visualization system in the operating room for early-stage phacoemulsification training. METHODS: A total of 10 ophthalmology residents of the first-year postgraduate were included. All the residents were novices to cataract surgery. Real-time cataract surgical observations were performed using a custom-built 3D visualization system. The training lasted 4wk (32h) in all. A modified International Council of Ophthalmology's Ophthalmology Surgical Competency Assessment Rubric (ICO-OSCAR) containing 4 specific steps of cataract surgery was applied. The self-assessment (self) and expert-assessment (expert) were performed through the microsurgical attempts in the wet lab for each participant. RESULTS: Compared with pre-training assessments (self 3.2±0.8, expert 2.5±0.6), the overall mean scores of post-training (self 5.2±0.4, expert 4.7±0.6) were significantly improved after real-time observation training of 3D visualization system (P<0.05). Scores of 4 surgical items were significantly improved both self and expert assessment after training (P<0.05). CONCLUSION: The 3D observation training provides novice ophthalmic residents with a better understanding of intraocular microsurgical techniques. It is a useful tool to improve teaching efficiency of surgical education.

Microcystin-LR Induces Estrogenic Effects at Environmentally Relevant Concentration in Black-Spotted Pond Frogs (Pelophylax nigromaculatus): In Situ, In Vivo, In Vitro, and In Silico Investigations.

Harmful cyanobacterial blooms are frequent and intense worldwide, creating hazards for aquatic biodiversity. The potential estrogen-like effect of Microcystin-LR (MC-LR) is a growing concern. In this study, we assessed the estrogenic potency of MC-LR in black-spotted frogs through combined field and laboratory approaches. In 13 bloom areas of Zhejiang province, China, the MC-LR concentrations in water ranged from 0.87 to 8.77 µg/L and were correlated with sex hormone profiles in frogs, suggesting possible estrogenic activity of MC-LR. Tadpoles exposed to 1 µg/L, an environmentally relevant concentration, displayed a female-biased sex ratio relative to controls. Transcriptomic results revealed that MC-LR induces numerous and complex effects on gene expression across multiple endocrine axes. In addition, exposure of male adults significantly increased the estradiol (E2)/testosterone (T) ratio by 3.5-fold relative to controls. Downregulation of genes related to male reproductive endocrine function was also identified. We also showed how MC-LR enhances the expression of specific estrogen receptor (ER) proteins, which induce estrogenic effects by activating the ER pathway and hypothalamic-pituitary-gonadal (HPG) axis. In aggregate, our results reveal multiple lines of evidence demonstrating that, for amphibians, MC-LR is an estrogenic endocrine disruptor at environmentally relevant concentrations. The data presented here support the need for a shift in the MC-LR risk assessment. While hepatoxicity has historically been the focus of MC-LR risk assessments, our data clearly demonstrate that estrogenicity is a major mode of toxicity at environmental levels and that estrogenic effects should be considered for risk assessments on MC-LR going forward.

Structural effects of water clusters on viscosity at high shear rates.

In this study, we use molecular dynamics simulations of liquid water to investigate how shear thinning affects the viscosity of liquid water by structural changes of the hydrogen bond network. The effect of shear on viscosity can be divided into two parts: shear-induced destruction of the hydrogen bond network and the influence of the water structure on shear viscosity. First, strong shear destroys tetrahedral structures and thus reduces the connectivity of the hydrogen bond network. It is mainly because shear deformation, characterized by compression and expansion axes, respectively, triggers the destruction and formation of hydrogen bonds, resulting in anisotropic effects on water structures. At the same time, shear destroys large clusters and enhances the formation of small ones, resulting in a decrease in average cluster sizes. Second, the change of viscosity obeys a power law relationship with the change of hydrogen bond structures, highlighting a one-to-one correspondence between structure and property. Meanwhile, in order to explain why the structure affects viscosity, we define hydrogen-bond viscosity and find that the cooperative motion of the water structures can promote momentum transfer in the form of aggregations. Hydrogen-bond viscosity accounts for 5%-50% of the total viscosity. Our results elucidate that water structures are the important structural units to explain the change of water properties.

Acupuncture Versus Oral Medications for Acute/Subacute Non-Specific Low Back Pain: A Systematic Review and Meta-Analysis.

PURPOSE OF REVIEW: Pharmacologic intervention do not always achieve benefits in the treatment of acute/subacute non-specific low back pain (NSLBP). We assessed efficacy and safety of acupuncture for acute/subacute NSLBP as alternative treatment. RECENT FINDINGS: We searched PubMed, Web of Science, Embase, Cochrane Library, Scopus, Epistemonikos, CNKI, Wan Fang Database, VIP database, CBMLD, CSTJ, clinical trials, EUCTR, World WHO ICTRP, and ChiCTR for randomized controlled trials, cross-over studies, and cohort studies of NSLBP treated by acupuncture versus oral medication from inception to 23th April 2022. A total of 6 784 records were identified, and 14 studies were included 1 263 participants in this review. The results of the meta-analysis indicated that acupuncture therapy was slightly more effective than oral medication in improving pain (P < 0.00001, I2 = 92%, MD = -1.17, 95% CI [-1.61, -0.72]). According to the results of the meta-analysis, acupuncture therapy exhibited a significant advantage over oral medication with a substantial effect (P < 0.00001, I2 = 90%, SMD = -1.42, 95% CI [-2.22, -0.62]). Based on the results of the meta-analysis, acupuncture therapy was associated with a 12% improvement rate compared to oral medication in patients with acute/subacute NSLBP (P < 0.0001, I2 = 54%, RR = 1.11, 95% CI [1.05, 1.18]). Acupuncture is more effective and safer than oral medication in treating acute/subacute NSLBP. This systematic review is poised to offer valuable guidance to clinicians treating acute/subacute NSLBP and potentially benefit the afflicted patients. REGISTRATION: This review was registered in PROSPERO ( http://www.crd.york.ac.uk/prospero ) with registration number CRD42021278346.

Electroacupuncture Ameliorates Depression-Like Behaviors in Post-Stroke Rats via Activating AMPK-Mediated Mitochondrial Function.

Background: Post-stroke depression (PSD) is one of the most common complications of stroke. Electroacupuncture (EA) is an effective traditional Chinese medicine treatment for PSD, which is widely used in clinical settings. EA has a significant therapeutic effect against PSD, but the mechanism is still unclear. This study aimed to determine whether EA ameliorates depression-like behaviors in PSD rats by regulating the adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK)-mediated mitochondrial function. Methods: Middle cerebral artery occlusion (MCAO) and chronic unpredictable mild stress (CUMS) were used to develop a PSD rat model. To elucidate the role of AMPK in EA treatment, compound C, a selective inhibitor of AMPK, was injected into the lateral ventricle of rats before EA treatment. EA treatment was performed for 14 consecutive days for 30 min per day after PSD modeling. A modified Zea-Longa five-point scale scoring system was used to determine neurologic function in MCAO rats. Behavioral tests were conducted to evaluate depression-like phenotypes in rats. Depression-like behaviors were tested by sucrose preference test (SPT), novelty suppressed feeding test (NSFT), and open-field test (OFT). The structure and morphology of the prefrontal cortex were observed by histopathological hematoxylin-eosin (HE) and Nissl staining. The mitochondrial morphology and function were analyzed by colorimetry, chemiluminescence, Western blotting, and quantitative real-time polymerase chain reaction (qRT-PCR). Results: EA treatment successfully ameliorated depression-like behaviors, upregulated AMPK expression, and improved mitochondrial function. However, AMPK inhibition by Compound C exacerbated depression-like behaviors and aggravated neuronal and mitochondrial injury in PSD rats. Conclusion: EA treatment improved depression-like behaviors in PSD rats and promoted mitochondrial function by activating AMPK.

Aggregation-caused dual-signal response of gold nanoclusters for ratiometric optical detection of cysteine.

Designing ratiometric sensors for cysteine (Cys) monitoring with high accuracy is of great significance for disease diagnosis and biomedical studies. The current ratiometric methods mainly rely on multiplex probes, which not only complicates the operation but also increases the cost, making it difficult for quantitative Cys detection in resource-limited areas. Herein, one-pot prepared gold nanoclusters (Au NCs) that glow red fluorescent were synthesized by employing glutathione as the stabilizer and reducing agent. When Fe3+ is present with Au NCs, the fluorescence is quenched and the scattering is strong because of the aggregation of Au NCs. With introduction of Cys, Cys can efficiently compete with glutathione-modified Au NCs for Fe3+, which leads to increase of fluorescence and decrease of scattering. The ratiometric determination of Cys can be thereby realized by collecting the fluorescence and SRS spectrum simultaneously. The linear range for Cys was 5-30 µM with a detection limit of 1.5 µM. In addition, the sensing system exhibits good selectivity for Cys and shows potential application in biological samples.

miR-342-5p promotes vascular smooth muscle cell phenotypic transition through a negative-feedback regulation of Notch signaling via targeting FOXO3.

AIM: Under various pathological conditions such as cancer, vascular smooth muscle cells (vSMCs) transit their contractile phenotype into phenotype(s) characterized by proliferation and secretion, a process called vSMC phenotypic transition (vSMC-PT). Notch signaling regulates vSMC development and vSMC-PT. This study aims to elucidate how the Notch signal is regulated. MAIN METHODS: Gene-modified mice with a SM22α-CreERT2 transgene were generated to activate/block Notch signaling in vSMCs. Primary vSMCs and MOVAS cells were cultured in vitro. RNA-seq, qRT-PCR and Western blotting were used to evaluated gene expression level. EdU incorporation, Transwell and collagen gel contraction assays were conducted to determine the proliferation, migration and contraction, respectively. KEY FINDINGS: Notch activation upregulated, while Notch blockade downregulated, miR-342-5p and its host gene Evl in vSMCs. However, miR-342-5p overexpression promoted vSMC-PT as shown by altered gene expression profile, increased migration and proliferation, and decreased contraction, while miR-342-5p blockade exhibited the opposite effects. Moreover, miR-342-5p overexpression significantly suppressed Notch signaling, and Notch activation partially abolished miR-342-5p-induced vSMC-PT. Mechanically, miR-342-5p directly targeted FOXO3, and FOXO3 overexpression rescued miR-342-5p-induced Notch repression and vSMC-PT. In a simulated tumor microenvironment, miR-342-5p was upregulated by tumor cell-derived conditional medium (TCM), and miR-342-5p blockade abrogated TCM-induced vSMC-PT. Meanwhile, conditional medium from miR-342-5p-overexpressing vSMCs significantly enhanced tumor cell proliferation, while miR-342-5p blockade had the opposite effects. Consistently, in a co-inoculation tumor model, miR-342-5p blockade in vSMCs significantly delayed tumor growth. SIGNIFICANCE: miR-342-5p promotes vSMC-PT through a negative-feedback regulation of Notch signaling via downregulating FOXO3, which could be a potential target for cancer therapy.

N-Trimethylated chitosan coating white adipose tissue vascular-targeting oral nano-system for the enhanced anti-obesity effects of celastrol.

Oral nanoparticles (NPs) are more suitable for obesity control compared to NPs administered intravenously, as their convenience increases patient compliance. Herein, we developed an oral nano-system to improve the anti-obesity efficacy of celastrol (Cel). The observed enhanced efficacy was mediated by zein core NPs decorated with adipose-homing peptides that were coated with N-trimethylated chitosan. The optimized Cel/AHP-NPs@TMC exhibited spherical morphology by TEM, as well as narrow size distribution (221.76 ± 6.73 nm) and adequate stability in a gastrointestinal environment. Based on the combined delivery advantages of AHP-NPs@TMC - i.e., improved cellular internalization within Caco-2 cells and enhanced white adipose tissue (WAT) vascular targeting - Cel/AHP-NPs@TMC significantly reduced the body weight, blood lipid levels, adipose inflammation, and WAT distribution in diet-induced obese mice without side-effects. In short, this study provides clear evidence that TMC-based oral NPs can effectively improve celastrol for obesity treatment.

Abnormal dynamics of brain functional networks in children with Tourette syndrome.

Tourette syndrome (TS) is a childhood-onset neurodevelopmental disorder characterized by the presence of multiple motor and vocal tics. Research using resting-state functional magnetic resonance imaging (rfMRI) have found aberrant static functional connectivity (FC) and its topological properties in the brain networks of TS. Our study is the first to investigate the dynamic functional connectivity (dFC) in the whole brain network of TS patients, focusing on the temporal properties of dFC states and the temporal variability of topological organization. The rfMRI data of 36 male children with TS and 27 matched healthy controls were collected and further analyzed by group spatial independent component analysis, sliding windows approach based dFC analysis, k-means clustering analysis, and graph theory analysis. The clustering analysis identified three dFC states. Of these states, state 2, characterized by increased inter-network connections in subcortical network (SCN), sensorimotor network (SMN), and default mode network (DMN), and decreased inter-network connections between salience network (SAN) and executive control network (ECN), was found to have higher fractional window and dwell time in TS, which was also positively correlated with tic severity. TS patients also exhibited higher temporal variability of whole-brain-network global efficiency and local efficiency, and higher temporal variability of nodal efficiency and local efficiency in SCN, DMN, ECN, SAN, and SMN. Additionally, temporal variability of the efficiency and local efficiency in insula was positively correlated with tic severity. Our findings revealed abnormal temporal property of dFC states and temporal variability of topological organization in TS, providing new insights into clinical diagnoses and neuropathology of TS.

Genome-wide identification glutathione-S-transferase gene superfamily in Daphnia pulex and its transcriptional response to nanoplastics.

Glutathione S-transferases (GSTs) are key multifunctional phase II detoxification enzymes involved in the regulation of growth, development, and stress responses. However, the knowledge of GSTs in the model invertebrate organism Daphnia pulex at the genomic level remains limited. In the present study, 35 GST genes were identified in D. pulex (Dp-GST), belonging to eight subfamilies, with the sigma, mu, and delta/epsilon subfamilies constituting approximately 29 %, 20 %, and 20 % of the GST superfamily, respectively. Chromosome tandem duplication of genes within the same subfamily was observed, which may be the main force driving GST expansion in D. pulex. DpGST genes showed different expression patterns in response to nanoplastic exposure for 96 h and 21 days. Some homologous GST genes in D. pulex showed similar expression patterns in response to nanoplastic exposure, likely owing to their unique motifs. For example, motif 9 is found in all delta/epsilon GST genes, whereas motifs 1, 2, 3, 5, and 7 are highly conserved in sigma GST genes. The characterization of D. pulex GSTs extending the knowledge of GST-mediated environmental contaminants, especially nanoplastics.

Perfluorooctanoic acid and perfluorooctanesulfonic acid induce immunotoxicity through the NF-κB pathway in black-spotted frog (Rana nigromaculata).

Perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS) are widely detected in the environment and wild animals, thus posing a threat to wildlife and public health; however, knowledge about their immunotoxicity and the underlying mechanism remains limited. In the present study, male black-spotted frogs (Rana nigromaculata) were exposed to environmentally relevant concentrations (0, 1, and 10 µg/L) of PFOA or PFOS for 21 days; subsequently, biochemical analysis, molecular docking, and gene expression determination were conducted. The results indicated that exposure to 10 µg/L PFOA decreased the serum levels of immunoglobulin A. PFOS exposure significantly increased the hepatic levels of interleukin-1ß, interleukin-6, tumor necrosis factor-α, interferon-γ, and nitric oxide; but PFOA significantly increased the levels of only tumor necrosis factor-α. Furthermore, PFOA and PFOS exposure significantly decreased the activity of inducible nitric oxide synthase and total nitric oxide synthase. IBRv2 analysis indicated that PFOA and PFOS had a similar effect on these immune indicators, but PFOS was more toxic than PFOA. Molecular docking revealed that PFOA and PFOS can bind to nuclear factor-κB (NF-κB) by forming stable hydrogen bonds. PFOA and PFOS exposure upregulated the gene expression of NF-κB and its downstream genes. Significant correlations between the expression of genes involved in the NF-κB pathway and immune-related indicators suggests that PFOA- and PFOS-induced immunotoxicity was associated with the activation of NF-κB. Our findings provide novel insights into the potential role of NF-κB in immunotoxicity induced by PFOA and PFOS in frogs.

Electrocatalytic oxidation of 5-hydroxymethylfurfural to 2,5-furandicarboxylic acid via metal-organic framework-structured hierarchical Co3O4 nanoplate arrays.

Electrocatalytic oxidation of 5-hydroxymethylfurfural (HMF) to 2,5-furandicarboxylic acid (FDCA) was examined as an alternative to thermocatalytic methods in which two-dimensional (2D) cobalt-metal-organic framework (Co-MOF, ZIF-L-Co) nanoplate arrays were prepared on nickel foam (NF) and then transformed into hierarchical porous Co3O4 nanostructures by chemical etching and low temperature annealing to form electrode materials. Hierarchical porous nanoarrays formed during synthesis enlarged the surface area of the as-prepared catalysts introduced a large number of defects and exposed active sites leading to reduced charge diffusion, improved mass transfer and efficient HMF oxidation. Co3O4/NF electrode materials were able to achieve a current density of 10 mA·cm-2 at an overpotential of 105 mV in 1 M KOH with 10 mM HMF, which was reduced by 175 mV compared with water oxidation. Electrocatalytic oxidation experiments afforded 100 % HMF conversion and 96.7 % FDCA yields with a minimum 96.5 % faradaic efficiency at 1.43 V vs RHE. The proposed MOF-structured synthesis method fundamentally reduces charge diffusion, improves mass transfer of electrodes and is generally applicable to fabrication of hierarchical porous nanostructured materials.

The sustainability of urbanized land: Impacts of the growth of urbanized land in prefecture-level cities in China.

The sustainability of land conversion to urban use is an important factor that promotes sustainable urbanization. However, China's local governments have relied too much on the land finance model for urbanization, which has led to disordered growth of urban land, ultimately posing a threat to ecosystem security and socioeconomic development. Effectively evaluating the factors that influence the growth of urban land is critical to improving the sustainability of urban land use. In this study, we explored the factors that affect the growth in the area of urban land using spatial analysis models based on panel data for Chinese prefecture-level cities from 2007 to 2019. We found that fiscal decentralization, infrastructure, industrialization, and industrial upgrading were the main factors responsible for the growth of urban land, but that their degrees of influence differed between regions. Our findings identify the necessity for local governments to design urban land-use policies and perform strategic planning for urban land use to improve sustainability based on the main factors that influenced the growth of urban land in each region.

LsARF3 mediates thermally induced bolting through promoting the expression of LsCO in lettuce (Lactuca sativa L.).

Lettuce (Lactuca sativa L.) is a leafy vegetable whose edible organs usually are leaf or stems, and thus high-temperature induced bolting followed by flower initiation is an undesirable trait in lettuce production. However, the molecular mechanism that controls lettuce bolting and flowering upon thermal treatments is largely unknown. Here, we identified a Lettuce auxin response factor 3 (LsARF3), the expression of which was enhanced by heat and auxin treatments. Interestingly, LsARF3 is preferentially expressed in stem apex, suggesting it might be associated with lettuce bolting. Transgenic lettuce overexpressing LsARF3 displayed early bolting and flowering, whereas knockout of LsARF3 dramatically delayed bolting and flowering in lettuce under normal or high temperature conditions. Furthermore, Exogenous application of IAA failed to rescue the late-bolting and -flowering phenotype of lsarf3 mutants. Several floral integrator genes including LsCO, LsFT, and LsLFY were co-expressed with LsARF3 in the overexpression and knockout lettuce plants. Yeast one-hybrid (Y1H) experiments suggested that LsARF3 could physically interact with the LsCO promoter, which was further confirmed by a dual luciferase assay in tobacco leaves. The results indicated that LsARF3 might directly modulate the expression of LsCO in lettuce. Therefore, these results demonstrate that LsARF3 could promote lettuce bolting in response to the high temperature by directly or indirectly activating the expression of floral genes such as LsCO, which provides new insights into lettuce bolting in the context of ARFs signaling and heat response.

An Efficient Clearing Protocol for the Study of Seed Development in Tomato (Solanum lycopersicum L.).

Tomato (Solanum lycopersicum L.) is one of the major cash crops worldwide. The tomato seed is an important model for studying genetics and developmental biology during plant reproduction. Visualization of finer embryonic structure within a tomato seed is often hampered by seed coat mucilage, multi-cell-layered integument, and a thick-walled endosperm, which needs to be resolved by laborious embedding-sectioning. A simpler alternative is to employ tissue clearing techniques that turn the seed almost transparent using chemical agents. Although conventional clearing procedures allow deep insight into smaller seeds with a thinner seed coat, clearing tomato seeds continues to be technically challenging, especially in the late developmental stages. Presented here is a rapid and labor-saving clearing protocol to observe tomato seed development from 3 to 23 days after flowering when embryonic morphology is nearly complete. This method combines chloral hydrate-based clearing solution widely used in Arabidopsis with other modifications, including the omission of Formalin-Aceto-Alcohol (FAA) fixation, the addition of sodium hypochlorite treatment of seeds, removal of the softened seed coat mucilage, and washing and vacuum treatment. This method can be applied for efficient clearing of tomato seeds at different developmental stages and is useful in full monitoring of the developmental process of mutant seeds with good spatial resolution. This clearing protocol may also be applied to deep imaging of other commercially important species in the Solanaceae.

Water clusters and density fluctuations in liquid water based on extended hierarchical clustering methods.

The microscopic structures of liquid water at ambient temperatures remain a hot debate, which relates with structural and density fluctuations in the hydrogen bond network. Here, we use molecular dynamics simulations of liquid water to study the properties of three-dimensional cage-like water clusters, which we investigate using extended graph-based hierarchical clustering methods. The water clusters can cover over 95% of hydrogen bond network, among which some clusters maximally encompass thousands of molecules extending beyond 3.0 nm. The clusters imply fractal behaviors forming percolating networks and the morphologies of small and large clusters show different scaling rules. The local favored clusters and the preferred connections between adjacent clusters correspond to lower energy and conformational entropy depending on cluster topologies. Temperature can destroy large clusters into small ones. We show further that the interior of clusters favors high-density patches. The water molecules in the small clusters, inside which are the void regarded as hydrophobic objects, have a preference for being more tetrahedral. Our results highlight the properties and changes of water clusters as the fundamental building blocks of hydrogen bond networks. In addition, the water clusters can elucidate structural and density fluctuations on different length scales in liquid water.