Multiview confocal super-resolution microscopy.

Confocal microscopy1 remains a major workhorse in biomedical optical microscopy owing to its reliability and flexibility in imaging various samples, but suffers from substantial point spread function anisotropy, diffraction-limited resolution, depth-dependent degradation in scattering samples and volumetric bleaching2. Here we address these problems, enhancing confocal microscopy performance from the sub-micrometre to millimetre spatial scale and the millisecond to hour temporal scale, improving both lateral and axial resolution more than twofold while simultaneously reducing phototoxicity. We achieve these gains using an integrated, four-pronged approach: (1) developing compact line scanners that enable sensitive, rapid, diffraction-limited imaging over large areas; (2) combining line-scanning with multiview imaging, developing reconstruction algorithms that improve resolution isotropy and recover signal otherwise lost to scattering; (3) adapting techniques from structured illumination microscopy, achieving super-resolution imaging in densely labelled, thick samples; (4) synergizing deep learning with these advances, further improving imaging speed, resolution and duration. We demonstrate these capabilities on more than 20 distinct fixed and live samples, including protein distributions in single cells; nuclei and developing neurons in Caenorhabditis elegans embryos, larvae and adults; myoblasts in imaginal disks of Drosophila wings; and mouse renal, oesophageal, cardiac and brain tissues.

Bio-friendly long-term subcellular dynamic recording by self-supervised image enhancement microscopy.

Fluorescence microscopy has become an indispensable tool for revealing the dynamic regulation of cells and organelles. However, stochastic noise inherently restricts optical interrogation quality and exacerbates observation fidelity when balancing the joint demands of high frame rate, long-term recording and low phototoxicity. Here we propose DeepSeMi, a self-supervised-learning-based denoising framework capable of increasing signal-to-noise ratio by over 12 dB across various conditions. With the introduction of newly designed eccentric blind-spot convolution filters, DeepSeMi effectively denoises images with no loss of spatiotemporal resolution. In combination with confocal microscopy, DeepSeMi allows for recording organelle interactions in four colors at high frame rates across tens of thousands of frames, monitoring migrasomes and retractosomes over a half day, and imaging ultra-phototoxicity-sensitive Dictyostelium cells over thousands of frames. Through comprehensive validations across various samples and instruments, we prove DeepSeMi to be a versatile and biocompatible tool for breaking the shot-noise limit.

Rapid detection of neurons in widefield calcium imaging datasets after training with synthetic data.

Widefield microscopy can provide optical access to multi-millimeter fields of view and thousands of neurons in mammalian brains at video rate. However, tissue scattering and background contamination results in signal deterioration, making the extraction of neuronal activity challenging, laborious and time consuming. Here we present our deep-learning-based widefield neuron finder (DeepWonder), which is trained by simulated functional recordings and effectively works on experimental data to achieve high-fidelity neuronal extraction. Equipped with systematic background contribution priors, DeepWonder conducts neuronal inference with an order-of-magnitude-faster speed and improved accuracy compared with alternative approaches. DeepWonder removes background contaminations and is computationally efficient. Specifically, DeepWonder accomplishes 50-fold signal-to-background ratio enhancement when processing terabytes-scale cortex-wide functional recordings, with over 14,000 neurons extracted in 17 h.

Incorporating the image formation process into deep learning improves network performance.

We present Richardson-Lucy network (RLN), a fast and lightweight deep learning method for three-dimensional fluorescence microscopy deconvolution. RLN combines the traditional Richardson-Lucy iteration with a fully convolutional network structure, establishing a connection to the image formation process and thereby improving network performance. Containing only roughly 16,000 parameters, RLN enables four- to 50-fold faster processing than purely data-driven networks with many more parameters. By visual and quantitative analysis, we show that RLN provides better deconvolution, better generalizability and fewer artifacts than other networks, especially along the axial dimension. RLN outperforms classic Richardson-Lucy deconvolution on volumes contaminated with severe out of focus fluorescence or noise and provides four- to sixfold faster reconstructions of large, cleared-tissue datasets than classic multi-view pipelines. We demonstrate RLN's performance on cells, tissues and embryos imaged with widefield-, light-sheet-, confocal- and super-resolution microscopy.

Unilateral biportal endoscopic decompression versus anterior cervical decompression and fusion for unilateral cervical radiculopathy or coexisting cervical myelopathy: a prospective, randomized, controlled, noninferiority trial.

BACKGROUND: Cervical spondylosis (CS), including myelopathy and radiculopathy, is the most common degenerative cervical spine disease. This study aims to evaluate the clinical outcomes of unilateral biportal endoscopy (UBE) compared to those of conventional anterior cervical decompression and fusion (ACDF) for treating unilateral cervical radiculopathy or coexisting cervical myelopathy induced by unilateral cervical herniated discs. METHODS: A prospective, randomized, controlled, noninferiority trial was conducted. The sample consisted of 131 patients who underwent UBE or ACDF was conducted between September 2021 and September 2022. Patients with cervical nerve roots or coexisting spinal cord compression symptoms and imaging-defined unilateral cervical radiculopathy or coexisting cervical myelopathy induced by unilateral cervical herniated discs were randomized into two groups: a UBE group (n = 63) and an ACDF group (n = 68). The operative time, blood loss, length of hospital stay after surgery, and perioperative complications were recorded. Preoperative and postoperative modified Japanese Orthopaedic Association (mJOA) scale scores, visual analog scale (VAS) scores, neck disability index (NDI) scores, and recovery rate (RR) of the mJOA were utilized to evaluate clinical outcomes. RESULTS: The hospital stay after surgery was significantly shorter in patients treated with UBE than in those treated with ACDF (p < 0.05). There were no significant differences in the neck or arm VAS score, NDI score, mJOA score, or mean RR of the mJOA between the two groups (p < 0.05). Only mild complications were observed in both groups, with no significant difference (p = 0.30). CONCLUSION: UBE can significantly relieve pain and disability without severe complications, and most patients are satisfied with this technique. Consequently, this procedure can be used safely and effectively as an alternative to ACDF for treating unilateral cervical radiculopathy or coexisting cervical myelopathy induced by unilateral cervical herniated discs. TRIAL REGISTRATION: This study was registered in the Chinese Clinical Trial Registry on 02/08/2023 ( http://www.chictr.org.cn , #ChiCTR2300074273).

WRKY15 Suppresses Tracheary Element Differentiation Upstream of VND7 During Xylem Formation.

Formation of the vascular cylinder, a structure critical to water and nutrient transport in higher plants, is highly regulated. Here we identify WRKY15 as an important regulator that suppresses tracheary element (TE) differentiation in Arabidopsis (Arabidopsis thaliana). Overexpression of WRKY15 resulted in discontinuous protoxylem vessel files and TEs with reduced spiral wall thickening/lignification. Expression of a dominant-negative WRKY15 variant, WRKY15-EAR, led to extra protoxylem vessels and ectopic TEs with increased spiral wall thickening/lignification. Ectopic TE formation in the root cortex and hypocotyl/leaf epidermis reveals that the suppression of WRKY15 is sufficient to trigger the transdifferentiation of other types of cells to TEs. Expression profiling, RT-qPCR, and reporter analyses revealed that WRKY15 suppresses the expression of VASCULAR-RELATED NAC DOMAIN7 (VND7), a master transcriptional regulator that promotes TE differentiation. We propose that WRKY15 negatively regulates VND7 expression indirectly based on (1) the absence of a W-box in the promoter of VND7 and (2) the observation that WRKY15 and VND7 are expressed in different cells in the vascular cylinder, with WRKY15 expressed in the procambial cells and VND7 in the protoxylem poles of procambium and differentiating TEs. Future research is needed to reveal the details underlying the interaction of WRKY15 and VND7 in plant vascular development.

Optimal insertion positions of anterior-posterior orientation sacroiliac screw.

PURPOSE: To explore the optimal insertion positions of anterior-posterior orientation sacroiliac screw (AP-SIS). METHODS: Pelvic CT data of 80 healthy adults were employed to measure the anatomical parameters including the insertable ranges of S1 and S2, the length, width and height of the channel with three different horizontal and vertical anterior insertion points starting from the ilium-acetabular recess. To compare pelvic stability by replicating a type C Tile lesions, fifteen synthetic pelvises were fixed with an anterior plate and a posterior AP-SIS employing different anterior insertion points, the whole specimen displacements and shifts in the sacroiliac gap under a cyclic vertical load of 300 N in a biomechanical machine recorded. RESULTS: The posterior and anterior insertable ranges averaged 17.9 × 8.5mm2 and 47.1 × 21.2 mm2, respectively. The channel lengths for three horizontal anterior insertion points gradually decreased from front to back with significant difference (p < 0.05), whereas the width and height for three horizontal anterior insertion points and the parameters for the three vertical anterior insertion points were similar (p > 0.05). The displacements and shifts for three horizontal insertion points gradually increased from front to back (p < 0.05) whereas the measurements involving the three vertical insertion points were similar (p > 0.05). CONCLUSION: The posterior insertable range is small, where the center between adjacent nerve roots (foramens) is the optimal posterior insertion point. The anterior insertable range is large, where the iliac-acetabular recess is the optimal anterior insertion point for S1 and S2, providing the longest channel and best stability.

Phospholipase D and phosphatidic acid mediate regulation in the biosynthesis of spermidine and ganoderic acids by activating GlMyb in Ganoderma lucidum under heat stress.

Polyamines are essential for all kinds of organisms and take part in the regulation of multiple biological processes. Our previous study revealed that heat stress promoted the conversion of putrescine to spermidine and subsequently promoted the accumulation of ganoderic acids (GAs). However, how heat stress affects polyamine homeostasis remains unclear. To explore the underlying mechanism by which heat stress promoted spermidine biosynthesis, we assessed the effects of signalling molecules that respond to heat stress on spermidine biosynthesis. Our data suggested that heat stress-induced spermidine biosynthesis and GAs accumulation via a phospholipase D (PLD)-mediated phosphatidic acid (PA) signal. Further research revealed that the transcription factor GlMyb promoted spermidine biosynthesis via regulating spermidine synthase genes (spds1 and spds2) expression by directly bonding to their promoters and it responded to heat stress and PA signal. In summary, heat stress activated GlMyb by PLD-mediated PA signalling and subsequently induced the expression of spds1 and spds2 to promote the biosynthesis of spermidine and the accumulation of GAs. Our findings firstly reveal a detailed mechanism by which heat signalling regulates polyamine homeostasis by PLD-mediated PA signal in fungi and provide a greater understanding of how organisms alter polyamine levels in response to environmental changes.

Spermidine Regulates Mitochondrial Function by Enhancing eIF5A Hypusination and Contributes to Reactive Oxygen Species Production and Ganoderic Acid Biosynthesis in Ganoderma lucidum.

Spermidine, a kind of polycation and one important member of the polyamine family, is essential for survival in many kinds of organisms and participates in the regulation of cell growth and metabolism. To explore the mechanism by which spermidine regulates ganoderic acid (GA) biosynthesis in Ganoderma lucidum, the effects of spermidine on GA and reactive oxygen species (ROS) contents were examined. Our data suggested that spermidine promoted the production of mitochondrial ROS and positively regulated GA biosynthesis. Further research revealed that spermidine promoted the translation of mitochondrial complexes I and II and subsequently influenced their activity. With a reduction in eukaryotic translation initiation factor 5A (eIF5A) hypusination by over 50% in spermidine synthase gene (spds) knockdown strains, the activities of mitochondrial complexes I and II were reduced by nearly 60% and 80%, respectively, and the protein contents were reduced by over 50%, suggesting that the effect of spermidine on mitochondrial complexes I and II was mediated through its influence on eIF5A hypusination. Furthermore, after knocking down eIF5A, the deoxyhypusine synthase gene (dhs), and the deoxyhypusine hydroxylase gene (dohh), the mitochondrial ROS level was reduced by nearly 50%, and the GA content was reduced by over 40%, suggesting that eIF5A hypusination contributed to mitochondrial ROS production and GA biosynthesis. In summary, spermidine maintains mitochondrial ROS homeostasis by regulating the translation and subsequent activity of complexes I and II via eIF5A hypusination and promotes GA biosynthesis via mitochondrial ROS signaling. The present findings provide new insight into the spermidine-mediated biosynthesis of secondary metabolites. IMPORTANCE Spermidine is necessary for organism survival and is involved in the regulation of various biological processes. However, the specific mechanisms underlying the various physiological functions of spermidine are poorly understood, especially in microorganisms. In this study, we found that spermidine hypusinates eIF5A to promote the production of mitochondrial ROS and subsequently regulate secondary metabolism in microorganisms. Our study provides a better understanding of the mechanism by which spermidine regulates mitochondrial function and provides new insight into the spermidine-mediated biosynthesis of secondary metabolites.

Heat stress promotes the conversion of putrescine to spermidine and plays an important role in regulating ganoderic acid biosynthesis in Ganoderma lucidum.

Heat stress (HS) is inescapable environmental stress that can induce the production of ganoderic acids (GAs) in Ganoderma lucidum. Our previous studies found that putrescine (Put) played an inhibitory role in GAs biosynthesis, which appeared to be inconsistent with the upregulated transcription of the Put biosynthetic gene GlOdc under HS. To uncover the mechanism underlying this phenomenon, two spermidine (Spd) biosynthetic genes, GlSpds1 and GlSpds2, were identified and upregulated under HS. Put and Spd increased by 94% and 160% under HS, respectively, suggesting that HS induces polyamine biosynthesis and promotes the conversion of Put to Spd. By using GlSpds knockdown mutants, it is confirmed that Spd played a stimulatory role in GAs biosynthesis. In GlOdc-kd mutants, Put decreased by 62-67%, Spd decreased by approximately 34%, and GAs increased by 15-22% but sharply increased by 75-89% after supplementation with Spd. In GlSpds-kd mutants, Put increased by 31-41%, Spd decreased by approximately 63%, and GAs decreased by 24-32% and were restored to slightly higher levels than a wild type after supplementation with Spd. This result suggested that Spd, rather than Put, is a crucial factor that leads to the accumulation of GAs under HS. Spd plays a more predominant and stimulative role than Put under HS, possibly because the absolute content of Spd is 10 times greater than that of Put. GABA and H2O2, two major catabolites of Spd, had little effect on GAs biosynthesis. This study provides new insight into the mechanism by which environmental stimuli regulate secondary metabolism via polyamines in fungi. KEY POINTS: • HS induces polyamine biosynthesis and promotes the conversion of Put to Spd in G. lucidum. • Put and Spd played the inhibitory and stimulatory roles in regulating GAs biosynthesis, respectively. • The stimulatory role of Spd was more predominant than the inhibitory role of Put in GAs biosynthesis.

Improving axial resolution of Bessel beam light-sheet fluorescence microscopy by photobleaching imprinting.

Light-sheet microscopy has been widely used in high-speed fluorescence imaging with low phototoxicity, while the trade-off between the field-of-view and optical sectioning capability limits its application in large-scale imaging. Although Bessel beam light-sheet microscopy greatly enhances the light-sheet length with the self-healing ability, it suffers from the strong side-lobe effect. To solve these problems, we introduce the photobleaching imprinting technique in Bessel beam light-sheet microscopy. By extracting the non-linear photobleaching-induced fluorescence decay, we get rid of the large concentric side lobe structures of the Bessel beam to achieve uniform isotropic resolution across a large field-of-view for large-scale fluorescence imaging. Both numerical simulations and experimental results on various samples are demonstrated to show our enhanced resolution and contrast over traditional Bessel-beam light-sheet microscopy.

Retraction of transporting bone segment during Ilizarov bone transport.

BACKGROUND: Retraction of transporting bone segment (TBS) may occur when the fixator of the TBS is removed prior to full consolidation of the distracted callus, which has adverse effect on the healing of the docking site. However, there are few reports on the retraction of TBS. The purpose of this study is to analyze the causes and risk factors of the retraction of TBS. METHODS: The clinical data of 37 cases with tibial bone defect treated by Ilizarov bone transport were analyzed retrospectively, in whom the TBS fixator was removed prior to full consolidation of the distracted callus and union of the docking site. Bivariate correlation was used to analyze relationship between the retraction distance of TBS and potential risk factors including age, gender, course, length of bone defect, number of operations, size of TBS, transport distance, timing and time interval of removal of TBS fixator. Risk factors with significant level were further identified using multivariate linear regression. RESULTS: Bivariate correlation showed that the timing of removal was negatively correlated with the retraction distance, and the time interval and transport distance were positively correlated with the retraction distance(p < 0.05), the age, gender, course, length of bone defect, size of TBS and number of operations were not correlated with the retraction distance(p > 0.05). Multivariate linear regression of the 3 risk factors showed that the timing of removal and time interval were the main risk factors affecting the retraction distance (p < 0.05), but the transport distance was not (p > 0.05). CONCLUSION: The traction forces of TBS endured from the soft tissues and the unconsolidated distracted callus have elastic properties, which can make retraction of TBS. The timing of removal and time interval are the main risk factors of the retraction of TBS. In the case of early removal, another external fixation or quickly converted to internal fixation should be performed to avoid the adverse effect of more retraction.

Regulation of GDSL Lipase Gene Expression by the MPK3/MPK6 Cascade and Its Downstream WRKY Transcription Factors in Arabidopsis Immunity.

Mitogen-activated protein kinase (MAPK) cascades serve as unified signaling modules in plant development and defense response. Previous reports demonstrated an essential role of Arabidopsis GLIP1, a member of the GDSL-like-motif lipase family, in both local and systemic resistance. GLIP1 expression is highly induced by pathogen attack. However, the one or more signaling pathways involved are unknown. Here, we report that two pathogen-responsive MAPKs, MPK3 and MPK6, are implicated in regulating gene expression of GLIP1 as well as GLIP3 and GLIP4. After gain-of-function activation, MPK3 and MPK6 can strongly induce the expression of GLIP1, GLIP3, and GLIP4. Both GLIP1 and GLIP3 contribute to the plant resistance to Botrytis cinerea. WRKY33, a MPK3/MPK6 substrate, is essential for the MPK3/MPK6-dependent GLIP1 induction. In addition, WRKY2 and WRKY34, two close homologs of WRKY33, have a minor effect in MPK3/MPK6-regulated GLIP1 expression in B. cinerea-infected plants. Chromatin immunoprecipitation-quantitative polymerase chain reaction analysis demonstrated that the GLIP1 gene is a direct target of WRKY33. In addition, we demonstrated that MPK3/MPK6-induced GLIP1 expression is independent of ethylene and jasmonic acid, two important hormones in plant defense. Our results provide insights into the regulation of the GLIP family at the transcriptional level in plant immunity.

miR-181a Participates in Contextual Fear Memory Formation Via Activating mTOR Signaling Pathway.

Long-term memory formation has been proven to require gene expression and new protein synthesis. MicroRNAs (miRNAs), as an endogenous small non-coding RNAs, inhibit the expression of their mRNA targets, through which involve in new memory formation. In this study, elevated miR-181a levels were found to be responsible for hippocampal contextual fear memory consolidation. Using a luciferase reporter assay, we indicated that miR-181a targets 2 upstream molecules of mTOR pathway, namely, PRKAA1 and REDD1. Upregulated miR-181a can downregulate the PRKAA1 and REDD1 protein levels and promote mTOR activity to facilitate hippocampal fear memory consolidation. These results indicate that miR-181a is involved in hippocampal contextual fear memory by activating the mTOR signaling pathway. This work provides a novel evidence for the role of miRNAs in memory formation and demonstrates the implication of mTOR signaling pathway in miRNA processing in the adult brain.

Solid/liquid phase microextraction of five bisphenol-type endocrine disrupting chemicals by using a hollow fiber reinforced with graphene oxide nanoribbons, and determination by HPLC-PDA.

A novel solid/liquid phase microextraction (SLPME) technique was developed and applied to the simultaneous determination of five bisphenol-type endocrine disrupting chemicals (EDCs) including bisphenol A, bisphenol S, bisphenol F, bisphenol B and bisphenol AF. The method is based on the use of a graphene oxide nanoribbon-reinforced hollow fiber (GONRs-HF) and 1-octanol. GONRs were longitudinally unzipped from multiwalled carbon nanotubes (MWCNTs) by means of chemical oxidation and carefully characterized. GONRs were dispersed in ultrapure water with the assistance of surfactant and then immobilized into the wall pores of HF. Compared with CNTs, the hydrophilicity of GONRs was improved, and a uniform dispersion was formed. This simplifies the preparation process and reduces waste of materials. The pores and lumen of GONRs-HF were filled with 1-octanol, and then the whole assembly was used for the extraction of the analytes. In comparison with different extraction modes, GONRs-HF-SLPME exhibits a more effective extraction performance for five bisphenol-type EDCs. Response surface methodology was used to optimize experimental parameters affecting the extraction efficiency. The enrichment factors are in the range from 76 to 127 with good inter-fiber and batch-to-batch reproducibility. The method shows good linearity with determination coefficients (R2) higher than 0.9985. The limits of detection are in the range of 0.1-0.4 µg L-1. Recoveries from spiked real samples are between 83% and 114% with relative standard deviations between 1.6% and 7.8%. Graphical abstract Schematic presentation of the preparation of graphene oxide nanoribbons (GONRs) with multiwalled carbon nanotubes (MWCNTs), immobilization of GONRs into the pores of hollow fiber (HF), and application for solid/liquid phase microextraction (SLPME) of five bisphenol-type endocrine disrupting chemicals in three kinds of real samples.

Mitogen-activated protein kinases and calcium-dependent protein kinases are involved in wounding-induced ethylene biosynthesis in Arabidopsis thaliana.

Ethylene, an important hormone in plant growth, development and response to environmental stimuli, is rapidly induced by mechanical injury or wounding. Although induction of ACS (1-aminocyclopropane-1-carboxylic acid synthase) gene expression has been associated with this process, the detailed regulatory mechanism is unclear. Here, we report that the wounding-induced ethylene production is modulated by both mitogen-activated protein kinase (MAPK) pathway and calcium-dependent protein kinase (CPK) pathway. Study using acs mutants demonstrated that four ACS isoforms, including ACS2, ACS6, ACS7 and ACS8, contribute to ethylene production in response to wounding. Loss-of-function analysis defines the role of MPK3 and MPK6, and their upstream MKK4 and MKK5, in wounding-induced ethylene production. They play an important role in the wounding-induced up-regulation of all four ACS genes expression. Independent of MAPK pathway, CPK5 and CPK6 are also involved in the wounding-induced ethylene production by regulating the expression of ACS2, ACS6 and ACS8 genes. Taken together, we demonstrate that two independent kinase signalling pathways, MPK3/MPK6 cascade and CPK5/CPK6, are involved in the wounding-induced ethylene biosynthesis via differential regulation of ACS genes at transcriptional level.

Enhancing axial resolution and background rejection in line-scanning temporal focusing microscopy by focal modulation.

Compared with two-photon point-scanning microscopy, line-scanning temporal focusing microscopy breaks the limitation on imaging rate and maintains the axial resolution, which makes it promising for various biomedical studies. However, for deep tissue imaging, it suffers from reduced axial resolution and increased background noise due to sample induced wavefront distortion. Here, we propose a spatio-spectral focal modulation technique to enhance axial resolution and background rejection by simply subtracting an aberrated image, which is induced by a spatial light modulator, from an unaberrated image. The proposed technique could improve the axial resolution by a factor of 1.3 in our implementation, verified by both simulations and experiments. Besides, we show that compared with spatial modulation alone, spatio-spectral modulation induces less peak intensity loss caused by image subtraction. We further demonstrate the performance of our technique on the enhanced axial resolution and background rejection by deep imaging of cleared mouse brains and in vivo imaging of living mouse brains.

Single-shot lensless imaging via simultaneous multi-angle LED illumination.

Lensless imaging is a technique that records diffraction patterns without using lenses and recovers the complex field of object via phase retrieval. Robust lensless phase retrieval process usually requires multiple measurements with defocus variation, transverse translation or angle-varied illumination. However, making such diverse measurements is time-consuming and limits the application of lensless setup for dynamic samples. In this paper, we propose a single-shot lensless imaging scheme via simultaneous multi-angle LED illumination. Diffraction patterns under multi-angle lights are recorded by different areas of the sensor within a single shot. An optimization algorithm is applied to utilize the single-shot measurement and retrieve the aliasing information for reconstruction. We first use numerical simulations to evaluate the proposed scheme quantitatively by comparisons with the multi-acquisition case. Then a proof-of-concept lensless setup is built to validate the method by imaging a resolution chart and biological samples, achieving ∼ 4.92 µm half-pitch resolution and ∼ 1.202mm2 field of view (FOV). We also discuss different design tradeoffs and present a 4-frame acquisition scheme (with ∼ 3.48 µm half-pitch resolution and ∼ 2.35 × 2.55 mm2 FOV) to show the flexibility of performance enhancement by capturing more measurements.

Wedelolactone Enhances Osteoblastogenesis through ERK- and JNK-mediated BMP2 Expression and Smad/1/5/8 Phosphorylation.

Our previous study showed that wedelolactone, a compound isolated from Ecliptae herba, has the potential to enhance osteoblastogenesis. However, the molecular mechanisms by which wedelolactone promoted osteoblastogenesis from bone marrow mesenchymal stem cells (BMSCs) remain largely unknown. In this study, treatment with wedelolactone (2 µg/mL) for 3, 6, and 9 days resulted in an increase in phosphorylation of extracellular signal-regulated kinases (ERKs), c-Jun N-terminal protein kinase (JNK), and p38. Phosphorylation of mitogen-activated protein kinases (MAPKs), ERK and JNK started to increase on day 3 of treatment, and p38 phosphorylation was increased by day 6 of treatment. Expression of bone morphogenetic protein (BMP2) mRNA and phosphorylation of Smad1/5/8 was enhanced after treatment of cells with wedelolactone for 6 and 9 days. The addition of the JNK inhibitor SP600125, ERK inhibitor PD98059, and p38 inhibitor SB203580 suppressed wedelolactone-induced alkaline-phosphatase activity, bone mineralization, and osteoblastogenesis-related marker genes including Runx2, Bglap, and Sp7. Increased expression of BMP2 mRNA and Smad1/5/8 phosphorylation was blocked by SP600125 and PD98059, but not by SB203580. These results suggested that wedelolactone enhanced osteoblastogenesis through induction of JNK- and ERK-mediated BMP2 expression and Smad1/5/8 phosphorylation.

Characterization of porcine cytokine inducible SH2-containing protein gene and its association with piglet diarrhea traits.

OBJECTIVE: The cytokine inducible SH2-containing protein (CISH), which might play a role in porcine intestine immune responses, was one of the promising candidate genes for piglet anti-disease traits. An experiment was conducted to characterize the porcine CISH (pCISH) gene and to evaluate its genetic effects on pig anti-disease breeding. METHODS: Both reverse transcription polymerase chain reaction (RT-PCR) and PCR were performed to obtain the sequence of pCISH gene. A pEGFP-C1-CISH vector was constructed and transfected into PK-15 cells to analysis the distribution of pCISH. The sequences of individuals were compared with each other to find the polymorphisms in pCISH gene. The association analysis was performed in Min pigs and Landrace pigs to evaluate the genetic effects on piglet diarrhea traits. RESULTS: In the present research, the coding sequence and genomic sequence of pCISH gene was obtained. Porcine CISH was mainly localized in cytoplasm. TaqI and HaeIII PCR restriction fragment length polymorphism (RFLP) assays were established to detect single nucleotide polymorphisms (SNPs); A-1575G in promoter region and A2497C in Intron1, respectively. Association studies indicated that SNP A-1575G was significantly associated with diarrhea index of Min piglets (p<0.05) and SNP A2497C was significantly associated with the diarrhea trait of both Min pig and Landrace piglets (p<0.05). CONCLUSION: This study suggested that the pCISH gene might be a novel candidate gene for pig anti-disease traits, and further studies are needed to confirm the results of this preliminary research.