A model that predicts a real-time tumour surface using intra-treatment skin surface and end-of-expiration and end-of-inhalation planning CT images.

OBJECTIVES: To develop a mapping model between skin surface motion and internal tumour motion and deformation using end-of-exhalation (EOE) and end-of-inhalation (EOI) 3D CT images for tracking lung tumours during respiration. METHODS: Before treatment, skin and tumour surfaces were segmented and reconstructed from the EOE and the EOI 3D CT images. A non-rigid registration algorithm was used to register the EOE skin and tumour surfaces to the EOI, resulting in a displacement vector field that was then used to construct a mapping model. During treatment, the EOE skin surface was registered to the real-time, yielding a real-time skin surface displacement vector field. Using the mapping model generated, the input of a real-time skin surface can be used to calculate the real-time tumour surface. The proposed method was validated with and without simulated noise on 4D CT images from 15 patients at Léon Bérard Cancer Center and the 4D-lung dataset. RESULTS: The average centre position error, dice similarity coefficient (DSC), 95%-Hausdorff distance and mean distance to agreement of the tumour surfaces were 1.29 mm, 0.924, 2.76 mm, and 1.13 mm without simulated noise, respectively. With simulated noise, these values were 1.33 mm, 0.920, 2.79 mm, and 1.15 mm, respectively. CONCLUSIONS: A patient-specific model was proposed and validated that was constructed using only EOE and EOI 3D CT images and real-time skin surface images to predict internal tumour motion and deformation during respiratory motion. ADVANCES IN KNOWLEDGE: The proposed method achieves comparable accuracy to state-of-the-art methods with fewer pre-treatment planning CT images, which holds potential for application in precise image-guided radiation therapy.

Profiling of RNA editing events in plant organellar transcriptomes with high-throughput sequencing.

RNA editing is a crucial post-transcriptional modification process in plant organellar RNA metabolism. rRNA removal-based total RNA-seq is one of the most common methods to study this event. However, the lack of commercial kits to remove rRNAs limits the usage of this method, especially for non-model plant species. DSN-seq is a transcriptome sequencing method utilizing duplex-specific nuclease (DSN) to degrade highly abundant cDNA species especially those from rRNAs while keeping the robustness of transcript levels of the majority of other mRNAs, and has not been applied to study RNA editing in plants before. In this study, we evaluated the capability of DSN-seq to reduce rRNA content and profile organellar RNA editing events in plants, as well we used commercial Ribo-off-seq and standard mRNA-seq as comparisons. Our results demonstrated that DSN-seq efficiently reduced rRNA content and enriched organellar transcriptomes in rice. With high sensitivity to RNA editing events, DSN-seq and Ribo-off-seq provided a more complete and accurate RNA editing profile of rice, which was further validated by Sanger sequencing. Furthermore, DSN-seq also demonstrated efficient organellar transcriptome enrichment and high sensitivity for profiling RNA editing events in Arabidopsis thaliana. Our study highlights the capability of rRNA removal-based total RNA-seq for profiling RNA editing events in plant organellar transcriptomes and also suggests DSN-seq as a widely accessible RNA editing profiling method for various plant species.

Risk Factors for Left Ventricular Hypertrophy in Patients with Diabetic Kidney Disease: A Multi-Center Study.

Objective: One of the earliest echocardiographic features of the left ventricle explored extensively was left ventricular hypertrophy (LVH). Numerous studies have identified a few risk factors for LVH, however, there are few for people with diabetic kidney disease (DKD). Therefore, we evaluated the risk factors in DKD patients with LVH by analyzing laboratory data and clinical traits. Methods: In total, 500 DKD patients in the Baoding area from February 2016 and June 2020 were admitted and classified as an experimental group (240 cases, LVH group) and a control group (260 cases, non-LVH group). The clinical parameters and laboratory tests of the participants were collected and analyzed retrospectively. Results: Compared with the control group, low-density lipoprotein (LDL), body mass index (BMI), intact parathyroid hormone (iPTH), systolic blood pressure, and 24-hour urine protein were higher in the experimental group (all P<0.01). Multivariable logistic regression analysis results confirmed that high BMI (OR=1.332, 95% CI 1.016-1.537, P=0.006), LDL (OR=1.279, 95% CI 1.008-1.369, P=0.014) and 24-hour urine proteins (OR=1.446, 95% CI 1.104-1.643, P=0.016) were statistically significant. The ROC analysis illustrated that the optimum cutoff value of BMI, LDL, and 24-hour urine proteins for diagnosis of LVH in patients with DKD was 27.36 kg/m2, 4.18 mmol/L, and 1.42 g respectively. Conclusion: The increase in BMI, LDL, and 24-hour urine proteins quantification are independent risk factors for LVH in patients with DKD.

Motionless volumetric structured light sheet microscopy.

To meet the increasing need for low-cost, compact imaging technology with cellular resolution, we have developed a microLED-based structured light sheet microscope for three-dimensional ex vivo and in vivo imaging of biological tissue in multiple modalities. All the illumination structure is generated directly at the microLED panel-which serves as the source-so light sheet scanning and modulation is completely digital, yielding a system that is simpler and less prone to error than previously reported methods. Volumetric images with optical sectioning are thus achieved in an inexpensive, compact form factor without any moving parts. We demonstrate the unique properties and general applicability of our technique by ex vivo imaging of porcine and murine tissue from the gastrointestinal tract, kidney, and brain.

Depth random-access two-photon Bessel light-sheet imaging in brain tissue.

Two-photon light-sheet fluorescence microscopy enables high-resolution imaging of neural activity in brain tissue at a high frame rate. Traditionally, light-sheet microscopy builds up a 3D stack by multiple depth scans with uniform spatial intervals, which substantially limits the volumetric imaging speed. Here, we introduce the depth random-access light-sheet microscopy, allowing rapid switching scanning depth for light-sheet imaging. With a low-cost electrically tunable lens and minimum modification of an existing two-photon light-sheet imaging instrument, we demonstrated fast random depth hopping light-sheet imaging at 100 frames per second in the live brain slice. Through depth random-access, calcium activities for an astrocyte were recorded on four user-selected detection planes at a refreshing rate of 25 Hz.

Fourier Multiplexed Fluorescence Lifetime Imaging.

Fluorescence lifetime imaging microscopy (FLIM) is a widely used functional imaging method in bioscience. Fourier multiplexed FLIM (FmFLIM), a frequency-domain lifetime measurement method, explores the principle of Fourier (frequency) multiplexing to achieve parallel lifetime detection on multiple fluorescence labels. Combining FmFLIM with a confocal scanning microscope allows multiplexed 3D lifetime imaging of cells and tissues. FmFLIM can also be integrated with the scanning laser tomography imaging method to perform 3D multiplex lifetime imaging of whole embryos and thick tissues.

Surgical treatment of sternum comminuted fracture with memory alloy embracing fixator.

BACKGROUND: Sternal fracture can result from multiple types of severe chest trauma and carries significant risk. Surgical fixation is an effective method for sternal fracture. METHODS: The clinical data of patients with sternal fractures who presented to our hospital between August 2016 and July 2019 were collected. The 42 patients were divided into three groups, with 15 patients treated by internal fixation with NI-TI memory alloy embracing fixator, 10 patients receiving steel wire fixation, and 17 who received non-surgical treatment and who was designated as a control (conservative) group. Differences in clinical indices included the duration of surgery, blood loss, hospitalization, wound healing, hospitalization expenses, VAS scores, and patient satisfaction scores between the three groups was compared. The analysis of variance and t-test were used for quantitative variables, which were approximately normally distributed. Dichotomous data were compared used Pearson χ2 or Fisher's exact test, and a P value less than 0.05 was considered as statistically significant. RESULTS: All patients were cured, and there were no significant differences in general clinical features between the three groups (P>0.05). Thoracic deformity in the surgical groups was corrected anatomically and received better pain scores, while patients in the NI-TI memory alloy embracing fixator group showed advantages of bleeding and patient satisfaction (P<0.05). CONCLUSIONS: Operative treatment for a sternal fracture is safe, effective and can quickly restore the stability of the thorax. Memory alloy embracing fixator is markedly superior to other fixator materials.

Silencing of rice PPR gene PPS1 exhibited enhanced sensibility to abiotic stress and remarkable accumulation of ROS.

Abiotic stresses widely constrain the development and reproduction of plant, especially impaired the yield of crops greatly. Recent researches presented pentatricopeptide repeat (PPR) proteins play crucial role in response to abiotic stress. However, the underlying mechanism of PPR genes in regulation of abiotic stress is still obscures. In our recent study, we found that the knockout of rice PPS1 causes pleiotropic growth disorders, including growth retardation, dwarf and sterile pollen, and finally leads to impaired C-U RNA editing at five consecutive sites on the mitochondrial nad3. In this study, we further investigate the roles of PPS1 in abiotic stress tolerance, we confirmed that pss1-RNAi line exhibited enhanced sensitivity to salinity and ABA stress at vegetative stage, specifically. While reactive oxygen species (ROS) accumulate significantly only at reproductive stage, which further activated the expression of several ROS-scavenging system related genes. These results implied that PPS1 functioned on ROS signaling network to contribute for the flexibility to abiotic stresses. Our research emphasizes the stress adaptability mediated by the PPR protein, and also provides new insight into the understanding of the interaction between cytoplasm and nucleus and signal transduction involved in RNA editing.

A simplified method to isolate rice mitochondria.

BACKGROUND: Mitochondria play critical roles in plant growth, development and stress tolerance. Numerous researchers have carried out studies on the plant mitochondrial genome structure, mitochondrial metabolism and nuclear-cytoplasmic interactions. However, classical plant mitochondria extraction methods are time-consuming and consist of a complicated ultracentrifugation procedure with expensive reagents. To develop a more rapid and convenient method for the isolation of plant mitochondria, in this study, we established a simplified method to isolate rice mitochondria efficiently for subsequent studies. RESULTS: To isolate rice mitochondria, the cell wall was first disrupted by enzymolysis to obtain the protoplast, which is similar to animal mitochondria. Rice mitochondria were then isolated with a modified method based on the animal mitochondria isolation protocol. The extracted mitochondria were next assessed according to DNA and protein levels to rule out contamination by the nucleus and chloroplasts. Furthermore, we examined the physiological status and characteristics of the isolated mitochondria, including the integrity of mitochondria, the mitochondrial membrane potential, and the activity of inner membrane complexes. Our results demonstrated that the extracted mitochondria remained intact for use in subsequent studies. CONCLUSION: The combination of plant protoplast isolation and animal mitochondria extraction methods facilitates the extraction of plant mitochondria without ultracentrifugation. Consequently, this improved method is cheap and time-saving with good operability and can be broadly applied in studies on plant mitochondria.

Structured illumination imaging with quasi-periodic patterns.

Structured illumination microscopy (SIM) is a well-established method for optical sectioning and super-resolution. The core of structured illumination is using a periodic pattern to excite image signals. This work reports a method for estimating minor pattern distortions from the raw image data and correcting these distortions during SIM image processing. The method was tested with both simulated and experimental image data from two-photon Bessel light-sheet SIM. The results proves the method is effective in challenging situations, where strong scattering background exists, signal-to-noise ratio (SNR) is low and the sample structure is sparse. Experimental results demonstrate restoring synaptic structures in deep brain tissue, despite the presence of strong light scattering and tissue-induced SIM pattern distortion.

Mechanosensing during directed cell migration requires dynamic actin polymerization at focal adhesions.

The mechanical properties of a cell's microenvironment influence many aspects of cellular behavior, including cell migration. Durotaxis, the migration toward increasing matrix stiffness, has been implicated in processes ranging from development to cancer. During durotaxis, mechanical stimulation by matrix rigidity leads to directed migration. Studies suggest that cells sense mechanical stimuli, or mechanosense, through the acto-myosin cytoskeleton at focal adhesions (FAs); however, FA actin cytoskeletal remodeling and its role in mechanosensing are not fully understood. Here, we show that the Ena/VASP family member, Ena/VASP-like (EVL), polymerizes actin at FAs, which promotes cell-matrix adhesion and mechanosensing. Importantly, we show that EVL regulates mechanically directed motility, and that suppression of EVL expression impedes 3D durotactic invasion. We propose a model in which EVL-mediated actin polymerization at FAs promotes mechanosensing and durotaxis by maturing, and thus reinforcing, FAs. These findings establish dynamic FA actin polymerization as a central aspect of mechanosensing and identify EVL as a crucial regulator of this process.

Anticancer effects of α-Bisabolol in human non-small cell lung carcinoma cells are mediated via apoptosis induction, cell cycle arrest, inhibition of cell migration and invasion and upregulation of P13K/AKT signalling pathway.

PURPOSE: Non-small lung cancer (NSLC) is one of the leading causes of cancer-related deaths world over. Excempting operable cases the treatments for NSCLC mainly include chemotherapy and radiotherapy. However, the survival rate for NSCLC is still far from satsifactory. Moreover, chemotherapy has lot of associated side effects. Therefore, there is an urgent need to look for novel and more viable treatment options. Against this background, the present study was designed to evaluate the anticancer activity of α-Bisabolol against NSCLC. METHODS: Cell viability was assessed by MTT assay. Apoptosis was determined by DAPI and annexin V/propidium iodide (PI) staining. Mitochondrial membrane potential (MMP) and cell cycle analysis were determined by flow cytometry. Cell migration was investigated by wound healing assay and protein expression was evaluated by western blotting. RESULTS: α-Bisabolol exerted significant anticancer activity on A549 NSCLC cells with IC50 of 15 µM. The anticancer effects of α-Bisabolol were found to be due to G2/M cell cycle arrest and mitochondrial apoptosis. α-Bisabolol also inhibited cell migration of A549 cells dose-dependently. Moreover, the results showed that α-Bisabolol could inhibit the PI3K/AKT signalling pathway in a dose-dependent manner. The results of the present study indicate that α-Bisabolol exerted selective anticancer effects on A549 cells via induction of cell cycle arrest, mitochondrial apoptosis and inhibition of PI3K/Akt signalling pathways. CONCLUSIONS: This molecule showed promising anticancer features and could be developed as a potent lead candidate for the management and treatment of NSCLC.

A rice dual-localized pentatricopeptide repeat protein is involved in organellar RNA editing together with OsMORFs.

In flowering plants, various RNA editing events occur in the mitochondria and chloroplasts as part of post-transcriptional processes. Although several pentatricopeptide repeat (PPR) proteins and multiple organellar RNA editing factors (MORFs) have been identified as RNA editing factors, the underlying mechanism of PPRs and the cooperation among these proteins are still obscure. Here, we identified a rice dual-localized PPR protein, OsPGL1. The loss of function of OsPGL1 resulted in defects in both chloroplast RNA editing of ndhD-878 and mitochondrial RNA editing of ccmFc-543, both of which could be restored in transgenic complementation lines. Despite synonymous editing of ccmFc-543, the loss of editing of ndhD-878 caused a failed conversion of serine to leucine, leading to chloroplast dysfunction and defects in the photosynthetic complex; the results of additional experiments demonstrated that OsPGL1 directly binds to both transcripts. Interactions between three OsMORFs (OsMORF2/8/9) and OsPGL1 both in vitro and in vivo were confirmed, implying that OsPGL1 functions in RNA editing via an editosome. These findings also suggested that OsMORFs assist with and contribute to a flexible PPR-RNA recognition model during RNA editing. These results indicate that, in cooperation with PPRs, OsPGL1 is required for RNA editing. In addition, our study provides new insights into the relationship between RNA editing and plant development.

Surgical versus conservative therapy for multiple rib fractures: a retrospective analysis.

BACKGROUND: In this study, we aimed to evaluate the clinical effects of multiple rib fracture treatments using a rib plate fixator. METHODS: From June 2014 to December 2016, 110 cases of patients with multiple rib fractures were collected for our study from the 105th Hospital of PLA; 59 patients were treated by surgery, and 51 patients received conservative treatment. We compared surgical rib plate fixation with conservative treatment for patients with multiple rib fractures. RESULTS: The hospital stay for the surgical group was 13.12±4.21 days, whereas for the conservative group, it was 18.57±5.39 days (P<0.001). The duration of the thoracic intensive care unit (ICU) stay was 4.02±1.41 days in the surgical group and 5.06±1.80 days in the conservative group (P=0.001). The indwelling thoracic drainage tube time was 5.85±1.52 days in the surgical group and 8.26±1.96 days in the conservative group (P<0.001). The index of partial pressure of arterial oxygen (PaO2) was 91.05±10.88 mmHg 24 h after surgery whereas 86.49±11.69 mmHg 24 h after conservative treatment (P=0.036). The index of partial pressure of carbon dioxide (PaCO2) was 37.80±2.86 mmHg 24 h after surgery and it was 39.08±2.46 mmHg 24 h after conservative treatment. The hospitalization cost was 6,206.44±371.42 USD for the surgical group and 4,544.61±524.79 USD for the conservative group (P<0.001). The number of rib displacement cases after treatment was 1 case in the surgical group and 2 cases in the conservative group (P=0.475). Atelectasis occurred in 10 cases in the surgical group and in 17 cases in the conservative group (P=0.046). Delayed hemopneumothorax occurred in 7 cases in the surgical group and in 16 cases in the conservative group (P=0.012). CONCLUSIONS: Surgical treatment is a safe and effective therapy for multiple rib fractures. Our data suggested that surgical treatment can significantly shorten the patients' pain-endurance time, hospitalization time, and can reduce complication incidence. Severe rib fracture patients might benefit from surgical fixation.

Three-dimensional live multi-label light-sheet imaging with synchronous excitation-multiplexed structured illumination.

Multiplexed imaging is a powerful tool for studying complex interactions inside biological systems. Spectral imaging methods that capture multiple fluorescent markers synchronously without sacrificing the imaging speed or resolution are most suitable for live imaging. We describe spectral-encoded structured illumination (spectral-SIM) light-sheet microscopy, which enables parallel multi-excitation-channel imaging in 3D. Spectral-SIM encodes the excitation wavelength as the phase of the illumination pattern, and allows synchronous image capture over multiple excitation channels at the same speed and spatial resolution as mono-channel structured light-sheet imaging. The technique retains structured light-sheet microscopy's ability in removing out-of-focus and scattered emission background, and generates clear 3D multiplexed images in thick tissue. The capability of this technique was demonstrated by the imaging of live triple-labeled transgenic zebrafish to over 300 µm deep with 0.5µm-by-2µm (lateral-by-axial) resolution.

Cellular resolution multiplexed FLIM tomography with dual-color Bessel beam.

Fourier multiplexed FLIM (FmFLIM) tomography enables multiplexed 3D lifetime imaging of whole embryos. In our previous FmFLIM system, the spatial resolution was limited to 25 µm because of the trade-off between the spatial resolution and the imaging depth. In order to achieve cellular resolution imaging of thick specimens, we built a tomography system with dual-color Bessel beam. In combination with FmFLIM, the Bessel FmFLIM tomography system can perform parallel 3D lifetime imaging on multiple excitation-emission channels at a cellular resolution of 2.8 µm. The image capability of the Bessel FmFLIM tomography system was demonstrated by 3D lifetime imaging of dual-labeled transgenic zebrafish embryos.

Multiplexed 3D FRET imaging in deep tissue of live embryos.

Current deep tissue microscopy techniques are mostly restricted to intensity mapping of fluorophores, which significantly limit their applications in investigating biochemical processes in vivo. We present a deep tissue multiplexed functional imaging method that probes multiple Förster resonant energy transfer (FRET) sensors in live embryos with high spatial resolution. The method simultaneously images fluorescence lifetimes in 3D with multiple excitation lasers. Through quantitative analysis of triple-channel intensity and lifetime images, we demonstrated that Ca(2+) and cAMP levels of live embryos expressing dual FRET sensors can be monitored simultaneously at microscopic resolution. The method is compatible with a broad range of FRET sensors currently available for probing various cellular biochemical functions. It opens the door to imaging complex cellular circuitries in whole live organisms.

FPGA-based multi-channel fluorescence lifetime analysis of Fourier multiplexed frequency-sweeping lifetime imaging.

We report a fast non-iterative lifetime data analysis method for the Fourier multiplexed frequency-sweeping confocal FLIM (Fm-FLIM) system [Opt. Express 22, 10221 (2014)]. The new method, named R-method, allows fast multi-channel lifetime image analysis in the system's FPGA data processing board. Experimental tests proved that the performance of the R-method is equivalent to that of single-exponential iterative fitting, and its sensitivity is well suited for time-lapse FLIM-FRET imaging of live cells, for example cyclic adenosine monophosphate (cAMP) level imaging with GFP-Epac-mCherry sensors. With the R-method and its FPGA implementation, multi-channel lifetime images can now be generated in real time on the multi-channel frequency-sweeping FLIM system, and live readout of FRET sensors can be performed during time-lapse imaging.

Microdynamics mechanism of thermal-induced hydrogel network destruction of poly(vinyl alcohol) in D2O studied by two-dimensional infrared correlation spectroscopy.

Microdynamics mechanism of thermal-induced hydrogel network destruction of poly(vinyl alcohol) (PVA) in D2O at heating (25-62 °C) was studied by in situ Fourier transform infrared (FTIR) spectroscopy combining with moving-window two-dimensional (MW2D) technique and two-dimensional (2D) correlation analysis. The temperature range of hydrogel destruction was determined within 34-52 °C by dynamic rheological test at first, and then also monitored by MW2D FTIR spectra. The motion of vs(-C-O-, microcrystals) was important in the entire hydrogel destruction process. The microdynamics mechanism of PVA molecular chains can be elaborated as follows: At 32 °C, the number of D2O molecules in the swollen amorphous remains unchanged. At 32-37 °C, more D2O molecules enter into the swollen amorphous region, and the groups of -C-O-, together with -CH2-, are partially hydrated. At 37 °C, the intramolecular or intermolecular hydrogen bonds of PVA are dissociated. The physical cross-linking points of hydrogel are broken due to the melting of PVA microcrystals. At 42 °C, the dissociated hydroxyls from PVA microcrystals rapidly integrate solid hydrogen bonds with D2O molecules. The groups of -C-O- and -CH- are completely hydrated by D2O simultaneously. At 45-55 °C, PVA molecules are surrounded by more D2O molecules. The partially hydrated -CH2- is completely hydrated, and all of the PVA molecules are fully dissolved in D2O.

Parallel excitation-emission multiplexed fluorescence lifetime confocal microscopy for live cell imaging.

We present a novel excitation-emission multiplexed fluorescence lifetime microscopy (FLIM) method that surpasses current FLIM techniques in multiplexing capability. The method employs Fourier multiplexing to simultaneously acquire confocal fluorescence lifetime images of multiple excitation wavelength and emission color combinations at 44,000 pixels/sec. The system is built with low-cost CW laser sources and standard PMTs with versatile spectral configuration, which can be implemented as an add-on to commercial confocal microscopes. The Fourier lifetime confocal method allows fast multiplexed FLIM imaging, which makes it possible to monitor multiple biological processes in live cells. The low cost and compatibility with commercial systems could also make multiplexed FLIM more accessible to biological research community.