The roles of FUS-RNA binding domain and low complexity domain in RNA-dependent phase separation.

Fused in sarcoma (FUS) is an archetypal phase separating protein asymmetrically divided into a low complexity domain (LCD) and an RNA binding domain (RBD). Here, we explore how the two domains contribute to RNA-dependent phase separation, RNA recognition, and multivalent complex formation. We find that RBD drives RNA-dependent phase separation but forms large and irregularly shaped droplets that are rescued by LCD in trans. Electrophoretic mobility shift assay (EMSA) and single-molecule fluorescence assays reveal that, while both LCD and RBD bind RNA, RBD drives RNA engagement and multivalent complex formation. While RBD alone exhibits delayed RNA recognition and a less dynamic RNP complex compared to full-length FUS, LCD in trans rescues full-length FUS activity. Likewise, cell-based data show RBD forms nucleolar condensates while LCD in trans rescues the diffuse nucleoplasm localization of full-length FUS. Our results point to a regulatory role of LCD in tuning the RNP interaction and buffering phase separation.

Advanced imaging techniques for studying protein phase separation in living cells and at single-molecule level.

Protein-protein and protein-RNA interactions are essential for cell function and survival. These interactions facilitate the formation of ribonucleoprotein complexes and biomolecular condensates via phase separation. Such assembly is involved in transcription, splicing, translation and stress response. When dysregulated, proteins and RNA can undergo irreversible aggregation which can be cytotoxic and pathogenic. Despite technical advances in investigating biomolecular condensates, achieving the necessary spatiotemporal resolution to deduce the parameters that govern their assembly and behavior has been challenging. Many laboratories have applied advanced microscopy methods for imaging condensates. For example, single molecule imaging methods have enabled the detection of RNA-protein interaction, protein-protein interaction, protein conformational dynamics, and diffusional motion of molecules that report on the intrinsic molecular interactions underlying liquid-liquid phase separation. This review will outline advances in both microscopy and spectroscopy techniques which allow single molecule detection and imaging, and how these techniques can be used to probe unique aspects of biomolecular condensates.

Comparative transcriptome analysis reveals key genes for polyphyllin difference in five Paris species.

Paris species accumulate a large amount of steroidal saponins, which have numerous pharmacological activities and have become an essential component in many patented drugs. However, only two among all Paris species. Paris are identified as official sources due to high level of bioactive compounds. To clarify the composition of steroidal saponins and the molecular basis behind the differences between species, we investigated transcriptome and metabolic profiles of leaves and rhizomes in Paris polyphylla var. chinensis (PPC), Paris polyphylla var. yunnanensis (PPY), Paris polyphylla var. stenophylla (PPS), Paris fargesii (PF), and Paris mairei (PM). Phytochemical results displayed that the accumulation of steroidal saponins was tissue- and species-specific. PF and PPS contained more steroidal saponins in leaves than rhizomes, while PPY accumulated more steroidal saponins in rhizomes than leaves. PPC and PM contained similar amounts of steroidal saponins in leaves and rhizomes. Transcriptome analysis illustrated that most differentially expressed genes related to the biosynthesis of steroidal saponins were abundantly expressed in rhizomes than leaves. Meanwhile, more biosynthetic genes had significant correlations with steroidal saponins in rhizomes than in leaves. The result of CCA indicated that ACAT, DXS, DWF1, and CYP90 constrained 97.35% of the variance in bioactive compounds in leaves, whereas CYP72, UGT73, ACAT, and GPPS constrained 98.61% of the variance in phytochemicals in rhizomes. This study provided critical information for enhancing the production of steroidal saponins by biotechnological approaches and methodologies.

The genome of Dioscorea zingiberensis sheds light on the biosynthesis, origin and evolution of the medicinally important diosgenin saponins.

Diosgenin saponins isolated from Dioscorea species such as D. zingiberensis exhibit a broad spectrum of pharmacological activities. Diosgenin, the aglycone of diosgenin saponins, is an important starting material for the production of steroidal drugs. However, how plants produce diosgenin saponins and the origin and evolution of the diosgenin saponin biosynthetic pathway remain a mystery. Here we report a high-quality, 629-Mb genome of D. zingiberensis anchored on 10 chromosomes with 30 322 protein-coding genes. We reveal that diosgenin is synthesized in leaves ('source'), then converted into diosgenin saponins, and finally transported to rhizomes ('sink') for storage in plants. By evaluating the distribution and evolutionary patterns of diosgenin saponins in Dioscorea species, we find that diosgenin saponin-containing may be an ancestral trait in Dioscorea and is selectively retained. The results of comparative genomic analysis indicate that tandem duplication coupled with a whole-genome duplication event provided key evolutionary resources for the diosgenin saponin biosynthetic pathway in the D. zingiberensis genome. Furthermore, comparative transcriptome and metabolite analysis among 13 Dioscorea species suggests that specific gene expression patterns of pathway genes promote the differential evolution of the diosgenin saponin biosynthetic pathway in Dioscorea species. Our study provides important insights and valuable resources for further understanding the biosynthesis, evolution, and utilization of plant specialized metabolites such as diosgenin saponins.

Genome-Wide Identification of CYP72A Gene Family and Expression Patterns Related to Jasmonic Acid Treatment and Steroidal Saponin Accumulation in Dioscorea zingiberensis.

Dioscorea zingiberensis is a medicinal herb containing a large amount of steroidal saponins, which are the major bioactive compounds and the primary storage form of diosgenin. The CYP72A gene family, belonging to cytochromes P450, exerts indispensable effects on the biosynthesis of numerous bioactive compounds. In this work, a total of 25 CYP72A genes were identified in D. zingiberensis and categorized into two groups according to the homology of protein sequences. The characteristics of their phylogenetic relationship, intron-exon organization, conserved motifs and cis-regulatory elements were performed by bioinformatics methods. The transcriptome data demonstrated that expression patterns of DzCYP72As varied by tissues. Moreover, qRT-PCR results displayed diverse expression profiles of DzCYP72As under different concentrations of jasmonic acid (JA). Likewise, eight metabolites in the biosynthesis pathway of steroidal saponins (four phytosterols, diosgenin, parvifloside, protodeltonin and dioscin) exhibited different contents under different concentrations of JA, and the content of total steroidal saponin was largest at the dose of 100 µmol/L of JA. The redundant analysis showed that 12 DzCYP72As had a strong correlation with specialized metabolites. Those genes were negatively correlated with stigmasterol and cholesterol but positively correlated with six other specialized metabolites. Among all DzCYP72As evaluated, DzCYP72A6, DzCYP72A16 and DzCYP72A17 contributed the most to the variation of specialized metabolites in the biosynthesis pathway of steroidal saponins. This study provides valuable information for further research on the biological functions related to steroidal saponin biosynthesis.

Simultaneous detection of cerebral blood perfusion and cerebral edema using swept-source optical coherence tomography.

The progression of ischemic cerebral edema (CE) is closely related to the level of cerebral blood perfusion (CBP) and affects each other. Simultaneous detection of CBP and CE is helpful in understanding the mechanisms of ischemic CE development. In this article, a wide field of view swept-source optical coherence tomography system was used to detect CE status and CBP levels simultaneously in middle cerebral artery occlusion rats. Images reflecting these two physiological states can be reconstructed with only one C-scan. We quantify these two physiological states into four parameters, which contain two vascular parameters (vascular displacement distance and vascular perfusion density) and two edema parameters (optical attenuation coefficient and edema area). The association between the two vascular parameters and the two edema parameters was analyzed. The results show that there is a strong linear relationship between blood flow parameters and edema parameters. This work provides a new option for CE in vivo detection, and is very likely to play an important role in the development of relevant drugs or in selection of treatment options.

Cerebral edema detection in vivo after middle cerebral artery occlusion using swept-source optical coherence tomography.

Cerebral edema is a severe complication of ischemic cerebrovascular disease, which can lead to microcirculation compression resulting in additional ischemic damage. Real-time and continuous in vivo imaging techniques for edema detection are of great significance to basic research on cerebral edema. We attempted to monitor the cerebral edema status in rats with middle cerebral artery occlusion (MCAO) over time, using a wide field-of-view swept-source optical coherence tomography (SS-OCT) system. Optical attenuation coefficients (OACs) were calculated by an optimized depth-resolved estimation method, and en face OAC maps covering the whole cortex were obtained. Then, the tissue affected by edema was segmented from the OAC maps, and the cortical area affected by edema was estimated. Both magnetic resonance image (MRI) and brain water content measurements were used to verify the presence of cerebral edema. The results showed that the average OAC of the ischemic area gradually decreased as cerebral edema progressed, and the edema area detected by SS-OCT had high similarity in position and shape to that obtained by MRI. This work extends the application of OCT and provides an option for detecting cerebral edema in vivo after ischemic stroke.

Whole-brain microcirculation detection after ischemic stroke based on swept-source optical coherence tomography.

The occurrence and development of ischemic stroke are closely related to cerebral blood flow. Real-time monitoring of cerebral perfusion level is very useful for understanding the mechanisms of the disease. A wide field of view (FOV) is conducive to capturing lesions and observing the progression of the disease. In this paper, we attempt to monitor the whole-brain microcirculation in middle cerebral artery occlusion (MCAO) rats over time using a wide FOV swept-source OCT (SS-OCT) system. A constrained image registration algorithm is used to remove motion artifacts that are prone to occur in a wide FOV angiography. During ischemia, cerebral perfusion levels in the left and right hemispheres, as well as in the whole brain were quantified and compared. Changes in the shape and location of blood vessels were also recorded. The results showed that the trend in cerebral perfusion levels of both hemispheres was highly consistent during MCAO, and the position of the blood vessels varied over time. This work will provide new insights of ischemic stroke and is helpful to assess the effectiveness of potential treatment strategies.

Optimized depth-resolved estimation to measure optical attenuation coefficients from optical coherence tomography and its application in cerebral damage determination.

The optical attenuation coefficient (OAC) reflects the optical properties of various tissues or tissues of the same type under different physiological conditions. Quantitative measurement of OAC from optical coherence tomography (OCT) signals can provide additional information and can increase the potential for OCT applications. We present an optimized depth-resolved estimation (ODRE) method that derives a precise mapping between the measured OCT signal and the OAC. In contrast to previous depth-resolved estimation (DRE) methods, the optimized method can estimate the OAC in any depth range and ignore whether the light is completely attenuated. Numerical simulations and phantom experiments are used to verify its validity, and this method is applied to detect cerebral damage. In combination with OCT angiography, real-time observation of the change of blood perfusion and the degree of cerebral damage in mice with focal cerebral ischemia provides important information to help us understand the temporal relationship between brain damage and ischemia.

Quantification of cerebral vascular perfusion density via optical coherence tomography based on locally adaptive regional growth.

Optical coherence tomography (OCT) angiography is a noninvasive imaging modality that produces volumetric views of blood flow perfusion in vivo with resolution at capillary level, which has been widely adopted to monitor cerebral perfusion status after stroke in experimental settings. Accurate quantification of cerebral perfusion from OCT angiograms is important for understanding the cerebral vascular pathophysiology and assessing the treatment of ischemic stroke. Quantification of blood vessels from OCT angiography faces some problems; one is uneven backscatter (which causes some blood vessels to be very bright, some very dark), and the other is that the brightness in the same blood vessel also changes due to the difference in diameter or depth. In this paper, we proposed a locally adaptive region growing algorithm to solve this problem. The algorithm, which confines the region growing process to a local region, is used to segment blood vessels in different images to cope well with the intensity changes in blood vessels. During segmentation, the initial seed pixels were selected with the aid of the Otsu algorithm, the growth criterion considered both global and local information, and the thresholds were also adjusted adaptively as local regions varied. After these processes are completed, we can calculate the percentage of segmented blood vessels across field of view of the images, named cerebral vascular perfusion density, and use it as an indicator to evaluate the cerebral blood perfusion of middle cerebral artery occlusion in mice. This paper demonstrates that the algorithm can produce satisfactory vascular segmentation results, and CVPD can be used as an effective indicator for evaluating post-ischemic injury.

Application of intraoperative computed tomography with or without navigation system in surgical correction of spinal deformity: a preliminary result of 59 consecutive human cases.

STUDY DESIGN: A retrospective analysis of patients undergoing spinal deformity correction surgery by the assistance of intraoperative computed tomography (iCT) with or without navigation system. OBJECTIVE: To share our preliminary experience and analysis of the iCT navigation system applied to spinal deformity surgery. SUMMARY OF BACKGROUND DATA: The iCT navigation system has been shown to improve accuracy and safety in posterior instrumentation. It not only decreased the operation time but also prevented excessive radiation exposure to the medical staff. To date, there are only few reports about the application of the iCT navigation system in spinal deformity surgery. METHODS: From April 2009 to September 2010, 59 patients who had a diagnosis of scoliosis, kyphosis, or scoliokyphosis and underwent iCT-assisted surgical correction were included. Without randomization, 28 patients were operated with the iCT-navigation system, and the other 31 patients were operated with standard procedure under iCT assistance. The detailed procedures, preoperative and intraoperative images were illustrated. The accuracy of screw placement, time for screw insertion, postoperative correction rate, and iCT scanning data were analyzed. RESULTS: There were significant differences between 2 groups in (1) the preoperative Cobb angle (76.2° and 62.6° in the navigation and non-navigation groups), (2) the accuracy and the revision rate of thoracic pedicle screws and total pedicle screws, and (3) the average screw insertion time. The breach rate and the revision rate of thoracic pedicle screws and total pedicle screws were significantly lower and the average screw insertion time was significantly lesser in the navigation group than in the non-navigation group. There were no statistically significant difference in (1) the breach rate and the revision rate of lumbar pedicle screws, (2) the mean iCT scanning time and time-out, (3) the mean number of fusion segments, (4) the mean number of iCT scans, and (5) the postoperative correction rate. Complications were encountered in 2 patients in the non-navigation group but none in the navigation group. There was no reoperation due to implant malposition in both groups. CONCLUSION: The iCT navigation system provides desirable accuracy of posterior spinal instrumentation for patients during surgical correction of spinal deformity without radiation exposure to the medical staff, especially in thoracic spine instrumentation. Meanwhile, the iCT in itself is an effective means of assessing complex instrumentation of the spinal deformity.

[Arthroscopy for the diagnosis and treatment of hip joint disease].

OBJECTIVE: To evaluate the value of arthroscopy for the diagnosis and treatment of hip joint disease. METHODS: Arthroscopy was performed in 54 patients (68 hips) from July 1998 to September 2001. Of the 54 patients, 36 were male and 18 female, aged from 17 to 64 years. The disease was located on the left side in 18 patients, the right side in 22, and both sides in 14. In this group, osteoarthritis was noted in 21 patients, avascular necrosis of the femoral head in 16, infection of the hip joint in 3, chondrosarcoma in 2, synovial-chondromatosis in 7, acetabular labral tears in 2, and spondylitis in 3. The procedures of arthroscopy consisted of removal of loose bodies, debridement, and biopsy. RESULTS: The operation made accurate diagnosis possible for chondrosarcoma, acetabular labral tears, and infection of the hip joint. After debridement of the hip joint for avascular necrosis of the femoral head, osteoarthritis and spondylitis, most patients in this group were free from pain, function and range of the motion were restored and the pathological process was delayed. The Harris hip scores increased by 27 on average. CONCLUSION: Arthroscopy plays an important role in accurate diagnosis and treatment of hip joint.