Morphological, anatomical, and transcriptomics analysis reveals the regulatory mechanisms of cassava plant height development.

BACKGROUND: Cassava is one of three major potato crops and the sixth most important food crop globally. Improving yield remains a primary aim in cassava breeding. Notably, plant height significantly impacts the yield and quality of crops; however, the mechanisms underlying cassava plant height development are yet to be elucidated. RESULTS: In this study, we investigated the mechanisms responsible for cassava plant height development using phenotypic, anatomical, and transcriptomic analyses. Phenotypic and anatomical analysis revealed that compared to the high-stem cassava cultivar, the dwarf-stem cassava cultivar exhibited a significant reduction in plant height and a notable increase in internode tissue xylem area. Meanwhile, physiological analysis demonstrated that the lignin content of dwarf cassava was significantly higher than that of high cassava. Notably, transcriptome analysis of internode tissues identified several differentially expressed genes involved in cell wall synthesis and expansion, plant hormone signal transduction, phenylpropanoid biosynthesis, and flavonoid biosynthesis between the two cassava cultivars. CONCLUSIONS: Our findings suggest that internode tissue cell division, secondary wall lignification, and hormone-related gene expression play important roles in cassava plant height development. Ultimately, this study provides new insights into the mechanisms of plant height morphogenesis in cassava and identifies candidate regulatory genes associated with plant height that can serve as valuable genetic resources for future crop dwarfing breeding.

Efficient preparation of high-purity and intact mesenchymal stem cell-derived extracellular vesicles.

Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) have shown great promise for regeneration and immunomodulation. However, efficient and scalable methods for their preparation are still lacking. In this study, we present the adoption of a label-free technique known as "EXODUS" to isolate and purify MSC-EVs from the conditioned medium. Our findings indicate that EXODUS can rapidly isolate EVs from 10 mL of conditioned medium with a 5-fold higher yield compared to conventional approaches, including ultracentrifugation (UC) and polyethylene glycol precipitation (PEG) methods. Additionally, pre-storing the conditioned medium at 4°C for 1 week resulted in a ~2-fold higher yield of MSC-EVs compared to the freshly prepared medium. However, storing the purified EV particles at 4°C for 1 month led to a 2-fold reduction in particle concentration. Furthermore, we found that MSC-EVs isolated using EXODUS exhibit higher expression levels of EV markers such as Alix, Flotillin1, CD81, and TSG101 in comparison to PEG and UC methods. We also discovered that MSC-EVs isolated using EXODUS are enriched in response to cytokine, collagen-containing extracellular matrix, and calcium ion binding compared to PEG method and enriched in extracellular structure organization, extracellular matrix, and extracellular matrix structure constituents compared to UC. Finally, we demonstrated that MSC-EVs isolated using EXODUS exhibit greater potential in animal organ development, tissue development, and anatomical structure morphogenesis compared to the UC. These findings suggest that EXODUS is a suitable method for the large-scale preparation of high-quality MSC-EVs for various clinical applications.

Genome-wide characterization and expression analysis of α-amylase and ß-amylase genes underlying drought tolerance in cassava.

BACKGROUND: Starch hydrolysates are energy sources for plant growth and development, regulate osmotic pressure and transmit signals in response to both biological and abiotic stresses. The α-amylase (AMY) and the ß-amylase (BAM) are important enzymes that catalyze the hydrolysis of plant starch. Cassava (Manihot esculenta Crantz) is treated as one of the most drought-tolerant crops. However, the mechanisms of how AMY and BAM respond to drought in cassava are still unknown. RESULTS: Six MeAMY genes and ten MeBAM genes were identified and characterized in the cassava genome. Both MeAMY and MeBAM gene families contain four genes with alternative splicing. Tandem and fragment replications play important roles in the amplification of MeAMY and MeBAM genes. Both MeBAM5 and MeBAM10 have a BZR1/BES1 domain at the N-terminus, which may have transcription factor functions. The promoter regions of MeAMY and MeBAM genes contain a large number of cis-acting elements related to abiotic stress. MeAMY1, MeAMY2, MeAMY5, and MeBAM3 are proven as critical genes in response to drought stress according to their expression patterns under drought. The starch content, soluble sugar content, and amylase activity were significantly altered in cassava under different levels of drought stress. CONCLUSIONS: These results provide fundamental knowledge for not only further exploring the starch metabolism functions of cassava under drought stress but also offering new perspectives for understanding the mechanism of how cassava survives and develops under drought.

Metabolomic investigation of urinary extracellular vesicles for early detection and screening of lung cancer.

Lung cancer is a prevalent cancer type worldwide that often remains asymptomatic in its early stages and is frequently diagnosed at an advanced stage with a poor prognosis due to the lack of effective diagnostic techniques and molecular biomarkers. However, emerging evidence suggests that extracellular vesicles (EVs) may promote lung cancer cell proliferation and metastasis, and modulate the anti-tumor immune response in lung cancer carcinogenesis, making them potential biomarkers for early cancer detection. To investigate the potential of urinary EVs for non-invasive detection and screening of patients at early stages, we studied metabolomic signatures of lung cancer. Specifically, we conducted metabolomic analysis of 102 EV samples and identified metabolome profiles of urinary EVs, including organic acids and derivatives, lipids and lipid-like molecules, organheterocyclic compounds, and benzenoids. Using machine learning with a random forest model, we screened for potential markers of lung cancer and identified a marker panel consisting of Kanzonol Z, Xanthosine, Nervonyl carnitine, and 3,4-Dihydroxybenzaldehyde, which exhibited a diagnostic potency of 96% for the testing cohort (AUC value). Importantly, this marker panel also demonstrated effective prediction for the validation set, with an AUC value of 84%, indicating the reliability of the marker screening process. Our findings suggest that the metabolomic analysis of urinary EVs provides a promising source of non-invasive markers for lung cancer diagnostics. We believe that the EV metabolic signatures could be used to develop clinical applications for the early detection and screening of lung cancer, potentially improving patient outcomes.

Could extended laminectomy effectively prevent spinal cord injury due to spinal shortening after 3-column osteotomy?

OBJECTIVE: To explore whether the laminectomy extension can effectively prevent spinal cord injury (SCI) due to spinal shortening after 3-column osteotomy in goat models. METHODS: A total of twenty healthy goats were included and done with 3-column osteotomy of T13 and L1 under the somatosensory evoked potential (SSEP) monitoring. The samples were divided into two groups. The first group underwent regular laminectomy while the second group underwent an extended laminectomy with an extra 10 mm-lamina cranial to L2. The SSEP measured after 3-column osteotomy was set as the baseline, and the SSEP decreased by 50% from the baseline amplitude and/or delayed by 10% relative to the baseline peak latency was set as positive results, which indicated spinal cord injury. The vertebral column was gradually shortened until the SSEP monitoring just did not show a positive result. The height of the initial osteotomy gap (the distance from the lower endplate of T12 to the upper endplate of L2), the shortened distance (△H), the number of spinal cord angulated and the changed angle of the spinal cord (△α) were measured and recorded in each group. Neurological function was evaluated by the Tarlov scores on day 2 postoperatively. RESULTS: All the goats except one of the first group due to changes in the SSEP during the osteotomy were included and analyzed. In the first group, the height of the initial osteotomy segment and the safe shortening distances were 61.6 ± 2.6 mm and 35.2 ± 2.6 mm, respectively; the spinal cord of 5 goats was angulated (46.4 ± 6.6°), the other four goats were kinked and not angulated. In the second group, the height of the initial osteotomy segment and the safe shortening distances were 59.8 ± 1.5 mm and 43.3 ± 1.2 mm, respectively, and the spinal cord of ten goats were angulated (97.6 ± 7.2°). There was no significant difference in the height of the initial osteotomy segment between the two groups by using Independent-Samples T-Test, P = 0.095 (P > 0.05); there were significant difference in the safe shortening distance and the changed angle of the spinal cord between the two groups by using Independent-Samples T-Test (both [Formula: see text]H and [Formula: see text]α of P < 0.001), the difference between their mean were 8.1 mm and 51.2°. Significant difference was found in the number of spinal cord angulation between the two groups through Fisher's exact test (5/9 vs. 10/10, P = 0.033). CONCLUSIONS: An additional resection of 10 mm-lamina cranial to L2 showed the satisfactory effect in alleviating SCI after 3-column osteotomy. Timely and appropriate extend laminectomy could be a promising therapeutic strategy for SCI attributable to facilitating spinal cord angulation rather than spinal cord kinking and increasing the safe shortening distance.

Lipidomic identification of urinary extracellular vesicles for non-alcoholic steatohepatitis diagnosis.

BACKGROUND AND AIMS: Non-alcoholic fatty liver disease (NAFLD) is a usual chronic liver disease and lacks non-invasive biomarkers for the clinical diagnosis and prognosis. Extracellular vesicles (EVs), a group of heterogeneous small membrane-bound vesicles, carry proteins and nucleic acids as promising biomarkers for clinical applications, but it has not been well explored on their lipid compositions related to NAFLD studies. Here, we investigate the lipid molecular function of urinary EVs and their potential as biomarkers for non-alcoholic steatohepatitis (NASH) detection. METHODS: This work includes 43 patients with non-alcoholic fatty liver (NAFL) and 40 patients with NASH. The EVs of urine were isolated and purified using the EXODUS method. The EV lipidomics was performed by LC-MS/MS. We then systematically compare the EV lipidomic profiles of NAFL and NASH patients and reveal the lipid signatures of NASH with the assistance of machine learning. RESULTS: By lipidomic profiling of urinary EVs, we identify 422 lipids mainly including sterol lipids, fatty acyl lipids, glycerides, glycerophospholipids, and sphingolipids. Via the machine learning and random forest modeling, we obtain a biomarker panel composed of 4 lipid molecules including FFA (18:0), LPC (22:6/0:0), FFA (18:1), and PI (16:0/18:1), that can distinguish NASH with an AUC of 92.3%. These lipid molecules are closely associated with the occurrence and development of NASH. CONCLUSION: The lack of non-invasive means for diagnosing NASH causes increasing morbidity. We investigate the NAFLD biomarkers from the insights of urinary EVs, and systematically compare the EV lipidomic profiles of NAFL and NASH, which holds the promise to expand the current knowledge of disease pathogenesis and evaluate their role as non-invasive biomarkers for NASH diagnosis and progression.

Relationship between the laminectomy extension and spinal cord injury caused by acute spinal shortening: goat in vivo experiment.

OBJECTIVE: To investigate the relationship between different types of laminectomy extension and spinal cord injury subsequent to acute spinal shorting after 3-column osteotomy in living goat model. METHODS: A total of 18 healthy goats were selected, and a procedure of bivertebral column resections and total laminectomy of T13 and L1 was completed followed by different laminectomy extensions under the somatosensory evoked potential (SSEP) monitoring. The samples were divided into three groups according to types of subsequent laminectomy extension. In the first group (enlarged resection of upper lamina group), laminectomy extension was performed on 10 mm caudal to T12; in the second group (equidistant enlarged resection of upper and lower lamina group), laminectomy extension was performed on 5 mm caudal to T12 and 5 mm cranial to L2 simultaneously; and in the third group (enlarged resection of lower lamina group), laminectomy extension was performed on 10 mm cranial to L2. The SSEP measured after vertebral resection was set as the baseline, and the SSEP decreased by 50% from the baseline amplitude and/or delayed by 10% relative to the baseline peak latency was set as positive results, which indicated spinal cord injury. Spinal column was gradually shortened until the SSEP monitoring just did not show a positive result. The shortened distance (ΔH) and the changed angle of the spinal cord buckling (Δα) were measured in each group. Neurologic function was recorded by the Tarlov scores at 2 days after the surgery. RESULTS: The safe shortening distances of three groups were 38.6 ± 1.2 mm, 41.5 ± 0.7 mm, 43.7 ± 0.8 mm, respectively; the corresponding changed angles of the spinal cord buckling were 62.8 ± 6.9°, 82.8 ± 7.5°, and 98.5 ± 7.0°. Significant differences of ΔH and Δα were found among the three groups by LSD multiple comparison test (P < 0.05). Strong correlation between ΔH and Δα was shown in each group by Pearson's correlation test. CONCLUSIONS: Different laminectomy extensions after 3-column osteotomy have different effects on the prevention of SCI caused by acute spinal shortening. The enlarged resection of lower lamina is superior to equidistant enlarged resection of upper and lower laminas which is superior to enlarged resection of upper lamina in preventing SCI. These slides can be retrieved under Electronic Supplementary Material.

Identification and detection of plasma extracellular vesicles-derived biomarkers for esophageal squamous cell carcinoma diagnosis.

Esophageal cancer is a malignant tumor with two-thirds of patients having a local recurrence or distant metastasis. To date, diagnostic biomarkers with high sensitivity and specificity are lacking. Extracellular vesicles (EVs) have shown their potential values as disease biomarkers as they carry specific proteins and RNAs derived from cancer cells. In this study, we investigate ESCC precision diagnostics from the insights of circulating EVs, and integrate the ultrafast EV isolation approach (EXODUS) and ELISA for fast detection and screening of ESCC patients. First, we isolate and characterize the high-purity plasma EVs with EXODUS and identify 401 proteins and 372 proteins from ESCC patient and healthy individuals, respectively. Further looking into the differentially expressed proteins (DEPs) of ESCC patients and enriched KEGG pathways, we discover EV-CD14 as a potential diagnostic biomarker for ESCC, which has been further validated as a significantly differentially expressed protein by Western Blot and immunogold labelling TEM. For fast screening and detection of ESCC towards clinical applications, we apply ELISA method to diagnose ESCC from 60 clinical samples based on circulating EV-CD14, which shows a high AUC value up to 96.0% for detection of ESCC in a test set (30 samples), and displays a high accuracy rate up to 90% for prediction of ESCC in a screening test (30 samples). Our results suggest that the circulating EV-CD14 may highly be related to the initiation and progression of ESCC, providing a novel method for the diagnosis and prognosis of ESCC towards clinical translations.

Robust Acute Pancreatitis Identification and Diagnosis: RAPIDx.

The occurrence of acute pancreatitis (AP) is increasing significantly worldwide. However, current diagnostic methods of AP do not provide a clear clinical stratification of severity, and the prediction of complications in AP is still limited. Here, we present a robust AP identification and diagnosis (RAPIDx) method by the proteomic fingerprinting of intact nanoscale extracellular vesicles (EVs) from clinical samples. By tracking analysis of circulating biological nanoparticles released by cells (i.e., EVs) via bottom-up proteomics, we obtain close phenotype connections between EVs, cell types, and multiple tissues based on their specific proteomes and identify the serum amyloid A (SAA) proteins on EVs as potential biomarkers that are differentially expressed from AP patients significantly. We accomplish the quantitative analysis of EVs fingerprints using MALDI-TOF MS and find the SAA proteins (SAA1-1, desR-SAA1-2, SAA2, SAA1-2) with areas under the curve (AUCs) from 0.92 to 0.97, which allows us to detect AP within 30 min. We further realize that SAA1-1 and SAA2, combined with two protein peaks (5290.19, 14032.33 m/z), can achieve an AUC of 0.83 for classifying the severity of AP. The RAPIDx platform will facilitate timely diagnosis and treatment of AP before severity development and persistent organ failure and promote precision diagnostics and the early diagnosis of pancreatic cancer.

Interaction network of extracellular vesicles building universal analysis via eye tears: iNEBULA.

Discovering the secrets of diseases from tear extracellular vesicles (EVs) is well-recognized and appreciated. However, a precise understanding of the interaction network between EV populations and their biogenesis from our body requires more in-depth and systematic analysis. Here, we report the biological profiles of different-size tear EV subsets from healthy individuals and the origins of EV proteins. We have identified about 1800 proteins and revealed the preferential differences in the biogenesis among distinct subsets. We observe that eye-related proteins that maintain retinal homeostasis and regulate inflammation are preferentially enriched in medium-size EVs (100 to 200 nm) fractions. Using universal analysis in combination with the Human Protein Atlas consensus dataset, we found the genesis of tear EV proteins with 37 tissues and 79 cell types. The proteins related to retinal neuronal cells, glial cells, and blood and immune cells are selectively enriched among EV subsets. Our studies in heterogeneous tear EVs provide building blocks for future transformative precision molecular diagnostics and therapeutics.

Safe Limit of Shortening of the Spinal Cord in Thoracolumbar Bivertebral Column Resections: An Experimental Study in Goats.

OBJECTIVE: To clarify the safe limit of shortening of the spinal cord in thoracolumbar bivertebral column resection in a goat model. METHODS: Ten healthy goats were selected for the experiment. Radiographs were taken before surgery to measure the height of T13, L1, and the initial osteotomy segment (distance from the lower end plate of T12 to the upper end plate of L2). A procedure of thoracolumbar bivertebral column resection (T13 and L1) was completed under the monitoring of somatosensory evoked potential (SSEP) monitoring. The SSEP measured after vertebral resection was set as the baseline. SSEPs decreased by 50% from the baseline amplitude and/or delayed by 10% relative to the baseline peak latency were set as positive results, indicating spinal cord injury. The initial height of the osteotomy gap was measured first and the spinal column was gradually shortened until the SSEP monitoring did not show a positive result. Then the height of the osteotomy gap was recorded again. The safe limit of shortening was measured and recorded when any morphologic change of the spinal cord was observed. Hindlimb function was evaluated by the Tarlov scores on day 2 postoperatively. RESULTS: The safe limit of shortening of the spinal cord in thoracolumbar bivertebral columns resection was 35.2 ± 2.6 mm, which was roughly equal to 127.6% of the mean osteotomy vertebral height and 57.1% of the initial osteotomy gap height. Pearson correlation test showed that the safe limit of shortening of the spinal cord was correlated with the height of T13, the height of L1, the mean height of T13 and L1, and the height of the initial osteotomy gap. CONCLUSIONS: The safe limit of shortening distance of the bivertebral column resection was roughly equal to 127.6% of the mean osteotomy vertebral height and 57.1% of the initial osteotomy gap height with good correlation. Moreover, the safe limit of shortening distance of the bivertebral column resection was longer than that in single vertebral column resection. Increasing the number of vertebrae resected may prevent spinal cord injury because of excessive shortening.