Association of Lipoprotein(a) Levels With Myocardial Infarction in Patients With Low-Attenuation Plaque.

BACKGROUND: Lipoprotein(a) (Lp[a]) is associated with an increased risk of myocardial infarction (MI). However, the mechanism underlying this association has yet to be fully elucidated. OBJECTIVES: This multicenter study aimed to investigate whether association between Lp(a) and MI risk is reinforced by the presence of low-attenuation plaque (LAP) identified by coronary computed tomography angiography (CCTA). METHODS: In a derivation cohort, a total of 5,607 patients with stable chest pain suspected of coronary artery disease who underwent CCTA and Lp(a) measurement were prospectively enrolled. In validation cohort, 1,122 patients were retrospectively collected during the same period. High Lp(a) was defined as Lp(a) ≥50 mg/dL. The primary endpoint was a composite of time to fatal or nonfatal MI. Associations were estimated using multivariable Cox proportional hazard models. RESULTS: During a median follow-up of 8.2 years (Q1-Q3: 7.2-9.3 years), the elevated Lp(a) levels were associated with MI risk (adjusted HR [aHR]: 1.91; 95% CI: 1.46-2.49; P < 0.001). There was a significant interaction between Lp(a) and LAP (Pinteraction <0.001) in relation to MI risk. When stratified by the presence or absence of LAP, Lp(a) was associated with MI in patients with LAP (aHR: 3.03; 95% CI: 1.92-4.76; P < 0.001). Mediation analysis revealed that LAP mediated 73.3% (P < 0.001) for the relationship between Lp(a) and MI. The principal findings remained unchanged in the validation cohort. CONCLUSIONS: Elevated Lp(a) augmented the risk of MI during 8 years of follow-up, especially in patients with LAP identified by CCTA. The presence of LAP could reinforce the relationship between Lp(a) and future MI occurrence.

Alternative Biological Material for Tissue Engineering of the Vagina: Porcine-Derived Acellular Vaginal Matrix.

BACKGROUND: Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome is a severe congenital disorder characterized by vaginal hypoplasia caused by dysplasia of the Müllerian duct. Patients with MRKH syndrome often require nonsurgical or surgical treatment to achieve satisfactory vaginal length and sexual outcomes. The extracellular matrix has been successfully used for vaginal reconstruction. METHODS: In this study, we developed a new biological material derived from porcine vagina (acellular vaginal matrix, AVM) to reconstruct the vagina in Bama miniature pigs. The histological characteristics and efficacy of acellularization of AVM were evaluated, and AVM was subsequently transplanted into Bama miniature pigs to reconstruct the vaginas. RESULTS: Macroscopic analysis showed that the neovaginas functioned well in all Bama miniature pigs with AVM implants. Histological analysis and electrophysiological evidence indicated that morphological and functional recovery was restored in normal vaginal tissues. Scanning electron microscopy showed that the neovaginas had mucosal folds characteristics of normal vagina. No significant differences were observed in the expression of CK14, HSP47, and α-actin between the neovaginas and normal vaginal tissues. However, the expression of estrogen receptor (ER) was significantly lower in the neovaginas than in normal vaginal tissues. In addition, AVM promoted the expression of ß-catenin, c-Myc, and cyclin D1. These results suggest that AVM might promotes vaginal regeneration by activating the ß-catenin/c-Myc/cyclin D1 pathway. CONCLUSION: This study reveals that porcine-derived AVM has potential application for vaginal regeneration.

Associations of MRI-Derived Glymphatic System Impairment With Global White Matter Damage and Cognitive Impairment in Mild Traumatic Brain Injury: A DTI-ALPS Study.

BACKGROUND: Assessing the glymphatic function using diffusion tensor image analysis along the perivascular space (DTI-ALPS) may be helpful for mild traumatic brain injury (mTBI) management. PURPOSE: To assess glymphatic function using DTI-ALPS and its associations with global white matter damage and cognitive impairment in mTBI. STUDY TYPE: Prospective. POPULATION: Thirty-four controls (44.1% female, mean age 49.2 years) and 58 mTBI subjects (43.1% female, mean age 48.7 years), including uncomplicated mTBI (N = 32) and complicated mTBI (N = 26). FIELD STRENGTH/SEQUENCE: 3-T, single-shot echo-planar imaging sequence. ASSESSMENT: Magnetic resonance imaging (MRI) was done within 1 month since injury. DTI-ALPS was performed to assess glymphatic function, and peak width of skeletonized mean diffusivity (PSMD) was used to assess global white matter damage. Cognitive tests included Auditory Verbal Learning Test and Digit Span Test (forward and backward). STATISTICAL TESTS: Neuroimaging findings comparisons were done between mTBI and control groups. Partial correlation and multivariable linear regression assessed the associations between DTI-ALPS, PSMD, and cognitive impairment. Mediation effects of PSMD on the relationship between DTI-ALPS and cognitive impairment were explored. P-value <0.05 was considered statistically significant, except for cognitive correlational analyses with a Bonferroni-corrected P-value set at 0.05/3 ≈ 0.017. RESULTS: mTBI showed lower DTI-ALPS and higher PSMD, especially in complicated mTBI. DTI-ALPS was significantly correlated with verbal memory (r = 0.566), attention abilities (r = 0.792), executive function (r = 0.618), and PSMD (r = -0.533). DTI-ALPS was associated with verbal memory (ß = 8.77, 95% confidence interval [CI] 5.00, 12.54), attention abilities (ß = 5.67, 95% CI 4.56, 6.97), executive function (ß = 2.34, 95% CI 1.49, 3.20), and PSMD (ß = -0.79, 95% CI -1.15, -0.43). PSMD mediated 46.29%, 20.46%, and 24.36% of the effects for the relationship between DTI-ALPS and verbal memory, attention abilities, and executive function. DATA CONCLUSION: Glymphatic function may be impaired in mTBI reflected by DTI-ALPS. Glymphatic dysfunction may cause cognitive impairment related to global white matter damage after mTBI. LEVEL OF EVIDENCE: 2 TECHNICAL EFFICACY: Stage 2.

Determination of microplastics in agricultural soil by double-shot pyrolysis-gas chromatography combined with two-step extraction.

A method for the determination of five microplastics in agricultural soil was established by double-shot pyrolysis-gas chromatography combined with two-step extraction. First, polycarbonate (PC), polystyrene (PS), polypropylene (PP), and polyethylene (PE) were extracted from soil samples using a mixed solvent of cyclohexanone and p-xylene, and then PE terephthalate was extracted with m-methylphenol. Subsequently, PC and PE terephthalate were analyzed by thermochemolysis, and PE, PP, and PS were investigated by direct pyrolysis at 600°C. The linearity of the method was satisfactory for five microplastics and the correlation coefficients were higher than 0.97 in the respective concentration range. The limits of detection and the limits of quantification were 0.2-10.0 and 0.5-20.0 µg/g, respectively. The method provided recoveries of 75.1%-141.5%, with acceptable repeatability within 20.0%. It was a supplementary method for the existing characterization of microplastics in agricultural soil.

Elevated plasma pyruvate kinase M2 concentrations are associated with the clinical severity and prognosis of coronary artery disease.

Introduction: Pyruvate kinase M2 (PKM2) was involved in the pathophysiology of atherosclerosis and coronary artery disease (CAD). We tested whether plasma PKM2 concentrations were correlated with clinical severity and major adverse cardiovascular events (MACEs) in CAD patients. Materials and methods: A total of 2443 CAD patients and 238 controls were enrolled. The follow-up time was two years. Plasma PKM2 concentrations were detected by enzyme-linked immunosorbent assay (ELISA) kits (Cloud-Clone, Wuhan, China) using SpectraMax i3x Multi-Mode Microplate Reader (Molecular Devices, San Jose, USA). The predictors of acute coronary syndrome (ACS) were assessed by logistic regression analysis. The association between PKM2 concentration in different quartiles and MACEs was evaluated by Kaplan-Meier (KM) curves with log-rank test and Cox proportional hazard models. The predictive value of PKM2 and a cluster of conventional risk factors was determined by Receiver operating characteristic (ROC) curves. The net reclassification improvement (NRI) and the integrated discrimination improvement (IDI) were utilized to evaluate the enhancement in risk prediction when PKM2 was added to a predictive model containing a cluster of conventional risk factors. Results: In CAD patients, PKM2 concentration was the independent predictor of ACS (P < 0.001). Kaplan-Meier cumulative survival curves and Cox proportional hazards analyses revealed that patients with a higher PKM2 concentration had higher incidence of MACEs compared to those with a lower PKM2 concentration (P < 0.001). The addition of PKM2 to a cluster of conventional risk factors significantly increased its prognostic value of MACEs. Conclusion: Baseline plasma PKM2 concentrations predict the clinical severity and prognosis of CAD.

Reversal of pulmonary veno-occlusive disease phenotypes by inhibition of the integrated stress response.

Pulmonary veno-occlusive disease (PVOD) is a rare form of pulmonary hypertension arising from EIF2AK4 gene mutations or mitomycin C (MMC) administration. The lack of effective PVOD therapies is compounded by a limited understanding of the mechanisms driving the vascular remodeling in PVOD. We show that the administration of MMC in rats mediates the activation of protein kinase R (PKR) and the integrated stress response (ISR), which lead to the release of the endothelial adhesion molecule VE-Cadherin in the complex with Rad51 to the circulation, disruption of endothelial barrier, and vascular remodeling. Pharmacological inhibition of PKR or ISR attenuates the depletion of VE-Cadherin, elevation of vascular permeability, and vascular remodeling instigated by MMC, suggesting potential clinical intervention for PVOD. Finally, the severity of PVOD phenotypes was increased by a heterozygous BMPR2 mutation that truncates the carboxyl tail of BMPR2, underscoring the role of deregulated BMP signal in the development of PVOD.

Alliance between titans: combination strategies of CAR-T cell therapy and oncolytic virus for the treatment of hematological malignancies.

There have been several clinical studies using chimeric antigen receptor (CAR)-T cell therapy for different hematological malignancies. It has transformed the therapy landscape for hematologic malignancies dramatically. Nonetheless, in acute myeloid leukemia (AML) and T cell malignancies, it still has a dismal prognosis. Even in the most promising locations, recurrence with CAR-T treatment remains a big concern. Oncolytic viruses (OVs) can directly lyse tumor cells or cause immune responses, and they can be manipulated to create therapeutic proteins, increasing anticancer efficacy. Oncolytic viruses have been proven in a rising number of studies to be beneficial in hematological malignancies. There are limitations that cannot be avoided by using either treatment alone, and the combination of CAR-T cell therapy and oncolytic virus therapy may complement the disadvantages of individual application, enhance the advantages of their respective treatment methods and improve the treatment effect. The alternatives for combining two therapies in hematological malignancies are discussed in this article.

Vaginal reconstruction with a double-sided biomembrane-a preclinical experimental study on large animals.

Tissue engineering techniques bring the promise of vaginal reconstruction with low invasiveness and fewer complications. However, existing biomaterial scaffolds remain limited in efficient vaginal recovery, focusing only on regenerating an epithelial layer, but muscle layers are missing or abnormal. The lack of a multi-tissue hierarchical structure in the reconstructed vagina leads to shrinking, stenosis, and fibrosis. Here, an acellular matrix named a double-sided biomembrane (DBM) is demonstrated for vaginal recovery. The regeneration of epithelial and muscle layers is achieved simultaneously since the smooth side of the DBM is helpful for guiding epithelial cell growth, while its loose and porous side guides muscle cell growth. In addition, the DBM demonstrates excellent mechanical properties similar to vaginal tissue, and hydrophilicity. Therefore, neovaginas were observed in the fourth and twelfth weeks after DBMs were transplanted to repair full-thickness vaginal defects (4 cm) that we established in large animals. The DBMs can effectively promote rapid epithelialization, the formation of large muscle bundles, higher rates of angiogenesis, and the restoration of physiological function in a neovagina. That is, the injured vagina achieves nearly complete recovery in anatomy and function, similar to a normal vagina. These preclinical results indicate that the DBM has prospects for vaginal injury repair.

Human umbilical cord mesenchymal stem cell­derived exosomes improve ovarian function in natural aging by inhibiting apoptosis.

Prolonging the reproductive lifespan is beneficial for preserving the physical and psychological health of women. The transplantation of mesenchymal stem cell (MSC)­derived exosomes (MSC­Exos) has been reported to be a promising regenerative therapeutic strategy for restoring the function of aging ovaries. The present study thus evaluated the therapeutic efficacy of exosomes derived from human umbilical cord­MSCs (hUCMSC­Exos) in a mouse model of natural ovarian aging (NOA), and further investigated the role of exosomal microRNAs (miRNAs/miRs) in the mechanisms of this creative therapy. Specifically, following the administration of hUCMSC­Exos in mice with NOA, ovarian function was found to improve, as indicated by the restoration of follicle numbers and hormone levels. These exosomes were found to exhibit the ability to inhibit PTEN expression and suppress apoptosis both in vivo and in vitro. Subsequently, miRNA sequencing of the exosomes was performed, following which bioinformatics analysis was used to identify the highly expressed miRNAs that are capable of targeting PTEN expression. Through high­throughput sequencing and molecular analyses, miR­21­5p was found to be the highest in ranking in terms of expression, suggesting that hUCMSC­Exos can preserve ovarian function by suppressing PTEN expression to inhibit apoptosis by delivering miR­21­5p. On the whole, the results of the present study suggest that the application of exosomes can be used to restore ovarian function in mice with NOA. These positive findings also suggest that the transplantation of exosomes derived from MSCs holds promise as an agent against ovarian aging.

The interspecific competition of tree plants in the presence of AM fungi and litter facilitates root morphological development and nutrition when compared with intraspecific competition.

Arbuscular mycorrhizal (AM) fungi can affect plant growth by regulating competition. Nutrient-deficient karst habitats contain abundant plants that compete for nutrients through interspecific or intraspecific competition, involving the nutritional transformation of litter decomposition. However, how plant competition in the presence of AM fungi and litter affects root development and nutrition remains unclear. A potted experiment was conducted, including AM fungus treatment with or without Glomus etunicatum, the competition treatment concerning intraspecific or interspecific competition through planting Broussonetia papyrifera and Carpinus pubescens seedlings, and the litter treatment with or without the mixture of B. papyrifera and C. pubescens litter leaves. The root morphological traits were analyzed, and nitrogen (N), phosphorus (P), and potassium (K) were measured. The results showed that AM fungus differently affected the root morphological development and nutrition of both competitive plants, significantly promoting B. papyrifera roots in the increase of dry weight, length, volume, surface area, tips, and branches as well as N, P, and K acquisitions regardless of litter addition. However, there was no apparent influence for C. pubescens roots, except for the diameter in the interspecific competition with litter. The root dry weight, length, volume, surface area, and tips of B. papyrifera under two competitive styles were significantly greater than C. pubescens regulated by AM fungus, presenting significant species differences. The responses of the relative competition intensity (RCI) on root morphological and nutritional traits indicated that AM fungus and litter both asymmetrically alleviated more competitive pressure for B. papyrifera than C. pubescens, and the interspecific competition facilitated more root morphological development and nutrition utilization by endowing B. papyrifera root superiority relative to C. pubescens compared with the intraspecific competition. In conclusion, interspecific competition is more beneficial for plant root development and nutrition than intraspecific competition in the presence of AM fungus and litter via asymmetrically alleviating competitive pressure for different plants.

Small intestinal submucosa promotes angiogenesis via the Hippo pathway to improve vaginal repair.

Vaginal reconstruction has incorporated the use of gastrointestinal segments for decades, but the technique is inevitably associated with complications. Tissue-engineering techniques, however, have brought great hope for vaginal reconstruction. This study aimed to evaluate the utility of small intestinal submucosa (SIS) in reconstructing clinically significant large vaginal defects in a porcine model and to investigate the role of the Hippo pathway in the vascular remodeling process. The composition and mechanical properties of SIS were characterized. Full-thickness vaginal defects were established in 10 minipig donors, with 4 cm lengths removed and replaced by an equal sized SIS scaffolds. The neovaginas were subjected to macroscopic, histological, immunohistochemical and molecular evaluations at 4 and 12 weeks after the surgery. Four weeks after the operation, extracellular matrix reorganization and complete coverage of the surface of the luminal matrix by vaginal epithelium were observed, accompanied by the formation of a microvascular network and the regeneration of smooth muscles, albeit disorderly arranged. Twelve weeks after implantation, enhancements were seen in the formation of the multi-layered squamous epithelium, angiogenesis, and large muscle bundle formation in the vagina. Additionally, the expression levels of angiogenesis-related proteins, proliferation-related proteins and Hippo pathway-related proteins in the neovagina were significantly increased. These results indicate that SIS could be used to reconstruct large vaginal defects and that the vascular remodeling process is potentially regulated by the Hippo pathway.

Damage and failure of underground subway stations under different soil constraint conditions.

Investigations from past earthquakes have shown that underground subway stations can suffer excessive deformation under strong seismic loads, leading to the damage of critical components and the collapse of structures. This study presents the results of finite element analyses on the seismic damage of underground subway stations installed under different soil constraint conditions. The plastic hinge distribution and damage characteristics of cut and cover double-storey and three-storey subway stations are analyzed using the finite element method software ABAQUS. Combined with the static analysis results of the column sections, a discriminant method for bending plastic hinges is presented. The numerical results show that the collapse of the subway stations begins with the failure of the bottom columns' bottom sections, which leads to the bending of the plates and the destruction of the whole structure. The bending deformation at the end section of columns has an approximatively linear relationship with the inter-storey drift ratio, and the change in soil conditions shows no apparent influence. The sidewall deformation behavior varies significantly under different soil conditions, and the bending deformation at the bottom section of sidewalls increases along with an increase in the soil-structure stiffness ratio at the same inter-storey drift deformation level. The sidewall bending ductility ratio of the double-storey and three-storey stations at the elastic-plastic drift ratio limit increases by 61.6% and 26.7%, respectively. In addition, the fitting curves between the component bending ductility ratio and inter-storey drift ratio based on the analysis results are also presented. The findings may provide a helpful reference for the seismic performance evaluation and design of underground subway stations.

De novo domestication: retrace the history of agriculture to design future crops.

Certain crops were domesticated from their wild progenitors and have served as the major staple food since then, but now suffered from the limited genetic diversity in breeding. Enormous wild species possess unique advantages such as stress tolerance, polyploidy, perennial habit, and natural nutrition. However, it remains a big challenge to utilize wild species in conventional breeding. With recent advances in biotechnologies, one new breeding strategy, de novo domestication, has emerged and been demonstrated by pioneer work. Here, we review the emergence and milestone progress of de novo domestication and discuss how wild relatives could be exploited into new types of crops. With the understanding of the genetic basis of crop domestication and the development of biotechnologies, various elite wild germplasms will be designed and practiced to fulfill particular breeding goals and create new types of crops. De novo domestication is paving a new way for breeding the future.

Development and validation of a MRI-based combined radiomics nomogram for differentiation in chondrosarcoma.

Objective: This study aims to develop and validate the performance of an unenhanced magnetic resonance imaging (MRI)-based combined radiomics nomogram for discrimination between low-grade and high-grade in chondrosarcoma. Methods: A total of 102 patients with 44 in low-grade and 58 in high-grade chondrosarcoma were enrolled and divided into training set (n=72) and validation set (n=30) with a 7:3 ratio in this retrospective study. The demographics and unenhanced MRI imaging characteristics of the patients were evaluated to develop a clinic-radiological factors model. Radiomics features were extracted from T1-weighted (T1WI) images to construct radiomics signature and calculate radiomics score (Rad-score). According to multivariate logistic regression analysis, a combined radiomics nomogram based on MRI was constructed by integrating radiomics signature and independent clinic-radiological features. The performance of the combined radiomics nomogram was evaluated in terms of calibration, discrimination, and clinical usefulness. Results: Using multivariate logistic regression analysis, only one clinic-radiological feature (marrow edema OR=0.29, 95% CI=0.11-0.76, P=0.012) was found to be independent predictors of differentiation in chondrosarcoma. Combined with the above clinic-radiological predictor and the radiomics signature constructed by LASSO [least absolute shrinkage and selection operator], a combined radiomics nomogram based on MRI was constructed, and its predictive performance was better than that of clinic-radiological factors model and radiomics signature, with the AUC [area under the curve] of the training set and the validation set were 0.78 (95%CI =0.67-0.89) and 0.77 (95%CI =0.59-0.94), respectively. DCA [decision curve analysis] showed that combined radiomics nomogram has potential clinical application value. Conclusion: The MRI-based combined radiomics nomogram is a noninvasive preoperative prediction tool that combines clinic-radiological feature and radiomics signature and shows good predictive effect in distinguishing low-grade and high-grade bone chondrosarcoma, which may help clinicians to make accurate treatment plans.

Doppler Echocardiography Combined with NTproBNP/BNP in the Diagnosis of Pulmonary Artery Hypertension Associated with Congenital Heart Disease.

Background: Pulmonary artery hypertension (PAH) is a common complication of congenital heart disease (CHD) and is associated with worse outcomes and increased mortality. The Doppler echocardiography (DE) is a commonly used imaging tool for both diagnosis and follow-up examination of PAH. Here is to evaluate the diagnostic performance of DE combined with NTproBNP/BNP as screening strategy in PAH patients with CHD. Methods: A retrospective study in 64 patients with CHD has been carried out to compare estimate pulmonary artery systolic pressure (PASP) measured with DE to that measured with right heart catheterization (RHC). The Pearson correlation analyses were used to calculate the correlation coefficients between RHC and DE. The Bland-Altman analyses were carried out to assess the agreement between the two methods. ROC analyses were used to evaluate the diagnostic performance of DE, NTproBNP/BNP, and DE combined with NTproBNP/BNP. Results: Our data have demonstrated that a mild correlation (r = 0.4401, P < 0.01) was observed between PASP (78.1 ± 29.0 mmHg) measured during RHC and PASP (74.9 ± 19.7 mmHg) as estimated using DE. The Bland-Altman analysis demonstrated that the bias for DE PASP estimates was 3.2 mmHg with 95% limits of agreement ranging from -49.53 to 55.90 mmHg. The results of DE showed an AUC of 0.848 (95% CI = 0.666-1; P < 0.001), the sensitivity of which was 98.3% and the specificity was 77.8%. The AUC of NTproBNP/BNP for the identification of PAH was 0.804 (95% CI = 0.651-0.956; P < 0.001), the sensitivity of which was 81.4% and the specificity was 87.5%. The AUC of DE combined with NTproBNP/BNP was 0.857 (95% CI = 0.676-1; P < 0.001), of which sensitivity was 100% and specificity was 77.8%. The positive predictive value (PPV) and negative predictive value (NPV) were 96.6% and 100%, respectively. Conclusions: Our study shows that the Doppler echocardiography combined with NTproBNP/BNP has better diagnostic performance in pulmonary artery hypertension associated with congenital heart disease, especially when DE negative screening in PAH patients.

Dysregulated miR-222-3p in plasma exosomes of preeclampsia patients and its In vitro effect on HTR8/SVneo extravillous trophoblast cells by targeting STMN1.

OBJECTIVE: To investigate the diagnostic efficiency of miR-222-3p in plasma exosomes (Exos) and plasma for preeclampsia (PE) and the effect of miR-222-3p targeting STMN1 in PE. METHODS: MiR-222-3p levels in total plasma and plasma Exos were detected in PE patients and healthy controls. A bioinformatics database and dual-luciferase reporter assay were employed to verify the targeting relationship between miR-222-3p and STMN1. Trophoblast HTR-8/Svneo cells were transfected with miR-222-3p inhibitors with/without STMN1 shRNA, followed by MTT, wound healing and Transwell invasion assays. The mRNA and protein expressions were measured by qRT‒PCR and Western blotting, respectively. RESULTS: MiR-222-3p levels in total plasma and plasma Exos were higher in PE patients than in healthy controls, particularly in severe PE patients. In addition, miR-222-3p levels in total plasma and plasma Exos from PE patients were positively correlated with diastolic and systolic blood pressure. The area under the curve (AUC) of miR-222-3p in total plasma for PE diagnostic efficiency was 0.798, with a sensitivity of 76.67% and specificity of 71.93%, while the AUC of miR-222-3p in plasma Exos was 0.708 (sensitivity: 61.67%; specificity: 78.95%). In vitro, miR-222-3p targeted STMN1 in HTR-8/Svneo cells. Low miR-222-3p expression reversed the inhibitory effect of STMN1 shRNA on the proliferation, invasion and migration of HTR/SVneo cells. CONCLUSION: PE patients had increased miR-222-3p expression in total plasma and plasma Exos, which both have high diagnostic efficiency for PE. MiR-222-3p can target STMN1 to promote the proliferation, invasion and migration of HTR-8/Svneo cells and is a potential therapeutic target of PE.

Protocol for genome editing in wild allotetraploid rice Oryza alta.

We present a protocol for Agrobacterium-mediated genetic transformation and genome editing in wild allotetraploid rice Oryza alta. We detail steps to induce embryogenic calli from mature seeds and co-cultivate with Agrobacterium after infection. We further describe how to select transformed cells that proliferated from infected calli based on hygromycin resistance, resulting in regeneration of transformed plantlets with multiple edited alleles. Edited lines produced via this protocol can be used for gene functions studies and de novo domestication of O. alta. For complete details on the use and execution of this protocol, please refer to Yu et al. (2021).

Ovary-derived Decellularized Extracellular Matrix-based Bioink for Fabricating 3D Primary Ovarian Cells-laden Structures for Mouse Ovarian Failure Correction.

Fertility preservation is becoming a clinical duty in practice. Three-dimensional (3D) bioprinting technology is potentially realize ovarian morphological repair and reproductive endocrine function rebuild. There is no published work on 3D bioprinting ovary using a decellularized extracellular matrix (dECM)-based bioink, though dECM is the preferred matrix choice for an artificial ovary. The study aimed to explore swine ovarian dECM-based bioink to fabricate 3D primary ovarian cells (POCs)-laden structures for mouse ovarian failure correction. In this study, the ovarian dECM was converted to dECM-based bioink by dECM solution mixed with a seaweed gelatin blend solution of bioink that was characterized using scanning electron microscopy, circular dichroism, rheology, hematoxylin and eosin staining, and immunohistochemistry. The 3D scaffolds were, then, printed with or without POCs by the extrusion 3D bioprinter. The laden POCs viability was detected with the live/dead assay kit. A female castrated mouse model was established, and the mice were treated with five different methods. The results revealed that the 3D scaffold encapsulating POCs group had more positive signals in neoangiogenesis, cell proliferation and survival than the 3D scaffold group, and ensured sex hormone secretion. Meanwhile, the expression of germ cells in the 3D scaffold encapsulating POCs group was more intensely than the non-printed hydrogel encapsulating POCs group. The work shows that the 3D bioprinting ovary employing ovarian dECM-based bioink is a promising approach for ovarian failure correction.

Joint action of binary mixtures based on parameter k·ECx from concentration-response curves in long-term toxicity assay.

A previous acute toxicity study of binary mixtures showed that the combined toxicity can be predicted with the parameter k∙ECx. To systematically investigate the ability of k∙ECx to predict the chronic combined toxicity of binary mixtures, the toxicity of six contaminants and five binary mixtures was determined by long-term microplate toxicity analysis (L-MTA) using Aliivibrio fischeri as the test organism. The independent action model (IA) and the relative model deviation ratio (rMDR) were employed to determine the relationship between the Δ(k∙ECx)% and rMDRx. The results showed that these two factors conformed to the exponential function in long-term toxicity. Owing to the time-dependence of toxicity, the mixture type of chronic toxicity changes to the relative type of acute toxicity. If the acute toxicity of binary mixtures changes their mode of joint action throughout the concentration range, the chronic toxicity will also change their mode of joint action, and vice versa. This study clarified the change rules of the joint action of binary mixtures in acute and chronic toxicity which can promote research on chronic toxicity of binary mixtures.

Drug-free in vitro activation combined with 3D-bioprinted adipose-derived stem cells restores ovarian function of rats with premature ovarian insufficiency.

BACKGROUND: Emerging drug-free in vitro activation (IVA) technique enables patients with premature ovarian insufficiency (POI) to restore ovarian function and conceive their own genetic offspring. However, various issues have greatly restricted its clinical application. Transplantation of adipose-derived stem cells (ADSCs) has promising roles in restoring ovarian function of rats with POI, but insufficient retention has greatly hampered their efficiency. Here, we designed a 3D-bioprinted engineering ovary composed of drug-free IVA and ADSCs, which may prolong the retention of ADSCs and construct an early vascular microenvironment, thus compensating for the disadvantages of drug-free IVA to some extent and ameliorating impaired ovarian function in the POI rats. METHODS: After intraperitoneal injection of cyclophosphamide, the POI model rats were randomized into 5 groups: (1) POI group; (2) ovarian fragments group; (3) 3D scaffold combined with ovarian fragments group; (4) ovarian fragments combined with ADSCs group; (5) 3D scaffold with ADSCs combined with ovarian fragments as 3D-bioprinted engineering ovary group. Normal rats were identified as the control group. The localization of CM-Dil-labeled ADSCs and co-localization with CD31 were observed to examine the distribution and underlying mechanism of differentiation. Histomorphological and immunohistochemical analyses were performed to calculate follicle number and assess proliferation and apoptosis of granulosa cells (GCs). Immunofluorescence staining was used to evaluate angiogenesis. Hormone levels were measured to evaluate the restoration of endocrine axis. Western blot analysis and RT-PCR were conducted to explore the potential mechanism. RESULTS: CM-Dil-labeled ADSCs were distributed in the interstitium of ovaries and had significantly higher retention in the 3D-bioprinted engineering ovary group. Several regions of the co-staining for CM-Dil and CD31 were in the area of vascular endothelial cells. Meanwhile, the follicle counts, GCs proliferation, neoangiogenesis, and hormone levels were significantly improved in the 3D-bioprinted engineering ovary group, as compared with other groups. Furthermore, the ovarian function was ameliorated and angiogenesis was promoted through regulating the PI3K/AKT pathway. CONCLUSION: Our results suggested that 3D-bioprinted engineering ovary had great potential for restoring impaired ovarian function of rats with POI, which could compensate for the disadvantages of drug-free IVA to some extent.