Prediction of matrilineal specific patatin-like protein governing in-vivo maternal haploid induction in maize using support vector machine and di-peptide composition.

The mutant matrilineal (mtl) gene encoding patatin-like phospholipase activity is involved in in-vivo maternal haploid induction in maize. Doubling of chromosomes in haploids by colchicine treatment leads to complete fixation of inbreds in just one generation compared to 6-7 generations of selfing. Thus, knowledge of patatin-like proteins in other crops assumes great significance for in-vivo haploid induction. So far, no online tool is available that can classify unknown proteins into patatin-like proteins. Here, we aimed to optimize a machine learning-based algorithm to predict the patatin-like phospholipase activity of unknown proteins. Four different kernels [radial basis function (RBF), sigmoid, polynomial, and linear] were used for building support vector machine (SVM) classifiers using six different sequence-based compositional features (AAC, DPC, GDPC, CTDC, CTDT, and GAAC). A total of 1170 protein sequences including both patatin-like (585 sequences) from various monocots, dicots, and microbes; and non-patatin-like proteins (585 sequences) from different subspecies of Zea mays were analyzed. RBF and polynomial kernels were quite promising in the prediction of patatin-like proteins. Among six sequence-based compositional features, di-peptide composition attained > 90% prediction accuracies using RBF and polynomial kernels. Using mutual information, most explaining dipeptides that contributed the highest to the prediction process were identified. The knowledge generated in this study can be utilized in other crops prior to the initiation of any experiment. The developed SVM model opened a new paradigm for scientists working in in-vivo haploid induction in commercial crops. This is the first report of machine learning of the identification of proteins with patatin-like activity.

Low expression of lipoxygenase 3 (LOX3) enhances the retention of kernel tocopherols in maize during storage.

BACKGROUND: Deficiency of vitamin E results in several neurological and age-related disorders in humans. Utilization of maize mutants with favourable vte4-allele led to the development of several α-tocopherol (vitamin E) rich (16-19 µg/g) maize hybrids worldwide. However, the degradation of tocopherols during post-harvest storage substantially affects the efficacy of these genotypes. METHODS AND RESULTS: We studied the role of lipoxygenase enzyme and Lipoxygenase 3 (LOX3) gene on the degradation of tocopherols at monthly intervals under traditional storage up to six months in two vte4-based contrasting-tocopherol retention maize inbreds viz. HKI323-PVE and HKI193-1-PVE. The analysis revealed significant degradation of tocopherols across storage intervals in both the inbreds. Lower retention of α-tocopherol was noticed in HKI193-1-PVE. HKI323-PVE with the higher retention of α-tocopherol showed lower lipoxygenase activity throughout the storage intervals. LOX3 gene expression was higher (~ 1.5-fold) in HKI193-1-PVE compared to HKI323-PVE across the storage intervals. Both lipoxygenase activity and LOX3 expression peaked at 120 days after storage (DAS) in both genotypes. Further, a similar trend was observed for LOX3 expression and lipoxygenase activity. The α-tocopherol exhibited a significantly negative correlation with lipoxygenase enzyme and expression of LOX3 across the storage intervals. CONCLUSIONS: HKI323-PVE with high tocopherol retention, low -lipoxygenase activity, and -LOX3 gene expression can act as a potential donor in the vitamin E biofortification program. Protein-protein association network analysis also indicated the independent effect of vte4 and LOX genes. This is the first comprehensive report analyzing the expression of the LOX3 gene and deciphering its vital role in the retention of α-tocopherol in biofortified maize varieties under traditional storage.

Regenerative Potential of Injured Spinal Cord in the Light of Epigenetic Regulation and Modulation.

A spinal cord injury is a form of physical harm imposed on the spinal cord that causes disability and, in many cases, leads to permanent mammalian paralysis, which causes a disastrous global issue. Because of its non-regenerative aspect, restoring the spinal cord's role remains one of the most daunting tasks. By comparison, the remarkable regenerative ability of some regeneration-competent species, such as some Urodeles (Axolotl), Xenopus, and some teleost fishes, enables maximum functional recovery, even after complete spinal cord transection. During the last two decades of intensive research, significant progress has been made in understanding both regenerative cells' origins and the molecular signaling mechanisms underlying the regeneration and reconstruction of damaged spinal cords in regenerating organisms and mammals, respectively. Epigenetic control has gradually moved into the center stage of this research field, which has been helped by comprehensive work demonstrating that DNA methylation, histone modifications, and microRNAs are important for the regeneration of the spinal cord. In this review, we concentrate primarily on providing a comparison of the epigenetic mechanisms in spinal cord injuries between non-regenerating and regenerating species. In addition, we further discuss the epigenetic mediators that underlie the development of a regeneration-permissive environment following injury in regeneration-competent animals and how such mediators may be implicated in optimizing treatment outcomes for spinal cord injurie in higher-order mammals. Finally, we briefly discuss the role of extracellular vesicles (EVs) in the context of spinal cord injury and their potential as targets for therapeutic intervention.

Biomarkers for parkinsonian disorders in CNS-originating EVs: promise and challenges.

Extracellular vesicles (EVs), including exosomes, microvesicles, and oncosomes, are nano-sized particles enclosed by a lipid bilayer. EVs are released by virtually all eukaryotic cells and have been shown to contribute to intercellular communication by transporting proteins, lipids, and nucleic acids. In the context of neurodegenerative diseases, EVs may carry toxic, misfolded forms of amyloidogenic proteins and facilitate their spread to recipient cells in the central nervous system (CNS). CNS-originating EVs can cross the blood-brain barrier into the bloodstream and may be found in other body fluids, including saliva, tears, and urine. EVs originating in the CNS represent an attractive source of biomarkers for neurodegenerative diseases, because they contain cell- and cell state-specific biological materials. In recent years, multiple papers have reported the use of this strategy for identification and quantitation of biomarkers for neurodegenerative diseases, including Parkinson's disease and atypical parkinsonian disorders. However, certain technical issues have yet to be standardized, such as the best surface markers for isolation of cell type-specific EVs and validating the cellular origin of the EVs. Here, we review recent research using CNS-originating EVs for biomarker studies, primarily in parkinsonian disorders, highlight technical challenges, and propose strategies for overcoming them.

Development of a Novel Electrochemiluminescence ELISA for Quantification of α-Synuclein Phosphorylated at Ser129 in Biological Samples.

Synucleinopathies are a group of neurodegenerative diseases including Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). These diseases are characterized by the aggregation and deposition of α-synuclein (α-syn) in Lewy bodies (LBs) in PD and DLB or as glial cytoplasmic inclusions in MSA. In healthy brains, only ∼4% of α-syn is phosphorylated at Ser129 (pS129-α-syn), whereas >90% pS129-α-syn may be found in LBs, suggesting that pS129-α-syn could be a useful biomarker for synucleinopathies. However, a widely available, robust, sensitive, and reproducible method for measuring pS129-α-syn in biological fluids is currently missing. We used Meso Scale Discovery (MSD)'s electrochemiluminescence platform to create a new assay for sensitive detection of pS129-α-syn. We evaluated several combinations of capture and detection antibodies and used semisynthetic pS129-α-syn as a standard for the assay at a concentration range from 0.5 to 6.6 × 104 pg/mL. Using the antibody EP1536Y for capture and an anti-human α-syn antibody (MSD) for detection was the best combination in terms of assay sensitivity, specificity, and reproducibility. We tested the utility of the assay for the detection and quantification of pS129-α-syn in human cerebrospinal fluid, serum, plasma, saliva, and CNS-originating small extracellular vesicles, as well as in mouse brain lysates. Our data suggest that the assay can become a widely used method for detecting pS129-α-syn in biomedical studies including when only a limited volume of sample is available and high sensitivity is required, offering new opportunities for diagnostic biomarkers, monitoring disease progression, and quantifying outcome measures in clinical trials.

Variation in anthocyanin pigmentation by R1-navajo gene, development and validation of breeder-friendly markers specific to C1-Inhibitor locus for in-vivo haploid production in maize.

BACKGROUND: In-vivo maternal haploids serve as the basis of doubled haploid (DH) breeding in maize. R1-navajo (R1-nj) gene governing anthocyanin colouration in the endosperm and embryo is widely used to identify haploid seeds. However, the expression of R1-nj depends on genetic-background of source-germplasm used for deriving DH-lines. Further, presence of C1-Inhibitor (C1-I) gene suppresses the expression of R1-nj, thus makes the selection of haploids difficult. METHODS: In the present study, 178 subtropically-adapted maize inbreds were crossed with two R1-nj donors 'that do not have haploid induction genes'. Of these, 76.4% inbreds developed purple colour in endosperm, while 23.6% did not show any colouration. In case of scutellum, 62.9% inbreds possessed colour and 37.1% were colourless. The anthocyanin intensity varied greatly, with 19.66% and 42.98% inbreds displayed the least intensity, while 16.85% and 0.84% inbreds showed the highest intensity in endosperm and scutellum, respectively. Two C1-I specific breeder-friendly markers (MGU-CI-InDel8 and MGU-C1-SNP1) covering (i) 8 bp InDel and (ii) A to G SNP, respectively, were developed. MGU-CI-InDel8 and MGU-C1-SNP1 markers predicted presence of C1-I allele with 92.9% and 84.7% effectiveness, respectively. However, when both markers were considered together, they provided 100% effectiveness. CONCLUSIONS: These markers of C1-I gene would help in saving valuable resources and time during haploid induction in maize. The information generated here assume great significance in DH breeding of maize.

A Comprehensive Cancer-Associated MicroRNA Expression Profiling and Proteomic Analysis of Human Umbilical Cord Mesenchymal Stem Cell-Derived Exosomes.

BACKGROUND: The mesenchymal stem cells (MSCs) have enormous therapeutic potential owing to their multi-lineage differentiation and self-renewal properties. MSCs express growth factors, cytokines, chemokines, and non-coding regulatory RNAs with immunosuppressive, anti-tumor, and migratory properties. MSCs also release several anti-cancer molecules via extracellular vesicles, that act as pro-apoptotic/tumor suppressor factors. This study aimed to identify the stem cell-derived secretome that could exhibit anti-cancer properties through molecular profiling of cargos in MSC-derived exosomes. METHODS: Human umbilical cord mesenchymal stem cells (hUCMSCs) were isolated from umbilical cord tissues and culture expanded. Subsequently, exosomes were isolated from hUCMSC conditioned medium and characterized by DLS, electron microscopy. Western blot for exosome surface marker protein CD63 expression was performed. The miRNA profiling of hUCMSCs and hUCMSC-derived exosomes was performed, followed by functional enrichment analysis. RESULTS: The tri-lineage differentiation potential, fibroblastic morphology, and strong expression of pluripotency genes indicated that isolated fibroblasts are MSCs. The isolated extracellular vesicles were 133.8 ± 42.49 nm in diameter, monodispersed, and strongly expressed the exosome surface marker protein CD63. The miRNA expression profile and gene ontology (GO) depicted the differential expression patterns of high and less-expressed miRNAs that are crucial to be involved in the regulation of apoptosis. The LCMS/MS data and GO analysis indicate that hUCMSC secretomes are involved in several oncogenic and inflammatory signaling cascades. CONCLUSION: Primary human MSCs released miRNAs and growth factors via exosomes that are increasingly implicated in intercellular communications, and hUCMSC-exosomal miRNAs have a critical influence in regulating cell death and apoptosis of cancer cells.

Current advances in the use of exosomes, liposomes, and bioengineered hybrid nanovesicles in cancer detection and therapy.

Three major approaches of cancer therapy can be enunciated as delivery of biotherapeutics, tumor image analysis, and immunotherapy. Liposomes, artificial fat bubbles, are long known for their capacity to encapsulate a diverse range of bioactive molecules and release the payload in a sustained, stimuli-responsive manner. They have already been widely explored as a delivery vehicle for therapeutic drugs as well as imaging agents. They are also extensively being used in cancer immunotherapy. On the other hand, exosomes are naturally occurring nanosized extracellular vesicles that serve an important role in cell-cell communication. Importantly, the exosomes also have proven their capability to carry an array of active pharmaceuticals and diagnostic molecules to the tumor cells. Exosomes, being enriched with tumor antigens, have numerous immunomodulatory effects. Much to our intrigue, in recent times, efforts have been directed toward developing smart, bioengineered, exosome-liposome hybrid nanovesicles, which are augmented by the benefits of both vesicular systems. This review attempts to summarize the contemporary developments in the use of exosome and liposome toward cancer diagnosis, therapy, as a vehicle for drug delivery, diagnostic carrier for tumor imaging, and cancer immunotherapy. We shall also briefly reflect upon the recent advancements of the exosome-liposome hybrids in cancer therapy. Finally, we put forward future directions for the use of exosome/liposome and/or hybrid nanocarriers for accurate diagnosis and personalized therapies for cancers.

mTOR Inhibition with Sirolimus in Multiple System Atrophy: A Randomized, Double-Blind, Placebo-Controlled Futility Trial and 1-Year Biomarker Longitudinal Analysis.

BACKGROUND: Multiple system atrophy (MSA) is a fatal neurodegenerative disease characterized by the aggregation of α-synuclein in glia and neurons. Sirolimus (rapamycin) is an mTOR inhibitor that promotes α-synuclein autophagy and reduces its associated neurotoxicity in preclinical models. OBJECTIVE: To investigate the efficacy and safety of sirolimus in patients with MSA using a futility design. We also analyzed 1-year biomarker trajectories in the trial participants. METHODS: Randomized, double-blind, parallel group, placebo-controlled clinical trial at the New York University of patients with probable MSA randomly assigned (3:1) to sirolimus (2-6 mg daily) for 48 weeks or placebo. Primary endpoint was change in the Unified MSA Rating Scale (UMSARS) total score from baseline to 48 weeks. (ClinicalTrials.gov NCT03589976). RESULTS: The trial was stopped after a pre-planned interim analysis met futility criteria. Between August 15, 2018 and November 15, 2020, 54 participants were screened, and 47 enrolled and randomly assigned (35 sirolimus, 12 placebo). Of those randomized, 34 were included in the intention-to-treat analysis. There was no difference in change from baseline to week 48 between the sirolimus and placebo in UMSARS total score (mean difference, 2.66; 95% CI, -7.35-6.91; P = 0.648). There was no difference in UMSARS-1 and UMSARS-2 scores either. UMSARS scores changes were similar to those reported in natural history studies. Neuroimaging and blood biomarker results were similar in the sirolimus and placebo groups. Adverse events were more frequent with sirolimus. Analysis of 1-year biomarker trajectories in all participants showed that increases in blood neurofilament light chain (NfL) and reductions in whole brain volume correlated best with UMSARS progression. CONCLUSIONS: Sirolimus for 48 weeks was futile to slow the progression of MSA and had no effect on biomarkers compared to placebo. One-year change in blood NfL and whole brain atrophy are promising biomarkers of disease progression for future clinical trials. © 2022 International Parkinson and Movement Disorder Society.

α-Synuclein in blood exosomes immunoprecipitated using neuronal and oligodendroglial markers distinguishes Parkinson's disease from multiple system atrophy.

The diagnosis of Parkinson's disease (PD) and atypical parkinsonian syndromes is difficult due to the lack of reliable, easily accessible biomarkers. Multiple system atrophy (MSA) is a synucleinopathy whose symptoms often overlap with PD. Exosomes isolated from blood by immunoprecipitation using CNS markers provide a window into the brain's biochemistry and may assist in distinguishing between PD and MSA. Thus, we asked whether α-synuclein (α-syn) in such exosomes could distinguish among healthy individuals, patients with PD, and patients with MSA. We isolated exosomes from the serum or plasma of these three groups by immunoprecipitation using neuronal and oligodendroglial markers in two independent cohorts and measured α-syn in these exosomes using an electrochemiluminescence ELISA. In both cohorts, α-syn concentrations were significantly lower in the control group and significantly higher in the MSA group compared to the PD group. The ratio between α-syn concentrations in putative oligodendroglial exosomes compared to putative neuronal exosomes was a particularly sensitive biomarker for distinguishing between PD and MSA. Combining this ratio with the α-syn concentration itself and the total exosome concentration, a multinomial logistic model trained on the discovery cohort separated PD from MSA with an AUC = 0.902, corresponding to 89.8% sensitivity and 86.0% specificity when applied to the independent validation cohort. The data demonstrate that a minimally invasive blood test measuring α-syn in blood exosomes immunoprecipitated using CNS markers can distinguish between patients with PD and patients with MSA with high sensitivity and specificity. Future optimization and validation of the data by other groups would allow this strategy to become a viable diagnostic test for synucleinopathies.

Elastoplastic Approach Based on Microscopic Insights for the Steady State and Transient Dynamics of Sheared Disordered Solids.

We develop a framework to study the mechanical response of athermal amorphous solids via a coupling of mesoscale and microscopic models. Using measurements of coarse-grained quantities from simulations of dense disordered particulate systems, we present a coherent elastoplastic model approach for deformation and flow of yield stress materials. For a given set of parameters, this model allows us to match consistently transient and steady state features of driven disordered systems with diverse preparation histories under both applied shear-rate and creep protocols.

The molecular tweezer CLR01 improves behavioral deficits and reduces tau pathology in P301S-tau transgenic mice.

BACKGROUND: Molecular tweezers (MTs) are broad-spectrum inhibitors of abnormal protein aggregation. A lead MT, called CLR01, has been demonstrated to inhibit the aggregation and toxicity of multiple amyloidogenic proteins in vitro and in vivo. Previously, we evaluated the effect of CLR01 in the 3 × Tg mouse model of Alzheimer's disease, which overexpresses mutant human presenilin 1, amyloid ß-protein precursor, and tau and found that subcutaneous administration of the compound for 1 month led to a robust reduction of amyloid plaques, neurofibrillary tangles, and microgliosis. CLR01 also has been demonstrated to inhibit tau aggregation in vitro and tau seeding in cell culture, yet because in Alzheimer's disease (AD) and in the 3 × Tg model, tau hyperphosphorylation and aggregation are thought to be downstream of Aß insults, the study in this model left open the question whether CLR01 affected tau in vivo directly or indirectly. METHODS: To determine if CLR01 could ameliorate tau pathology directly in vivo, we tested the compound similarly using the P301S-tau (line PS19) mouse model. Mice were administered 0.3 or 1.0 mg/kg per day CLR01 and tested for muscle strength and behavioral deficits, including anxiety- and disinhibition-like behavior. Their brains then were analyzed by immunohistochemical and biochemical assays for pathological forms of tau, neurodegeneration, and glial pathology. RESULTS: CLR01 treatment ameliorated muscle-strength deterioration, anxiety-, and disinhibition-like behavior. Improved phenotype was associated with decreased levels of pathologic tau forms, suggesting that CLR01 exerts a direct effect on tau in vivo. Limitations of the study included a relatively short treatment period of the mice at an age in which full pathology is not yet developed. In addition, high variability in this model lowered the statistical significance of the findings of some outcome measures. CONCLUSIONS: The findings suggest that CLR01 is a particularly attractive candidate for the treatment of AD because it targets simultaneously the two major pathogenic proteins instigating and propagating the disease, amyloid ß-protein (Aß), and tau, respectively. In addition, our study suggests that CLR01 can be used for the treatment of other tauopathies in the absence of amyloid pathology.

Low expression of carotenoids cleavage dioxygenase 1 (ccd1) gene improves the retention of provitamin-A in maize grains during storage.

Provitamin-A (proA) is essentially required for vision in humans but its deficiency affects children and pregnant women especially in the developing world. Biofortified maize rich in proA provides new opportunity for sustainable and cost-effective solution to alleviate malnutrition, however, significant loss of carotenoids during storage reduces its efficacy. Here, we studied the role of carotenoid cleavage dioxygenase 1 (ccd1) gene on degradation of carotenoids in maize. A set of 24 maize inbreds was analyzed for retention of proA during storage. At harvest, crtRB1-based maize inbreds possessed significantly high proA (ß-carotene: 12.30 µg/g, ß-cryptoxanthin: 4.36 µg/g) than the traditional inbreds (ß-carotene: 1.74 µg/g, ß-cryptoxanthin: 1.28 µg/g). However, crtRB1-based inbreds experienced significant degradation of proA carotenoids (ß-carotene: 20%, ß-cryptoxanthin: 32% retention) following 5 months. Among the crtRB1-based genotypes, V335PV had the lowest retention of proA (ß-carotene: 1.63 µg/g, ß-cryptoxanthin: 0.82 µg/g), while HKI161PV had the highest retention of proA (ß-carotene: 4.17 µg/g, ß-cryptoxanthin: 2.32 µg/g). Periodical analysis revealed that ~ 60-70% of proA degraded during the first three months. Expression analysis revealed that high expression of ccd1 led to low retention of proA carotenoids in V335PV, whereas proA retention in HKI161PV was higher due to lower expression. Highest expression of ccd1 was observed during first 3 months of storage. Copy number of ccd1 gene varied among yellow maize (1-6 copies) and white maize (7-35 copies) while wild relatives contained 1-4 copies of ccd1 gene per genome. However, copy number of ccd1 gene did not exhibit any correlation with proA carotenoids. We concluded that lower expression of ccd1 gene increased the retention of proA during storage in maize. Favourable allele of ccd1 can be introgressed into elite maize inbreds for higher retention of proA during storage.

Two-step method for isolation of high-quality RNA from stored seeds of maize rich in starch.

A modified SDS-Trizol method was optimized for isolation of total RNA from the stored maize seeds at regular interval of one month for 4 months. Use of SDS extraction buffer before the use of Trizol reduced the co-precipitation problem associated with high carbohydrate content in the seed. Recorded mean RNA yield from seeds across the storage intervals was 978.6 ± 65.46 ng/µl. Average spectrophotometric values (A 260/280) of isolated RNA varied from 1.974 ± 0.033 to 1.998 ± 0.022. Attempts to isolate RNA from green leaves using Trizol method also ensured comparable quality and quantity of the isolated RNA. RNA yield from fresh leaves was recorded 1008.2 ± 77.088 ng/µl which is slightly higher than the mean RNA yield from seeds across months. Observed mean A 260/280 values of isolated RNA were 1.984 ± 0.030. DNase treatment further improved the A 260/280 ratio in both seeds (2.003 ± 0.006) and leaves (2.012 ± 0.037). High quality and quantity along with integrity of the isolated RNA was ensured through downstream analysis after RNA extraction such as first-strand cDNA synthesis and normal PCR. Extraction of RNA from the stored seeds using modified SDS-based Trizol method and from fresh leaves using Trizol method opened new possibility of understanding role of key genes involving developmental steps especially in the stored seeds.

Length-scales of dynamic heterogeneity in a driven binary colloid.

Here we study the characteristic length scales in an aqueous suspension of a symmetric oppositely charged colloid subjected to a uniform electric field by Brownian dynamics simulations. We consider the in-plane structure in the presence of a sufficiently strong electric field where the like charges in the system form macroscopic lanes. We construct spatial correlation functions characterizing the structural order and that of particles of different mobilities in the plane transverse to the electric field at a given time. We call these functions equal time density correlation functions (ETDCFs). The ETDCFs between particles of different charges, irrespective of mobilities, are the structural ETDCFs, while those between particles of different mobilities are the dynamic ETDCFs. We extract the characteristic length of correlation by fitting the envelopes of the ETDCFs to exponential dependences. We find that the correlation length scales of the structural ETDCFs and the dynamic ETDCFs of the slow particles increase with time in a concurrent manner. This suggests that the clustering of particles tends to build up dynamically correlated slow particles in the plane transverse to the lanes. The ETDCFs can be measured for colloidal systems by directly following the particle motion by video-microscopy and may be useful to understand the patterns out of equilibrium.