Evaluation of safety and early efficacy of AAV gene therapy in mouse models of vanishing white matter disease.

Leukoencephalopathy with vanishing white matter (VWM) is a progressive incurable white matter disease that most commonly occurs in childhood and presents with ataxia, spasticity, neurological degeneration, seizures, and premature death. A distinctive feature is episodes of rapid neurological deterioration provoked by stressors such as infection, seizures, or trauma. VWM is caused by autosomal recessive mutations in one of five genes that encode the eukaryotic initiation factor 2B complex, which is necessary for protein translation and regulation of the integrated stress response. The majority of mutations are in EIF2B5. Astrocytic dysfunction is central to pathophysiology, thereby constituting a potential therapeutic target. Herein we characterize two VWM murine models and investigate astrocyte-targeted adeno-associated virus serotype 9 (AAV9)-mediated EIF2B5 gene supplementation therapy as a therapeutic option for VWM. Our results demonstrate significant rescue in body weight, motor function, gait normalization, life extension, and finally, evidence that gene supplementation attenuates demyelination. Last, the greatest rescue results from a vector using a modified glial fibrillary acidic protein (GFAP) promoter-AAV9-gfaABC(1)D-EIF2B5-thereby supporting that astrocytic targeting is critical for disease correction. In conclusion, we demonstrate safety and early efficacy through treatment with a translatable astrocyte-targeted gene supplementation therapy for a disease that has no cure.

Transcriptome analysis reveals the key role of overdominant expression of photosynthetic and respiration-related genes in the formation of tobacco(Nicotiana tabacum L.) biomass heterosis.

BACKGROUND: Leaves are the nutritional and economic organs of tobacco, and their biomass directly affects tobacco yield and the economic benefits of farmers. In the early stage, our research found that tobacco hybrids have more leaves and larger leaf areas, but the performance and formation reasons of biomass heterosis are not yet clear. RESULTS: This study selected 5 parents with significant differences in tobacco biomass and paired them with hybrid varieties. It was found that tobacco hybrid varieties have a common biomass heterosis, and 45 days after transplantation is the key period for the formation of tobacco biomass heterosis; By analyzing the biomass heterosis of hybrids, Va116×GDH94 and its parents were selected for transcriptome analysis. 76.69% of the differentially expressed genes between Va116×GDH94 and its parents showed overdominant expression pattern, and these overdominant expression genes were significantly enriched in the biological processes of photosynthesis and TCA cycle; During the process of photosynthesis, the overdominant up-regulation of genes such as Lhc, Psa, and rbcl promotes the progress of photosynthesis, thereby increasing the accumulation of tobacco biomass; During the respiratory process, genes such as MDH, ACO, and OGDH are overedominantly down-regulated, inhibiting the TCA cycle and reducing substrate consumption in hybrid offspring; The photosynthetic characteristics of the hybrid and its parents were measured, and the net photosynthetic capacity of the hybrid was significantly higher than that of the parents. CONCLUSION: These results indicate that the overdominant expression effect of differentially expressed genes in Va116×GDH94 and its parents plays a crucial role in the formation of tobacco biomass heterosis. The overdominant expression of genes related to photosynthesis and respiration enhances the photosynthetic ability of Va116×GDH94, reduces respiratory consumption, promotes the increase of biomass, and exhibits obvious heterosis.

Non-additive expression genes play a critical role in leaf vein ratio heterosis in Nicotiana tabacum L.

Heterosis, recognized for improving crop performance, especially in the first filial (F1) generation, remains an area of significant study in the tobacco industry. The low utilization of leaf veins in tobacco contributes to economic inefficiency and resource waste. Despite the positive impacts of heterosis on crop genetics, investigations into leaf-vein ratio heterosis in tobacco have been lacking. Understanding the mechanisms underlying negative heterosis in leaf vein ratio at the molecular level is crucial for advancing low vein ratio leaf breeding research. This study involved 12 hybrid combinations and their parental lines to explore heterosis associated with leaf vein ratios. The hybrids displayed diverse patterns of positive or negative leaf vein ratio heterosis across different developmental stages. Notably, the F1 hybrid (G70 × Qinggeng) consistently exhibited substantial negative heterosis, reaching a maximum of -19.79% 80 days after transplanting. A comparative transcriptome analysis revealed that a significant proportion of differentially expressed genes (DEGs), approximately 39.04% and 23.73%, exhibited dominant and over-dominant expression patterns, respectively. These findings highlight the critical role of non-additive gene expression, particularly the dominance pattern, in governing leaf vein ratio heterosis. The non-additive genes, largely associated with various GO terms such as response to abiotic stimuli, galactose metabolic process, plant-type cell wall organization, auxin-activated signaling pathway, hydrolase activity, and UDP-glycosyltransferase activity, were identified. Furthermore, KEGG enrichment analysis unveiled their involvement in phenylpropanoid biosynthesis, galactose metabolism, plant hormone signal transduction, glutathione metabolism, MAPK signaling pathway, starch, and sucrose metabolism. Among the non-additive genes, we identified some genes related to leaf development, leaf size, leaf senescence, and cell wall extensibility that showed significantly lower expression in F1 than in its parents. These results indicate that the non-additive expression of genes plays a key role in the heterosis of the leaf vein ratio in tobacco. This study marks the first exploration into the molecular mechanisms governing leaf vein ratio heterosis at the transcriptome level. These findings significantly contribute to understanding leaf vein ratios in tobacco breeding strategies.

The formation of nicotine heterosis is mainly achieved by enhancing the nicotine transport capacity in hybrids.

Nicotine exhibits obvious heterosis, which can be used to create Nicotiana tabacum L. (tobacco) varieties with varying nicotine content. However, the reasons for the formation of nicotine heterosis and its relationship to nicotine transport and accumulation remain unknown. This study conducted a comprehensive analysis of six tobacco hybrids with varying heterosis levels and their parent materials from various aspects, such as phenotype, physiology, and transcriptomics. The results showed that the direct path coefficient of transport heterosis to nicotine heterosis was highest in hybrids, at 0.98, and a highly significant positive correlation between the two. The plant height, thick stalk circumference, large flow of tissue fluid in the stalk, and high nicotine concentration of tobacco were the underlying factors that led to the strong nicotine transport capacity of hybrids. The formation of nicotine transport heterosis in hybrids was mainly influenced by non-additive gene effects (accounting for 89.93%), with over-dominant effects playing a dominant role (accounting for 58.79%). Among non-additive expression DEGs, nicotine transporter related multi antimicrobial extrusion protein, drug/metabolite transporter, ABC family transporter, and glutathione S-transferase were significantly upregulated in hybrid strains. The RT-qPCR results indicated that these genes related nicotine transport also exhibited heterosis at the expression level. Our results revealed that the formation of nicotine heterosis is mainly achieved by enhancing the nicotine transport capacity in hybrids. The results are not only beneficial for promoting the theoretical study of nicotine heterosis in tobacco and the breeding and utilization of hybrids, but are also of great significance for guiding nicotine production and promoting its multipurpose utilization.

Neuromodulatory Responses Elicited by Intermittent versus Continuous Transcranial Focused Ultrasound Stimulation of the Motor Cortex in Rats.

Transcranial focused ultrasound stimulation (tFUS) has emerged as a promising neuromodulation technique that delivers acoustic energy with high spatial resolution for inducing long-term potentiation (LTP)- or depression (LTD)-like plasticity. The variability in the primary effects of tFUS-induced plasticity could be due to different stimulation patterns, such as intermittent versus continuous, and is an aspect that requires further detailed exploration. In this study, we developed a platform to evaluate the neuromodulatory effects of intermittent and continuous tFUS on motor cortical plasticity before and after tFUS application. Three groups of rats were exposed to either intermittent, continuous, or sham tFUS. We analyzed the neuromodulatory effects on motor cortical excitability by examining changes in motor-evoked potentials (MEPs) elicited by transcranial magnetic stimulation (TMS). We also investigated the effects of different stimulation patterns on excitatory and inhibitory neural biomarkers, examining c-Fos and glutamic acid decarboxylase (GAD-65) expression using immunohistochemistry staining. Additionally, we evaluated the safety of tFUS by analyzing glial fibrillary acidic protein (GFAP) expression. The current results indicated that intermittent tFUS produced a facilitation effect on motor excitability, while continuous tFUS significantly inhibited motor excitability. Furthermore, neither tFUS approach caused injury to the stimulation sites in rats. Immunohistochemistry staining revealed increased c-Fos and decreased GAD-65 expression following intermittent tFUS. Conversely, continuous tFUS downregulated c-Fos and upregulated GAD-65 expression. In conclusion, our findings demonstrate that both intermittent and continuous tFUS effectively modulate cortical excitability. The neuromodulatory effects may result from the activation or deactivation of cortical neurons following tFUS intervention. These effects are considered safe and well-tolerated, highlighting the potential for using different patterns of tFUS in future clinical neuromodulatory applications.

Genome-wide identification and characterization of Glutathione S-Transferases (GSTs) and their expression profile under abiotic stresses in tobacco (Nicotiana tabacum L.).

BACKGROUND: Glutathione S-transferases (GSTs) are large and multifunctional proteases that play an important role in detoxification, protection against biotic and abiotic stresses, and secondary metabolite transportation which is essential for plant growth and development. However, there is limited research on the identification and function of NtGSTs. RESULTS: This study uses K326 and other six tobacco varieties (Hongda, HG, GDH11, Va116, VG, and GDH88) as materials to conduct comprehensive genome-wide identification and functional characterization of the GST gene in tobacco. A total of 59 NtGSTs were identified and classified into seven subfamilies via the whole-genome sequence analysis, with the Tau type serving as the major subfamily. The NtGSTs in the same branch of the evolutionary tree had similar exon/intron structure and motif constitution. There were more than 42 collinear blocks between tobacco and pepper, tomato, and potato, indicating high homology conservation between them. Twelve segmental duplicated gene pairs and one tandem duplication may have had a substantial impact on the evolution and expansion of the tobacco GST gene family. The RT-qPCR results showed that the expression patterns of NtGSTs varied significantly among tissues, varieties, and multiple abiotic stresses, suggesting that NtGST genes may widely respond to various abiotic stresses and hormones in tobacco, including NtGSTF4, NtGSTL1, NtGSTZ1, and NtGSTU40. CONCLUSIONS: This study provides a comprehensive analysis of the NtGST gene family, including structures and functions. Many NtGSTs play a critical regulatory role in tobacco growth and development, and responses to abiotic stresses. These findings offer novel and valuable insights for understanding the biological function of NtGSTs and the reference materials for cultivating highly resistant varieties and enhancing the yield and quality of crops.

Weak Ultrasound Contributes to Neuromodulatory Effects in the Rat Motor Cortex.

Transcranial focused ultrasound (tFUS) is a novel neuromodulating technique. It has been demonstrated that the neuromodulatory effects can be induced by weak ultrasound exposure levels (spatial-peak temporal average intensity, ISPTA < 10 mW/cm2) in vitro. However, fewer studies have examined the use of weak tFUS to potentially induce long-lasting neuromodulatory responses in vivo. The purpose of this study was to determine the lower-bound threshold of tFUS stimulation for inducing neuromodulation in the motor cortex of rats. A total of 94 Sprague-Dawley rats were used. The sonication region aimed at the motor cortex under weak tFUS exposure (ISPTA of 0.338-12.15 mW/cm2). The neuromodulatory effects of tFUS on the motor cortex were evaluated by the changes in motor-evoked potentials (MEPs) elicited by transcranial magnetic stimulation (TMS). In addition to histology analysis, the in vitro cell culture was used to confirm the neuromodulatory mechanisms following tFUS stimulation. In the results, the dose-dependent inhibitory effects of tFUS were found, showing increased intensities of tFUS suppressed MEPs and lasted for 30 min. Weak tFUS significantly decreased the expression of excitatory neurons and increased the expression of inhibitory GABAergic neurons. The PIEZO-1 proteins of GABAergic neurons were found to involve in the inhibitory neuromodulation. In conclusion, we show the use of weak ultrasound to induce long-lasting neuromodulatory effects and explore the potential use of weak ultrasound for future clinical neuromodulatory applications.

Overdominant expression of related genes of ion homeostasis improves K+ content advantage in hybrid tobacco leaves.

BACKGROUND: Potassium(K+) plays a vital role in improving the quality of tobacco leaves. However, how to improve the potassium content of tobacco leaves has always been a difficult problem in tobacco planting. K+ content in tobacco hybrid is characterized by heterosis, which can improve the quality of tobacco leaves, but its underlying molecular genetic mechanisms remain unclear. RESULTS: Through a two-year field experiment, G70×GDH11 with strong heterosis and K326×GDH11 with weak heterosis were screened out. Transcriptome analyses revealed that 80.89% and 57.28% of the differentially expressed genes (DEGs) in the strong and weak heterosis combinations exhibited an overdominant expression pattern, respectively. The genes that up-regulated the overdominant expression in the strong heterosis hybrids were significantly enriched in the ion homeostasis. Genes involved in K+ transport (KAT1/2, GORK, AKT2, and KEA3), activity regulation complex (CBL-CIPK5/6), and vacuole (TPKs) genes were overdominant expressed in strong heterosis hybrids, which contributed to K+ homeostasis and heterosis in tobacco leaves. CONCLUSIONS: K+ homeostasis and accumulation in tobacco hybrids were collectively improved. The overdominant expression of K+ transport and homeostasis-related genes conducted a crucial role in the heterosis of K+ content in tobacco leaves.

Synaptic loss and progression in mice infected with Angiostrongylus cantonensis in the early stage.

BACKGROUND: Angiostrongylus cantonensis is also known as rat lungworm. Infection with this parasite is a zoonosis that can cause eosinophilic meningitis and/or eosinophilic meningoencephalitis in humans and may lead to fatal outcomes in severe cases. In this study, we explored the mechanisms of the impairments in the cognitive functions of mice infected with A. cantonensis. METHODS: In infected mice with different infective intensities at different timepoint postinfection, loss and recovery of cognitive functions such as learning and memory abilities were determined. Neuronal death and damage to synaptic structures were analyzed by Western blotting and IHC in infected mice with different infection intensities at different timepoint postinfection. RESULTS: The results of behavioral tests, pathological examinations, and Golgi staining showed that nerve damage caused by infection in mice occurred earlier than pathological changes of the brain. BDNF was expressed on 14 day post-infection. Cleaved caspase-3 increased significantly in the late stage of infection. However, IHC on NeuN indicated that no significant changes in the number of neurons were found between the infected and uninfected groups. CONCLUSIONS: The synaptic loss caused by the infection of A. cantonensis provides a possible explanation for the impairment of cognitive functions in mice. The loss of cognitive functions may occur before severe immunological and pathological changes in the infected host.

Significance of Hypermethylation of Tumor-Suppressor Genes PTGER4 and ZNF43 at CpG Sites in the Prognosis of Colorectal Cancer.

The status of DNA methylation in primary tumor tissue and adjacent tumor-free tissue is associated with the occurrence of aggressive colorectal cancer (CRC) and can aid personalized cancer treatments at early stages. Tumor tissue and matched adjacent nontumorous tissue were extracted from 208 patients with CRC, and the correlation between the methylation levels of PTGER4 and ZNF43 at certain CpG loci and the prognostic factors of CRC was determined using the MassARRAY System testing platform. The Wilcoxon signed-rank test, a Chi-square test, and McNemar's test were used for group comparisons, and Kaplan-Meier curves and a log-rank test were used for prediction. The hypermethylation of PTGER4 at the CpG_4, CpG_5, CpG_15, and CpG_17 tumor tissue sites was strongly correlated with shorter recurrence-free survival (RFS), progression-free survival (PFS), and overall survival (OS) [hazard ratio (HR) = 3.26, 95% confidence interval (CI) = 1.38-7.73 for RFS, HR = 2.35 and 95% CI = 1.17-4.71 for PFS, HR = 4.32 and 95% CI = 1.8-10.5 for OS]. By contrast, RFS and PFS were significantly longer in the case of increased methylation of ZNF43 at the CpG_5 site of normal tissue [HR = 2.33, 95% CI = 1.07-5.08 for RFS, HR = 2.42 and 95% CI = 1.19-4.91 for PFS]. Aberrant methylation at specific CpG sites indicates tissue with aggressive behavior. Therefore, the differential methylation of PTGER4 and ZNF43 at specific loci can be employed for the prognosis of patients with CRC.

Human Papillomavirus and Risk of Colorectal Cancer: An Analysis of Nationwide Claims Data.

Background and Objectives: Although human papillomavirus (HPV) is a major etiology of cervical and anogenital cancers, whether it is associated with colorectal carcinogenesis is yet undetermined. Materials and Methods: The longitudinal association of HPV infection with colorectal cancer (CRC) was evaluated using 2000-2013 data from a nationwide Taiwanese claims database. In this retrospective cohort study, 358 patients with primary HPV diagnoses (HPV-infected cohort) and 1432 patients without such a diagnosis (HPV-uninfected cohort) were recruited between 2000 and 2006. Both cohorts were followed up to identify CRC incidences from 2006 to 2013. Hazard ratios (HRs) and their 95% confidence intervals (CIs) derived from Cox proportional hazards models were used to estimate the association between HPV and CRC risk. Results: The HPV-infected cohort had a significantly higher cumulative incidence of CRC than the HPV-uninfected cohort. The presence of HPV was associated with an increased risk of CRC (adjusted HR, 1.63; 95% CI, 1.02-3.62). Furthermore, the significant HPV-CRC risk association was evident in both sexes. Conclusions: This population-based cohort study reveals longitudinal evidence that HPV is associated with an increased risk of CRC. Further studies are required to verify the role of HPV in colorectal carcinogenesis.

Association between gastroesophageal reflux disease and colorectal cancer risk: a population-based cohort study.

PURPOSE: Several studies have investigated the association between gastroesophageal reflux disease (GERD) and colorectal cancer (CRC) risk, but the presented scientific results are highly debatable. This study examined the longitudinal association between GERD and CRC in an Asian population. METHODS: A retrospective cohort study was performed using the National Health Insurance Research Database of Taiwan. The study cohort comprised 45,828 individuals with newly diagnosed GERD (the GERD cohort) and 229,140 age, sex, and date of enrollment-matched patients without GERD (the comparison cohort) from 2000 to 2006. The primary outcome was the incidence of CRC. To estimate the effect of GERD on the risk of CRC, the Cox proportional hazards model was fitted to estimate hazard ratios (HRs) and 95% confidence intervals (CIs). RESULTS: There were 785 newly diagnosed CRC patients in the 45,828 patients with GERD. Relatively, there were 2375 incident CRC cases in 229,140 patients without GERD. The incidence rate of CRC for the GERD cohort (17.60 per 10,000 person-years) was significantly higher than the corresponding incidence rate for the comparison cohort (10.22 per 10,000 person-years). After adjustment for confounders, GERD was associated with a significantly increased risk of CRC (adjusted HR,1.76; 95% CI, 1.62-2.90). Of note, a significant association between GERD and CRC risk was evident in both genders. CONCLUSIONS: In conclusion, this nationwide population-based cohort study supports the hypothesis that GERD was associated with a significantly increased risk of CRC. Our findings warrant still further investigation of the underlying mechanisms related to carcinogenic effect of GERD on colorectal carcinoma.

Early Repetitive Transcranial Magnetic Stimulation Exerts Neuroprotective Effects and Improves Motor Functions in Hemiparkinsonian Rats.

Repetitive transcranial magnetic stimulation (rTMS) is a popular noninvasive technique for modulating motor cortical plasticity and has therapeutic potential for the treatment of Parkinson's disease (PD). However, the therapeutic benefits and related mechanisms of rTMS in PD are still uncertain. Accordingly, preclinical animal research is helpful for enabling translational research to explore an effective therapeutic strategy and for better understanding the underlying mechanisms. Therefore, the current study was designed to identify the therapeutic effects of rTMS on hemiparkinsonian rats. A hemiparkinsonian rat model, induced by unilateral injection of 6-hydroxydopamine (6-OHDA), was applied to evaluate the therapeutic potential of rTMS in motor functions and neuroprotective effect of dopaminergic neurons. Following early and long-term rTMS intervention with an intermittent theta burst stimulation (iTBS) paradigm (starting 24 h post-6-OHDA lesion, 1 session/day, 7 days/week, for a total of 4 weeks) in awake hemiparkinsonian rats, the effects of rTMS on the performance in detailed functional behavioral tests, including video-based gait analysis, the bar test for akinesia, apomorphine-induced rotational analysis, and tests of the degeneration level of dopaminergic neurons, were identified. We found that four weeks of rTMS intervention significantly reduced the aggravation of PD-related symptoms post-6-OHDA lesion. Immunohistochemically, the results showed that tyrosine hydroxylase- (TH-) positive neurons in the substantia nigra pars compacta (SNpc) and fibers in the striatum were significantly preserved in the rTMS treatment group. These findings suggest that early and long-term rTMS with the iTBS paradigm exerts neuroprotective effects and mitigates motor impairments in a hemiparkinsonian rat model. These results further highlight the potential therapeutic effects of rTMS and confirm that long-term rTMS treatment might have clinical relevance and usefulness as an additional treatment approach in individuals with PD.

Dopamine D3 receptor and GSK3ß signaling mediate deficits in novel object recognition memory within dopamine transporter knockdown mice.

BACKGROUND: Over-stimulation of dopamine signaling is thought to underlie the pathophysiology of a list of mental disorders, such as psychosis, mania and attention-deficit/hyperactivity disorder. These disorders are frequently associated with cognitive deficits in attention or learning and memory, suggesting that persistent activation of dopamine signaling may change neural plasticity to induce cognitive or emotional malfunction. METHODS: Dopamine transporter knockdown (DAT-KD) mice were used to mimic a hyper-dopamine state. Novel object recognition (NOR) task was performed to assess the recognition memory. To test the role of dopamine D3 receptor (D3R) on NOR, DAT-KD mice were treated with either a D3R antagonist, FAUC365 or by deletion of D3R. Total or phospho-GSK3 and -ERK1/2 signals in various brain regions were measured by Western blot analyses. To examine the impact of GSK3 signal on NOR, wild-type mice were systemically treated with GSK3 inhibitor SB216763 or, micro-injected with lentiviral shRNA of GSK3ß or GSK3α in the medial prefrontal cortex (mPFC). RESULTS: We confirmed our previous findings that DAT-KD mice displayed a deficit in NOR memory, which could be prevented by deletion of D3R or exposure to FAUC365. In WT mice, p-GSK3α and p-GSK3ß were significantly decreased in the mPFC after exposure to novel objects; however, the DAT-KD mice exhibited no such change in mPFC p-GSK3α/ß levels. DAT-KD mice treated with FAUC365 or with D3R deletion exhibited restored novelty-induced GSK3 dephosphorylation in the mPFC. Moreover, inhibition of GSK3 in WT mice diminished NOR performance and impaired recognition memory. Lentiviral shRNA knockdown of GSK3ß, but not GSK3α, in the mPFC of WT mice also impaired NOR. CONCLUSION: These findings suggest that D3R acts via GSK3ß signaling in the mPFC to play a functional role in NOR memory. In addition, treatment with D3R antagonists may be a reasonable approach for ameliorating cognitive impairments or episodic memory deficits in bipolar disorder patients.

Protective Effects of Rhodiola Crenulata Extract on Hypoxia-Induced Endothelial Damage via Regulation of AMPK and ERK Pathways.

Rhodiola crenulata root extract (RCE) has been shown to possess protective activities against hypoxia both in vitro and in vivo. However, the effects of RCE on response to hypoxia in the endothelium remain unclear. In this study, we aimed to examine the effects of RCE in endothelial cells challenged with hypoxic exposure and to elucidate the underlying mechanisms. Human umbilical vein endothelial cells were pretreated with or without RCE and then exposed to hypoxia (1% O2) for 24 h. Cell viability, nitric oxide (NO) production, oxidative stress markers, as well as mechanistic readouts were studied. We found that hypoxia-induced cell death, impaired NO production, and oxidative stress. These responses were significantly attenuated by RCE treatment and were associated with the activation of AMP-activated kinase and extracellular signal-regulated kinase 1/2 signaling pathways. In summary, we showed that RCE protected endothelial cells from hypoxic insult and suggested that R. crenulata might be useful for the prevention of hypoxia-associated vascular dysfunction.

RETRACTED: Cheng et al. Using Comorbidity Pattern Analysis to Detect Reliable Methylated Genes in Colorectal Cancer Verified by Stool DNA Test. Genes 2021, 12, 1539.

The Genes journal retracts the article "Using Comorbidity Pattern Analysis to Detect Reliable Methylated Genes in Colorectal Cancer Verified by Stool DNA Test" [...].

Overdominant expression of genes plays a key role in root growth of tobacco hybrids.

Heterosis has greatly improved the yield and quality of crops. However, previous studies often focused on improving the yield and quality of the shoot system, while research on the root system was neglected. We determined the root numbers of 12 F1 hybrids, all of which showed strong heterosis, indicating that tobacco F1 hybrids have general heterosis. To understand its molecular mechanism, we selected two hybrids with strong heterosis, GJ (G70 × Jiucaiping No.2) and KJ (K326 × Jiucaiping No.2), and their parents for transcriptome analysis. There were 84.22% and 90.25% of the differentially expressed genes were overdominantly expressed. The enrichment analysis of these overdominantly expressed genes showed that "Plant hormone signal transduction", "Phenylpropanoid biosynthesis", "MAPK signaling pathway - plant", and "Starch and sucrose metabolism" pathways were associated with root development. We focused on the analysis of the biosynthetic pathways of auxin(AUX), cytokinins(CTK), abscisic acid(ABA), ethylene(ET), and salicylic acid(SA), suggesting that overdominant expression of these hormone signaling pathway genes may enhance root development in hybrids. In addition, Nitab4.5_0011528g0020、Nitab4.5_0003282g0020、Nitab4.5_0004384g0070 may be the genes involved in root growth. Genome-wide comparative transcriptome analysis enhanced our understanding of the regulatory network of tobacco root development and provided new ideas for studying the molecular mechanisms of tobacco root development.

Negative regulation of tobacco cold stress tolerance by NtPhyA.

Cold stress is a non-biological stressor that adversely affects tobacco yield and leaf quality. Plant photoreceptor proteins, which function as dual light-temperature sensors, play a vital role in temperature changes, making them crucial for responses to non-biological stressors. However, the regulatory mechanisms of PhyA in tobacco remain poorly understood. Therefore, in this study, we aimed to clone the NtPhyA gene from tobacco and generate overexpression (OE-NtPhyA) and mutant (KO-NtPhyA) constructs of NtPhyA. By assessing the physiological and biochemical responses of the mutants under cold stress and performing transcriptome sequencing, we determined the signalling mechanism of NtPhyA under cold stress. Comparative analysis with wild-type (WT) NtPhyA revealed that KO-NtPhyA exhibited increased seed germination rates and reduced wilting under cold stress. In additional, the degree of damage to leaf cells, cell membranes, and stomatal structures was mitigated, and the levels of reactive oxygen species (ROS) were significantly decreased. Antioxidant enzyme activity, net photosynthetic rate, and Fv/Fm were significantly enhanced in KO-NtPhyA, whereas the opposite effects were observed in OE-NtPhyA. These findings indicate that KO-NtPhyA augments tobacco tolerance to cold stress, implying a negative regulatory role of NtPhyA in tobacco during cold stress. Transcriptome analysis revealed that NtPhyA governs the expression of a cascade of genes involved in the response to oxygen-containing compounds, hydrogen peroxide (H2O2), ROS, temperature stimuli, photosystem II oxygen-evolving complex assembly, water channel activity, calcium channel activity, and carbohydrate transport. Collectively, our findings indicate that NtPhyA activates downstream gene expression to enhance the resilience of tobacco to cold stress.

Integrated transcriptome and proteome analysis provides insights into the mechanism of cytoplasmic male sterility (CMS) in tobacco (Nicotiana tabacum L.).

Cytoplasmic male sterility (CMS) is critical in maximizing crop yield and quality by utilizing tobacco heterosis. However, the mechanism of tobacco CMS formation remains unknown. Using paraffin section observation, transcriptome sequencing, and TMT proteomic analysis, this study describes the differences in expression profiles in morphology, transcription, and translation between the sua-CMS tobacco line (MSYY87) and its corresponding maintainer line (YY87). According to the microspore morphology, MSYY87 began to exhibit abnormal microspore development during the early stages of germination and differentiation (androgynous primordium differentiation stage). According to transcriptomic and proteomic analyses, 17 genes/proteins involved in lipid transport/binding and phenylpropane metabolism were significantly down-regulated at both the mRNA and protein levels. Through further analysis, we identified some key genes that may be involved in tobacco male sterility, including ß-GLU related to energy metabolism, 4CL and bHLHs related to anther wall formation, nsLTPs related to pollen germination and anther cuticle, and bHLHs related to pollen tapetum degradation. We speculate that the down-regulation of these genes affects the normal physiological metabolism, making tobacco plants show male sterility. SIGNIFICANCE: Cytoplasmic male sterility (CMS) plays a vital role in utilizing tobacco heterosis and enhancing crop yield and quality. We observed paraffin sections and conducted transcriptome sequencing and mitochondrial proteomics to examine the tobacco CMS line Yunyan 87 (MSYY87) and its maintainer line Yunyan 87 (YY87). The down-regulation expression of ß-GLU resulted in insufficient ATP supply, which resulted in disordered energy metabolism. The down-regulation expression of 4CL, nsLTPs and bHLHs may affect the formation of anther wall and anther cuticle, pollen germination, as well as the degradation of pollen tapetum. These various abnormal physiological processes, the male sterility of tobacco is finally caused. The findings shed light on the molecular mechanisms of tobacco CMS and serve as a model for fertility research in other flowering plants.

Polygonum barbatum extract reduces colorectal cancer cell proliferation, migration, invasion, and epithelial-mesenchymal transition via YAP and ß-catenin pathway regulation.

Colorectal cancer (CRC) is the third most common cancer worldwide with novel therapeutic developmental challenges. Polygonum barbatum has anticancer potential, but its mechanism(s) are unclear. This study investigates the inhibitory effect of P. barbatum on human CRC cells. Polygonum barbatum extract (PBE) and quercetin standard HPLC fingerprints were determined using analytical RP-HPLC and evaluations were completed using the human colon cancer cell line HCT-116 (KRASG13D mutation) and HT-29 (BRAF mutation) cells. Post-PBE treatment, cell viability, colony formation, migration, invasion, and apoptosis, as well as changes in the whole-transcriptome of cells were analyzed. PBE significantly reduced CRC cell growth, migration, and invasion, and the genes responsible for extracellular matrix (ECM) organization, cell motility, and cell growth were suppressed by PBE. The differentially expressed genes revealed that PBE treatment exerted a significant effect on the ECM interaction and focal adhesion pathways. Epithelial-to-mesenchymal transition markers, N-cadherin, vimentin, SLUG, and SNAIL, were shown to be regulated by PBE. These effects were associated with blockade of the Yes-associated protein and the GSK3ß/ß-catenin axis. PBE exerts a significant inhibitory effect on CRC cells and may be applicable in clinical trials.