Peripheral blood RNA biomarkers can predict lesion severity in degenerative cervical myelopathy

Degenerative cervical myelopathy is a common cause of spinal cord injury,with longer symptom duration and higher myelopathy severity indicating a worse prognosis.While numerous studies have investigated serological biomarkers for acute spinal cord injury,few studies have explored such biomarkers for diagnosing degenerative cervical myelopathy.This study involved 30 patients with degenerative cervical myelopathy(51.3±7.3 years old,12 women and 18 men),seven healthy controls(25.7±1.7 years old,one woman and six men),and nine patients with cervical spondylotic radiculopathy(51.9±8.6 years old,three women and six men).Analysis of blood samples from the three groups showed clear differences in transcriptomic characteristics.Enrichment analysis identified 128 differentially expressed genes that were enriched in patients with neurological disabilities.Using least absolute shrinkage and selection operator analysis,we constructed a five-gene model(TBCD,TPM2,PNKD,EIF4G2,and AP5Z1)to diagnose degenerative cervical myelopathy with an accuracy of 93.5%.One-gene models(TCAP and SDHA)identified mild and severe degenerative cervical myelopathy with accuracies of 83.3%and 76.7%,respectively.Signatures of two immune cell types(memory B cells and memory-activated CD4^(+)T cells)predicted levels of lesions in degenerative cervical myelopathy with 80%accuracy.Our results suggest that peripheral blood RNA biomarkers could be used to predict lesion severity in degenerative cervical myelopathy.

The autophagy-lysosome pathway:a potential target in the chemical and gene therapeutic strategies for Parkinson’s disease

Parkinson’s disease is a common neurodegenerative disease with movement disorders associated with the intracytoplasmic deposition of aggregate proteins such asα-synuclein in neurons.As one of the major intracellular degradation pathways,the autophagy-lysosome pathway plays an important role in eliminating these proteins.Accumulating evidence has shown that upregulation of the autophagy-lysosome pathway may contribute to the clearance ofα-synuclein aggregates and protect against degeneration of dopaminergic neurons in Parkinson’s disease.Moreover,multiple genes associated with the pathogenesis of Parkinson’s disease are intimately linked to alterations in the autophagy-lysosome pathway.Thus,this pathway appears to be a promising therapeutic target for treatment of Parkinson’s disease.In this review,we briefly introduce the machinery of autophagy.Then,we provide a description of the effects of Parkinson’s disease–related genes on the autophagy-lysosome pathway.Finally,we highlight the potential chemical and genetic therapeutic strategies targeting the autophagy–lysosome pathway and their applications in Parkinson’s disease.

Dysregulation of genes involved in the long-chain fatty acid transport in pancreatic ductal adenocarcinoma

BACKGROUND Pancreatic ductal adenocarcinoma(PDAC)is an aggressive lethal malignancy with limited options for treatment and a 5-year survival rate of 11%in the United States.As for other types of tumors,such as colorectal cancer,aberrant de novo lipid synthesis and reprogrammed lipid metabolism have been suggested to be associated with PDAC development and progression.AIM To identify the possible involvement of lipid metabolism in PDAC by analyzing in tumoral and non-tumoral tissues the expression level of the most relevant genes involved in the long-chain fatty acid(FA)import into cell.METHODS A gene expression analysis of FASN,CD36,SLC27A1,SLC27A2,SLC27A3,SLC27A4,SLC27A5,ACSL1,and ACSL3 was performed by qRT-PCR in 24 tumoral PDAC tissues and 11 samples from non-tumoral pancreatic tissues obtained via fine needle aspiration or via surgical resection.The genes were considered significantly dysregulated between the groups when the p value was<0.05 and the fold change(FC)was≤0.5 and≥2.RESULTS We found that three FA transporters and two long-chain acyl-CoA synthetases genes were significantly upregulated in the PDAC tissue compared to the non-tumoral tissue:SLC27A2(FC=5.66;P=0.033),SLC27A3(FC=2.68;P=0.040),SLC27A4(FC=3.13;P=0.033),ACSL1(FC=4.10;P<0.001),and ACSL3(FC=2.67;P=0.012).We further investigated any possible association between the levels of the analyzed mRNAs and the specific characteristics of the tumors,including the anatomic location,the lymph node involvement,and the presence of metastasis.A significant difference in the expression of SLC27A3(FC=3.28;P=0.040)was found comparing patients with and without lymph nodes involvement with an overexpression of this transcript in 17 patients presenting tumoral cells in the lymph nodes.CONCLUSION Despite the low number of patients analyzed,these preliminary results seem to be promising.Addressing lipid metabolism through a broad strategy could be a beneficial way to treat this malignancy.Future in vitro and in vivo studies on these genes may offer important insights into the mechanisms linking PDAC with the long-chain FA import pathway.

Recovery of the injured neural system through gene delivery to surviving neurons in Parkinson’s disease

A critical unaddressed problem in Parkinson’s disease is the lack of therapy that slows or hampers neurodegeneration.While medications effectively manage symptoms,they offer no long-term benefit because they fail to address the underlying neuronal loss.This highlights that the elusive goals of halting progression and restoring damaged neurons limit the long-term impact of current approaches.Recent clinical trials using gene therapy have demonstrated the safety of various vector delivery systems,dosages,and transgenes expressed in the central nervous system,signifying tangible and substantial progress in applying gene therapy as a promising Parkinson’s disease treatment.Intriguingly,at diagnosis,many dopamine neurons remain in the substantia nigra,offering a potential window for recovery and survival.We propose that modulating these surviving dopamine neurons and axons in the substantia nigra and striatum using gene therapy offers a potentially more impactful therapeutic approach for future research.Moreover,innovative gene therapies that focus on preserving the remaining elements may have significant potential for enhancing long-term outcomes and the quality of life for patients with Parkinson’s disease.In this review,we provide a perspective on how gene therapy can protect vulnerable elements in the substantia nigra and striatum,offering a novel approach to addressing Parkinson’s disease at its core.

AAV2-PDE6B restores retinal structure and function in the retinal degeneration 10 mouse model of retinitis pigmentosa by promoting phototransduction and inhibiting apoptosis

Retinitis pigmentosa is a group of inherited diseases that lead to retinal degeneration and photoreceptor cell death.However,there is no effective treatment for retinitis pigmentosa caused by PDE6B mutation.Adeno-associated virus(AAV)-mediated gene therapy is a promising strategy for treating retinitis pigmentosa.The aim of this study was to explore the molecular mechanisms by which AAV2-PDE6B rescues retinal function.To do this,we injected retinal degeneration 10(rd10)mice subretinally with AAV2-PDE6B and assessed the therapeutic effects on retinal function and structure using dark-and light-adapted electroretinogram,optical coherence tomography,and immunofluorescence.Data-independent acquisition-mass spectrometry-based proteomic analysis was conducted to investigate protein expression levels and pathway enrichment,and the results from this analysis were verified by real-time polymerase chain reaction and western blotting.AAV2-PDE6B injection significantly upregulated PDE6βexpression,preserved electroretinogram responses,and preserved outer nuclear layer thickness in rd10 mice.Differentially expressed proteins between wild-type and rd10 mice were closely related to visual perception,and treating rd10 mice with AAV2-PDE6B restored differentially expressed protein expression to levels similar to those seen in wild-type mice.Kyoto Encyclopedia of Genes and Genome analysis showed that the differentially expressed proteins whose expression was most significantly altered by AAV2-PDE6B injection were enriched in phototransduction pathways.Furthermore,the phototransductionrelated proteins Pde6α,Rom1,Rho,Aldh1a1,and Rbp1 exhibited opposite expression patterns in rd10 mice with or without AAV2-PDE6B treatment.Finally,Bax/Bcl-2,p-ERK/ERK,and p-c-Fos/c-Fos expression levels decreased in rd10 mice following AAV2-PDE6B treatment.Our data suggest that AAV2-PDE6B-mediated gene therapy promotes phototransduction and inhibits apoptosis by inhibiting the ERK signaling pathway and upregulating Bcl-2/Bax expression in retinitis pigmentosa.

Heterogeneity of mature oligodendrocytes in the central nervous system

Mature oligodendrocytes form myelin sheaths that are crucial for the insulation of axons and efficient signal transmission in the central nervous system.Recent evidence has challenged the classical view of the functionally static mature oligodendrocyte and revealed a gamut of dynamic functions such as the ability to modulate neuronal circuitry and provide metabolic support to axons.Despite the recognition of potential heterogeneity in mature oligodendrocyte function,a comprehensive summary of mature oligodendrocyte diversity is lacking.We delve into early 20th-century studies by Robertson and Río-Hortega that laid the foundation for the modern identification of regional and morphological heterogeneity in mature oligodendrocytes.Indeed,recent morphologic and functional studies call into question the long-assumed homogeneity of mature oligodendrocyte function through the identification of distinct subtypes with varying myelination preferences.Furthermore,modern molecular investigations,employing techniques such as single cell/nucleus RNA sequencing,consistently unveil at least six mature oligodendrocyte subpopulations in the human central nervous system that are highly transcriptomically diverse and vary with central nervous system region.Age and disease related mature oligodendrocyte variation denotes the impact of pathological conditions such as multiple sclerosis,Alzheimer's disease,and psychiatric disorders.Nevertheless,caution is warranted when subclassifying mature oligodendrocytes because of the simplification needed to make conclusions about cell identity from temporally confined investigations.Future studies leveraging advanced techniques like spatial transcriptomics and single-cell proteomics promise a more nuanced understanding of mature oligodendrocyte heterogeneity.Such research avenues that precisely evaluate mature oligodendrocyte heterogeneity with care to understand the mitigating influence of species,sex,central nervous system region,age,and disease,hold promise for the development of therapeutic interventions targeting varied central nervous system pathology.

Pan-TRK positive uterine sarcoma in immunohistochemistry without neurotrophic tyrosine receptor kinase gene fusions:A case report

BACKGROUND The classification of uterine sarcomas is based on distinctive morphological and immunophenotypic characteristics,increasingly supported by molecular genetic diagnostics.Data on neurotrophic tyrosine receptor kinase(NTRK)gene fusionpositive uterine sarcoma,potentially aggressive and morphologically similar to fibrosarcoma,are limited due to its recent recognition.Pan-TRK immunohistochemistry(IHC)analysis serves as an effective screening tool with high sensitivity and specificity for NTRK-fusion malignancies.CASE SUMMARY We report a case of a malignant mesenchymal tumor originating from the uterine cervix,which was pan-TRK IHC-positive but lacked NTRK gene fusions,accompanied by a brief literature review.A 55-year-old woman presented to the emergency department with abdominal pain and distension,exhibiting significant ascites and multiple solid pelvic masses.Pelvic examination revealed a tumor encompassing the uterine cervix,extending to the vagina and uterine corpus.A punch biopsy of the cervix indicated NTRK sarcoma with positive immunochemical pan-TRK stain.However,subsequent next generation sequencing revealed no NTRK gene fusion,leading to a diagnosis of poorly differentiated,advanced-stage sarcoma.CONCLUSION The clinical significance of NTRK gene fusion lies in potential treatment with TRK inhibitors for positive sarcomas.Identifying such rare tumors is crucial due to the potential applicability of tropomyosin receptor kinase inhibitor treatment.

Genetic and epigenetic alterations associated with gestational diabetes mellitus and adverse neonatal outcomes

Gestational diabetes mellitus(GDM)is a metabolic disorder,recognised during 24-28 weeks of pregnancy.GDM is linked with adverse newborn outcomes such as macrosomia,premature delivery,metabolic disorder,cardiovascular,and neurological disorders.Recent investigations have focused on the correlation of genetic factors such asβ-cell function and insulin secretary genes(transcription factor 7 like 2,potassium voltage-gated channel subfamily q member 1,adipo-nectin etc.)on maternal metabolism during gestation leading to GDM.Epigenetic alterations like DNA methylation,histone modification,and miRNA expression can influence gene expression and play a dominant role in feto-maternal meta-bolic pathways.Interactions between genes and environment,resulting in differ-ential gene expression patterns may lead to GDM.Researchers suggested that GDM women are more susceptible to insulin resistance,which alters intrauterine surroundings,resulting hyperglycemia and hyperinsulinemia.Epigenetic modi-fications in genes affecting neuroendocrine activities,and metabolism,increase the risk of obesity and type 2 diabetes in offspring.There is currently no treatment or effective preventive method for GDM,since the molecular processes of insulin resistance are not well understood.The present review was undertaken to un-derstand the pathophysiology of GDM and its effects on adverse neonatal out-comes.In addition,the study of genetic and epigenetic alterations will provide lead to researchers in the search for predictive molecular biomarkers.

Autophagy-targeting modulation to promote peripheral nerve regeneration

Nerve regeneration following traumatic peripheral nerve injuries and neuropathies is a complex process modulated by diverse factors and intricate molecular mechanisms.Past studies have focused on factors that stimulate axonal outgrowth and myelin regeneration.However,recent studies have highlighted the pivotal role of autophagy in peripheral nerve regeneration,particularly in the context of traumatic injuries.Consequently,autophagy-targeting modulation has emerged as a promising therapeutic approach to enhancing peripheral nerve regeneration.Our current understanding suggests that activating autophagy facilitates the rapid clearance of damaged axons and myelin sheaths,thereby enhancing neuronal survival and mitigating injury-induced oxidative stress and inflammation.These actions collectively contribute to creating a favorable microenvironment for structural and functional nerve regeneration.A range of autophagyinducing drugs and interventions have demonstrated beneficial effects in alleviating peripheral neuropathy and promoting nerve regeneration in preclinical models of traumatic peripheral nerve injuries.This review delves into the regulation of autophagy in cell types involved in peripheral nerve regeneration,summarizing the potential drugs and interventions that can be harnessed to promote this process.We hope that our review will offer novel insights and perspectives on the exploitation of autophagy pathways in the treatment of peripheral nerve injuries and neuropathies.

AAV-mediated expression of p65shRNA and bone morphogenetic protein 4 synergistically enhances chondrocyte regeneration

BACKGROUND:Adeno-associated virus(AAV)gene therapy has been proven to be reliable and safe for the treatment of osteoarthritis in recent years.However,given the complexity of osteoarthritis pathogenesis,single gene manipulation for the treatment of osteoarthritis may not produce satisfactory results.Previous studies have shown that nuclear factorκB could promote the inflammatory pathway in osteoarthritic chondrocytes,and bone morphogenetic protein 4(BMP4)could promote cartilage regeneration.OBJECTIVE:To test whether combined application of AAV-p65shRNA and AAV-BMP4 will yield the synergistic effect on chondrocytes regeneration and osteoarthritis treatment.METHODS:Viral particles containing AAV-p65-shRNA and AAV-BMP4 were prepared.Their efficacy in inhibiting inflammation in chondrocytes and promoting chondrogenesis was assessed in vitro and in vivo by transfecting AAV-p65-shRNA or AAV-BMP4 into cells.The experiments were divided into five groups:PBS group;osteoarthritis group;AAV-BMP4 group;AAV-p65shRNA group;and BMP4-p65shRNA 1:1 group.Samples were collected at 4,12,and 24 weeks postoperatively.Tissue staining,including safranin O and Alcian blue,was applied after collecting articular tissue.Then,the optimal ratio between the two types of transfected viral particles was further investigated to improve the chondrogenic potential of mixed cells in vivo.RESULTS AND CONCLUSION:The combined application of AAV-p65shRNA and AAV-BMP4 together showed a synergistic effect on cartilage regeneration and osteoarthritis treatment.Mixed cells transfected with AAV-p65shRNA and AAV-BMP4 at a 1:1 ratio produced the most extracellular matrix synthesis(P<0.05).In vivo results also revealed that the combination of the two viruses had the highest regenerative potential for osteoarthritic cartilage(P<0.05).In the present study,we also discovered that the combined therapy had the maximum effect when the two viruses were administered in equal proportions.Decreasing either p65shRNA or BMP4 transfected cells resulted in less collagen II synthesis.This implies that inhibiting inflammation by p65shRNA and promoting regeneration by BMP4 are equally important for osteoarthritis treatment.These findings provide a new strategy for the treatment of early osteoarthritis by simultaneously inhibiting cartilage inflammation and promoting cartilage repair.

PtrHB7, a class III HD-Zip Gene, Plays a Critical Role in Regulation of Vascular Cambium Differentiation in Populus

A key question in the secondary growth of trees is how differentiation of the vascular cambium cells is directed to concurrently form two different tissues： xylem or phloem, class III homeodomain-leucine zipper （HD-Zip III） genes are known to play critical roles in the initiation, patterning, and differentiation of the vascular system in the process of primary and secondary growth. However, the mechanism of how these genes control secondary vascular dif- ferentiation is unknown. Here, we show that a Populus class III HD-Zip gene, PtrHB7, was preferentially expressed in cambial zone. PtrHB7-suppressed plants displayed significant changes in vascular tissues with a reduction in xylem but increase in phloem. Transcriptional analysis revealed that genes regulating xylem differentiation were down-regulated, whereas genes regulating phloem differentiation were up-regulated. Correspondingly, PtrHB7 overexpression enhanced differentiation of cambial cells toward xylem cells but inhibited phloem differentiation. PtrHB7 regulation on cambial cell differentiation was associated with its transcript abundance. Together, the results demonstrated that PtrHB7 plays a critical role in controlling a balanced differentiation between secondary xylem and phloem tissues in the process of Populus secondary growth in a dosage-dependent manner.

工业大麻HD-Zip基因家族鉴定与干旱胁迫下的表达分析

HD-Zip在植物抗逆过程中起着重要的调节作用,为了解大麻基因组中HD-Zip基因家族的特征和潜在功能,并为进一步研究CsHDZ基因对干旱胁迫下的调控提供重要线索,根据大麻全基因组数据库中的参考序列,利用生物信息学的方法对大麻HD-Zip基因家族进行全基因组鉴定,并系统分析其理化特性、进化发育、基因结构、启动子顺式结合元件并测定干旱胁迫下的表达模式。结果表明,大麻HD-Zip基因家族共筛选出20个CsHDZs基因,分别定位在7条染色体上,属于4个亚家族,编码氨基酸204~837个,理论等电点4.54~9.04,属不稳定蛋白,亚细胞定位预测全部定位于细胞核;蛋白保守基序分析显示,CsHDZs共有10个保守基序,其中motif 1和motif 9为各成员共有;启动子调控元件分析发现,CsHDZs富含多种逆境胁迫应答相关元件。实时荧光定量分析结果显示,CsHDZ1/8/10基因转录水平在干旱前期无显著差异,而后期显著上调。研究表明CsHDZs基因参与了胁迫应答过程并能够积极响应干旱。

Expression Level of a Transcription Factor Gene Mdhd-Zip Up-regulated during Apple Fruit Senescence

[Objective] This study aimed to explore the role of one apple transcription factor of homeodomain-leucine zipper(MdHD-Zip) during apple fruit senescence.[Method] Postharvest Red Fuji fruits(Malus domestica Borkh ‘Red Fuji') were stored at room temperature(18 ℃-20 ℃) and cold condition(0 ± 1 ℃) separately.Fruit firmness and ethylene production during storage process were analyzed.Transcript level of Md HD-Zip was detected by real-time quantitative PCR during apple fruit storage under room temperature and cold condition.[Result] Expression level of Md HD-Zip was found up-regulated in later stage of apple fruit senescence at room temperature,while it showed a peak level after one month of cold storage.[Conclusion] The results of the present study suggest that Md HD-Zip may play a role in regulating apple fruit senescence.

海稻86转录因子HD-ZIP的克隆及表达分析

同源结构域亮氨酸拉链(HD-ZIP)转录因子是植物独有的转录因子,分为四个不同的亚家族(HDZIPⅠ~Ⅳ),其中HD-ZIPⅠ在植物发育过程的调控、信号网络和对环境胁迫的反应中发挥关键作用。海稻86是一种耐盐碱能力较高的特异水稻种质资源。为解析HD-ZIPⅠ在盐胁迫应答过程中的功能,本研究以海稻86为材料,利用RT-PCR技术克隆了一个HD-ZIPⅠ基因,命名为OsHDZ1,并对其进行生物信息学分析、亚细胞定位及盐胁迫下表达分析。结果表明,OsHDZ1基因的CDS序列全长831 bp,编码276个氨基酸,蛋白分子量为30.65 kDa,理论等电点为5.19,为亲水性不稳定蛋白;OsHDZ1蛋白不含跨膜结构,含有25个磷酸化位点,二级结构主要由α-螺旋和无规则卷曲组成;同源性分析发现该蛋白序列与禾本科植物弯叶画眉草和小麦具有较高的亲缘关系;对OsHDZ1启动子元件预测发现其具有脱落酸、茉莉酸等激素响应元件,同时具有环境胁迫等响应元件;细胞定位分析显示该蛋白定位于细胞核。荧光定量PCR结果发现,在盐胁迫处理下,Os-HDZ1表达量整体呈上升,在60 h之前逐渐上升,在60 h达到最高,在72 h表达量下降,表明该基因可能在海稻耐盐性中具有调控功能。本研究结果可为后续深入解析OsHDZ1基因调控海稻耐盐的功能奠定基础,为培育耐盐作物提供基因资源。

枇杷HD-ZipⅠ转录因子家族全基因组鉴定及表达模式分析

【目的】同源结构域-亮氨酸拉链(HD-Zip)转录因子参与多种植物的非生物胁迫响应过程。然而,在枇杷中,HD-ZipⅠ基因家族尚未被鉴定。【方法】利用生物信息学方法对全基因组范围内枇杷HD-ZipⅠ基因家族成员进行鉴定和综合分析,通过qRT-PCR法分析基因家族成员在枇杷不同组织和干旱胁迫下的表达特征。【结果】在枇杷基因组中筛选出20个HD-ZipⅠ家族成员。共线性分析结果发现了3对串联复制序列和22对片段复制序列,表明串联复制和片段复制可能促进了枇杷HD-ZipⅠ基因家族的扩张。蛋白序列比对分析表明,所有的HD-ZipⅠ家族成员均具有高度保守的HD和Zip结构域。系统发育分析表明,枇杷HD-ZipⅠ家族可以分为7个分支。HD-ZipⅠ各基因在枇杷不同组织中的表达模式有所差异。启动子序列分析表明,HD-ZipⅠ家族成员的启动子上含有多个与干旱胁迫和胁迫相关激素信号响应的顺式作用元件。干旱处理能够诱导EjHB9、EjHB10、EjHB17、EjHB18和EjHB20在叶片中的表达显著上调,预示着这些基因参与枇杷对干旱胁迫的响应。【结论】鉴定出5个受干旱胁迫显著诱导表达的枇杷HD-ZipⅠ基因,为进一步研究HD-ZipⅠ基因在响应枇杷干旱胁迫中的分子功能提供理论依据。

丹参HD-ZIP基因家族的鉴定与表达分析

为筛选丹参(Salvia miltiorrhiza)中响应高温胁迫的HD-ZIP基因,利用生物信息学研究方法,对丹参全基因组的HD-ZIP基因进行了鉴定和分析,并以天丹1号为材料,通过qPCR技术检测了丹参HDZIP基因在高温胁迫下的表达模式。结果表明,丹参全基因组中包含44个HD-ZIP基因(SmHD-ZIP),不均匀地分布于8条染色体上。SmHD-ZIP可分为HD-ZIPⅠ、HD-ZIPⅡ、HD-ZIPⅢ和HD-ZIPⅣ4个亚家族,序列分析结果表明,这些蛋白质的氨基酸残基数为180~982个,相对分子质量为20.947~109.620 ku。SmHD-ZIP均为亲水蛋白,无跨膜结构域,绝大多数不含信号肽,等电点为4.48~10.91,且几乎都在细胞核表达,蛋白质稳定性较差。44个SmHD-ZIP基因中有10个基因复制事件,均为纯化选择。结构和模体分析结果表明,同一亚家族成员的外显子数目基本一致,HD-ZIPⅢ亚家族成员的外显子数目最多,模体数也最多。模体1、6是SmHD-ZIP的保守模体,模体10、11、12、13、15为HD-ZIPⅢ亚家族特有,模体5为HD-ZIPⅣ亚家族特有。通过分析高温(37℃)胁迫下SmHD-ZIP的表达模式发现,表达量升高和下降的基因数基本一致,在上调表达的基因中,SmHD-ZIP1.11、SmHD-ZIP1.13、SmHD-ZIP2.2、SmHD-ZIP2.5、SmHD-ZIP3.1、SmHD-ZIP3.4、SmHD-ZIP4.9、SmHD-ZIP4.10和SmHD-ZIP4.12的表达量提高了10倍以上,可作为提高丹参耐热性的候选基因资源。

RPLP0/TBP are the most stable reference genes for human dental pulp stem cells under osteogenic differentiation

BACKGROUND Validation of the reference gene(RG)stability during experimental analyses is essential for correct quantitative real-time polymerase chain reaction(RT-qPCR)data normalisation.Commonly,in an unreliable way,several studies use genes involved in essential cellular functions[glyceraldehyde-3-phosphate dehydro-genase(GAPDH),18S rRNA,andβ-actin]without paying attention to whether they are suitable for such experimental conditions or the reason for choosing such genes.Furthermore,such studies use only one gene when Minimum Information for Publication of Quantitative Real-Time PCR Experiments guidelines recom-mend two or more genes.It impacts the credibility of these studies and causes dis-tortions in the gene expression findings.For tissue engineering,the accuracy of gene expression drives the best experimental or therapeutical approaches.We cultivated DPSCs under two conditions:Undifferentiated and osteogenic dif-ferentiation,both for 35 d.We evaluated the gene expression of 10 candidates for RGs[ribosomal protein,large,P0(RPLP0),TATA-binding protein(TBP),GAPDH,actin beta(ACTB),tubulin(TUB),aminolevulinic acid synthase 1(ALAS1),tyro-sine 3-monooxygenase/tryptophan 5-monooxygenase activation protein,zeta(YWHAZ),eukaryotic translational elongation factor 1 alpha(EF1a),succinate dehydrogenase complex,subunit A,flavoprotein(SDHA),and beta-2-micro-globulin(B2M)]every 7 d(1,7,14,21,28,and 35 d)by RT-qPCR.The data were analysed by the four main algorithms,ΔCt method,geNorm,NormFinder,and BestKeeper and ranked by the RefFinder method.We subdivided the samples into eight subgroups.RESULTS All of the data sets from clonogenic and osteogenic samples were analysed using the RefFinder algorithm.The final ranking showed RPLP0/TBP as the two most stable RGs and TUB/B2M as the two least stable RGs.Either theΔCt method or NormFinder analysis showed TBP/RPLP0 as the two most stable genes.However,geNorm analysis showed RPLP0/EF1αin the first place.These algorithms’two least stable RGs were B2M/GAPDH.For BestKeeper,ALAS1 was ranked as the most stable RG,and SDHA as the least stable RG.The pair RPLP0/TBP was detected in most subgroups as the most stable RGs,following the RefFinfer ranking.CONCLUSION For the first time,we show that RPLP0/TBP are the most stable RGs,whereas TUB/B2M are unstable RGs for long-term osteogenic differentiation of human DPSCs in traditional monolayers.

RNA sequencing of exosomes secreted by fibroblast and Schwann cells elucidates mechanisms underlying peripheral nerve regeneration

Exosomes exhibit complex biological functions and mediate a variety of biological processes,such as promoting axonal regeneration and functional recove ry after injury.Long non-coding RNAs(IncRNAs)have been reported to play a crucial role in axonal regeneration.Howeve r,the role of the IncRNA-microRNAmessenger RNA(mRNA)-competitive endogenous RNA(ceRNA)network in exosome-mediated axonal regeneration remains unclear.In this study,we performed RNA transcriptome sequencing analysis to assess mRNA expression patterns in exosomes produced by cultured fibroblasts(FC-EXOs)and Schwann cells(SCEXOs).Diffe rential gene expression analysis,Gene Ontology analysis,Kyoto Encyclopedia of Genes and Genomes analysis,and protein-protein intera ction network analysis were used to explo re the functions and related pathways of RNAs isolated from FC-EXOs and SC-EXOs.We found that the ribosome-related central gene Rps5 was enriched in FC-EXOs and SC-EXOs,which suggests that it may promote axonal regeneration.In addition,using the miRWalk and Starbase prediction databases,we constructed a regulatory network of ceRNAs targeting Rps5,including 27 microRNAs and five IncRNAs.The ceRNA regulatory network,which included Ftx and Miat,revealed that exsosome-derived Rps5 inhibits scar formation and promotes axonal regeneration and functional recovery after nerve injury.Our findings suggest that exosomes derived from fibro blast and Schwann cells could be used to treat injuries of peripheral nervous system.

Genetic dissection and validation of a major QTL for grain weight on chromosome 3B in bread wheat(Triticum aestivum L.)

Grain weight is one of the key components of wheat(Triticum aestivum L.)yield.Genetic manipulation of grain weight is an efficient approach for improving yield potential in breeding programs.A recombinant inbred line(RIL)population derived from a cross between W7268 and Chuanyu 12(CY12)was employed to detect quantitative trait loci(QTLs)for thousand-grain weight(TGW),grain length(GL),grain width(GW),and the ratio of grain length to width(GLW)in six environments.Seven major QTLs,QGl.cib-2D,QGw.cib-2D,QGw.cib-3B,QGw.cib-4B.1,QGlw.cib-2D.1,QTgw.cib-2D.1 and QTgw.cib-3B.1,were consistently identified in at least four environments and the best linear unbiased estimation(BLUE)datasets,and they explained 2.61 to 34.85%of the phenotypic variance.Significant interactions were detected between the two major TGW QTLs and three major GW loci.In addition,QTgw.cib-3B.1 and QGw.cib-3B were co-located,and the improved TGW at this locus was contributed by GW.Unlike other loci,QTgw.cib-3B.1/QGw.cib-3B had no effect on grain number per spike(GNS).They were further validated in advanced lines using Kompetitive Allele Specific PCR(KASP)markers,and a comparison analysis indicated that QTgw.cib-3B.1/QGw.cib-3B is likely a novel locus.Six haplotypes were identified in the region of this QTL and their distribution frequencies varied between the landraces and cultivars.According to gene annotation,spatial expression patterns,ortholog analysis and sequence variation,the candidate gene of QTgw.cib-3B.1/QGw.cib-3B was predicted.Collectively,the major QTLs and KASP markers reported here provide valuable information for elucidating the genetic architecture of grain weight and for molecular marker-assisted breeding in grain yield improvement.

Genetic and epigenetic targets of natural dietary compounds as anti-Alzheimer's agents

Alzheimer’s disease is a progressive neurodegenerative disorder and the most common cause of dementia that principally affects older adults.Pathogenic factors,such as oxidative stress,an increase in acetylcholinesterase activity,mitochondrial dysfunction,genotoxicity,and neuroinflammation are present in this syndrome,which leads to neurodegeneration.Neurodegenerative pathologies such as Alzheimer’s disease are considered late-onset diseases caused by the complex combination of genetic,epigenetic,and environmental factors.There are two main types of Alzheimer’s disease,known as familial Alzheimer’s disease(onset<65 years)and late-onset or sporadic Alzheimer’s disease(onset≥65 years).Patients with familial Alzheimer’s disease inherit the disease due to rare mutations on the amyloid precursor protein(APP),presenilin 1 and 2(PSEN1 and PSEN2)genes in an autosomaldominantly fashion with closely 100%penetrance.In contrast,a different picture seems to emerge for sporadic Alzheimer’s disease,which exhibits numerous non-Mendelian anomalies suggesting an epigenetic component in its etiology.Importantly,the fundamental pathophysiological mechanisms driving Alzheimer’s disease are interfaced with epigenetic dysregulation.However,the dynamic nature of epigenetics seems to open up new avenues and hope in regenerative neurogenesis to improve brain repair in Alzheimer’s disease or following injury or stroke in humans.In recent years,there has been an increase in interest in using natural products for the treatment of neurodegenerative illnesses such as Alzheimer’s disease.Through epigenetic mechanisms,such as DNA methylation,non-coding RNAs,histone modification,and chromatin conformation regulation,natural compounds appear to exert neuroprotective effects.While we do not purport to cover every in this work,we do attempt to illustrate how various phytochemical compounds regulate the epigenetic effects of a few Alzheimer’s disease-related genes.