Molecular Biology Reports最新文献

Background: Cashew (Anacardium occidentale L.) is a widely cultivated tree with great economic significance. In cashew, several elite cultivars have been developed for commercial cultivation, which form the underpinning for the cashew-based industries and the several billion-dollar world trade. However, frequently the genetic purity of the planting material is not maintained, resulting in great economic losses. Therefore, there is a need to develop a reliable method for the identification of cultivars to avoid economic losses to the cultivators and the protection of cultivars by the breeders.

Methods and results: In this study, 35 new microsatellite/simple sequence repeat (SSR) markers were developed, and a set of 20 highly polymorphic and reproducible markers were used for DNA fingerprinting and genetic diversity analysis in 32 cashew cultivars. The polymorphic information content (PIC) of newly developed markers varied from 0.19 to 0.67, with an average of 0.44. The probability of identifying any two genotypes with identical fingerprints using the 20 SSR markers used for fingerprinting here in cashew cultivars was less than 2.8 × 10^-11. Of the set of 20 markers, eight were sufficient for uniquely fingerprinting all the cultivars. Genetic diversity analysis by the neighbor-joining (NJ) method grouped 32 cultivars into three main clusters, and the grouping had no relationship to the geographic regions or the pedigree.

Conclusions: The findings of this study are useful for the conservation and protection of cultivars under the PVP Act for ensuring the trading of quality planting material and are also useful for cashew breeding programs.

Background: Dyggve-Melchior-Clausen (DMC) disease is a rare autosomal recessive disorder primarily characterized by spondylo-epimetaphyseal dysplasia, intellectual disability, and distinctive facial features. Patients typically present with severe developmental delays and cognitive impairments, defining features of the syndrome.

Methods and results: This case report examines a 13-year-old Moroccan child diagnosed with DMC disease, presenting classical skeletal abnormalities, including spondylo-epimetaphyseal dysplasia, as confirmed through exome sequencing. Notably, the child exhibited a mutation recurrently identified in the Moroccan population. However, the patient showed no signs of developmental delay or intellectual disability, a marked deviation from the traditionally described phenotype. This finding suggests a broader clinical variability associated with DMC disease, emphasizing the importance of individualized assessments.

Conclusions: This atypical presentation expands the phenotypic spectrum of DMC disease, challenging its conventional diagnostic criteria. Further research is required to elucidate the factors influencing phenotypic variability in DMC and to explore potential genotype-phenotype correlations. Early identification and documentation of such atypical cases are critical for refining diagnostic and management strategies for rare disorders.

Background: Whole genome resequencing (WGRS) platforms provide exceptional fingerprinting of the entire genome but are expensive and less flexible to use as a routine genotyping tool for targeting causal polymorphisms within a germplasm collection or breeding program. Therefore, there has been a continuous effort to develop small-scale genotyping platforms that facilitate robust and quick assessments of the allelic status of causal variants for important traits within soybean breeding programs. The objective was to develop a comprehensive panel of soybean cyst nematode (SCN) resistance TaqMan^® assays via selecting the causative genes and analyzing their associated alleles.

Methods: The Soybean Allele Catalog was utilized to investigate WGRS-derived variants which are predicted to cause a change in the amino acid sequence of a gene product. This panel of TaqMan^® assays reflects current knowledge about known SCN resistance-causing genes and their associated alleles: GmSNAP18-a and -b, GmSNAP11, GmSHMT08, GmSNAP15, GmNSF_RAN07, and GmSNAP02-ins and -del. Developed assays were tested using elite breeding lines and segregating populations. TaqMan assays were compared to other currently available KASP and CAPS assays.

Conclusion: All assays showed excellent allele determination efficiencies. This SCN genotyping assay panel can be utilized as a simplified, accurate and reliable genotyping platform further equipping the updated soybean breeding toolbox.

Background: Hematopoietic stem cell transplantation (HSCT) is one of the most effective ways to treat hematological malignant diseases, but the traditional culture of hematopoietic stem cells (HSCs) in vitro will soon lose their ability to self-renewal or differentiate into multilineage blood cells.

Methods: To determine whether Forkhead boxO1 (FoxO1) is implicated in the development of HSCs, lentiviral vectors expressing knockdown (KD) or overexpression (OE) of FoxO1 were utilized in fetal liver-derived hematopoietic stem and progenitor cells (FL-HSPCs). The impacts on the proliferation and hematopoietic differentiation of FL-HSPCs were subsequently evaluated via flow cytometry (FCM). Furthermore, the effect of FoxO1-OE on the self-renewal of cord blood-derived hematopoietic stem and progenitor cells (CB-HSPCs) was investigated. Additionally, the transplantation ability of hematopoietic stem cells derived from these CB-HSPCs in mice after secondary transplantation was also assessed by FCM.

Result: After knocking down FoxO1 in FL-HSPCs, the apoptosis rate was significantly increased, and the expression of hCD45 was significantly decreased. Conversely, overexpression of FoxO1 reversed this phenomenon, effectively promoting the expansion and differentiation of FL-HSPCs in vitro. Similarly, it was found that FoxO1-OE could effectively enhance the expansion of CB-HSPCs. Furthermore, upon transplantation of CB-HSPCs overexpressing FoxO1 into NSG mice, multilineage human hematopoietic reconstruction was promoted. Notably, the results of secondary transplantation revealed that only the FoxO1-OE group exhibited multilineage reconstitution.

Conclusion: In conclusion, our study confirmed that FoxO1-OE could enhance the self-renewal and engraftment of CB-HSPCs.

Sepsis, a systemic inflammatory response syndrome resulting from an uncontrolled inflammatory reaction to infection, remains without a definitive cure despite therapeutic advancements. Mesenchymal stem cells (MSCs), renowned for their capacity to alleviate inflammation and modulate the immune system, have emerged as a potential treatment avenue for sepsis. In sepsis pathophysiology, hyperactivated neutrophils release extracellular neutrophil traps (NETs). NETs are essential for eradicating pathogens; however, excessive formation leads to tissue damage. Given the limited knowledge regarding the impact of NETs on MSCs used in sepsis therapy and the established interaction between MSCs and NETs, this study investigates the effects of NETs on MSCs in vitro. NETs were isolated from stimulated neutrophils, and MSCs were sourced from umbilical cord blood. After co-culturing MSCs with isolated NETs, MSCs' viability, migration, intracellular antioxidant capacity, and changes in gene expression were analyzed. Following exposure to NETs, MSCs exhibited obvious apoptosis and necrosis. NETs disrupt MSCs' mitochondrial activity. Also, NETs upregulate the pro-apoptotic gene BAX and downregulate the anti-apoptotic gene BCL2 in MSCs. Additionally, NETs reduce MSCs' intracellular antioxidant capacity. Furthermore, MSC migration is significantly impaired by NETs. This study collectively demonstrates that NETs have toxic and detrimental effects on MSCs. These effects on MSCs indicate a potential barrier to their functionality and therapeutic efficacy. Therefore, it appears that reducing the undesirable effects of NETs could serve as a novel target to enhance the therapeutic efficacy of MSCs in septic patients.

Background: Despite the development of novel therapeutic modalities, lung cancer persists as the leading cause of cancer-related mortality. Platinum-based treatments represent the most prominent treatment option, with cisplatin being the most frequently utilized chemotherapeutic agent. However, cisplatin has several serious side effects. A substantial body of evidence has emerged in recent years indicating that boron compounds exhibit anti-cancer properties when administered as monotherapy or in combination with chemotherapy agents. The objective of this study is to examine the anti-cancer effects of Cisplatin (Cis) and sodium pentaborate pentahydrate (NaB), both individually and in combination, on non-small cell lung cancer (NSCLC) cell line A-549 cells and small cell lung cancer (SCLC) cell line DMS-114 cells under in vitro conditions.

Methods and results: The effects of cisplatin and NAB on cell survival, apoptosis, the cell cycle, and the expression levels of apoptotic, anti-apoptotic, and tumor suppressor genes were determined by an MTS assay, an Annexin-V assay, a cell cycle analysis, and real-time PCR (qPCR). It was found that the IC-50 value of cisplatin, which was 10 µM when used alone, decreased to 2.5 µM when combined with a non-toxic dose of NaB on the A-549 cell line. BAX and TP53 gene expression levels were elevated by the Nab-Cisplatin combination in the A-549 cell line. The combination was observed to result in an approximately 19-fold increase in CDK2 gene expression in the A-549 cell line and an approximately 6-fold increase in the DMS-114 line, which resulted in S phase and/or G2 phase arrest on the cell cycle. Gene expression levels of Survivin and Ki-67 were decreased by the combination on both cell lines when compared with cisplatin alone. The findings demonstrate that NaB exerts an anti-cancer effect on the A-549 and DMS-114 cell lines. Moreover, when combined with cisplatin, it produces a synergistic anti-cancer effect on the A-549 cell line, whereby apoptosis is activated and cell proliferation is inhibited.

Conclusion: The combination of NaB and cisplatin represents a novel approach to the treatment of NSCLC. This is due to the fact that it reduces the IC-50 value of cisplatin and also results in a greater inhibition of cell division and a stronger induction of cell death when used in the context of a combined treatment. Further insight into the effects of the NaB-cisplatin combination will be gained from in vivo experiments and clinical studies.

Breast cancer remains the most commonly diagnosed cancer in female worldwide, marked by its molecular diversity and complex subtypes. Despite progress in targeted therapies, tumor heterogeneity and treatment resistance continue to present major challenges. Recent studies emphasize the crucial role of RNA modifications in cancer biology, with nearly 200 distinct modifications identified. Among these, methylation is particularly significant, with methylation-related factors emerging as key regulators of RNA metabolism, influencing cancer progression, metastasis, and treatment resistance. This review focuses on the roles of key RNA methylation in breast cancer, particularly N6-methyladenosine (m6A), N7-methylguanosine (m7G), 5-methylcytosine (m5C), N1-methyladenosine (m1A), and N3-methylcytidine (m3C). We examine the functions of m6A "writers" like METTL3 and METTL14, and "readers" such as the YTH domain family in modulating tumor behavior. Dysregulation of m6A "erasers" like FTO and ALKBH5 are noticed too, highlighting their impact on cancer stem cell phenotypes, chemoresistance, and immune evasion. Additionally, the role of m7G modifications in mRNA stability and translation, facilitated by METTL1/WDR4 and RNMT, is discussed as a potential therapeutic target. The involvement of m5C, m1A, and m3C modifications, particularly those mediated by NSUN2 and NSUN6, in breast cancer tumorigenesis and prognosis is also reviewed. Despite coding RNAs, the interplay between these RNA methylations and non-coding RNAs, such as lncRNAs and miRNAs, is explored, shedding light on their roles in cancer cell proliferation, invasion, and immune response modulation. This review highlights the potential of RNA methylations as novel therapeutic targets in breast cancer, offering insights for precision medicine and improved patient outcomes.

Background: Botulinum neurotoxin serotypes E and F (BoNT/E and BoNT/F) produced by the bacteria Clostridium botulinum (C. botulinum) found in a wide variety of foods cause poisoning in humans with high mortality rates. Mouse bioassays (MBAs), the gold standard method for BoNT detection, have a low detection limit; however, require experienced personnel and take a long time to obtain results. Therefore, it has been gradually replaced by nucleic acid amplification tests (NAATs) with primers targeting species-specific genes.

Methods and results: In this study, for each serotype E and F, six LAMP primers were designed based on multiple sequence alignments of the conserved regions of the bont/E and bont/F genes collected from 180 serotype E strains and 23 serotype F strains published in NCBI. In silico PCR amplification with the outer primer pairs showed successful amplification of the target fragments. To validate the LAMP method, we constructed two synthetic plasmids containing the target sequences extended approximately 10-50 bp to both ends. The specificity of the primers was further evaluated using six different Clostridium species and eight strains belonging to other common food poisoning-related bacterial species. Employing the synthetic plasmids, the optimal temperatures and limits of detection (LODs) were determined for bont/E (63 °C, LOD ≤ 10¹ copies/reaction) and bont/F (65 °C, LOD ≤ 10² copies/reaction) within 30 min. In addition, the LAMP primer set for BoNT/F was redesigned with degenerate nucleotides that improved the coverage from 15 to 45%.

Conclusions: For future directions, applications of the established method, especially with the degenerate primers, could be used as an alternative assay for the rapid and sensitive detection of C. botulinum.

Background: The purity of the extracted DNA is critical for successful molecular testing. This study aimed to compare the effect of various DNA extraction methods, extraction processes, and sources of consumables, such as microtubes, on PCR results.

Methods: DNA extraction from whole blood was performed using four different approaches utilizing four types of microtubes: chloroform-based, sodium perchlorate-based, heat-assisted salting out, and solid-phase extraction. Extracted DNA was evaluated by nanodrop spectrophotometry and used in PCR-based methods for human leukocyte antigens (HLA) typing.

Results: The lowest and highest concentrations of extracted DNA were observed in the column-based and heat-assisted methods, respectively. The mean ratios of A260/A230, represents chemical contamination, were significantly lower in all extraction methods using all types of microtubes versus "A" microtube. We observed the highest mean ratio of A260/A230 (> 1.9) in the sodium perchlorate method by using the "A" microtube compared to other types of microtubes and extraction methods. Extracted DNA samples using microtube "A" showed acceptable results for HLA typing compared to other microtubes that represented uninterpretable results in the PCR using the sequence-specific oligonucleotide probe (PCR-SSOP) method.

Conclusion: Our findings indicate that the chemical contaminations derived mainly from microtubes can decrease the quality of DNA and consequently interfere with the amplification or hybridization reactions in the PCR-SSOP method for HLA typing. Although, technical issues and extraction processes may also influence the quality of DNA.