Southern Blot Technique Research Articles

Detection of Thermoacidophiles from Yellowstone Hot Spring

Aerobic methanotrophic bacteria maintain an unrivalled capability of utilizing methane as their sole carbon and energy source and have been retrieved from various environments. Phylogenetically, true aerobic thermoacidophilic methane oxidizers capable of growing below pH 3 have hitherto been associated only with the phylum Verrucomicrobia. In this report, the initial detection of a moderately thermoacidophilic Methylococcus-like Type Ib methanotroph of the class Gammaproteobacteria from an acidic thermal spring (50oC and pH 2.8) in the Yellowstone National Park, USA is presented. The isolate, termed YT-MC, was identified in a methane enrichment (55°C), which may represent a novel strain in the family Methylococcaceae Type Ib. The existence of this bacterium in the enrichments was demonstrated by the detection of pmoA gene, Southern blotting technique, phase-contrast, and electron microscopy. The coccus-typed cells showed tubular membranes instead of intracytoplasmic membrane systems (ICM). The soluble methane monooxygenase (sMMO) was not detected by PCR, indicating that the biotransformation of methane to methanol is oxidized by the particulate methane monooxygenase (pMMO). Moreover, YT-MC performs in a formerly undiscovered active biological methane sink in geothermal acidic environments, magnifying our knowledge of its ecological role in methane cycling, diversity, and coexistence of aerobic methanotrophy. Furthermore, the present study also reports the isolation and identification of an alphaproteobacterial heterotroph (strain YT-AC) and a verrucomicrobial methanotroph (strain YT-VM) from the same environment. Bangladesh J Microbiol, Volume 40, Number 2, December 2023, pp 86-94

T-DNA characterization of genetically modified 3-R-gene late blight-resistant potato events with a novel procedure utilizing the Samplix Xdrop® enrichment technology.

Before the commercialization of genetically modified crops, the events carrying the novel DNA must be thoroughly evaluated for agronomic, nutritional, and molecular characteristics. Over the years, polymerase chain reaction-based methods, Southern blot, and short-read sequencing techniques have been utilized for collecting molecular characterization data. Multiple genomic applications are necessary to determine the insert location, flanking sequence analysis, characterization of the inserted DNA, and determination of any interruption of native genes. These techniques are time-consuming and labor-intensive, making it difficult to characterize multiple events. Current advances in sequencing technologies are enabling whole-genomic sequencing of modified crops to obtain full molecular characterization. However, in polyploids, such as the tetraploid potato, it is a challenge to obtain whole-genomic sequencing coverage that meets the regulatory approval of the genetic modification. Here we describe an alternative to labor-intensive applications with a novel procedure using Samplix Xdrop® enrichment technology and next-generation Nanopore sequencing technology to more efficiently characterize the T-DNA insertions of four genetically modified potato events developed by the Feed the Future Global Biotech Potato Partnership: DIA_MSU_UB015, DIA_MSU_UB255, GRA_MSU_UG234, and GRA_MSU_UG265 (derived from regionally important varieties Diamant and Granola). Using the Xdrop® /Nanopore technique, we obtained a very high sequence read coverage within the T-DNA and junction regions. In three of the four events, we were able to use the data to confirm single T-DNA insertions, identify insert locations, identify flanking sequences, and characterize the inserted T-DNA. We further used the characterization data to identify native gene interruption and confirm the stability of the T-DNA across clonal cycles. These results demonstrate the functionality of using the Xdrop® /Nanopore technique for T-DNA characterization. This research will contribute to meeting regulatory safety and regulatory approval requirements for commercialization with small shareholder farmers in target countries within our partnership.

Senescence State in Mesenchymal Stem Cells at Low Passages: Implications in Clinical Use.

Mesenchymal stem cells (MSCs) are multipotent cells found in various tissues and are easily cultivated. For use in clinical protocols, MSCs must be expanded to obtain an adequate number of cells, but a senescence state may be instituted after some passages, reducing their replicative potential. In this study, we report a case where MSC derived from an elderly donor acquired a senescence state after three passages. The bone marrow was aspirated from a female patient submitted to a cell therapy for the incontinency urinary protocol; MSCs were cultivated with DMEM low glucose, supplemented with 10% autologous serum (AS) plus 1% L-glutamine and 1% antibiotic/antimycotic. Senescence analysis was performed by β-galactosidase staining after 24 and 48 h. Controls were established using BM-MSC from healthy donors and used for senescence and gene expression assays. Gene expression was performed using RT-PCR for pluripotency genes, such as SOX2, POU5F1, NANOG, and KLF4. MSC telomere length was measured by the Southern blotting technique, and MSCs were also analyzed for their capacity to differentiate into adipocytes, chondrocytes, and osteocytes. The patient’s MSC expansion using AS displayed an early senescence state. In order to understand the role of AS in senescence, MSCs were then submitted to two different culture conditions: 1) with AS or 2) with FBS supplementation. Senescence state was assessed after 24 h, and no statistical differences were observed between the two conditions. However, patients’ cells cultured with AS displayed a higher number of senescence cells than FBS medium after 48 h (p = 0.0018). Gene expression was performed in both conditions; increased expression of KLF4 was observed in the patient’s cells in comparison to healthy controls (p = 0.0016); reduced gene expression was observed for NANOG (p = 0.0016) and SOX2 (p = 0.0014) genes. Telomere length of the patient’s cells was shorter than that of a healthy donor and that of a patient of similar age. Osteocyte differentiation seemed to be more diffuse than that of the healthy donor and that of the patient of similar age. MSCs could enter a senescence state during expansion in early passages and can impact MSC quality for clinical applications, reducing their efficacy when administered.

Body mass index and shortened telomere length in middle-aged female and male RUNNING HEAD: Middle-aged and shortened telomere length

Obesity is a risk factor associated with age-related disorders that accelerate aging, and it increases the risk of metabolic diseases. Therefore, this study was conducted to investigate the association of leukocyte telomere length (LTL) with the presence of higher body weight in middle-aged females and males. The study subjects comprised 160 (80 control and 80 higher body mass index BMI groups) with ranging ages of 30-50 years included and stratified for BMI. The physio-biochemical analysis was measured using enzymatic determination. Mean telomere length was determined by using the southern blotting technique. The association analysis revealed a significant variance (P < 0.01) in biochemical parameters between higher BMI groups and control including waist, lipid profile, and the level of estradiol, testosterone, follicle-stimulating hormone, and luteinizing hormone. Mean telomere length was shorter in middle-aged males compared to the females of higher BMI groups and control groups for both age groups. LTL was shorter in the overweight and obese patients compared with the control group, and these differences in LTL obese group were shorter compared to the overweight group. In conclusion, shorter telomere length was observed in middle-aged males associated with higher body weight and lipid abnormalities. Lipid/lipoprotein abnormalities can be used as a predictor for the shortened telomere length and the reduction in adiposity indices can improvement the telomere length in both overweight and obese subjects.

Genetic engineering of novel yellow color african violet (Saintpaulia ionantha) produced by accumulation of Aureusidin 6-O-glucoside

BackgroundFlower color is one of the main characteristics of ornamental plants. Aurones are light yellow flavonoids produced in the petals of a limited number of plant species including snapdragon (Antirrhinum majus). As a commercially-recognized species, African violet can be found in various colors except yellow. This research, aiming at changing the petals’ color of African violet from white to yellow, was conducted using the simultaneous expressions of chalcone 4’-O-glucosyltransferase (4’CGT) and aureusidin synthase (AS1) genes without the need for silencing anthocyanin biosynthesis pathway genes via both transient and stable transfer methods.ResultsThe transient gene transfer among transgenic plants led to a clear change of petals’ color from white to light yellow. This occurs while no change was observed in non-transgenic (Wild type) petals. In total, 15 positive transgenic plants, produced via stable gene transfer, were detected. Moreover, since their flower color was yellow, both genes were present. Meanwhile, the corresponding transformation yield was determined 20-30%. The transformation, expression and integration of genes among T0 transgenic plants were verified using the PCR, qRT-PCR and Southern blotting techniques, respectively. Furthermore, the probable color change of petals’ cross-section and existence of Aureusidin 6-O-glucoside (AOG) compound were determined using a light microscope and HPLC-DAD-MSn analysis, correspondingly.ConclusionsGenerally, the creation of aurones biosynthesis pathway is only viable through the simultaneous expression of genes which leads to color change of African violet’s petal from white to yellow. This conclusion can lead to an effective strategy to produce yellow color in ornamental plant species.

Orofacial Muscle Weakening in Facioscapulohumeral Muscular Dystrophy (FSHD) Patients.

Background: Facioscapulohumeral muscular dystrophy is the third most commonly found type of muscular dystrophy. The aim of this study was to correlate the D4Z4 repeat array fragment size to the orofacial muscle weakening exhibited in a group of patients with a genetically supported diagnosis of FSHD. Methods: Molecular genetic analysis was performed for 52 patients (27 female and 25 male) from a group that consisted of 36 patients with autosomal dominant pedigrees and 16 patients with either sporadic or unknown family status. The patients were tested with the southern blotting technique, using EcoRI/Avrll double digestion, and fragments were detected by a p13E-11 telomeric probe. Spearman’s correlation was used to compare the fragment size with the degree of muscle weakening found in the forehead, periocular and perioral muscles. Results: A positive non-significant correlation between the DNA fragment size and severity of muscle weakness was found for the forehead (r = 0.27; p = 0187), the periocular (r = 0.24; p = 0.232) and the left and right perioral (r = 0.29; p = 0.122), (r = 0.32; p = 0.085) muscles. Conclusions: Although FSHD patients exhibited a decrease in muscular activity related to the forehead, perioral, and periocular muscles the genotype–phenotype associations confirmed a weak to moderate non-significant correlation between repeat size and the severity of muscle weakness. Orofacial muscle weakening and its association with a D4Z4 contraction alone may not have the significance to serve as a prognostic biomarker, due to the weak to moderate association. Further studies with larger sample sizes are needed to determine the degree of genetic involvement in the facial growth in FSHD patients.

Telomere length in dromedary camels (Camelus dromedarius) produced by somatic cell nuclear transfer (SCNT) and their age-matched naturally produced counterparts

There are controversial reports on the restoration of eroded telomere length in offspring produced by somatic cell nuclear transfer (SCNT) in different animal species. To the best of our knowledge, no earlier studies report the telomere length in naturally produced or cloned animals in any of the camelid species. Therefore, the present study was conducted to estimate the telomere length in dromedary camels produced by SCNT, the donor cells, and their age-matched naturally produced counterparts by Terminal Restriction Fragment (TRF) length analysis and real-time Q PCR T/S ratio methods. Genomic DNA was extracted from venous blood collected from 6 cloned animals and their age-matched counterparts. Using the southern blot technique, digested DNA was blotted onto a positively charged nylon membrane, and its hybridization was carried out using telomere (TTAGGG)n specific, DIG-labeled hybridization probe (Roche Diagnostics, Germany) at 42 °C for 4 h. Stringent washes were carried out at the same temperature, followed by a chemiluminescence reaction. The signals were captured using the Azure Biosystems C600 gel documentation system. A TeloTool program from MATLAB software with a built-in probe intensity correction algorithm was used for TRF analysis. The experiment was replicated three times, and the data, presented as mean ± SEM, were analyzed using a two-sample t-test (MINITAB statistical software, Minitab ltd, CV3 2 TE, UK). No difference was found in the mean telomere length of cloned camels when compared to their naturally produced age-matched counterparts. However, the telomere length was more (P < 0.05) than that of the somatic cells used for producing the SCNT embryos. A moderate positive Pearson correlation coefficient (r = 0.6446) was observed between the telomere lengths estimated by TRF and Q PCR T/S ratio method. In conclusion, this is the first study wherein we are reporting telomere length in naturally produced and cloned dromedary camels produced by somatic cell nuclear transfer. We found that telomere lengths in cloned camels were similar to their age-matched naturally produced counterparts, suggesting that the camel cytoplast reprograms the somatic cell nucleus and restores the telomere length to its totipotency stage.

Association of antidiabetic therapy with shortened telomere length in middle-aged Type 2 diabetic patients.

A wide range of antidiabetic therapies have been developed to manage diabetes and limit its lifespan but each of them have adverse long-term drug reactions. This study was performed for the investigation of the possible association of antidiabetic therapy with shortened telomere length in middle-aged Type 2 diabetic patients. The subjects in this case-control study included 100 non-diabetic patients and 300 patients with Type 2 diabetes with ages in the range of 30-50years. The treated patients were further subdivided into diabetic patients using Doanil, those using insulin and those using both the therapies. The mean telomere length was determined using the southern-blotting technique. A logistic regression analysis was performed to predict the relationship between antidiabetic therapy and shortened telomere length. The results revealed a significant increase (P < 0.01) in the fasting blood glucose and lipid profile in non-treatment diabetic patients compared to diabetic patients with treatment, and also in diabetic patients with insulin therapy, compared to diabetic patients with Doanil or both therapies. The results showed that non-treatment diabetic patients had shorter telomere length, compared to the diabetic patients with treatment, and patients treated with insulin therapy had shorter telomere length, compared to the diabetic patients with Doanil or both therapies. The logistic regression analysis confirmed that insulin therapy was closely related to diabetic risk factors and shortened telomere length. The results revealed that Doanil therapy was more effective in managing diabetic risk and limiting the shortening telomere length than insulin therapy.

Selection of glufosinate-resistant cotton lines (Gossypium hirsutum L.) among bar transgenic lines

Basta-herbicide was tested at a concentration of 0.6 kg ai./ha for confirming resistance of 116 bar transgenic T1 lines; many lines with tolerance were obtained. Evaluation of selected lines using PCR, the integration and expression of transgenes in genome of transgenic plants was determined by southern blot and northern blot techniques. The combination of molecular and biological assessments resulted in the selection of 5 lines, i.e., B1, B6, B9, B18, and BF17 contained 01 target-gene copy which expressed transcription activities and showed uniform growth and best tolerance to glufosinate. Two T2 transgenic cotton lines, i.e., B9 and BF17, carried one copy of the gene which transmitted to the next generation according to the Mendel's rules of inheritance. These transgenic lines were highly resistant to Basta herbicide at a concentration of 0.6 kg ai./ha and had no difference in botanical characteristics and disease resistance in comparison with original non-transgenic Coker310 cotton plant

Construction and transformation of peroxisome proliferator activated receptor gamma (RnPPARγ) gene using Agrobacterium tumefaciens into Glycine max L. Merr.

The PPARγ gene encodes a member of the peroxisome proliferator-activated receptor (PPAR) nuclear receptors subfamily which forms heterodimers with retinoid X receptors (RXRs) and they regulate transcription of various genes. In the present study, the binary vector of pBI121 was constructed with RnPPARγ gene (1428 bp) of Rattus norvegicus under CaMV35s promoter and also kanamycin resistance gene available for selecting the transforms. Transformation of soybean was conducted for half seed explants of JS335 variety using strain EHA105 harboring pBI121- RnPPARγ. Transformation conditions were standardized for soybean, and the results demonstrate that L-Cysteine (1 mM) has improved the regeneration of transformants. The combination of TDZ (10 μM) and L-Cysteine (1 mM) induced a maximum of 80% shooting, whereas IBA (15 μM) and L-Cysteine (1 mM) combination induced a maximum of 83% rooting frequency. PCR and Southern Blotting techniques confirmed the transgene integration. These study findings suggest that L-Cysteine (1 mM) involvement increased the transfer of T-DNA during transformation and the gene delivered under the suitable condition for regeneration and also enhanced the transformation efficiency in soybean through direct (1.6%), indirect (1.2%) and in planta (2.0%) method of transformation.

Impact of direct-acting antivirals on leukocytic DNA telomere length in hepatitis C virus-related hepatic cirrhosis.

Direct-acting antiviral (DAAs) represent advancement in the management of hepatitis C virus (HCV)-related hepatic cirrhosis. A high proportion of patients achieve a sustained virologic response; eradication of HCV is coupled with a decreased risk of hepatocellular carcinoma. Recent evidence suggests that shortening of the DNA telomere may be linked to cellular senescence as well as predisposition to malignant transformation. This study aimed to assess pretreatment leukocytic DNA telomere length in HCV-related cirrhosis and post viral eradication using DAAs. This study included 24 patients with HCV-related cirrhosis, Child-Pugh A. Whole-blood samples were obtained from patients before treatment and 12 weeks after the end of treatment, as well as from 24 healthy controls. Terminal restriction fragment, corresponding to telomere length, was measured using a nonradioactive Southern blot technique, detected by chemiluminescence. DNA telomere length was significantly shorter before treatment compared with 12 weeks after end of treatment in HCV-related cirrhotic patients. Also, it was significantly shorter in patients before treatment compared with healthy individuals. Telomere elongation in blood leukocytes can be considered a marker of recovery of inflammation after DAAs-induced HCV eradication. Still, the possibility of activation by cancer initiation cannot be excluded.

The Role of Shelterin Complex and Post-Translational Non-Enzymatic Modification in Telomere Maintenance in Chronic Myeloid Leukemia

The introduction of tyrosine kinase inhibitors (TKI) revolutionized therapy of chronic myeloid leukemia (CML) and gives long-lasting remission in majority of patients. However, a significant proportion of patients develops resistance and are at risk of progression. Genomic instability is one of the key features associated with CML progression and as in majority of human malignancies, reduction in telomere length characterize transition from chronic phase (CML-CP) to the blastic phase (CML-BP). Telomere maintenance depends on telomerase nucleoprotein complex consisting of reverse transcriptase TERT, RNA template TERC and structural proteins, such as dyskerin (DKC1), but also on telomere-associated proteins including but not limited to shelterin complex(TRF1, TRF2, RAP1, TIN2, TPP1, POT1) and other telomeric-associated proteins (such as TEP1 and Tankyrase). The role of shelterin complex and its post-translational non-enzymatic modifications in BCR/ABL1-mediated genomic instability in CML progression and resistance to TKIs is not fully elucidated, thus we decided to examine the possible role of different components of shelterin complex and its glycation and carbonylation in promoting genomic instability and CML progression. We employed CML CD34+ primary cells isolated from peripheral blood leukocytes or bone marrow of CML patients at different stages of disease: CML-CP, CML-BP and CML-TKIres (TKI-resistant cells). Blood samples were taken after informed consent. The length of telomeres was determined by Southern blotting and PNA-FISH technique. Enzymatic activity of telomerase, as well as protein glycation was measured immunoenzymatically, while expression of subunits of telomerase and shelterin complexes was examined by RT-qPCR and by Western Blot.We confirmed that reduction in telomere length was positively correlated with CML progression. However, in samples from CD34+ CML-TKIres patients in comparison to CML-CP patients, an increase in telomere length was observed (Fig 1). In addition, the negative correlation between mean telomere length and expression of BCR-ABL1 in CML samples was observed. This suggests involvement of alternative telomere lengthening mechanisms, since dynamic changes in telomere length were neither associated with enzymatic activity of telomerase nor with gene copy number/expression of TERT/TERC. Therefore we postulate that telomere dynamics in CML may have a bi-phasic scenario. We found that the expression of RAP1, DKC1 and Tankyrase1 was increased in CML-BP as compared to CML-CP. Moreover, RAP1 is involved in increased metabolism, which is associated with elevated glycolytic and oxidative rates leading to post-translational non-enzymatic modification of proteins and damage of cellular proteostasis which may indirectly influence telomere length in CML. We found that telomere shortening was associated with accumulation of post-translational non-enzymatic modification ofselected proteins of shelterin complex, namely glycation and carbonylation.In conclusion, we postulate that aberrant expression of members of the shelterin complex such as RAP1 may be responsible for aberrant telomere maintenance mechanisms in CML cells and may play role in genomic instability associated with CML progression as well as the clonal selection and resistance to TKIs. In addition, the changes in the level of protein glycation may influence telomere length in CML. DisclosuresNo relevant conflicts of interest to declare.

Agrobacterium-Mediated Transformation of the Oryza sativa Thaumatin-Like Protein to Canola (R Line Hyola308) for Enhancing Resistance to Sclerotinia sclerotiorum.

Background: Canola is an agro-economically oilseed crop. Yield loss due to fungal disease of stem rot caused by Sclerotinia sclerotiorum is a serious problem in canola cultivation. Thaumatin-like proteins are large groups of the pathogenesis-related proteins which provide resistance to the fungal infection in response to invading pathogens and play a key role in plant defense system. Objectives: Transformation of the rice tlp into canola via Agrobacterium-mediated transformation and evaluation of the antifungal activity of the expressed TLP in the transgenic events on the S. sclerotiorum growth was subject to investigation. Materials and methods: The canola (R line Hyola308) was used for transformation experiment. The vector, pBITLPRA1, was used for the stable transformation. The PCR and southern blotting techniques were used to confirm transgene's presence in the transgenic canola events. Antifungal activity of transgenic plants was evaluated by the radial diffusion and spore germination assays. T2 transgenic plants were evaluated by the intact leaf inoculation method in greenhouse assay. Results: In this study, pBITLPRA1 construct containing tlp gene was introduced into canola and the transformed plants were verified by PCR. The glucanase activity of tlp gene in T0 generation was measured and transgenic plants with high activity were assessed by Southern blot analysis to confirm the copy number of the gene. Also, antifungal activity of the single copy T0 transgenic plants against Sclerotinia sclerotiorum was evaluated by radial diffusion and spore germination assays. In greenhouse assay, evaluation of T2 transgenic plants by the intact leaf inoculation method demonstrated that following the infection with S. sclerotiorum, there was a significant reduction in the lesion's diameter in transgenic lines compared to the non-transgenic ones. Conclusions: These results revealed that expression of TLP has an inhibitory effect against fungus compared to non-transgenic plants both in vitro and in vivo (i.e., greenhouse condition). These transgenic lines could be used as the additional sources of disease resistance for canola breeding program.

Molecular combing reveals complex 4q35 rearrangements in Facioscapulohumeral dystrophy.

Facioscapulohumeral dystrophy (FSHD), one of the most common hereditary neuromuscular disorders, is associated with a complex combination of genetic variations at the subtelomeric 4q35 locus. As molecular diagnosis relying on Southern blot (SB) might be challenging in some cases, molecular combing (MC) was recently developed as an additional technique for FSHD diagnosis and exploration of the genomic organization of the 4q35 and 10q26 regions. In complement to the usual SB, we applied MC in a large cohort of 586 individuals with clinical FSHD. In 332 subjects, the two 4q alleles were normal in size, allowing exclusion of FSHD1 while we confirmed FSHD1 in 230 patients. In 14 patients from 10 families, we identified a recurrent complex heterozygous rearrangement at 4q35 consisting of a duplication of the D4Z4 array and a 4qA haplotype, irresolvable by the SB technique. In five families, we further identified variations in the SMCHD1 gene. Impact of the different mutations was tested using a minigene assay and we analyzed DNA methylation after sodium bisulfite modification and NGS sequencing. We discuss the involvement of this rearrangement in FSHD since all mutations in SMCHD1 are not associated with D4Z4 hypomethylation and do not always segregate with the disease.

Television Watching and Telomere Length Among Adults in Southwest China.

To explore the independent associations of sedentary behavior and physical activity with telomere length among Chinese adults. Data on total time of sedentary behavior, screen-based sedentary behavior (including television watching and computer or phone use), moderate to vigorous physical activity, and dietary intake of 518 adults in Chengdu, Guizhou, and Xiamen in China (54.25% women) aged 20 to 70 years were obtained between 2013 and 2015 through questionnaires. Height, weight, and waist circumference were measured to calculate body mass index and percentage of body fat. Telomere length was measured through Southern blot technique. Television watching was inversely related to adjusted telomere length (-71.75 base pair; SE = 34.40; P = .04). Furthermore, a similar trend between telomere length and television watching was found in the group aged 20 to 40 years after adjusting for all covariates. Adults aged 20 to 40 years in the highest tertile of daily time spent on watching television had 4.0% shorter telomere length than adults in the lowest tertile (P = .03). Although the association is modest, television watching is inversely related to telomere length among Chinese adults, warranting further investigation in large prospective studies.

Telomere length analysis in Down syndrome birth

Human reproductive fitness depends upon telomere chemistry. Maternal age, meiotic nondisjunction error and telomere length of mother of trisomic child are someway associated. Reports exhibiting maternal inheritance of telomere length in Down syndrome child are very scanty. To investigate this, we collected peripheral blood from 170 mothers of Down syndrome child and 186 age matched mothers of euploid child with their newly born babies. Telomere length was measured by restriction digestion – southern blotting technique. Meiotic nondisjunction error was detected by STR genotyping. Subjects are classified by age (old >35 years and young ˂35 years) and by meiotic error (MI and MII). Linear regression was run to explore the age – telomere length relationship in each maternal groups. The study reveals that with age, telomere erodes in length. Old MII mothers carry the shortest (p˂0.001), control mothers have the longest telomere and MI lies in between. Babies from older mother have longer telomere (p˂0.001) moreover; telomeres are longer in Down syndrome babies than control babies (p˂0.001). To conclude, this study represents not only the relation between maternal aging and telomere length but also explore the maternal heritability of telomere length in families with Down syndrome child.

Towards a Better Molecular Diagnosis of FMR1-Related Disorders—A Multiyear Experience from a Reference Lab

The article summarizes over 20 years of experience of a reference lab in fragile X mental retardation 1 gene (FMR1) molecular analysis in the molecular diagnosis of fragile X spectrum disorders. This includes fragile X syndrome (FXS), fragile X-associated primary ovarian insufficiency (FXPOI) and fragile X-associated tremor/ataxia syndrome (FXTAS), which are three different clinical conditions with the same molecular background. They are all associated with an expansion of CGG repeats in the 5′UTR of FMR1 gene. Until 2016, the FMR1 gene was tested in 9185 individuals with the pre-screening PCR, supplemented with Southern blot analysis and/or Triplet Repeat Primed PCR based method. This approach allowed us to confirm the diagnosis of FXS, FXPOI FXTAS in 636/9131 (6.96%), 4/43 (9.3%) and 3/11 (27.3%) of the studied cases, respectively. Moreover, the FXS carrier status was established in 389 individuals. The technical aspect of the molecular analysis is very important in diagnosis of FXS-related disorders. The new methods were subsequently implemented in our laboratory. This allowed the significance of the Southern blot technique to be decreased until its complete withdrawal. Our experience points out the necessity of implementation of the GeneScan based methods to simplify the testing procedure as well as to obtain more information for the patient, especially if TP-PCR based methods are used.

Whole-genome analysis of herbicide-tolerant mutant rice generated by Agrobacterium-mediated gene targeting.

Gene targeting (GT) is a technique used to modify endogenous genes in target genomes precisely via homologous recombination (HR). Although GT plants are produced using genetic transformation techniques, if the difference between the endogenous and the modified gene is limited to point mutations, GT crops can be considered equivalent to non-genetically modified mutant crops generated by conventional mutagenesis techniques. However, it is difficult to guarantee the non-incorporation of DNA fragments from Agrobacterium in GT plants created by Agrobacterium-mediated GT despite screening with conventional Southern blot and/or PCR techniques. Here, we report a comprehensive analysis of herbicide-tolerant rice plants generated by inducing point mutations in the rice ALS gene via Agrobacterium-mediated GT. We performed genome comparative genomic hybridization (CGH) array analysis and whole-genome sequencing to evaluate the molecular composition of GT rice plants. Thus far, no integration of Agrobacterium-derived DNA fragments has been detected in GT rice plants. However, >1,000 single nucleotide polymorphisms (SNPs) and insertion/deletion (InDels) were found in GT plants. Among these mutations, 20–100 variants might have some effect on expression levels and/or protein function. Information about additive mutations should be useful in clearing out unwanted mutations by backcrossing.

The role of mitochondrial reactive oxygen species in cartilage matrix destruction.

Upregulation of matrix metalloproteinases (MMPs) is a hallmark of osteoarthritis progression; along with the role reactive oxygen species (ROS) may play in this process. Moreover, mitochondrial DNA damage and dysfunction are also present in osteoarthritic chondrocytes. However, there are no studies published investigating the direct relationship between mitochondrial ROS, mitochondrial DNA damage, and MMP expression. Therefore, the purpose of the present study was to evaluate whether mitochondrial DNA damage and mitochondrial-originated oxidative stress modulates matrix destruction through the upregulation of MMP protein levels. MitoSox red was utilized to observe mitochondrial ROS production while a Quantitative Southern blot technique was conducted to analyze mitochondrial DNA damage. Additionally, Western blot analysis was used to determine MMP protein levels. The results of the present study show that menadione augmented mitochondrial-generated ROS and increased mitochondrial DNA damage. This increase in mitochondrial-generated ROS led to an increase in MMP levels. When a mitochondrial ROS scavenger was added, there was a subsequent reduction in MMP levels. These studies reveal that mitochondrial integrity is essential for maintaining the cartilage matrix by altering MMP levels. This provides new and important insights into the role of mitochondria in chondrocyte function and its potential importance in therapeutic approaches.

A blinded international study on the reliability of genetic testing for GGGGCC-repeat expansions in C9orf72 reveals marked differences in results among 14 laboratories

BackgroundThe GGGGCC-repeat expansion in C9orf72 is the most frequent mutation found in patients with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Most of the studies on C9orf72 have relied...