Transcription-polymerase Chain Reaction Research Articles

Two novel deep intronic variants cause Duchenne muscular dystrophy by splice-altering mechanism

Duchenne muscular dystrophy (DMD) is a genetic disorder characterized by progressive muscle degeneration and weakness, due to mutations in the DMD gene, which encodes the dystrophin protein. While mutations within the coding regions of DMD have been extensively studied, recent focus has shifted to deep intronic variants for their potential impact on disease severity. Here, we characterize two deep intronic variants, c.8669-19_8669-24del and c.6439-1016_6439-3376del, in unrelated DMD patients. These variants were identified using targeted long-read sequencing on patients' DNA. RNA sequencing/reverse transcription polymerase chain reaction on RNA extracted from muscle biopsies revealed the presence of a pseudoexon or retention of part of the intron in the transcript, resulting in the introduction of premature termination codons. This study enhances our understanding of pseudoexon activation mechanisms in DMD and underscores the diverse genetic abnormalities contributing to the disease's complexity.

Morinda Officinalis Polysaccharides Inhibit Osteoclast Differentiation by Regulating miR-214-3p/NEDD4L in Postmenopausal Osteoporosis Mice

To investigate the potential mechanism of Morinda officinalis F. C. How polysaccharides (MOPs) in regulating osteoclast differentiation and apoptosis through miR-214-3p and its target protein. Ovariectomy was performed in 8-week female C57BL6 mice to establish the postmenopausal osteoporosis (PMOP) model. Mice were treated immediately with 500 mg/kg of MOPs (prevention group); others were treated 2 weeks after operation (treatment group). Left femur bone mineral density (BMD) was examined. RAW264.7 cells were administered with receptor activator of NF-κB ligand (RANKL) to establish the osteoclast (OC) model and treated with serum containing 1 or 2 g/kg of MOPs. Apoptosis-related indexes, miR-214-3p, and Expressed Developmentally Down-regulated 4-Like (NEDD4L) were detected by western blot, quantitative real-time-reverse transcription polymerase chain reaction (qRT-PCR), and flow cytometry. OC received a miR-214-3p inhibitor or NEDD4L small interfering RNA (siRNA). MOPs reversed the PMOP-induced changes in bones. Compared with the RANKL group, MOPs increased the apoptosis and related markers in OCs. MOPs decreased the femur miR-214-3p of PMOP mice (P < 0.001). Higher concentrations of MOPs reversed the upregulation of miR-214 mRNA in OCs (P < 0.001). miR-214-3p inhibitor increased the expression of Bax and CC3 (P < 0.01) and decreased the expression of Bcl-2 (P < 0.05). NEDD4L is targeted by miR-214. NEDD4L was upregulated in the RANKL + MOPs group (P < 0.01). miR-214-3p inhibitor increased the upregulation of NEDD4L induced by MOPs (P < 0.05). siRNA NEDD4L significantly reversed the inhibition of MOPs on osteoclast differentiation with miR-214-3p inhibitor (P < 0.01). MOPs effectively prevent PMOP by inhibiting osteoclastogenesis and inducing OC apoptosis through the miR-214-3p/NEDD4L pathway.

DNA tweezer-assembled, regenerated nanosensor for one-step and sustainable detection of viral RNA and Hg2+

Transcription-polymerase chain reaction (RT-qPCR) is the golden standard to detect viral RNA. However, it suffers from several inherent defects such as high false-negative rates, time-consuming and expensive. To exploit better analytical methods for future global pandemics, it is vital to develop rapid, sustainable, and environmentally-friendly sensing methods. Here, a one-step, regenerated nanosensor assembled by DNA tweezers is developed for rapid and sustainable detection of viral RNA. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA is selected as model analyte. The DNA tweezer is designed with a central strand and two arm strands containing overhangs complementary to SARS-CoV-2 RNA. The SARS-CoV-2 RNA hybridizes with the two arm overhangs and strains the DNA tweezer to closed conformation, resulting in the close proximity of the donor and acceptor fluorophores. With the addition of an anti-strand complementary to the SARS-CoV-2 RNA, the closed DNA tweezer is relaxed to open conformation through a strand displacement process, leading to the separation of the dual fluorophores. Thus, the DNA tweezer is revived for fresh SARS-CoV-2 RNA. The proposed nanosensor can be regenerated for three cycles with the same performance. Moreover, the viral RNA can be detected within 30 min with a detection limit of 0.04 nM. By altering the sequences of the DNA tweezer, rapid, sensitive, and sustainable detection of Hg2+ is achieved. The proposed nanosensor provides a novel approach for rapid, flexible, and sustainable detection of viral RNA and Hg2+, exhibiting great potential for routine preliminary coronavirus disease tests, epidemic control, and environmental monitoring.

Ventilation improvement for effective protection of healthcare workers in negative pressure airborne infectious isolation room from viral aerosols

A negative pressure airborne infectious isolation room (AIIR) is the primary healthcare air contamination control system used for the treatment of severe respiratory infectious patients. Effects of the ventilation system configuration and conditions on airflow pattern, aerosol distribution and ventilation performance were investigated using computational fluid dynamics (CFD). The field measurement by SARS-CoV-2 environmental surface test was also conducted. The cycle threshold values from transcription polymerase chain reaction (RT-PCR) method showed inverse relation to the simulated number of particles trapped on the surfaces indicating a good agreement. Modification of the present AIIR to have alignment between air inlet and outlet where the aspect ratio of the air outlet, Width (W): Height (H) = 1:1 (Improved case: IC#1) showed a 78 % reduction of aerosol concentration in healthcare workers (HCWs) zones. Aerosol concentrations were increased when the openings of the air outlet were enlarged. Addition of air outlet led to large swirling air, resulting in more aerosols being trapped and suspended in the air. Results suggested that AIIR with alignment air inlet on the ceiling and air outlet at the wall over the patient's head with W:H = 1:1 be the most suitable configuration to maximize the ventilation performance and minimize exposure risk to aerosolized viral infection for HCWs. Air change rate plays a more important role than the differential pressure on the removal efficiency. The differential pressure value should be at least −2.5 Pa and the air supply rate 12 ACH for effective protection of HCWs in the negative pressure AIIR.

Investigation of circulating infectious agents in experimental Beagle dogs of a production colony and three research facilities in China from June 2021 to May 2022.

To understand the epizootiologic characteristics of pathogens and opportunistic infections in one Beagle dog production colony and three research facilities, viruses and mycoplasma were detected in 1777 samples collected from Beagle dogs in China by polymerase chain reaction/reverse transcription polymerase chain reaction, and bacteria were isolated and identified by 16S rRNA sequence analysis. In addition, genotyping of the major circulating viruses was carried out by amplification of gene fragments and homology analysis. Canine coronavirus (CCoV), Escherichia coli, canine parvovirus (CPV), Bordetella bronchiseptica, Clostridium perfringens, Mycoplasma cynos, Klebsiella pneumoniae, Streptococcus canis, canine astrovirus (CaAstV), canine kobuvirus (CaKV), Pseudomonas aeruginosa, Proteus mirabilis, Macrococcus canis, Pasteurella canis, canine bocavirus (CBoV) and canine adenovirus (CAdV) were detected in the samples. Single, double, triple and quadruple infections accounted for 6.6%, 1.4%, 1.2% and 0.96% of samples, respectively. CCoV strains in 81 samples included three genotypes, CCoV-I, CCoV-IIa and CCoV-IIb, by analysis of S gene. The rate of single infection of CCoV-I, CCoV-IIa or CCoV-IIb was 19%, 38% or 7.4% respectively. The double and triple infection rates of CCoV were 32.8% and 2.5% respectively. All CPV strains in 36 samples belonged to CPV-2c. There were three amino acid differences in the Fiber protein of CAdV-positive sample QD2022, compared with the reference strain Toronto A26/61 and the vaccine strain YCA-18. These results suggest that CCoV and CPV are primary infectious agents, and that these two viruses were often identified in mixed infections, or coinfections alongside mycoplasma or other bacteria. These results will provide the basis for improvements in prevention and control of naturally occurring infectious diseases in Beagle dog production colonies and research facilities.

COVID-19 vaccine booster doses provide increased protection against COVID-19 hospitalization compared with previously vaccinated individuals: Interim findings from the REFORCO-Brazil real-world effectiveness study during Delta and Omicron

BackgroundAlthough COVID-19 booster vaccination is widely recommended, there is limited long-term, population-level, real-world evidence on the magnitude of improved protection against severe COVID-19 conferred by boosting with monovalent COVID-19 vaccines developed against ancestral SARS-CoV-2, especially in low- or middle-income countries. We present interim results from the first large-scale assessment of the relative vaccine effectiveness (rVE) of first and second booster doses against severe COVID-19 in a low-/middle-income country. MethodsREFORCO-Brazil is an ongoing, test-negative case-control study (NCT05697705) utilizing Brazil national severe acute respiratory syndrome (SARS) surveillance and vaccination data. In SARS hospitalizations from August 1, 2021 to July 31, 2022, we matched test-positive (via SARS-CoV-2 antigen/reverse transcription polymerase chain reaction [RT-PCR]) cases and test-negative case-controls (via RT-PCR) based on admission date, preceding vaccinations, and age. We evaluated the rVEs of four monovalent COVID-19 vaccines (AZD1222, Ad26.COV2.S, CoronaVac, and BNT162b2) as second boosters compared with any first boosters received ≥4 months previously, and as first boosters compared with primary-series vaccinations completed ≥4 months previously. ResultsThe overall rVE of second boosters, from 5668 (2238 test-positive) evaluated hospitalizations, was 24.7 % (95 % confidence interval [CI]: 12.6–35.1); the overall rVE of first boosters, from 30,272 (12,063 test-positive) hospitalizations, was 46.8 % (95 % CI: 43.3–50.0). The rVEs of AZD1222 and BNT162b2 were similar: 29.4 % (95 % CI: 8.6–45.5) and 25.5 % (95 % CI: 4.2–42.2), respectively, for second boosters; and 42.5 % (95 % CI: 28.0–54.0) and 50.8 % (95 % CI: 47.5–54.0), respectively, for first boosters. In general, rVEs were higher in elderly (≥80 years) and immunocompromised/high-risk individuals. ConclusionsOur results support the use of AZD1222 and other adenoviral/mRNA vaccine boosters to maintain protection against COVID-19 hospitalization from Omicron subvariants, including in elderly and immunocompromised individuals at increased risk of accelerated waning or severe outcomes.

Analysis of Coconut cadang-cadang viroid variants on field samples exhibiting variation in orange spotting symptom expression and severity.

Sequence variation has been attributed to symptom variations but has not been investigated in Orange Spotting-Coconut cadang-cadang viroid (OS-CCCVd) infected palms. Likewise, the relationship between Coconut cadang-cadang viroid (CCCVd) variants, Orange Spotting (OS) severity and the accumulation of the viroid in the palms have not been elucidated. This paper describes the characterization of CCCVd variants by cloning and sequencing, followed by correlation with symptom expression. Total nucleic acids were extracted from leaf samples harvested from frond 20 of seven Dura×Pisifera (D×P) African oil palm (Elaeis guineensis Jacq.) aged between 13 and 21years old collected from local plantations. The nucleic acids were fractionated using 5% non-denaturing polyacrylamide gel electrophoresis (PAGE) before being subjected to detection by reverse transcribed polymerase chain reaction (RT-PCR). The PCR products were cloned into a plasmid vector and the sequence of the clones was analyzed. CCCVd variants were quantified using real-time qPCR assay with CCCVd specific primers. Sixteen randomly selected clones of (OP246) had an arbitrary 100% identity with CCCVdOP246 (GeneBank Accession No: HQ608513). Meanwhile, four clones had >93% similarity with several minor sequence variations forming variants of OP234, OP235, OP251 and OP279. The OS symptoms observed in the field were characterized into three categories based on the size and morphology of the orange spots on the affected fronds. In addition, there was no direct correlation between disease severity and the accumulation of CCCVd variants in oil palm. This finding is the first report describing the sequence variation of the CCCVd RNA and symptom variation in OS oil palm field samples.

Sublethal doses of glyphosate modulates mitochondria and oxidative stress in honeybees by direct feeding.

Honeybees are essential for the ecosystem maintenance and for plant production in agriculture. Glyphosate is a broad-spectrum systemic herbicide widely used in crops to control weeds and could affect honeybees' health in sublethal doses. Our aim was to study how sublethal doses of glyphosate affects to oxidative stress and mitochondrial homeostasis in honeybees. We exposed honeybees to glyphosate at 5 and 10 mg·l-1 for 2 and 10 h for the gene expression analysis by reverse transcription polymerase chainreaction and for 48 and 72 h for the antioxidant enzymes activity and lipid peroxidation determination. We observed a general increase in antioxidant- and mitochondrial-related genes expression in honeybees after 2 h of exposition to glyphosate, as well as a rise in catalase and superoxide dismutase enzymatic activity after 48 h and an increment in lipid peroxidation adducts generation after 72 h. These results suggest a direct effect of glyphosate on honeybees' health, with an insufficient response of the antioxidant system to the generated oxidative stress, resulting in an increase in lipid peroxidation and, therefore, oxidative damage. Altogether, results obtained in this work demonstrate that sublethal treatments of glyphosate could directly affect honeybee individuals under laboratory conditions. Therefore, it is necessary to investigate alternatives to glyphosate to determine if they are less harmful to non-target organisms.

The Predictors of Pneumonia in Children with COVID-19

The purpose of this study was to evaluate the relationship between the presence of pneumonia and blood parameters in casesof Coronavirus disease (COVID-19) and to examine their predictive characteristics in terms of pneumonia. We reviewed the filerecords of 151 pediatric patients with a diagnosis of COVID-19 confirmed by the real time-reverse transcription polymerase chainreaction test in nasopharyngeal swabs. The patients were divided into two groups based on direct chest X-ray and computedtomography results in [Group 1 (n:41), with pneumonia findings, and Group 2 (n:110), with no pneumonia findings]. The groups’demographic data, clinical and laboratory findings were compared. Pulmonary involvement was determined in 41 (27.1%) ofthe 151 patients. The [body mass index (BMI) Z-score], red blood cell distribution width (RDW), mean platelet volume (MPV),neutrophil lymphocyte ratio, passive leg raise, and D-dimer levels were significantly higher in patients with pneumonia than thosewithout pneumonia in our study. Based on multivariate logistic regression analysis, BMI Z-score, MPV, and RDW were found tobe independent risk factors of pneumonia in patients. The current study showed higher levels of blood parameters in patients withcoronavirus disease 2019 (COVID -19) presenting with pneumonia than those without pneumonia. We suggest that BMI-Z scoreand MPV value may assist in predicting pulmonary involvement in patients with COVID-19.

Gastric cancer-derived exosomes facilitate pulmonary metastasis by activating ERK-mediated immunosuppressive macrophage polarization.

Gastric cancer (GC) with pulmonary metastasis is one of the deadliest diseases in the world; however, the underlying pathological mechanisms and potential therapeutic targets remain to be elucidated. As exosomes play indispensable roles in the formation of premetastatic niches (PMN) and cancer metastasis. Therefore, investigating the underlying mechanisms of exosome-mediatedpulmonary metastasis of GC may shed new light on identifying novel therapeutic targets for GC treatment.GC-derived exosomes were isolated from the conditioned medium of mouse forestomach carcinoma(MFC) cell line. The effects of MFC-derived exosomeson pulmonary macrophage polarization were analyzed byreverse- transcription polymerase chain reactionand flow cytometry. Expression of PD-L1 and other proteins was evaluated by Western blot. Exosomal microRNAs (miRNAs) were analyzed by microarray.GC-derived exosomes (GC-exo) accumulated in high numbers in the lungs and were ingested by macrophages. The extracellular-signal-regulated kinase (ERK) signaling pathway was activated by GC-exo, inducing macrophage immunosuppressive-phenotype differentiation and increased PD-L1 expression. miRNA-sequencing identified 130 enriched miRNAs in GC-exo. Among the enriched miRNAs, miR-92a-3p plays a major role in activating ERK signaling via inhibition of PTEN expression. In addition, inhibiting ERK signaling with PD98059 significantly reduced the expression of PD-L1 in macrophages and, therefore, reversed the immunosuppressive PMN and inhibited the colonization of GC cells in the lungs. This study identified a novel mechanism of GC-exo mediated PD-L1 expression in lung macrophages that facilitates lung PMN formation and GC pulmonary metastasis, which also provided a potential therapeutic target for GC with pulmonary metastasis treatment.

Double Regulation of Supplement-Detoxification on Diabetic Atherosclerosis by Inhibiting Excessive Autophagy

<i>Objecive</i> To explore the mechanism of Huoxue Jiedu Jiangtang recipe (HJJR) in regulating the hyperactivation of endoplasmic reticulum stress (ERS) autophagy PERK-eIF2a-LC3 pathway, and reveal its prevention for diabetic atherosclerosis. <i>Methods</i> First, the rats were injected with streptozotocin intraperitoneally to make the diabetic models, and then the aortic balloon injury combined with high-fat feeding were used to make the models of atherosclerosis who were randomly divided into model group, western medicine group (Gliquidone + Benazepril), low-dose HJJR group (HJJR₁), high-dose HJJR group (HJJR₂), and accepted corresponding drugs for 8 weeks respectively. Another blank group was used as control. The changes of serum glycosylated hemoglobin (GHb), homocysteine (Hcy) and angiotensinII (Ang1I) were compared. HE staining was used to detect aortic pathology. The mRNA transcription of PRK-like endoplasmic reticulum kinase (PERK), eucaryotic translation initation factor 2α (eIF2α) and CCAAT / Enhance-Binding Protein Homologous Protein (CHOP) were tested by reverse- transcription polymerase chain reaction (RT-PCR). Western blot were used to detect the expression of autophagy activating protein ATG6 (Beclin-1), autophagy protein microtubule-associated protein light chain LC3-1, LC3-II and LC3-II/LC3-I. <i>Results </i>Compared with the model group, after the drug intervention, the aortic tissue injury in each drug group were reduced, and the improvement in the Chinese medicine group was significantly better than that of the western medicine group (P < 0.05). All treatment groups could significantly lowed the levels of serum GHb, Hcy, and Ang1I (P < 0.05), could down regulate the transcription level of PERK, eIF2α and CHOP mRNA (P < 0.05); and decreased the amount of Beclin 1, LC3-II and LC3-II/LC3-1 autophagy protein in aortic cells (P < 0.05). The effect of HJJF group was better, and it was more significant with the increase of HJJF dose. <i>Conclusion</i> HJJR can protect arteries by inhibiting ERS caused by metabolic disorder in diabetes, correcting the homeostasis affecting ER, reducing the overreaction of PERK-eIF2-LC3, and alleviating autophagy effect.

Inhibition Effect of Adipogenesis and Lipogenesis via Activation of AMPK in Preadipocytes Treated with Canavalia gladiata Extract.

The aim of this study was to investigate the effect of Canavalia gladiata extract (CGE) on the regulation of AMP-activated protein kinase (AMPK) in 3T3-L1 preadipocytes and evaluate the adipogenesis and lipogenesis mechanisms. In 3T3-L1 preadipocytes, lipid accumulation and differentiation were suppressed by 1.1, 1.3, and 1.4 times under the CGE treatment at 0.25, 0.5, and 1.0 mg/mL, respectively. The expression of the main genes involved in the inhibition of adipogenesis was evaluated at the mRNA level via a transcription-polymerase chain reaction. The extract at 1.0 mg/mL increased the mRNA expressions of AMPK and carnitine palmitoyl transferase-1 (CPT-1) by 1.9 and 1.2 times, respectively, while it decreased the expression of sterol regulatory element binding proteins-1c (SREBP-1c), peroxisome proliferator activated receptor-γ (PPAR-γ), CCAAT enhancer binding protein-α (C/EBP-α), and fatty acid synthase (FAS) by 1.1, 1.2, 1.8, and 1.5 times, respectively, indicating inhibition of the adipogenesis and lipogenesis potential of CGE. Gallic acid (4.02 mg/g) was identified as the main component of the CGE via LC-MS/MS and HPLC analysis. The results of this study suggested that CGE can be utilized as an anti-obesity food additive or medication by activating the AMPK-induced regulation and suppressing adipogenesis transcription factors.

Genome analysis of human respiratory syncytial virus in Fujian Province, Southeast China.

Respiratory syncytial virus (RSV) is one of the main causes of acute respiratory infections (ARI)leading to a heavy disease burden. Reports on RSV in China are limited, especially in Fujian Province, and RSV whole-genome sequences in Fujian Province are not reported. This study aimed to explore the genomic characteristics of RSV to provide evidence for the development of vaccines and medicines. Reverse transcription-polymerase chain reaction (RT-PCR) was used to amplify and obtain the attachment (G) gene and whole-genome fragments from the positive samples. Genetic evolution as well as nucleotide and amino acid homology analyses of the virus sequences were conducted to identify any amino acid mutations. A total of 72 RSV-positive cases were collected, and 53G gene sequences were obtained using polymerase chain reaction (PCR) amplification. The ON1 and BA11 genotypes were found to be dominant using the Basic Local Alignment Search Tool (BLAST) on the NCBI website. The 40 genotype ON1 sequences had high nucleotide identity (95.3%-99.8%) and amino acid similarity (92.5%-100%), whereas the 13 BA11 genotype sequenceshad 97.3%-99.6% nucleotide identity and 94.8%-99.7% amino acid similarity. Compared to the ON1 prototype (JN257693) and BA11 prototype (AY333364), the obtained sequences had no nucleotide insertions or deletions, indicating high similarity among the samples. A total of 17 RSV whole genome sequences were obtained, 10 of which were genotype ON1 and seven were genotype BA11. Certain amino acid mutations were found in the antigen site and epitope of the fusion (F) protein but not in the G protein. Glycosylation analyses of specific RSV genes revealed high positive selection rates for the gene, and the N- and O-linked glycosylation sequences in the F gene were relatively conserved. From July 2018 to January 2020, ON1 and BA11 were the most prevalent RSV genotypes in Fujian Province. A high nucleotide identity and amino acid similarity were observed between the reference strain and the obtained strains, as well as among the sequences of the obtained isotypes. Certain amino acid mutations occur at the antigen site and the epitope of the F protein.

Gene Dysregulation in the Adult Rat Paraventricular Nucleus and Amygdala by Prenatal Exposure to Dexamethasone.

Fetal programming is the concept that maternal stressors during critical periods of fetal development can alter offspring phenotypes postnatally. Excess glucocorticoids can interact with the fetus to effect genetic and epigenetic changes implicated in adverse developmental outcomes. The present study investigates how chronic exposure to the synthetic glucocorticoid dexamethasone during late gestation alters the expression of genes related to behavior in brain areas relevant to the regulation and function of the hypothalamic–pituitary–adrenal axis. Pregnant Wistar Kyoto rats received subcutaneous injections of dexamethasone (100 μg/kg) daily from gestational day 15–21 or vehicle only as sham controls. The amygdala and paraventricular nucleus (PVN) were micro-punched to extract mRNA for reverse transcription and quantitative polymerase chain reaction for the analysis of the expression of specific genes. In the PVN, the expression of the glucocorticoid receptor NR3C1 was downregulated in female rats in response to programming. The expression of CACNA1C encoding the Cav1.2 pore subunit of L-type voltage-gated calcium channels was downregulated in male and female rats prenatally exposed to dexamethasone. Collectively, the results suggest that prenatal exposure to elevated levels of glucocorticoids plays a role in the dysregulation of the hypothalamic–pituitary–adrenal axis and potentially learning and memory by altering the expression of specific genes within the amygdala and PVN.

Performance evaluation of a non-invasive one-step multiplex RT-qPCR assay for detection of SARS-CoV-2 direct from saliva

Polymerase chain reaction (PCR) has proven to be the gold-standard for SARS-CoV-2 detection in clinical settings. The most common approaches rely on nasopharyngeal specimens obtained from swabs, followed by RNA extraction, reverse transcription and quantitative PCR. Although swab-based PCR is sensitive, swabbing is invasive and unpleasant to administer, reducing patient compliance for regular testing and resulting in an increased risk of improper sampling. To overcome these obstacles, we developed a non-invasive one-step RT-qPCR assay performed directly on saliva specimens. The University of Nottingham Asymptomatic Testing Service protocol simplifies sample collection and bypasses the need for RNA extraction, or additives, thus helping to encourage more regular testing and reducing processing time and costs. We have evaluated the assay against the performance criteria specified by the UK regulatory bodies and attained accreditation (BS EN ISO/IEC 17,025:2017) for SARS-CoV-2 diagnostic testing by the United Kingdom Accreditation Service. We observed a sensitivity of 1 viral copy per microlitre of saliva, and demonstrated a concordance of > 99.4% between our results and those of other accredited testing facilities. We concluded that saliva is a stable medium that allows for a highly precise, repeatable, and robust testing method.

Therapeutic Potential of Pretreatment with Exosomes Derived from Stem Cells from the Apical Papilla against Cisplatin-Induced Acute Kidney Injury.

Acute kidney injury (AKI) is the most serious side effect of treatment with cisplatin in clinical practice. The aim of this study was to investigate the therapeutic effect of exosomes derived from stem cells from the apical papilla (SCAPs) on AKI. The medium from a SCAP culture was collected after 2 d of culture. From this, SCAP-derived exosomes (SCAP-ex), which were round (diameter: 30–150 nm) and expressed the characteristic proteins CD63 and CD81, were collected via differential ultracentrifugation. Rat renal epithelial cells (NRK-52E) were pretreated with SCAP-ex for 30 min and subsequently treated with cisplatin to induce acute injury. The extent of oxidative stress, inflammation, and apoptosis were used to evaluate the therapeutic effect of SCAP-ex against cisplatin-induced nephrotoxicity. The viability assay showed that the survival of damaged cells increased from 65% to 89%. The levels of reactive oxygen species decreased from 176% to 123%. The glutathione content increased by 78%, whereas the levels of malondialdehyde and tumor necrosis factor alpha (TNF-α) decreased by 35% and 9%, respectively. These results showed that SCAP-ex can retard oxidative stimulation in damaged kidney cells. Quantitative reverse transcription–polymerase chain-reaction gene analysis showed that they can also reduce the expression of nuclear factor-κβ (NF-κβ), interleukin-1β (IL-1β), and p53 in AKI. Further, they increased the gene expression of antiapoptotic factor B-cell lymphoma-2 (Bcl-2), whereas they reduced that of proapoptotic factors Bcl-2-associated X (Bax) and caspase-8 (CASP8), CASP9, and CASP3, thereby reducing the risk of cell apoptosis.

Changes in expression of miRNAs from the DLK1-DIO3 locus are characteristic of relapsing-remitting multiple sclerosis regardless of the disease activity

The role of miRNAs – small, regulatory, noncoding RNAs – in the multiple sclerosis (MS) development is being intensively investigated. Previously, we the first who observed a significant increase in the expression of 26 microRNA genes localized in the DLK1-DIO3 locus in men with relapsing-remitting MS (RRMS) in peripheral blood mononuclear cells (PBMCs), using RNA sequencing.Objective: to evaluate the possible involvement of miRNAs in the regulation of the activity of the pathological process in RRMS by comparing the miRNA genes expression levels from this locus in patients in remission, relapse, and healthy individuals, separately for men and women.Patients and methods. Analysis of miR-431-5p, miR-127-3p, miR-379, miR-376c, miR-381, miR-410 and miR-656-3p microRNA expression was performed by reverse transcription and subsequent real-time polymerase chain reaction in PBMCs of 16 patients in the relapse stage, 20 patients in remission who did not receive immunomodulatory drugs, and 20 healthy individuals.Results and discussion. The expression levels of all studied miRNAs did not differ in patients in remission and relapse stages, in men and women. At the same time, men (in remission and relapse) showed a significant increase in the levels of all miRNAs compared with healthy men; in women, no changes in expression were observed. A high level of correlation of miRNA expression from the DLK1-DIO3 locus was shown in both patients and healthy individuals from the control group, regardless of gender. Coexpression was observed not only for miRNA genes from the same cluster (14q32.2 or 14q32.31), but also for genes from different clusters.Conclusion. The miRNAs genes from the DLK1-DIO3 locus are involved in the pathophysiology of RMS onset, but not in the processes associated with the transition from remission to relapses. The high consistency of miRNA expression, regardless of the localization of their genes within this region, suggests the presense of a common mechanism that regulates their transcription.

Establishment of Multiplex RT-PCR for Differentiating between the CD1901 and Lederle Strains of Canine Distemper Virus

Canine distemper virus (CDV) is a life-threatening pathogen in dogs. Clinical, pathological and molecular methods are required to diagnose CDV infection, and it is important to differentiate between the Korean CDV strain CD1901 and Lederle CDV vaccine strain. Therefore, in this study, we used multiplex reverse- transcription polymerase chain reaction (RT-PCR) to differentiate between the CD1901 and Lederle strains. A primer set was designed based on the CDV nucleoprotein gene and nucleotide sequence variation in the fusion (F) gene. First, 224-bp DNA bands were amplified from viral RNA of the CD1901 and Lederle strains. Then, 428- and 326-bp DNA bands were amplified in the CD1901 and Lederle strain, respectively. The multiplex RT-PCR detection limits were 2.53 and 0.8 median tissue culture infectious dose/reaction for the CD1901 and Lederle strains, respectively. No cross-reactions were detected in non-CDV reference viruses, including rabies virus, parvovirus, canine adenovirus types 1 and 2, and parainfluenza virus. The results indicate that our one-step multiplex RT-PCR is useful for differentiating between wildtype and vaccine CDV strains.

Molecular markers of cardiac fibrosis after myocardial infarction

Aim. To perform a screening for molecular markers of cardiac fibrosis upon myocardial infarction.Materials and Methods. We carried out echocardiography-guided endomyocardial biopsy of affected and intact interventricular septum segments of 7 patients with anterior myocardial infarction. Fibrotic and adjacent intact cardiac tissue was dissected into 2 equal segments and: 1) homogenized with the further RNA extraction, reverse transcription, and quantitative polymerase chain reaction; 2) fixed in formalin and embedded into paraffin with the further van Gieson staining for the histological verification of cardiac fibrosis.Results. We found that the expression of ACTA2, VIM, CTGF, COL1A1, TGFB1, TGFBR1, AGTR1, CCL2 and TNF genes in fibrotic cardiac tissue was ≥ 3-fold higher as compared with the adjacent intact myocardium reflective of active extracellular matrix production by fibroblast-derived myofibroblasts.Conclusion. We have for the first time shown AGTR1, CCL2, and TNF genes as candidates for post-infarction cardiac fibrosis in addition to ACTA2, VIM, CTGF, COL1A1, TGFB1, and TGFBR1 genes.

Identification and validation of suitable housekeeping genes for gene expression studies in BCR-ABL1 positive B-lineage acute lymphoblastic leukemia.

The stability of the housekeeping gene (HKG) expression is an absolute prerequisite for accurate normalization of target gene expression in a quantitative real-time polymerase chain reaction (RQ-PCR). In RQ-PCR, the widely used normalization approach involves the standardization of target genes to the most stable HKG control genes. According to the recent literature, in different experimental conditions the HKGs exhibit either up or down-regulation and thus affecting the gene expression profiles of target genes which leads to erroneous results. This implies that it is very important to select the appropriate HKG and verify the expression stability of the HKG before quantification of the target gene. The present study aims to analyze six different HKGs for their expression profiles and stability in BCR-ABL1 negative cases and validate them in BCR-ABL1 positive cases, detected by multiplex reverse transcribed polymerase chain reaction (RT-PCR). Six commonly used reference genes (GAPDH, ABL1, RNA18S, ACTB, GUSB, and EEF2) were selected in this study. RQ-PCR was performed on 24 BCR-ABL1 negative cases and the outcomes were validated on 24 BCR-ABL1 positive cases. RefFinder™, a web-based composite software was used to check the stability of HKG genes by different algorithms and comprehensive ranking of each HKG gene in BCR-ABL1 negative cases and finally validated in BCR-ABL1 positive cases. It was found that RNA18S, ABL1 and GUSB are good stable HKG genes, which showed minimum variability in gene expression compared to GAPDH, EEF2, and ACTB, the most commonly used HKG.