Effect of miR-125b on dermal papilla cells of goat secondary hair follicle

BackgroundMicroRNAs (miRNAs) are endogenous noncoding RNAs that regulate various biological processes. miR-125b is a miRNA that has been reported to be critical for hair follicle (HF) morphogenesis and development. We identified that the expression of miR-125b varies during an individual hair cycle (anagen, catagen, and telogen) in the skin of cashmere goats. We constructed a gain model (by overexpressing miR-125b) and a loss model (by inhibiting endogenous miR-125b) based on dermal papilla cells (DPCs) to further investigate the role of miR-125b in HF cycle. In addition, we used a dual-luciferase system to highlight the predicated target genes of miR-125b.ResultsWe found that miR-125b affects the expression of FGF5, IGF-1, SHH, TNF-α, MSX2, LEF-1, FGF7, NOGGIN, BMP2, BMP4, TGF-β1, and β-catenin. The dual-luciferase assay further validated a direct interaction between miR-125b and FGF5 and TNF-α.ConclusionmiR-125b affects the expression levels of genes related to hair cycle and may also play a critical role in regulating the periodic development of HF.     

A fast and efficient protocol for small RNA extraction in Japanese plum and other Prunus species

BackgroundSmall ribonucleic acids represent an important repertoire of mobile molecules that exert key roles in several cell processes including antiviral defense. Small RNA based repertoire includes both small interfering RNA (siRNA) and microRNA (miRNA) molecules. In the Prunus genus, sharka disease, caused by the Plum pox virus (PPV), first occurred on European plum (Prunus domestica) and then spread over among all species in this genus and thus classified as quarantine pathogen. Next-generation sequencing (NGS) was used for the study of siRNA/miRNA molecules; however, NGS relies on adequate extraction protocols. Currently, knowledge of PPV-Prunus interactions in terms of siRNA populations and miRNA species is still scarce, and siRNA/miRNA extraction protocols are limited to species such as peach, almond, and sweet cherry.ResultsWe describe a reliable procedure for siRNA/miRNA purification from Prunus salicina trees, in which previously used protocols did not allow adequate purification. The procedure was based on a combination of commercially available RNA purification kits and specific steps that yielded high quality purifications. The resulting molecules were adequate for library construction and NGS, leading to the development of a pipeline for analysis of both siRNAs and miRNAs in the PPV–P. salicina interactions. Results showed that PPV infection led to altered siRNA profiles in Japanese plum as characterized by decreased 24-nt and increased 21- and 22-nt siRNAs. Infections showed miR164 and miR160 generation and increased miR166, miR171, miR168, miR319, miR157, and miR159.ConclusionWe propose this protocol as a reliable and reproducible small RNA isolation procedure for P. salicina and other Prunus species.     

MicroRNAs: Potential biomarkers in cancer

microRNAs (miRNAs) are evolutionarily conserved small noncoding RNAs, also known as micromanagers of gene expression. Polymorphisms in the miRNA pathway (miR-polymorphisms) are emerging as powerful tools to study the biology of a disease and have the potential to be used in disease prognosis and diagnosis. Advancements in the miRNA field also indicate a clear involvement of deregulated miRNA gene signatures in cancers, and several polymorphisms in pre-miRNA, miRNA binding sites or targets have been found to be associated with various cancers. The miRNA polymorphisms have also been reported to influence tumor aggressiveness as well as survival of cancer patients. miRNAs have a revolutionary impact on cancer research over recent years. They emerge as important players in tumorigenesis, leading to a paradigm shift in oncology. The extensive and comprehensive use of miRNA microarrays has enabled the identification of a number of miRNAs as potential biomarkers for cancer. Many miRNAs have been identified to act as oncogenes, tumor suppressors, or even modulators of cancer stem cells and metastasis. Some studies not only reported the identified miRNA biomarkers, but also deciphered their target genes and the underlying mechanisms. The rapid discovery of many miRNA targets and their relevant pathways has contributed to the development of miRNA-based therapeutics.     

CRISPR/Cas9-mediated activation of CDH1 suppresses metastasis of breast cancer in rats

BackgroundCancer is a life-threatening disease that affects approximately 18 million individuals worldwide. Breast cancer is the most common female neoplasm globally with more than 276,480 new cases of invasive breast cancer expected to be diagnosed in women in the U.S. alone in 2020. Genetic and epigenetic factors play role in the carcinogenesis and progression of this disease. In this study, MCF-7 adenocarcinoma cells were transfected with CRISPR/Cas9 plasmid to either knock out CDK11 or to activate CDH1. Treated cells were allografted into the mammary glands of female rats (150–190 g, 6–8 weeks) to evaluate the capability of these cells to control cancer progression and metastasis.ResultsqPCR data revealed a significant downregulation of CDK11 and upregulation of CDH1. Cell cycle analysis and apoptosis assays indicated the knockout of CDK11 and simultaneous activation of CDH1 resulted in cell cycle arrest at G2/M phase and accumulation of cells at G2. Meanwhile, the percentage of cells that underwent late apoptosis increased in both genome editing hits. Histopathological sectioning data indicated that untransfected MCF-7 cells were capable of developing tumors in the mammary gland and initiation g angiogenesis. Transfected cells significantly restricted cancer cell infiltration/invasion by minimally localizing tumors and inhibiting angiogenesis.ConclusionsAlthough further investigation is needed, the present data indicate the potentiality of using CRISPR/Cas9-based therapy as a promising approach to treat breast cancer. Impact: these data indicate targeting cancer-related genes via any genome editing tool might represent a novel approach to combat cancer.How to cite: Al-Mulhim F, Alqosaibi AI, Al-Muhnna A, et al. CRISPR/Cas9-mediated activation of CDH1 suppresses metastasis of breast cancer in rats. Electron J Biotechnol 2021;53. https://doi.org/10.1016/j.ejbt.2021.06.002     

Improved protocol for plasma microRNA extraction and comparison of commercial kits

IntroductionMicroRNAs are small, non-coding RNA molecules that are becoming popular biomarkers in several diseases. However, their low abundance in serum/plasma poses a challenge in exploiting their potential in clinics. Several commercial kits are available for rapid isolation of microRNA from plasma. However, reports guiding the selection of appropriate kits to study downstream assays are scarce. Hence, we compared four commercial kits to evaluate microRNA-extraction from plasma and provided a modified protocol that further improved the superior kit’s performance.Materials and methodsWe compared four kits (miRNeasy Serum/Plasma, miRNeasy Mini Kit from Qiagen; RNA-isolation, and Absolutely-RNA MicroRNA Kit from Agilent technologies) for quality and quantity of microRNA isolated, extraction efficiency, and cost-effectiveness. Bioanalyzer-based Agilent Small RNA kit was used to evaluate quality and quantity of microRNA. Extraction efficiency was evaluated by detection of four endogenous control microRNA using real-time-PCR. Further, we modified the manufacturer’s protocol for miRNeasy Serum/Plasma kit to improve yield.ResultsmiRNeasy Serum/Plasma kit outperformed the other three kits in microRNA-quality (P < 0.005) and yielded maximum microRNA-quantity. Recovery of endogenous control microRNA i.e. hsa-miR-24-3p, hsa-miR-191-5p, hsa-miR-423-5p and hsa-miR-484 was higher as well. Modification with the inclusion of a double elution step enhanced yield of microRNA extracted with miRNeasy Serum/Plasma kit significantly (P < 0.001).ConclusionWe demonstrated that miRNeasy Serum/Plasma kit outperforms other kits and can be reliably used with a limited plasma quantity. We have provided a modified microRNA-extraction protocol with improved microRNA output for downstream analyses.     

Prostate Specific Antigen in Cord Blood

Recent studies have demonstrated the presence of prostate specific antigen (PSA) in cord blood of male as well as female babies. The placental progesterone and estradiol up-regulate the synthesis and secretion of PSA in Placenta. This PSA is presumed to play a role in intrauterine growth of fetus by virtue of its proteolytic action on several substrates including insulin-like-growth-factor-binding-protein-3, insulin chains and Interleukin-2. This study was planned with the objective of correlating the levels of PSA in cord blood to gestation at delivery, the type of delivery and gender of the fetus. Fifty-seven cord blood samples were collected from the umbilical cord during delivery or mid-trimester abortion and analyzed for PSA using ‘Active PSA DSL-9700 ultra sensitive’ kit employing two-site immuno-radiometric assay principle and having a detection limit of 0.001 ng/ml. Mean PSA levels in cord blood were found to be 0.112 ± 0.027 ng/ml. The concentration of PSA in cord blood was found to be higher in case of higher gestational age, male baby and operative delivery. 50 % of cord bloods for female babies had PSA below detection limit (range <0.001–0.460 ng/ml), while all the male samples had detectable PSA (range 0.11–0.973 ng/ml). Higher Progesterone levels found in prenatal maternal blood in case of male babies may be responsible for the higher cord blood PSA. Mean cord blood PSA was 0.150 ± 0.150 ng/ml in forceps delivery and 0.078 ± 0.012 ng/ml in normal vaginal delivery. Forceps delivery causes much more stress and strain as compared to a normal vaginal delivery, resulting in increased levels of adrenal glucocorticoids, and therefore, higher cord blood PSA.     

Correlation of Cord Blood Lipid Heterogeneity in Neonates with Their Anthropometry at Birth

Objective: Fetus with intrauterine stress may exhibit programmed changes that can alter its metabolism and bear severe risk for diseases in adult life. The current study was designed to assess the correlation between cord blood lipid profile with the anthropometric data in neonates. Materials and methods: 146 newborn babies born at Dr. T M A Pai Hospital, Udupi were screened and their birth weight, length, head circumference and abdominal circumference were noted at birth. Umbilical cord blood samples were analyzed for total cholesterol, triglycerides (TG), high density lipoprotein (HDL) and low density lipoprotein (LDL). Infants were also grouped further based on gestational age (GA) and sex-adjusted birth weight percentiles into three groups i.e. Small for gestational age (SGA), Appropriate for gestational age (AGA) and Large for gestational age (LGA) for comparison of their lipid profiles. Inclusion criteria were normal fetal heart rate at birth and an APGAR score >7. Statistical significance of relation between lipid profile and anthropometry was done using ANOVA and Pearson correlation coefficient. Results: Triglycerides were significantly higher in babies with higher ponderal index (PI) than those with lower PI (P = 0.011). The TG level of SGA babies were significantly higher as compared to AGA group (P = 0.001). The LDL levels in neonates with higher abdominal circumference were significantly lower than those with lower AC (P = 0.019). Mean HDL levels were higher in neonates with larger AC, but not statistically significant. Maternal BMI had no influence on neonates’ lipid profile. Conclusion: Abnormal intrauterine milieu created by maternal changes during gestation may bear a profound impact on lipid metabolism in neonates, which may account for their differences in lipid profile and anthropometry at birth.     

Identification of key genes and construction of CircRNA–miRNA–mRNA regulatory networks in osteoarthritis

BackgroundOsteoarthritis (OA) is one of the most frequent degenerative joint diseases with high rate of disability, but its mode of action remains largely unclear. The current study was aimed at identifying key genes and molecular mechanism in OA. Gene expression datasets (GSE1919, GSE55235, GSE55457) were downloaded from Gene Expression Omnibus for integrated bioinformatics analysis. Differentially expressed genes (DEGs) in OA synovial tissues were identified using GeoDiver and GEO2R. Gene ontology enrichment analyses were undertaken via FunRich and Metascape. Also, Gene Set Enrichment Analysis was performed using miRWalk3.0. Subsequently, pathways interrelation analysis of hub genes was carried out using plug-in ClueGO v2.3.3. Additionally, circRNA–miRNA–mRNA regulatory networks were visualized using Cytoscape.ResultsA total of 508 DEGs were obtained from three GSE datasets, of these five intersection DEGs (TNFAIP3, VEGFA, GADD45B, SIK1, KLF9) were shared by three GSE datasets. Intersection DEGs were significantly enriched in LKB1 signaling events, signaling events mediated by focal adhesion kinase, and PDGFR-beta signaling pathway. Enrichment analysis for all the DEGs showed that they mainly enriched in inflammatory response, cytokine production, blood vessel development, stress response, osteoclast differentiation, and MAPK signaling pathway. A total of 39 genes were regarded as hub genes and pathways interrelation analysis indicated that hub genes mainly enriched in TNF signaling pathway, IL-17 signaling pathway, and NF-kappa B signaling pathway.ConclusionsThe current study revealed the potential key genes, pathways, and circRNA–miRNA–mRNA regulatory networks in OA, which may contribute to a more comprehensive understanding of OA pathogenesis.How to cite: Li HZ, Xu XH, Lu HD. Identification of key genes and construction of CircRNA–miRNA–mRNA regulatory networks in osteoarthritis. Electron J Biotechnol 2019;37. https://doi.org/10.1016/j.ejbt.2018.11.004.     

Study of the Concentration of Trace Elements Fe,Zn, Cu,Se and Their Correlation in Maternal Serum,Cord Serum and Colostrums

A study of iron, zinc, copper and selenium concentration levels was carried out in three compartments namely, maternal serum (MS), colostrums and cord blood serum (CS) of healthy Indian mothers (n = 42) who delivered healthy normal neonates without any congenital anomalies at Bhabha Atomic Research Centre hospital, Mumbai. Fe, Zn, Cu in maternal serum, cord blood and colostrums were estimated by flame atomic absorption spectrometry while Se was determined by graphite furnace absorption spectrometry. It was seen that there was a significant difference in the level of trace elements in the three compartments. The average levels of Fe in the three compartments were 1,132 ± 519, 2,312 ± 789 and 1,183 ± 602 μg/L while Zn was 514 ± 149, 819 ± 224 and 7,148 ± 2,316 μg/L respectively. Mean Cu values were 1,614 ± 295, 301 ± 77 and 392 ± 174 μg/L respectively while Se values were 70 ± 15, 36 ± 10 and 23 ± 8 μg/L respectively. The results indicated a positive correlation of Fe and Zn concentrations in MS versus CS which were (r = 0.386), (r = 0.572) respectively and Fe levels in MS and colostrums (r = 0.235). A few inter element correlations were found within compartments. Zn and Se showed a negative correlation in both MS (r = −0.489) and colostrums (r = −0.258) while a positive inter correlation of Fe and Zn was seen in MS (r = 0.44) and in CS (r = 0.54). This study gave us an overview of the serum and colostrum values of mother and neonates in Indian population, data of which are scarce.     

A quantitative PCR approach for determining the ribosomal DNA copy number in the genome of Agave tequila Weber

BackgroundAgave tequilana has a great economic importance in Mexico in order to produce alcoholic beverages and bioenergy. However, in this species the structure and organization of the rDNAs in the genome are limited, and it represents an obstacle both in their genetic research and improvement as well. rDNA copy number variations per eukaryotic genome have been considered as a source of genetic rearrangements. In this study, the copy number of 18S and 5S rDNAs in the A. tequilana genome was estimated, and an absolute quantitative qPCR assay and genome size was used. In addition, an association between the rDNAs copy number and physical mapping was performed to confirm our results.ResultsThe analysis were successfully applied to determine copy number of 18S and 5S rDNAs in A. tequilana genome, showing high reproducibility with coefficient of variation (CV) values of 0.014–0.0129%, respectively. A variation of 51 times in the copy number the 18s regarding 5s rDNA was found, thus contributing to genome size of 1.47 and 8.38 × 10^-3%, respectively. Similarly, data show a linear relationship (R [2] = 0.992) between rDNA copy number and the detected signals for each of the loci by FISH. The comparison of the rDNA copy number of agave showed differential relationship with other organisms and it may be due to evolutionary ecology.ConclusionsResults show that the proposed method a) can correctly detect the rDNA copy number, b) could be used as species-specific markers and c) might help in understanding the genetic diversity, genome organization and evolution of this species.     

Indirect reference intervals for haematological parameters in capillary blood of pre-school children

IntroductionIndirect estimation of reference intervals (RIs) is straightforward and inexpensive procedure for determination of intra-laboratory RIs. We applied the indirect approach to assess RIs for haematological parameters in capillary blood of pre-school children, using results stored in our laboratory database.Materials and methodsWe extracted data from laboratory information system, for the results obtained by automatic haematology analyser in capillary blood of 154 boys and 146 girls during pre-school medical examination. Data distribution was tested, and logarithmic transformation was applied if needed. Reference intervals were calculated by the nonparametric percentile method.ResultsReference intervals were calculated for: RBC count (4.2-5.4 x10¹²/L), haemoglobin (114-146 g/L), MCH (25.0-29.4 pg), MCHC (321-368 g/L), RDW-SD (36.1-43.5 fL), WBC count (4.5-12.3 x10⁹/L), neutrophils count (1.7-6.9 x10⁹/L) and percentage (29.0-69.0%), lymphocytes count (1.6-4.4 x10⁹/L) and percentage (21.9-60.7%), PLT (165-459 x10⁹/L), MPV (8.1-11.4 fL) and PDW (9.2-14.4%). Gender specific RIs were calculated for monocytes count (male (M): 0.2-1.6 x10⁹/L; female (F): 0.1-1.4 x10⁹/L) and percentage (M: 2.5-18.3%; F: 1.8-16.7%), haematocrit (M: 0.34-0.42 L/L; F: 0.34-0.43 L/L), MCV (M: 73.4-84.6 fL; F: 75.5-84.2 fL) and RDW (M: 12.1-14.3%; F: 11.7-13.9%), due to observed gender differences in these parameters (P = 0.031, 0.028, 0.020, 0.012 and 0.001; respectively). Estimated RIs markedly varied from the literature based RIs that are used in the laboratory.ConclusionsIndirect method employed in this study enables straightforward assessment of RIs in pre-school children. Herein derived RIs differed from the literature-based ones, indicating the need for intra-laboratory determination of RIs for specific populations and sample types.     

Idiopathic Fetal Growth Restriction: Repercussion of Modulation in Oxidative Stress

Oxidative stress has been proposed as one of the causes involved in idiopathic fetal growth restriction (IFGR). However, the exact relationship between oxidative stress and IFGR is not understood. This study aimed at understanding the role of oxidative stress and antioxidant status in IFGR materno-fetal dyads and matched controls. 75 materno-fetal dyads with IFGR were enrolled with equal number of normal low risk controls. Malondialdehyde (MDA) levels were measured as marker of oxidative stress, while paraoxonase-1 (PON1) activity and total antioxidant capacity (TAC) of serum were measured as markers of antioxidant status. MDA levels were increased in both maternal and cord blood of IFGR neonates as compared to controls (p < 0.001). TAC of serum were found to be decreased in IFGR (both maternal and cord blood) as compared to controls (p < 0.001; p < 0.05, respectively). PON1 activity was found to be decreased in the IFGR mothers while it was found increased in IFGR cord blood (p < 0.01; p < 0.001)). IFGR is a state of increased oxidative stress. Decreased PON1 enzymatic activity in mothers is also associated with IFGR.     

Utility of icteric index in clinical laboratories: more than a preanalytical indicator

IntroductionTotal bilirubin tests are highly demanded in clinical laboratories. Since icteric index (I-index) has zero cost, we aimed to evaluate its clinical utility and cost-effectiveness to determine if total bilirubin is necessary to be tested. We took into account if haemolysis could interfere to icteric index determination.Material and methodsRetrospectively we reviewed I-index results in two cohorts (43,372 and 8507 non-haemolysed and haemolysed samples, respectively). All determinations were done using Alinity c chemistry analysers (Abbott Diagnostics). Receiver operating characteristic (ROC) curve was used to determine the optimal index cut-off to discriminate between normal and abnormal bilirubin concentration (20.5 µmol/L).ResultsThe ROC curve analysis suggested 21.4 µmol/L as the optimal I-index cut-off but differences in sensitivity and specificity were detected between patient derivation. For rejecting purpose, 15.4 µmol/L and 17.1 µmol/L I-index thresholds were selected based on patient derivation (inpatients and emergency room; and primary care and outpatients, respectively) with 97% sensitivity and 0.25% false negative results. Sensitivity was much lower in haemolysed samples. We selected 34.2 µmol/L I-index as threshold to detect hyperbilirubinemia with 99.7% specificity and 0.26% false positive results, independent of haemolysis. With the icteric index cut-offs proposed, we would save 66% of total bilirubin requested and analyse total bilirubin in around 2% of samples without total bilirubin requested.ConclusionsThis study supports the use of I-index to avoid bilirubin determination and to identify patients with hyperbilirubinemia. This work considers that the economic and test savings could help to increase the efficiency in clinical laboratories.     

The comparison of the three assays for determination of fecal calprotectin in inflammatory bowel disease

IntroductionFecal calprotectin is a biomarker for monitoring inflammatory bowel disease (IBD) activity. Our aim, therefore, was to evaluate two new assays, the point of care test Quantum Blue and the Liaison Calprotectin with respect to the Calprest, commonly used assay, and to determine their performance for IBD diagnosis.Materials and methodsWe included 73 prospective patients with IBD. Fecal calprotectin was measured and analysed with the routine Calprest assay and two recently introduced assays, the Quantum Blue and the Liaison Calprotectin. Furthermore, we compared the results by Bland and Altman analysis, and Passing-Bablok regression.ResultsWe observed no difference in median calprotectin values obtained by the Calprest (94.6 µg/g, 95%CI 66.5 to 166.1) and Liaison assay (101.0 µg/g, 95%CI 48.1 to 180.1) whereas significantly higher concentrations were obtained with the Quantum Blue assay (240.0 µg/g, 95%CI 119.9 to 353.2). The mean absolute and relative difference between the Calprest and Quantum Blue methods was statistically significant (- 162.3 µg/g and
- 143.1%). Mean absolute difference between the Calprest and Liaison calprotectin methods was positive (2.2 µg/g). The agreement between assays revealed that Quantum Blue and Calprest have fair agreement with Kappa coefficient of 0.38 (95%CI 0.26 to 0.51). Liaison Calprotectin and Calprest revealed moderate agreement with a weak Kappa coefficient of 0.47 (95%CI 0.32 to 0.62).ConclusionClinicians should be aware of these differences between the assays and avoid comparison of their respective results.     

Possible Role of microRNA-122 in Modulating Multidrug Resistance of Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is a hypervascular primary liver cancer characterized by rapid progression, besides, resistance to traditional chemotherapeutic agents. It has been shown that microRNAs play critical roles in regulation of tumor cell sensitivity to drugs through modulating the expression of genes involved in drug transport. The present study investigated whether restoration of miR-122 in HCC cells could alter the cell cycle distribution and the expression of multidrug resistance (MDR)-related genes (ABCB1, ABCC1, ABCG2 and ABCF2). After overexpression of miR-122 in HepG2 cells treated or untreated with doxorubicin doses, total RNAs and protein extracts were isolated for application of QRT-PCR and western blotting techniques. Moreover, cell cycle distribution was monitored by flow cytometry. Our results revealed that, the over expression of miR-122 in HepG2 cells treated or untreated with doxorubicin could modulate the sensitivity of cells to chemotherapeutic drug through downregulation of MDR-related genes, ABCB1 and ABCF2. Interpretation of cell cycle distribution revealed that, the anti-proliferative effect of miR-122 is associated with the accumulation of cells in G₀/G1 phase. Moreover, treatment with miR-122 and doxorubicin resulted in high percentage of HCC cells in G₀/G1 phase. Taken together, our findings revealed that, overexpression of miR-122 inhibited HCC cell growth by inducing cell cycle arrest and this arrest is associated with down-regulation of MDR-related genes.     

Assessment of oxidative stress and antioxidant status in age related cataract in a rural population

Oxidative stress was assesed by estimating lipid peroxidation product (LPO) in the form of thiobarbituric acid reactive substances (TBARS), enzymatic antioxidants in the form of superoxide dismutase (SOD), catalase and nonenzymatic antioxidant vitamins e.g. vitamin C, β carotene and vitamin E in either serum or plasma or erythrocytes in 190 cases of age related cataract in the age group of 50–80 years. 190 cases were grouped into three morphological types namely, 73 cases of cortical, 77 cases of posterior subcapsular and 40 cases of nuclear cataract and values of LPO and antioxidants were compared with 78 cases of age matched healthy control groups. Plasma TBARS levels were cataract cases when compared with control groups. There were no significant differences in the erythrocyte levels of catalase and plasma levels of Vit E between cataract cases and control groups. No significant changes of parameters were seen among three different morphological types of age related cataract. The present study shows that the oxidative stress may play an important role in the age related cataract.