mRNA Ratio Research Articles

MBD2-NuRD but Not MBD3-NuRD Mediates Strong Silencing of Fetal γ-Globin Gene Expression in Human Adult Erythroid Cells

Elevated fetal hemoglobin (HbF) levels are effective in ameliorating the underlying pathophysiologic defects in β-thalassemia and sickle cell anemia. Studies aimed at a full understanding of the mechanisms that enforce silencing of HbF expression in adult erythroid cells offer the promise of targeted molecular therapy. Here we show that depletion of Methyl CpG Binding Domain Protein 2 (MBD2) or disruption of MBD2-Nucleosome Remodeling and Deacetylose Complex (NuRD) interactions results in high levels of HbF in HUDEP-2 cell progenitor derived primary erythroblasts and CD34 + progenitor derived primary adult erythroid cells.We have previously shown that depletion of MBD2 causes a 10-20 fold increase in γ-globin gene expression in adult erythroblasts of β-YAC transgenic mice. To determine the effect of total depletion of MBD2 in human erythroid cells, CRISPR/Cas9 mediated knockout was carried out in HUDEP-2 cells. The absolute level of γ-globin gene expression increased by > 100 fold and the γ/ γ+β mRNA ratio increased from 1-3% in CRISPR/Cas9 scrambled guide control cells to 50-60% in MBD2 knockout HUDEP-2 cells after erythroid differentiation, equivalent to the highest levels reported with knockout of other silencing factors. In contrast CRISPR/Cas9 mediated knockout of Methyl CpG Binding Domain Protein 3 (MBD3) had no significant effect on γ-globin gene expression levels in HUDEP-2 cells after erythroid differentiation.Partial depletion of several strong γ-globin gene silencers, including BCL11A, LRF and KLF-1 has been shown to relieve silencing in CD34+ progenitor derived primary adult erythroid cells, but differences in the differentiation states reported in individual studies make comparisons of the relative effects of these silencer factors difficult. We show here that knockdown of MBD2 by shRNA in CD34+ progenitor derived primary adult erythroid cells consistently increased γ/ γ+β mRNA rations by 7-10 fold across different stages of differentiation without affecting the level of differentiation as compared to shRNA scrambled control cells. MBD2 knockdown led to ~30-40% γ-globin mRNA levels compared to 3%-4% in scrambled shRNA control cells, a level comparable to that typically observed in sickle cell patient reticulocytes. A comparable increase in HbF levels was observed in the MBD2 shRNA knockdown CD34+ progenitor derived erythroid cells compared to scrambled shRNA controls.We have previously shown that MBD2 interacts with the CHD4 chromatin remodeling component of the NuRD complex through a coiled-coil domain, and with the histone deacetylase component of the complex via an intrinsically disordered region (IDR) within MBD2. To test the effects of disrupting these domains in a human adult erythroid cell model, MBD2 knockout HUDEP-2 cells were infected with lentiviral expression vectors containing either wild type MBD2, coiled-coil mutant MBD2, or IDR mutant MBD2. Wild type MBD2 expression resulted in significant silencing of γ-globin gene expression while neither the coiled-coil mutant nor the IDR mutant MBD2 had any silencing effect.These results establish MBD2-NuRD as a major silencer of fetal hemoglobin expression in adult human erythroid cells. Given the minimal phenotype of MBD2 knockout mice, these results suggest that the coiled-coil and IDR domains of the MBD2-NuRD complex are potential targets for therapeutic agents aimed at increasing HbF levels in patients with β-hemoglobinopathies. DisclosuresNo relevant conflicts of interest to declare.

Protective role of ABCA1 in ischemic preconditioning is mediated by downregulation of miR-33-5p and miR-135-5p

Ischemic preconditioning (IPC) significantly reduces ischemia–reperfusion injury in the brain by inducing ischemic tolerance. Although emerging evidence suggests that microRNAs (miRNAs) contribute to the pathogenesis of brain ischemia and IPC-induced neuroprotection, the role of miRNAs and their underlying mechanisms are still unclear. IPC was induced in male C57BL/6 mice by brief bilateral common carotid artery occlusion. After 24 h, mice underwent transient middle cerebral artery occlusion followed by 3 h of reperfusion. Expression levels of messenger RNAs (mRNAs) and proteins were examined in the ipsilateral cortex, and mimics and inhibitors of selective miRNAs were transfected into Neuro-2a cells before oxygen–glucose deprivation (OGD). Post-IPC miRNA expression profiling identified neuroprotection-associated changes in miRNA expression in the ipsilateral cortex after ischemic stroke. Among them, miR-33-5p and miR-135b-5p were significantly downregulated by IPC. Inhibition of miR-33-5p and miR-135b-5p expression protected Neuro-2a cells from OGD-induced apoptosis. Inhibition of these two miRNAs significantly increased mRNA and protein levels of ATP-binding cassette subfamily A member 1 (ABCA1), and a binding assay showed that these two miRNAs showed specificity for Abca1 mRNA. Overexpression of ABCA1 decreased the Bax/Bcl2 mRNA ratio and activation of caspase-9 and caspase-3, whereas knockdown of ABCA1 expression increased the Bax/Bcl2 mRNA ratio and the percentage of Neuro-2a cells with a loss of mitochondrial membrane potential after OGD-treatment. In conclusion, ABCA1 expression is regulated by miR-33-5p and miR-135b-5p. Increased ABCA1 expression following IPC exerts a protective influence against cerebral ischemia via suppression of a mitochondria-dependent apoptosis pathway.

Distinct expression of select and transcriptome-wide isolated 3'UTRs suggests critical roles in development and transition states.

Mature mRNA molecules are expected to be comprised of a 5’UTR, a 3’UTR and a coding region (CDS). Unexpectedly, however, there have been multiple recent reports of widespread differential expression of mRNA 3’UTRs and their cognate coding regions (CDS), reflecting the expression of isolated 3’UTRs (i3’UTRs); these i3’UTRs can be highly expressed, often in reciprocal patterns to their cognate CDS. As with other long non-coding (lncRNAs), isolated 3’UTRs are likely to play an important role in gene regulation, but little is known about the contexts in which they are deployed. To illuminate the functions of i3’UTRs, here we carry out in vitro, in vivo and in silico analyses of differential 3’UTR/CDS mRNA ratio usage across tissues, development and cell state changes both for a select list of developmentally important genes as well as by unbiased transcriptome-wide analyses. Across two developmental paradigms we find a distinct switch from high i3’UTR expression for stem cell related genes in proliferating cells to high CDS for these genes in newly differentiated cells. Unbiased transcriptome analysis across multiple gene sets shows that regardless of tissue, genes with high 3’UTR to CDS ratios belong predominantly to gene ontology categories related to cell-type specific functions. In contrast, the gene ontology categories of genes with low 3’UTR to CDS ratios are similar across tissues and relate to common cellular functions. We further show that, at least for some genes, traditional transcriptional start site genomic elements correspond to identified RNAseq 3’UTR peak regions, suggesting that some i3’UTRs may be generated by de novo transcription. Our results provide critical information from which detailed hypotheses for individual i3’UTRs can be tested, with a common theme that i3’UTRs appear poised to regulate cell-specific gene expression and state.

The impact of lentiviral vector genome size and producer cell genomic to gag-pol mRNA ratios on packaging efficiency and titre

Lentiviral vectors are showing success in the clinic, but producing enough vector to meet the growing demand is a major challenge. Furthermore, next-generation gene therapy vectors encode multiple genes resulting in larger genome sizes, which is reported to reduce titers. A packaging limit has not been defined. The aim of this work was to assess the impact of genome size on the production of lentiviral vectors with an emphasis on producer cell mRNA levels, packaging efficiency, and infectivity measures. Consistent with work by others, vector titers reduced as genome size increased. While genomic infectivity accounted for much of this effect, genome sizes exceeding that of clinical HIV-1 isolates result in low titers due to a combination of both low genomic infectivity and decreased packaging efficiency. Manipulating the relative level of genomic RNA to gag-pol mRNA in the producer cells revealed a direct relationship between producer cell mRNA levels and packaging efficiency yet could not rescue packaging of oversized genomes, implying a de facto packaging defect. However, independent of genome size, an equimolar ratio between wild-type gag-pol mRNA and vector genomic RNA in producer cells was optimal for titer.

LncRNA XIST/miR-137 axis strengthens chemo-resistance and glycolysis of colorectal cancer cells by hindering transformation from PKM2 to PKM1.

Glycolysis was an essential driver of chemo-resistance in colorectal cancer (CRC), albeit with limited molecular explanations. We strived to elucidate the involvement of lncRNA XIST/miR-137/PKM axis in chemo-tolerance and glycolysis of CRC. Altogether 212 pairs of tumor tissues and adjacent normal tissues were collected from CRC patients. Moreover, human CRC epithelial cell lines, including HT29, SW480, SW620 and LoVo, were purchased in advance, and their activity was estimated after transfection of si-XIST or miR-137 mimic. Furthermore, 5-FU/cisplatin-resistance of CRC cells was determined through MTT assay, and glycolytic potential of CRC cells was appraised based on oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). Highly-expressed XIST were predictive of severe symptoms and unfavorable 3-year survival of CRC patients (P< 0.05). Besides, silencing of XIST not only diminished proliferative, migratory and invasive power of CRC cells (P< 0.05), but also enhanced sensitivity of CRC cells responding to 5-FU/cisplatin (P< 0.05). Glycolytic potency of CRC cells was also undermined by si-XIST, with decreased maximal respiration and maximal glycolytic capacity in the si-XIST group as relative to NC group (P< 0.05). Nevertheless, miR-137 mimic attenuated the facilitating effect of pcDNA3.1-XIST on proliferation, migration, invasion, 5-FU/cisplatin-resistance and glycolysis of CRC cells (P< 0.05). Ultimately, ratio of PKM2 mRNA and PKM1 mRNA, despite being up-regulated by pcDNA3.1-XIST, was markedly lowered when miR-137 mimic was co-transfected (P< 0.05). LncRNA XIST/miR-137 axis reinforced glycolysis and chemo-tolerance of CRC by elevating PKM2/PKM1 ratio, providing an alternative to boost chemo-therapeutic efficacy of CRC patients.

MRNA expression analysis confirms CD44 splicing impairment in systemic lupus erythematosus patients.

Systemic Lupus Erythematosus (SLE) is a complex chronic autoimmune disease characterized by several immunological alterations. T cells have a peculiar role in SLE pathogenesis, moving from the bloodstream to the peripheral tissues, causing organ damage. This process is possible for their increased adherence and migration capacity mediated by adhesion molecules, such as CD44. Ten different variant isoforms of this molecule have been described, and two of them, CD44v3 and CD44v6 have been found to be increased on SLE T cells compared to healthy controls, being proposed as biomarkers of disease and disease activity. The process of alternative splicing of CD44 transcripts is not fully understood. We investigated the mRNA expression of CD44v3 and CD44v6 and also analyzed possible CD44 splicing regulators (ESRP1 molecule and rs9666607 CD44 polymorphism) in a cohort of SLE patients compared to healthy controls. This study involved 18 SLE patients and 18 healthy controls. Total RNA and DNA were extracted by peripheral blood mononuclear cells. The expression study was conducted by quantitative RT-polymerase chain reaction, using SYBR Green protocol. Genotyping of rs9666607 SNP was performed by direct sequencing. CD44v6 mRNA expression was higher in SLE patients compared to healthy controls (p = 0.028). CD44v3/v6 mRNA ratio in healthy controls was strongly unbalanced towards isoform v3 compared to SLE patients (p = 0.002) and decreased progressively from healthy controls to the SLE patients in remission and those with active disease (p = 0.015). The expression levels of CD44v3 and CD44v6 mRNA correlated with the disease duration (p = 0.038, Pearson r = 0.493 and p = 0.038, Pearson r = 0.495, respectively). Splicing regulator ESRP1 expression positively correlated with CD44v6 expression in healthy controls (p = 0.02, Pearson r = 0.532) but not in SLE patients. The variant A allele of rs9666607 of CD44 was associated with higher level of global CD44 mRNA (p = 0.04) but not with the variant isoforms. In SLE patients, the increase in CD44v6 protein correlates with a higher transcript level of this isoform, confirming an impairment of CD44 splicing in the disease, whose regulatory mechanisms require further investigation.

Protective effect of fermented Diospyros lotus L. extracts against the high glucose-induced apoptosis of MIN6 cells.

Date plum persimmon (Diospyros lotus L.) is a fruit crop from the Ebenaceae family. Its microorganism-fermented extract (DPEML) was shown to exhibit a hypoglycemic effect in our previous work. Here, we investigated the effects of DPEML fermented by Microbacterium flavum YM18-098 and Lactobacillus plantarum B7 on the high glucose-induced apoptosis of MIN6 cells and explored its potential cell protective mechanisms. DPEML ameliorated the apoptosis of MIN6 cells cultured under high glucose conditions, thereby improving cell viability. DPEML upregulated the Bcl-2/Bax mRNA ratio to obstruct an intrinsic apoptotic pathway and concomitantly downregulated the expression of the apoptosis-linked proteins, AIF, and Cyt-C, in high glucose-induced MIN6 cells. Furthermore, DPEML promoted the insulin secretion of MIN6 cells grown under chronically high-glucose conditions by upregulating Ins mRNA expression. In summary, our study suggested that DPEML is a promising functional food for the development of therapeutics for the treatment of Type 2 diabetes mellitus. PRACTICAL APPLICATIONS: We investigated the effects of DPEML fermented by Microbacterium flavum YM18-098 and Lactobacillus plantarum B7 on the high glucose-induced apoptosis of MIN6 cells and explored its potential cell protective mechanisms. DPEML ameliorated the apoptosis of MIN6 cells cultured under high glucose conditions, thereby improving cell viability. DPEML upregulated the Bcl-2/Bax mRNA ratio to obstruct an intrinsic apoptotic pathway and concomitantly downregulated the expression of the apoptosis-linked proteins, AIF and Cyt-C, in high glucose-induced MIN6 cells. Furthermore, DPEML promoted the insulin secretion of MIN6 cells grown under chronically high-glucose conditions by upregulating Ins mRNA expression. We suggested that DPEML is a promising functional food for the development of therapeutics for the treatment of Type 2 diabetes mellitus.

Thermodynamic and anticancer properties of inorganic zinc oxide nanoparticles synthesized through co-precipitation method

In this paper, inorganic zinc oxide nanoparticles (ZnO NPs) were synthesized through co-precipitation method and after evaluating their physicochemical properties by using TEM and XRD techniques, the bio-thermodynamic and structural parameters upon interaction of ZnO NPs with human serum albumin (HSA) were determined by using fluorescence, UV–vis, CD and molecular docking studies. Also, the selective anticancer effects of ZnO NPs on leukemia (K562) cells were explored by using different cellular and molecular assays. It was determined that the crystalline structure of ZnO NPs has a diameter of about 30 nm. Thermodynamic parameters and docking analysis indicated that the hydrophobic forces mediate the formation of static complex between ZnO NPs and HSA molecules. UV–vis and CD spectroscopic methods showed that the melting temperature (Tm) and secondary structure of HSA, respectively remain substantially unchanged by increase of ZnO NPs. Anticancer assays demonstrated that ZnO NPs trigger an inhibitory impact on the proliferation of K562 cells while being safe against lymphocyte normal cells. Exposure of K562 cells to ZnO NPs resulted in membrane leakage, activation of caspase-9, -3, overexpression of Bax/Bcl-2 mRNA ratio, increase in the level of ROS, cell cycle arrest, and apoptosis/necrosis. In conclusion, it may be suggested that ZnO NPs can be utilized as potential agents in the development of anticancer platforms.

Bsep expression\xa0in hilar cholangiocarcinoma of rat model

Develop a rat model of hilar cholangiocarcinoma for detecting bile salt export pump (Bsep) expression in hilar cholangiocarcinoma tissues, in order to provide a new therapeutic target for the gene therapy of hilar cholangiocarcinoma. Sixty male Wistar rats (body weight, 190 ± 8 g) were randomly divided into three groups (the experimental group, the control group and the sham operation group, n = 20 each) as follows: The three groups were fed a standard diet, the experimental group was injected by cholangiocarcinoma QBC939 cell suspension along the hilar bile duct into the bile duct bifurcation with microsyringe, the control group was injected by normal saline, the sham operation group did not inject anything. Every day assess the rats’ mental state, diet, and motion by using Basso–Beattie–Bresnahan and combined behavioral score. At 4 weeks, one rat of the experimental group was sacrificed after it was administered anesthesia, and we recorded changes in hilar bile duct size, texture, and form. This procedure was repeated at 6 weeks. After 6 weeks, hilar cholangiocarcinoma developed only in the experimental group, thereby establishing an experimental model for studying QBC939-induced hilar cholangiocarcinoma. Tumor formation was confirmed by pathological examination, and hilar bile duct tissues were harvested from both the groups. A real-time polymerase chain reaction assay and an immunohistochemical assay were used to analyze the expression of Bsep in hilar bile duct tissues of each group. From the second week, the rats in experimental group began to eat less, and their body mass decreased compared with control group and sham operation group. After 6 weeks, we detected hilar cholangiocarcinoma in the hilar bile duct tissues of 18 rats (90%) in the experimental group. In the experimental group with hilar cholangiocarcinoma, we found that the levels of total cholesterol, total bilirubin, and direct bilirubin were higher compared with those in the control group and sham operation group. Simultaneously, muddy stones emerged from the bile ducts of rats in the experimental group. The Bsep/Gapdh mRNA ratio in hilar cholangiocarcinoma, control group and sham operation group differed markedly. Light microscopy revealed a granular pattern of Bsep protein expression which reacted with the anti-Bsep antibody. Each section was randomly divided into six regions, with 80 cells were observed in every region. Sections with > 10% positive cells were designated positive, Sections with < 10% positive cells were designated negative. Each group included 4800 cells. In the experimental group, 1200 cells (25%) were positive, in the control group, 3648 cells (76%) were positive and in the sham operation group 3598 cells (75%) were positive, and this difference was statistically significant. Bsep expression significantly decreased in hilar cholangiocarcinoma of rats than those in control group and sham operation group, suggesting that drugs targeting Bsep are a new strategy for hilar cholangiocarcinoma.

374 Thioredoxin, thioredoxin interacting protein and galectin-3 in preeclampsia

Preeclampsia (PE) is a pregnancy-related syndrome characterized by the onset of hypertension and proteinuria and may lead to end-organ dysfunction. Thioredoxin (TXN), acts as antioxidant enzyme in several cellular processes, regulating inflammation, promoting cell proliferation and inhibiting apoptosis. TXNIP is an endogenous inhibitor of TXN. Galectin-3 involves in cell growth, differentiation, inflammation, and fibrosis. We evaluated changes in the inflammatory response of TXNIP, TXN, and Galectin-3 at the level of maternal serum, placenta, and umbilical blood of women with preeclampsia, compared to normal pregnancies (NP). 10 women with PE and 15 with NP were recruited during admission for delivery. Blood samples were obtained from parturients and umbilical cords, as well as placental tissue for mRNA and protein extraction. We evaluated changes in the inflammatory response of TXNIP, TXN, and Galectin-3 at the level of maternal serum, placenta, and umbilical blood of women with preeclampsia, compared to normal pregnancies (NP). TXNIP and Gal-3 mRNA expression were higher in PE maternal serum mRNA compared to NP. TXNIP and Gal-3 mRNA expression were lower in PE placentas and cord blood compared to PE maternal serum mRNA expression. TXN/TXNIP mRNA ratio had high absolute values in placental and cord blood (2.06 and 1.06, respectively), compared to maternal ratio (0.304). TXN/TXNIP placental protein ratio had similar values between PE and NP (1.04 and 1.01). Gal-3 protein was higher in PE placentas compare to NP (3.79±0.66; P<0.0001). We revealed that patients with preeclmapsia had Pro-inflammatory changes that are expressed by high Gal-3 expression and a low TXN/TXNIP mRNA ratio in maternal blood of women with PE, but not in their placental and umbilical cord blood samples. These findings imply that the placenta may have a role in protecting the fetus from the damages of oxidation and the inflammatory response, which are more common in preeclampsia than in normal pregnancies.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Protamine 1 and 2 mRNA Abundance in Human Spermatozoa and Its Relation to Semen Quality and Sperm DNA Fragmentation among Fertility Clinic Patients

Sperm contain a complex population of coding and noncoding RNA, and the utility of sperm RNA in fertility research is currently being explored. The aim of this study was to estimate the content of transcripts of protamines 1 and 2 in human ejaculated spermatozoa in relation to semen quality and sperm DNA fragmentation between fertile and infertile patients. Human ejaculates were obtained from 33 patients and semen analyzes were assessed by WHO criteria (2010). We evaluated the sperm DNA fragmentation measured by TUNEL assay. The ejaculates of patients were purified by density-gradient centrifugation, sperm cells were lysed, and mRNA was extracted, reverse transcribed, and subjected to real-time qPCR using specific primer pairs for protamine-1, protamine-2 RNA. The sperm protamine mRNA ratio in normozoospermic men (n = 19; 2.86 ± 0.16) differed significantly from that of patozoospermic patients (n = 14; 3.43 ± 0.22; p < 0.05). A significant correlation was shown between sperm DNA fragmentation and the PRM2/PRM1 mRNA ratio (r = 0.33; p < 0.05). In the group of patients with an increased sperm DNA fragmentation (n = 14; 2.74 ± 0.18), the PRM2/PRM1 ratio was significantly higher than in the group of patients with normal rates (n = 19; 3.26 ± 0.19; p < 0.05). An abnormal sperm protamine ratio was associated with poor semen quality and DNA fragmentation. Finding sperm-quality markers would help to understand the causes of male infertility and to improve male reproductive health.

Clonal Expansion of Tumor-Infiltrating T Cells and Analysis of the Tumor Microenvironment within Esophageal Squamous Cell Carcinoma Relapsed after Definitive Chemoradiation Therapy.

(1) Background: Comparable prognoses after definitive chemoradiation therapy (CRT) to surgery alone for esophageal squamous cell carcinoma (ESCC) have been previously reported; however, no robust prognostic markers have been established. The clonality of tumor-infiltrating lymphocytes (TILs) and tumor microenvironments (TMEs) in ESCC relapsed after CRT were examined to explore prognostic markers. (2) Methods: Clonality of TIL and TME were examined in ESCC with and without preceding CRT, as well as oral squamous cell carcinoma (OSCC) and healthy volunteers as controls. The clonality of TIL was assessed by T-cell receptor (TCR) α and β repertoire analyses and evaluated by diversity indices. The TME was assessed by quantitative polymerase chain reaction evaluating PD-L1 and CD8B. (3) Results: The clonal expansion of TIL was significantly induced within ESCCs and OSCCs, when compared to healthy volunteers, and was mostly induced within ESCCs after definitive CRT. Diversity indices of TIL were not associated with the prognosis, but the ratio of PD-L1 mRNA to CD8B mRNA in TME was significantly associated with a poor prognosis after salvage surgery (p = 0.007). (4) Conclusions: The clonal expansion of TIL is induced after definitive CRT for ESCC, and the ratio of PD-L1 mRNA to CD8B mRNA within tumor tissues is a prognostic marker candidate for salvage esophagectomy after CRT.

Tobacco smoking and its impact on the expression level of sperm nuclear protein genes: H2BFWT, TNP1, TNP2, PRM1 and PRM2.

The aim of this current study was to investigate the influence of tobacco smoke on sperm quality determined by standard parameters, on sperm DNA maturity tested by chromomycin A3 (CMA3) staining, on sperm DNA fragmentation tested by TUNEL assay and on the transcript level of sperm nuclear proteins H2BFWT, PRM1, PRM2, TNP1 and TNP2 genes quantified by RT-PCR. One hundred forty-one (141) sperm samples (43 nonsmokers (G.1) and 98 heavy smokers (G.2)) of couples undergoing ICSI were enrolled in this study. In G2, a significant decrease in standard semen parameters in comparison with nonsmokers was shown (p<.01). In contrast, protamine deficiency (CMA3 positivity) and sperm DNA fragmentation (sDF) were significantly higher in G2 than in G1 (p<.01). Furthermore, the studied genes were differentially expressed (p<.01), down-regulated in the spermatozoa of G.2 compared to that of G.1 (fold change <0.5) and were significantly correlated between each other (p<.01). Moreover, in comparison with G1, the protamine mRNA ratio in G2 was significantly higher (p<.01). It can therefore be concluded that smoking alters mRNA expression levels of H2BFWT, TNP1, TNP2, PRM1 and PRM2 genes and the protamine mRNA ratio and consequently alters normal sperm function.

Protamine 1/Protamine 2 mRNA ratio in nonobstructive azoospermic patients.

Altered protamine 1 (PRM1)/ protamine 2 (PRM2) mRNA ratio in testicular biopsy samples correlates with sperm quality and its fertilising ability. This study is planned to assess PRM1/ PRM2 mRNA ratio in subgroups of azoospermia to suggest a more reliable and accurate marker for assessing sperm quality in nonobstructive azoospermia (NOA). A cross-sectional study was done on testicular biopsy samples, taken from 106 azoospermic patients. Samples were histologically classified into subgroups: 36 obstructive azoospermia (OA), and two groups of NOA: 41 round spermatid maturation arrest (SMA) and 29 Sertoli cell-only syndrome (SCOS). OA samples showed histologically normal spermatogenesis and serve as a positive control. mRNA expression of jumonji domain-containing 1A (JMJD1A), PRM1, PRM2 and transition nuclear proteins (TNP1, TNP2) genes was determined, by RT-qPCR. Significantly lower expression of JMJD1A (p<.001), PRM1 (p=.0265) and PRM2 (p=.0032) has been seen in the SCOS group of NOA. We found significant (p<.001) increase in PRM1/PRM2 mRNA ratio in testicular biopsy samples of SCOS group of NOA patients and significant negative correlation of PRM1/PRM2 mRNA ratio with JMJD1A. Hence, PRM1/PRM2 mRNA ratio may represent a more reliable and accurate marker to assess sperm quality in NOA in addition to standard semen parameters.

Relationships between the Bone Expression of Alzheimer's Disease-Related Genes, Bone Remodelling Genes and Cortical Bone Structure in Neck of Femur Fracture.

Neck of femur (NOF) fracture is a prevalent fracture type amongst the ageing and osteoporotic populations, commonly requiring total hip replacement (THR) surgery. Increased fracture risk has also been associated with Alzheimer's disease (AD) in the aged. Here, we sought to identify possible relationships between the pathologies of osteoporosis and dementia by analysing bone expression of neurotropic or dementia-related genes in patients undergoing THR surgery for NOF fracture. Femoral bone samples from 66 NOF patients were examined for expression of the neurotropic genes amyloid precursor protein (APP), APP-like protein-2 (APLP2), Beta-Secretase Cleaving Enzyme-1 (BACE1) and nerve growth factor (NGF). Relationships were examined between the expression of these and of bone regulatory genes, systemic factors and bone structural parameters ascertained from plain radiographs. We found strong relative levels of expression and positive correlations between APP, APLP2, BACE1 and NGF levels in NOF bone. Significant correlations were found between APP, APLP2, BACE1 mRNA levels and bone remodelling genes TRAP, RANKL, and the RANKL:OPG mRNA ratio, indicative of potential functional relationships at the time of fracture. Analysis of the whole cohort, as well as non-dementia (n = 53) and dementia (n = 13) subgroups, revealed structural relationships between APP and APLP2 mRNA expression and lateral femoral cortical thickness. These findings suggest that osteoporosis and AD may share common molecular pathways of disease progression, perhaps explaining the common risk factors associated with these diseases. The observation of a potential pathologic role for AD-related genes in bone may also provide alternative treatment strategies for osteoporosis and fracture prevention.

FLT3-ITD DNA allelic burden, but not mRNA levels, influences the biological characteristics of AML patients

Abstract FMS-like tyrosine kinase 3 gene internal tandem (FLT3-ITD) mutations represent one of the most frequent genetic lesions in acute myeloid leukemia (AML) and imparts a negative prognostic. For an optimal patient management, current clinical guidelines recommend the evaluation of the allelic ratio (AR), expressed as the DNA FLT3-ITD/WT mutational burden. We sought to evaluate the differences between the AR and FLT3-ITD/WT mRNA ratio (RR) and their respective impact on the biological characteristics of AML patients. A total of 32 DNA and mRNA samples from AML patients with FLT3-ITD were evaluated. There was a good correlation between the AR and RR (Spearman’s rho= 0.652, P &lt;0.001). None of the biological characteristics were influenced by the RR values, whereas patients with high AR values (≥0.5) had higher WBC counts (Mann-Whitney, P= 0.01), LDH levels (Mann-Whitney, P= 0.037), and circulating blasts levels (Mann-Whitney, P= 0.023) than patients with low AR values (&lt;0.5). Also, there was a good correlation between AR values and WBC count (Spearman’s correlation, P= 0.001), and LDH levels (Spearman’s correlation, P= 0.007). In our study population the AR, but not the RR, influenced the biological characteristic of patients suggesting a dose-independent effect of FLT3-ITD mutations.

Leptin regulates OPG and RANKL expression in Gingival Fibroblasts and Tissues of Chronic Periodontitis Patients.

Objective: Chronic periodontitis is a bone-destructive disease affecting periodontal support structures. Although leptin has a protective effect against periodontitis, the underlying mechanism remains unclear. Therefore, this study aimed to investigate the possible role of leptin by examining its relationship with OPG and RANKL in human gingival tissues obtained from patients with chronic periodontitis.Method: Twenty-two patients with chronic periodontitis were enrolled (10 with moderate periodontitis and 12 with severe periodontitis) in the experimental group, and 12 healthy individuals were enrolled in the control group. Gingival tissue samples were collected, and the protein levels and localization of leptin, OPG, and RANKL were studied using immunohistochemistry (IHC). The staining intensities of leptin, OPG, and RANKL were correlated with the periodontal clinical index. Moreover, real-time quantitative PCR (RT-qPCR) was used to determine OPG and RANKL mRNA levels in gingival fibroblasts stimulated with gradient concentrations of leptin protein in vitro.Result: Leptin, OPG, and RANKL were located in the cytoplasm of gingival epithelial cells and the connective tissue. Leptin was widely and significantly expressed in the control group, whereas it was lightly stained in the severe group. RANKL was lightly stained in the control group, whereas it was widely and significantly expressed in the severe group. The control and the moderate groups had similar OPG levels, which were significantly higher than that in the severe group. Leptin was positively correlated with OPG(r = 0.905, p < 0.01) and negatively correlated with RANKL (r = -0.635, p < 0.01). In vitro low concentrations of leptin led to an increased OPG/RANKL mRNA ratio, whereas the opposite effect was observed at high concentrations.Conclusion: Leptin can regulate OPG and RANKL expression in gingival fibroblasts and may thus play a role in the development of chronic periodontitis by modulating the OPG/RANKL ratio.

Elucidating the interaction of propofol as an intravenous anesthetic drug with blood components: IgG and peripheral blood mononuclear cell as targets

Propofol is known as an intravenous anesthetic drug utilized for stimulation and prolongation of general anesthesia. However, the side effects of propofol are still elusive in the blood system. Therefore, in this study the interaction of propofol with immunoglobulin G (IgG) and peripheral blood mononuclear cells (PBMCs) as a model of blood system was explored by different spectroscopic, theoretical, cellular and molecular assays. The UV–Vis, intrinsic/ANS/synchronous fluorescence spectroscopic studies indicated the static quenching of IgG and corresponding protein unfolding in the presence of propofol. Far- and near-circular dichroism (CD) study further demonstrated that thestructure of IgG was unfolded after interaction with propofol. Molecular docking study indicated that formation of IgG-propofol complex occurs through involvement of hydrogen bonding and van der Waals forces. Moreover, the cellular assay determined that propofol led to an increase in the cell mortality, membrane leakage, upregulation of intracellular ROS, elevation of Caspase-3 and -9 protein levels, and overexpression of Bax/Bcl-2 mRNA ratio, in PBMCs. This paper may pave the way for exploring the nature of the reaction and side effects between propofol and the biological system.

Fabrication of inorganic alumina particles at nanoscale by a pulsed laser ablation technique in liquid and exploring their protein binding, anticancer and antipathogenic activities

The interaction of nanoparticles with biological systems can provide useful information about their therapeutic applications. The aluminum nanoparticles (Al2O3 NPs) were synthesized by laser ablation technique and well-characterized by different methods. Fluorescence spectroscopy, circular dichroism (CD) spectroscopy, and molecular docking studies were employed to evaluate the effect of Al2O3 NPs on the protein structure. Growth inhibitory and apoptotic effects of the Al2O3 NPs against K562 cancer cells and lymphocyte cells were assessed using [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT), flow cytometry, and real time polymerase chain reaction (PCR) assays. The antipathogenic activity of Al2O3 NPs against a diverse range of Gram-negative and Gram-positive pathogens was explored through a disk diffusion method. The characterization techniques determined that the Al2O3 NPs were successfully synthesized in the nanoscales. Intrinsic, 1-anilino-8-naphthalenesulfonate (ANS) and acrylamide fluorescence spectroscopy studies disclosed that Al2O3 NPs can partially change the tertiary structure of human serum albumin (HSA), whereas CD spectroscopy investigation depicted that the secondary structure of HSA remained intact. Molecular docking investigation also manifest that the Al2O3 nano-clusters preferably bind to electrostatic residues. Al2O3 NPs exhibited promising and selective anticancer features through reactive oxygen species (ROS) production, apoptosis induction, and elevation of Bax/Bcl-2 mRNA ratio. Furthermore, the Al2O3 NP showed a remarkable antibacterial activity against both Gram-negative and Gram-positive pathogens. In conclusion, it may be suggested that the synthesized Al2O3 NPs can be integrated in the development of anticancer and antipathogenic agents.

Relative expression of KLK5 to LEKTI is associated with aggressiveness of oral squamous cell carcinoma

BackgroundOral squamous cell carcinoma (OSCC) remains a challenging cancer to treat despite all the advances of the last 50 years. Kallikrein 5 (KLK5) is among the serine proteases implicated in OSCC development. However, whether the activity of KLK5 promotes carcinogenesis is still controversial. Moreover, knowledge regarding the role of the KLK5 cognate inhibitor, Lympho-Epithelial Kazal-Type related Inhibitor (LEKTI), in OSCC is scarce. We have, thus, sought to investigate the importance of KLK5 and LEKTI expression in premalignant and malignant lesions of the oral cavity. MethodsKLK5 and LEKTI protein expression was evaluated in 301 human samples, which were comprised of non-malignant and malignant lesions of the oral cavity. Moreover, a bioinformatic analysis of the overall survival rate from 517 head and neck squamous cell carcinoma (HNSCC) samples was performed. Additionally, to mimic the uncovered KLK5 to serine peptidase inhibitor (SPINK5) imbalance, the KLK5 gene was abrogated in an OSCC cell line using CRISPR-Cas9 technology. The generated cell line was then used for in vivo and in vitro carcinogenesis related experiments. ResultsLEKTI was found to be statistically downregulated in OSCCs, with increased KLK5/SPINK5 mRNA ratio being associated with a shorter overall survival (p = 0.091). Indeed, disruption of KLK5 to SPINK5 balance through the generation of KLK5 null OSCC cells led to smaller xenografted tumors and statistically decreased proliferation rates following multiple time points of BrdU treatment in vitro. ConclusionThe association of increased enzyme/inhibitor ratio with poor prognosis indicates KLK5 to SPINK5 relative expression as an important prognostic marker in OSCC.