Research in Molecular Medicine
Volume:4 Issue: 1, Feb 2016

There are significant challenges associated with qualitative and quantitative nucleic acid tests performed in diagnostic laboratories. The development of internationally available certified reference materials which can be traced to reference measurements will contribute to a better understanding of the performance characteristics of nucleic acid tests and enhance reliability and comparability of clinical data.
Next generation sequencing may have a future role in the identification and resistance detection of clinical pathogens, however, the current complexity of bioinformatics to support this technology makes its routine use in a diagnostic laboratory problematic. However, next generation sequencing is starting to impact epidemiological studies used to investigate the pathways of disease transmission in outbreaks and to determine microbial populations in metagenomics studies.

Hepatic ischemia-reperfusion (I/R) is a common phenomenon during liver surgery, transplantation, infection and trauma which results in damage and necrosis of the hepatic tissue through different pathways. Mechanisms involved in I/R damage are very intricate and cover several aspects. Several factors are involved in I/R-induced damages; briefly, decrease in sinusoidal perfusion and ATP generation because of low or no O2 supply, increase in production of reactive oxygen species (ROS) and inflammatory factors and destruction of parenchymal cells resulted by these molecules are of the main causes of liver tissue injury during reperfusion. Melatonin’s antioxidant effect, and regulatory roles in the expression of different genes in the I/R insulted liver have been investigated by several studies. Melatonin and its metabolites are of the powerful direct scavengers of free radicals and ROS, so it can directly protect liver cell impairment from oxidative stress following I/R. In addition, this bioactive molecule up-regulates anti-oxidant enzyme genes like superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT). Tumor necrosis factors (TNF-α) and interleukin-1 (IL-1), as potent pro-inflammatory factors, are generated in huge amounts during reperfusion. Melatonin is able to alleviate TNF-α generation and has hepatoprotective effect during I/R. It reduces the production of pro-inflammatory cytokines and chemokines via reducing the binding of NF-κB to DNA. Imbalance between vasodilators (nitric oxide, NO) and vasoconstrictors (endothelin, ET) during I/R was shown to be the primary cause of liver microcirculation disturbance. Melatonin helps maintaining the stability of liver circulation and reduces hepatic injury during I/R through preventing alteration of the normal balance between ET and NO. The aim of this review was to explore the mechanisms of liver I/R injuries and the protective effects of melatonin against them.

Lactoferrin (Lf) is a glycoprotein, a member of the transferrin family.From ten known mechanisms of anti-cancer chemoperotecive compounds, Lf alone, has six of these functions and inhibits cancer. In this study, the effect of lactoferrin purified from bovine colostrum was studied as an anti-cancer agent on esophageal cancer cell line.
Bovine colostrum were collected immediately after giving birth. At first, the fat, casein, and some of the milk proteins were removed. Then, lactoferrin was purified using CM-Sephadex-C50 cation exchange chromatography by FPLC system. Purified lactoferrin with 80 kDa molecular weight and 2mg/ml concentration was obtained. Esophageal cancer cell line KYSE-30 and normal cell line HEK were cultured. After appropriate confluency, different concentrations of Lf were added to KYSE-30 and HEK for 20 h and its anti-cancer effect was evaluated by MTT and flow cytometric methods. The maximum concentration inhibitory effect was studied at different times using MTT method.
MTT test determined that 500 µg/ml of lactoferrin reduced cell viability in esophageal cancer cell lines KYSE by 53% and 80% after 20 and 62 hours, respectively, but had no effect on normal cells. Also, flow cytometric analysis determined that lactoferrin was able to induce apoptosis in KYSE-30 cell line.
The isolated lactoferrin from bovine milk showed inhibitory effect on esophageal cancer cell line whereas; it did not have any significant effect on normal cells.

The capability of embryonic carcinoma cells P19 in differentiation to Cardiomyocyte was examined through inducing effects of Oxytocin (OT) and 5-Azacytidin (5Az) individually and compared with each other in laboratory condition.
P19 Embryoid Bodies (EBs) was formed through hanging drops method. Then, EBs were treated with (5Az) or (OT) and the EB medium (Ctrl) until 12 days. Morphology and beating number per minute were recorded every two days. Viability was carried out every three days. The expression of several cardiomyocyte-associated genes was assessed by RT-PCR.
The beating area percentage of EBs in OT treatment groups was more than that of the 5Az group in all days of experiment. However, only in final stage, a significant increase was observed in beating area of OT group. There was no significant difference in viability and morphological changes. OT induction expressed three more specific proteins in cell culture than 5Az.
Statistical analysis revealed that response to OT inducer was more excessive than 5Az in all treatment groups. The Oxytocin was found to be effective inducer of cardiomyocytes differentiation from embryonic carcinoma cells P19 than 5-azacytidine.

The ability of polyclonal antibodies to react with many epitopes of an antigen makes them valuable reagents in research and diagnosis. The aim of this study was purification of mouse IgG2a and production of polyclonal antibody against purified mouse IgG2a subclass.
Mouse IgG2a was purified by ProA affinity. Verification method of the purified antibody was SDS-PAGE and ELISA by a mouse isotyping Kit. Rabbit was immunized with purified IgG2a. The production of antibody in rabbit was investigated by direct ELISA method. Rabbit serum was collected and precipitated at the final concentration of 50% ammonium sulfate. Polyclonal antibody was purified by ion-exchange chromatography and labeled with HRP. The titre and cross reactivity of product was detected by direct ELISA method.
The results of SDS-PAGE in reduced and non-reduced conditions showed bands with 50-KDa, 25-30 KDa MW and a distinct band with 150 KDa MW. Isotype determination showed the presence of mouse IgG2a in related fraction. The titer of Anti-mouse polyclonal antibody was 200000. The optimum titer of prepared HRP conjugated IgG was 4000. Conjugated rabbit IgG has more cross reactivity with mouse IgG2b.
Taking together, affinity chromatography and ion-exchange chromatography are appropriate techniques for purification of mouse IgG subclasses and rabbit IgG, respectively.

Increasing worldwide contamination with hydrocarbons has urged environmental remediation using biological agents such as bacteria. Our goal here was to study the phylogenetic relationship of two crude oil degrader bacteria and investigation of their ability to degrade hydrocarbons.
Phylogenetic relationship of isolates was determined using morphological and biochemical characteristics and 16S rDNA gene sequencing. Optimum conditions of each isolate for crude oil degradation were investigated using one factor in time method. The rate of crude oil degradation by individual and consortium bacteria was assayed via Gas chromatography–mass spectrometry (GC-MS) analysis. Biosurfactant production was measured by Du Noüy ring method using Krüss-K6 tensiometer.
The isolates were identified as Dietzia cinnamea KA1 and Dietzia cinnamea AP and clustered separately, while both are closely related to each other and with other isolates of Dietzia cinnamea. The optimal conditions for D. cinnamea KA1 were 35°C, pH9.0, 510 mM NaCl, and minimal requirement of 46.5 mM NH4Cl and 2.10 mM NaH2PO4. In the case of D. cinnamea AP, the values were 30°C, pH8.0, 170 mM NaCl, and minimal requirement of 55.8 mM NH4Cl and 2.10 mM NaH2PO4, respectively. Gas chromatography – Mass Spectroscopy (GC-MS) analysis showed that both isolates were able to utilize various crude oil compounds, but D. cinnamea KA1 was more efficient individually and consortium of isolates was the most. The isolates were able to grow and produce biosurfactant when cultured in MSM supplemented with crude oil and optimization of MSM conditions lead to increase in biosurfactant production.
To the best of our knowledge this is the first report of synergistic relationship between two strains of D. cinnamea in biodegradation of crude oil components, including poisonous and carcinogenic compound in a short time.

As the third most frequent cause of cancer death, breast cancer is a common disease worldwide. Most of the patients are being diagnosed in the stage that conventional treatments are not effective, and invasion and metastases lead to death. Therefore, identification of novel molecular markers to improve early diagnosis, prognosis and treatment of the breast cancer is a necessity. Zinc finger X-linked (ZFX) gene is a member of ZFY family, which they upregulation has been demonstrated in several types of cancer. The aim of this study was to assess ZFX gene expression in Formalin-fixed, paraffin-embedded (FFPE) tissues of the breast cancer invasive ductal carcinoma and to investigate its correlation with clinicopathological parameters.
A total of 52 tumor and non-tumor breast specimens were evaluated for ZFX gene expression using quantitative real-time RT-PCR. Total RNA extraction was performed using RNeasy FFPE kit (Qiagene). complementary DNA (cDNA) synthesis was performed using PrimeScript-RT Master Mix (Takara). The PCR mixture containing SYBR® Premix Ex Taq ™ II (Takara Bio Inc., Otsu, Japan), was run on the Rotor-gene 3000 (Qiagen, Hilden, Germany)
The ZFX expression increased significantly in breast tumor tissues compared with non-tumor breast tissues. We further showed that there was a positive correlation between the ZFX gene expression level and lymphatic invasion.
ZFX might be used as a potential biomarker to monitor breast carcinoma progression. Further studies to determine the mechanism of action of ZFX is needed to unravel the role of this gene in breast cancer pathogenesis.

Current microscopic experimental methods cannot diagnose DNA damages present in spermatozoa .Therefore, some methods are needed to address the abnormality of the genetic material status on the sperm samples. As reported by many investigators aniline blue staining technique has been used for identifying sperm chromatin condensation. Also, chromomycin A3 is used for evaluation of the degree of protamination of spermatozoa. This study aimed at evaluating these two different staining techniques on human sperm protamine status.
Sperm samples were collected from 72 males [including 37 infertile men: (seven asetenotratospermic, two trato-espermic, and one azo-spermic) and 35 healthy fertile men] attending the research and clinical center for infertility affiliated with Babol University of Medical Sciences. Measurement of sperm motility, volume and density of semen samples were carried out in andrology laboratory. In estimation with light microscopy aniline blue tool, in each slide, blue stained were assumed as normal spermatozoa, but dark blue stained were regarded as abnormal spermatozoa. Bright yellow stained chromomycin-reacted spermatozoa (CMA3) were observed under fluorescent microscope with 460 nm filter considered as normal and yellowish green were assumed as abnormal. Statistical analysis results were expressed as mean ± SD.
The rate of reacted spermatozoa to aniline blue in the infertile group was higher than that of the healthy control group 42.8% ±8.7 vs. 17.9% ±6.4. Also, the rate of reacted spermatozoa to CMA3 in infertile and normal group was [53.6 ± 8.7 and 24.7% ±5.1], respectively.
Infertility status could be assessed by staining the spermatozoa via aniline blue and CMA3 techniques. Combination of these two staining methods had the best predictive values for semen analysis compared to using just one method. Our results showed that both CMA3 and AB staining methods were successful in detecting sperm chromatin defects.