keivan majidzadeh

Several types of cancer can be detected early through thermography, which uses thermal profiles to image tissues in recent years, thermography has gained increasing attention due to its non-invasive and radiation-free nature. There is a growing need for thermographic images of breast cancer lesions in different nationalities and ages to develop this technique, however. This study aims to introduce a dataset of breast thermograms. In this study, thermographic images of breast cancer from Iranian samples were prepared and confirmed due to the limited number of breast thermogram databases.  The prepared database was tested using artificial intelligence and another well-known DMR database (Database for Mastology Research) in this study to determine its reliability. A variety of deep learning architectures and transfer learning are used to evaluate these databases for accuracy, sensitivity, speed, training compliance, and validation compliance. According to best-fitted structures for both types of databases, the database obtained from this study has a quality comparable to the DMR reference database, with minimum accuracy, sensitivity, specificity, precision, and F-score of 80%, 86%, 86%, 88%, and 87%, respectively. Using thermography as a method of early breast screening is demonstrated to be effective. In comparison to DMR, the lower statistics of the proposed database (between 2 and 7 percent) indicates that more diverse breast thermograms should be captured in conjunction with improvements to imaging equipment as well as adherence to thermography recording protocols in order to improve the reliability and efficiency of the database.

Liquid biopsy is a novel diagnostic tool that investigates biological biomarkers within the blood and other body liquids in order to provide information about the genetics of the tumor and its treatment response. The current review study aimed to highlight the role of liquid biopsy in breast cancer and precision medicine.

In the current review study, we attempted to evaluate the recent innovations in breast cancer diagnosis by investigating liquid biopsy biomarkers in related databases, including Scopus, Web of Science, PubMed, and Google Scholar, from 2018 to 2024.

The assessment of the published articles in this field revealed that the applications of biomarkers in liquid biopsy have a significant role in personalized medicines, highlighting their reliability for use in personalized medicine.  

The liquid biopsy biomarkers seem to be a promising approach in breast cancer early detection and remarkable reduction of mortality caused by this disease in the near future.

 The Salmonella origin diseases is wide which could be from gastroenteritis, enteric fever, bacteraemia and focal infection. The infection of salmonella usually comes from the consumption of the contaminated food or water with animal or human excrement. This study aimed to design a Quantitative-LAMP method to real-time detection of Salmonella enterica serovar Typhi (S. Typhi), the causative agent of Typhoid fever.

A new LAMP primer set specifically was designed based on ViaB gene and used for detection of S. Typhi. To evaluate the analytical specificity, the genome of some Salmonella-related and non-related bacteria were subjected to the S. Typhi LAMP assay. Also, the analytical sensitivity or limit of detection of the assay was evaluated. Furthermore, in the experiment, turbidity in tubes was assessed by a turbidimeter and then a standard curve was depicted by plotting time threshold values against the log of ViaB gene copy number. With this standard curve, we could use the method to make a quantitative.

The Salmonella Typhi LAMP assay specifically assessed the ViaB gene. The analytical sensitivity of the assay when using agarose gel electrophoresis or amplification plot obtained from Loop amp real-time turbidimeter system was 0.28 fg whereas with direct observation of fluorescent color change to evaluate amplification was 2.8 fg. So, the lower limit of detection of the assay when applying the revealing methods was ~1 and 8 copies of the ViaB gene respectively. 

The Salmonella Typhi LAMP assay is a simple and accurate tool to detect the causative agent of Typhoid fever that may designate to apply in clinical laboratories

Yersinia pestis, a gram-negative rod belonging to the Enterobacteriaceae family, is the causative agent of plague. Classical methods of detecting the organisms are time-consuming, expensive and dangerous. The aim of the study was to design a Real-time PCR assay on the basis of the pla gene of Yersinia pestis. In this research the Real- time PCR test was optimized by using special primers for targeting pla gene. After preparing 10-fold serial dilutions of the pla and their analysis by the assay, the last dilution showing a fluorescent signal was confirmed as the limit of detection (LOD). A standard curve based on the Ct values was depicted, so the assay was developed to quantify the target gene. The analytical specificity was determined by subjecting the genome of some control negative bacteria to the assay. In this experiment, negative control genomes did not show detectable signals in the assay. The last dilution of pla plasmid which showed a fluorescent signal was 4.5 fg. So, the lower detectable copy numbers of the gene in a 20 μl PCR reaction was calculated as 1×103.

Recent advances in DNA sequencing techniques have led to an increase in the identification of single nucleotide polymorphisms (SNPs) in BRCA1 and BRCA2 genes, but no further information regarding the deleterious probability of many of them is available (Variants of Unknown Significance/VUS). As a result, in the current study, different sequence- and structure-based computational tools including SIFT, PolyPhen2, PANTHER, SNPs&GO, FATHMM, SNAP, PhD-SNP, Align-GVGD, and I-Mutant were utilized for determining how resulted BRCA protein is affected by corresponding missense mutations. FoldX was used to estimate mutational effects on the structural stabilityof BRCA proteins. Variants were considered extremely deleterious only when all tools predicted them to be deleterious. A total of 10 VUSs in BRCA1 (Cys39Ser, Cys64Gly, Phe861Cys, Arg1699Pro, Trp1718Cys, Phe1761Ser, Gly1788Asp, Val1804Gly, Trp1837Gly, and Trp1837Cys) and 12 in BRCA2 (Leu2510Pro, Asp2611Gly, Tyr2660Asp, Leu2686Pro, Leu2688Pro, Tyr2726Cys, Leu2792Pro, Gly2812Glu, Gly2813Glu, Arg2842Cys, Asp3073Gly, and Gly3076Val) were considered as extremely deleterious. Results suggested that deleterious variants were mostly enriched in theN- and C-terminal domain of the BRCA1 and BRCA2 C-terminus. Utilizing evolutionary conservation analysis, we demonstrated that the majority of deleterious SNPs ensue in highly conserved regions of BRCA genes. Furthermore, utilizing FoldX, we demonstrated that alterations in the function of proteins are not always together with stability alterations.

Mutations in the BRCA1 gene are known to be a major cause of hereditary breast cancer. However, characterizing the point mutationsassociated with cancer in BRCA1 is challenging because the functional impact of most of them is still unknown. Nowadays, a variety of methods are employed to identify cancer-associated mutations in BRCA1. This study is aimed to assess the functional effects of two mutations, Asp1733Gly and Val1714Gly, using a combination of in silico tools and yeast functional transcription activator assay. Our computational analysis showed that theVal1714Gly mutation was deleterious, while the other one, Asp1733Gly, predicted as neutral. Also using yeast functional transcription activator assay, we found that the Asp1733Gly mutation displayed similar ability with positive controls. In contrast, the Val1714Gly mutation completely abrogated transcriptional activity in the yeast. These results suggested that Val1714Gly and Asp1733Gly can be classified as pathogenic and benign mutations for the BRCA1, respectively.

Loop-mediated isothermal amplification (LAMP) is a cost-effective, rapid, and specific method for the detection of bacterial pathogens within a sample. We designed and evaluated a new quantitative LAMP primer set specific to the ctxB gene for the rapid measurement of the load of toxigenic V. cholerae DNA in the reaction. Unlike the previously reported LAMP assays for V. cholerae detection, our LAMP primer set was able to detect and quantify toxigenic V. cholerae DNA with a detection limit of 2.8 pg of the ctxB gene equivalent to 8.3 copies of genomic DNA per reaction. Our LAMP assay was found to be highly specific with no amplification detected in non-toxigenic V. cholerae bacterial strains. Thus, the ctxB-based LAMP assay developed in this study can serve as a simple, sensitive, specific, and quantitative molecular tool for the monitoring and epidemiological study of cholera. Overall, the low cost and simplicity of the LAMP assay can make it a preferable molecular method in the detection of pathogenic organisms.

Mycoplasmas are major contaminants of cell culture and affect in vitro biological and diagnostic tests. Mycoplasma detection is conducted using culture and molecular methods. These methods vary in terms of accuracy, reliably and sensitivity. Loop-mediated isothermal amplification (LAMP) is used to amplify target DNA in a highly specific and rapid manner. This study aimed to develop a LAMP method for rapid detection of Mycoplasma in culture samples. In this descriptive laboratory study, for LAMP detection of Mycoplasma contaminations in cell culture, we used primers specifically designed for targeting the 16S rRNA conserved gene of Mycoplasma spp. For a positive control structure, 16S rRNA amplified based on PCR, was cloned in a plasmid vector and sequenced. The assay specificity was evaluated using Mycoplasma genomic DNA and a panel containing genomes of gram-positive and gram-negative organisms. In this study, the method developed for detection of Mycoplasma contamination of cell cultures was a rapid, sensitive and cost-effective LAMP approach. The results demonstrated that this method benefits from high specificity (100%) for amplification of Mycoplasma strains and high speed (multiplication within 60 minutes), while it does not require expensive laboratory equipment compared to those needed for polymerase chain reaction (PCR)-based detection. Our study is the first report about application of LAMP assay based on 16S rRNA gene for detection of Mycoplasma strains; this technique could be considered a useful tool for rapid detection of contamination of cell culture.

Mycoplasmas are one of the most serious contaminants of cellular cultures and their biological productions. Mycoplasma diagnosis is conducted on the basis of culture and molecular methods. These methods are different from each other in terms of accuracy, reliability, and sensitivity. This study aimed to trace the mycoplasma contaminations in culture samples using 16S rRNA specific primers based on the PCR method.
Methods and Materials: 16S rRNA gene sequence of cell culture contaminant mycoplasmas were extracted from gen bank and were sorted using gene sequence pattern. Specific primers were designed using obtained synonymies. To build positive control structure, 16S rRNA gene proliferated based on the PCR methods then cloned in a plasmid vector.
Electrophorus of PCR product of 16S rRNA revealed a band of 219bp length on Agarose gel. This pattern was not observed in negative control samples. Moreover, the fragment proliferation with the same size in recombinant plasmid confirmed the authenticity of cloning.
Discussion and Due to its high speed and accuracy, the method has potential for using in cell culture laboratories.

On behalf of the editorial and executive board members, it is my great pleasure to present the Multidisciplinary
Cancer Investigation Journal. The rapid growth of medical sciences has necessitated the development of platforms to distribute and publish the results of research projects, and declare new discoveries.
Among all medical disciplines, cancer is the one that involves the largest number of scientists, and is counted among the most important areas of research and discovery, for a number of reasons, including: 1. Its high impact on public health. Globally, cancer is identified among the leading causes of morbidity and mortality. Recent statistics have shown some 14.1 million new cancer cases and 8.2 million cancer deaths annually, while some 32.6 million people were living with cancer in 2012 around the world [1].
2. Cancer is a mysterious disease, and this mystery in itself is an attraction. Many scientists around the world are striving to identify the root causes of cancer, and they generally believe cancer to be a genetic disease, with its complex processes commencing with a sole mutation and proceeding with other mutations [2]. Each mutation or group of mutations confers a new characteristic to this fast growing cellular mass, including angiogenesis, anti-apoptosis, extracellular matrix break and etc. [3]. A minority of scientists, on the other hand, believe that malignancy originates from the micro-environment of the cell and mutational processes occur afterward [4, 5]. Moreover, there are sporadic attitudes and observations in parapsychology and pseudoscience regarding the origin of cancer, which claim that the human body possesses dimensions beyond its corporal composition, some of which are physical and others metaphysical [6]. According to these unproved approaches that can be named dimensional medicine, tumorigenic events begin from these dimensions, and mutational processes or micro-environmental changes follow. Therefore, major mysteries are still to be uncovered, and that has made cancer attractive in the eyes of many scientists seeking the answer to such questions.
3. Many novel approaches to the treatment and even eradication of cancer have recently emerged. Some of which are: The targeted therapy approach, in which lots of monoclonal antibodies have been developed to target the principal role players in cancer signaling pathways [7]; the cancer stem cell approach, which suggests the extermination of tumor to be dependent on the annihilation power of cancer stem cells; the immunotherapy and cell therapy approach that relies on training of the host’s immune system to defend against cancer cells [8]; and finally, the personalized medicine approach, the great achievement of pharmacogenomics and biomarker discovery, especially with the help of high throughput technology, which has faced the challenge of big data in its primary phases of introduction to the community [9]. This challenge has opened a new window for the involvement of many other professionals, such as bioinformaticians, statisticians, and software designers to solve this recent challenge. Statistical model-based software would be the main outcome of the efforts of such professionals. Such software is able to predict sensitivity and resistance to each specific treatment for each specific patient, and also predict prognosis and survival of cancer patients based on their biomarker profile.
4. Integrative Medicine in cancer have brought many professionals of alternative and complementary medicine to the scene to present their potential, especially to raise the quality of life of cancer patients [10].
5. There are also numerous professionals of non-medical specialties, such as electromagnetic, photonic, biomaterial and many other engineers, which have entered the field of cancer and created an interdisciplinary atmosphere of research. Recently, microfluidic-based platforms have shown the potential of paradigm change in critical areas of cancer research, such as biomarker discovery, molecular diagnostics, tumor biology and high-throughput screening for therapeutics; liquid biopsy for isolation of circulating tumor cells (CTC), cell-free DNA and nano-vesicles, such as exosomes, is one of its important potentials. Furthermore, as an alternative to conventional monolayer cell culture and animal models, modeling of primary tumors on microfluidic-based chips will change the face of cancer research in the near future. These three-dimensional models can be applied to investigate cancer growth and metastasis, drug target discovery for immunotherapy purposes, testing drug compounds, and also associated companion diagnostics [11, 12].
All of the above approaches to various aspects of cancer have engaged many different professionals and scientists worldwide. Cancer has thus encompassed diverse areas of science and technology to encounter challenges. However, the separate involvement of each discipline would not be greatly beneficial, and what is required is a suitable atmosphere where disciplines can influence one another. This highlights the urgent need to gather all the relevant professionals to discuss and reach a mutual understanding of the interdisciplinary points of view and new insights into cancer. That is, we need a multidisciplinary cancer investigation to achieve the target. In this journal, we attempt to highlight and facilitate the spreading of such approaches. We look forward to receiving meritorious manuscripts and works from around the world, and disseminating them for the benefit of all enthusiastic scientists thirsty for progress.

Achievement of new drugs with minimal side effects is a important goal in the cancer treatment due to irreversible side effects of conventional drugs. Tricalcium phosphates as natural bone components have unique characteristics including excellent biocompatibility, high biosorption and superior bioactivity. This research aimed at investigating the inhibitory effect of tricalcium phosphate (TCP) sintered at different temperatures.
TCP nanoparticles (nTCP) were sintered at three temperatures of 700°C, 900°C and 1000°C, and their structural characterization was examined. Heat treatment of TCP at 900°C was found optimal due to its morphological properties, such as particle size and its crystallinity. The inhibitory effect of optimized nTCP sintered at 900°C was surveyed through in vitro tests.
Cell culture assay studies exhibited that such effect depended on the concentration of nTCP. Moreover, the results depicted that the effect was 80%, which could be attributed to the 50 mg.L-1 dose of nTCP.
In lower concentrations, higher inhibitory effect of nTCP was observed. In comparison with hydroxyapatite, at low concentrations, anti-cancer properties of TCP were far greater than other calcium phosphates.

Calcium phosphate nanoparticles such as nano hydroxyapatite have shown excellent physical, chemical and biological properties and have allocated special place in regenerative medicine applications. Recently, the inhibitory effect of hydroxyapatite, as a calcium phosphate, on many cancers has been reported.
In this article β-tricalcium phosphate was synthesized using co-precipitation method and its physicochemical properties were studied (SEM, FTIR and XRD). Inhibitory effect of beta-tricalcium phosphate nanoparticles on the growth and proliferation of breast cancer cell line MCF-7 was investigated.
In vitro MTT assay studies showed that the inhibitory effect was dependent on the concentration of tricalcium phosphate nanoparticles. In addition, the results demonstrated that the inhibitory effect was 78% attributed to 50 mg. L-1 concentration of tricalcium phosphate nanoparticles.
In lowest concentrations, the inhibitory effect of tricalcium phosphate nanoparticles was higher than others.

Vitamin D and insulin play an important role in susceptibility to polycystic ovary syndrome (PCOS), and therefore vitamin D receptor (VDR), parathyroid hormone (PTH), and insulin receptor (INSR) gene variants might be involved in the pathogenesis of PCOS.

The present study was designed to investigate the possible associations between polymorphisms in VDR, PTH, and INSR genes and the risk of PCOS.

VDR, PTH, and INSR gene variants were genotyped in 35 women with PCOS and 35 controls using Polymerase chain reaction – Restriction fragment length polymorphism method. Furthermore, serum levels of glucose and insulin were measured in all participants.

No significant differences were observed for the VDR FokI, VDR Tru9I, VDR TaqI, PTH DraII, INSR NsiI, and INSR PmlI gene polymorphisms between the women with PCOS and controls. However, after adjustment for confounding factors, the VDR BsmI “Bb” genotype and the VDR ApaI "Aa" genotype were significantly under transmitted to the patients (p= 0.016; OR= 0.250; 95% CI= 0.081-0.769, and p= 0.017; OR= 0.260; 95% CI= 0.086-0.788, respectively). Furthermore, in the women with PCOS, insulin levels were lower in the participants with the INSR NsiI "NN" genotype compared with those with the "Nn + nn" genotypes (P= 0.045).

The results showed an association between the VDR gene BsmI and ApaI polymorphisms and PCOS risk. These data also indicated that the INSR "NN" genotype was a marker of decreased insulin in women with PCOS. Our findings, however, do not lend support to the hypothesis that PTH gene DraII variant plays a role in susceptibility to PCOS.

Due to the limitations of the classical methods to detect Coxiella burnetii, direct diagnosis of the pathogen using PCR techniques is still the preferable approach. However, false negative results owing to the presence of PCR inhibitors are troublesome.. In order to identify the inhibitors during PCR assay, an internal positive control (IPC) was designed based on 16SrRNA gene of C. burnetii.. In the current study, the initial and ending parts of the target gene in an external positive control plasmid (pTZ57R/T-16S) amplified using internal primers which had a BglII restriction site on the 5´ends. Both PCR products (fragments 1 and 2) were cloned into pTZ57R/T vector. Following BglII enzyme digestion, the two obtained linear plasmids were ligated. The ligation product was transformed into Escherichia coli Top10Fʹ. Screening of the desired recombinant clone was carried out using colony PCR.. The size of the PCR product was equal to the sum of the first and second fragments. Sequencing confirmed the presence of the desire insert (IPC sequence) in recombinant plasmid. Consequently, the IPC fragment was longer than the target gene while both ends had similar attachments to the same primer pair.. The results showed that direct fusion of the recombinant plasmids containing the initial and ending parts of the target gene are simple and cost-effective techniques for increasing the length of the fragment and constructing IPC.

Q-fever in humans and coxiellosis in animals. This bacterium can survive in the environment out of the Coxiella burnetii is the etiologicagent of a zoonotic disease which named specific host. Accordingly، it categorized by the CDC in bioterrorism agents ‘category B. Consequently، rapid detection of the bacteriumadministratesthe treatment of disease.

world and Iran scientific databases was conducted. This paper includes biological safety، cultivation and A review study on the different researches on laboratory diagnosis field in the detection assays.

Detection of Coxiella burnetii can be done byclassical methods (isolation، cultivation in the appropriate cell line such as P388D1، J774، and L929)، serologic tests (immunofluorescence، micro immunofluorescence، complement fixation test and ELISA) and molecular biology methods (PCR، Nested PCR and Real Time PCR).

While have existed kinds of detection methods for this agent، costly، need to specific laboratory and time consuming are the limitation of the mentioned techniques. Molecular methods due to accuracy and high rapidity in detection can be effective.

Mesenchymal stem cells (MSCs), also known as mesenchymal stromal cells, are considered as an important source of adult stem cells in tissue engineering and cell therapy. They are present in various tissues such as, endometrium as the supportive cells. According to anatomical position of endometrial mesenchymal stem cells that put them in neighborhood of the fetus, they may have a significant role in fetus tolerance during pregnancy. This study was conducted to evaluate the molecular mechanism of immunosuppressive affect of endometrial mesenchymal stem cells. Mesenchymal Cells from bone marrow and endometrium were cultured at density of 2 ×105 cells/ml at presence of 100IU/ml and 500IU/ml INF-γ (IFN-gamma) and expression of indoleamine 2 3-dioxygenase (IDO) were studied after 72 hours by real-time PCR and immunocytochemistry. The study showed that IDO expression in cells exposed to IFN-γ was increased compared to the cells in the absence of IFN-γ (p<0.05). Additionally, up regulation of IDO expression was higher in endometrial cells than bone marrow cells. From these results it is concluded that endometrial mesenchymal stem cells may be used as a good candidate for cell therapy.

HER-2/neu is one of the major molecular prognostic markers in breast cancer and due to treatment decision guidelines، the amplification of this gene is very important in breast cancer patients treatment. The most commonly used method for detecting HER-2/neu gene amplification is immunohistochemistry (IHC). Unquantifiable results، lack of reproducibility and long procedure time are among the drawbacks of this method which have persuaded researchers to seek alternative modern molecular based techniques such as Real-Time PCR. The aim of our study was the comparison of HER-2 gene amplification assessed by Real-Time PCR and IHC method.

After DNA extraction، 41 formalin-fixed paraffin-embedded (FFPE) breast carcinoma with known IHC results، had been assessed using Real-Time PCR and gene copy number of HER-2 were measured and compared with the results of IHC. Fluorescent in situ hybridization (FISH) for equivocal IHC and unreliable results were performed. The Fisher''s Exact Test and Phi Cramer''s V Coefficient were used for statistical analysis.

According to the results، 39% of samples tested by Real-Time PCR have HER-2/neu gene amplification and 36% of samples was positive by IHC. The correlation of two methods in positive and negative samples were 85% and 87% respectively. Overrall correlation between two methods was 71%. Also، IHC2+ results have been assessed by FISH technique and all of them were negative، which was in concordance with the results of the Real-Time PCR.

About 71% concordance had been observed between two methods. In the case of IHC2+ results، FISH was performed and proved the Real-Time PCR results. Real-Time PCR could precisely measure the amount of tissue HER2 amplification and also were in agreement with IHC results. Since Real-Time PCR has high precision، also is cheaper and more accessible than other methods، we suggest it to be used to assess this indicator.

In recent years, recombinant monoclonal antibodies and their derivatives have emerged as important targeted therapy agents. Monoclonal antibodies are ex-tremely difficult to produce. So, the cost of production is very high and many people cannot afford these drugs. In this regard, choosing inexpensive and easy ways to manipulate production systems such as bacterial hosts to reduce the cost of manufacturing these critical components are considered as vital step for developmental issues in recombinant expression systems. We, therefore, at-tempted to generate a polycistronic construct of anti HER-2 F(ab')2 fragment antibody for insertion in an expression bacterial plasmid. Also some modifica-tions were made in the hinge region to express antibody F(ab')2 fragment in its authentic form preventing from multiple varieties of disulfide bond formation. Finally, synthesized construct was cloned in pET-32 Ek/LIC vector without using restriction enzyme digestion or ligation reactions. The results of this study show-ed that modified F(ab')2 fragment was simply and successfully inserted in Escherichia coli (E.coli) using the Ligation Independent Cloning technology.

Recombinant tissue plasminogen activator (rt-PA) is one of the most important thrombolytic agents used in patients with vascular occlusions such as acute ischemic stroke or myocardial infarction. A variety of recombinant protein expression systems have been developed for heterologous gene expression in prokaryotic and eukaryotic hosts. In recent years, Leishmania tarentolae (L. tarentolae), a non-pathogenic trypanosomatid protozoa, has come under consideration because of its safety and immunogenicity as a vaccine vector and special attributes in the expression of complex proteins. This study was done to improve rt-PA expression in this protozoon and create the opportunity for the replacement of rt-PA gene with other genes for the production of other complex proteins.

Two expression cassettes were used for the integration of two copies of t-PA cDNA, one copy in each cassette, into the parasite genome by electroporation. The transformed clones were selected by antibiotic resistancy. The expression of active secreted rt-PA was confirmed by Western blot analysis and Chromolize assay.

Appearance of a 64 kD band in nitrocellulose membrane in the Western blot analysis confirmed the presence of full-length rt-PA in the culture media. Chromolize assay showed the expression levels of active recombinant t-PA in single and double transfected L. tarentolae clones- 375 IU/ml and 480 IU/ml of the culture media, respectively.

The produced rt-PA in the culture media containing the transfected cells was at least seven times higher than what has been reported in previous studies on L. tarentolae or on some other eukaryotic systems.

Yersinia pestis, the causative agent of the zoonotic plague infection, is a major public health concern both as a threat and potential bioweapon. The objective of the present study was to establish a uniplex and multiplex - polymerase chain reaction (PCR) test for the specific detection of Y. pestis. PCR reactions performed by three pair primers which targeted the caf and pla genes located on the pFra and pPst plasmids and the irp chromosomal gene located on the ‘pathogenicity island’. After TA cloning of the PCR products, the test’s limit of detection (LOD) was determined. For evaluating the specificity, PCR reactions were performed with negative control bacteria. Assays were performed with the genome of Y. pestis which produced three DNA fragments of the expected sizes 00, 400 and 50 bp which corresponded to the irp, caf and pla genes, respectively. The lower LoD was 70 copy numbers for the caf gene and for the pla gene. In PCR reactions that used negative control bacteria, detectable fragments were not observed. Our method clearly discriminated Y. pestis DNA. The rapidity, specificity and sensitivity of this procedure suggest that it can serve as a useful alternative method for the inoculation of laboratory animals or the use of specific culture media for routine plaque surveillance and outbreak investigations. Another vital result of this study was the establishment of Y. pestis molecular detection technique in Iran.

Tissue plasminogen activator (tPA) is a serine protease, which is composed of five distinct structural domains with 17 disulfide bonds, representing a model of high-disulfide proteins in human body. One of the most important limitations for high yield heterologous protein production in Escherichia coli (E. coli) is the expression of complex proteins with multiple disulfide bridges. In this study the combination of two distinct strategies, manipulated cytoplasm and native periplasm, was applied to produce the functional full length tPA enzyme in E. coli. Using a PelB signal peptide sequence at 5'' site of tPA gene, the expression cassette was prepared and subsequently was transformed into a strain with manipulated oxidizing cytoplasm. Then the induction was made to express the protein of interest. The SDS-PAGE analysis and gelatin hydrolysis confirmed the successful expression of functional tPA. The results of this study showed that complex proteins can be produced in E. coli using the cumulative power of both cytoplasm and periplasm.