Journal of Genes and Cells

21-26

Authors: Adel Ghorani-Azam, Seyed Ahmad Mohajeri, Bamdad Riahi-Zanjani, Sona Sepahi, Samaneh Sepahi

Amylase is an enzyme with broad hydrolysis activities that catalyzes the hydrolysis of starch into glucose and other small molecules such as maltose. It is an enzyme of glycoside family with surface layer homology (SLH) domain. SLH, which is known as cell wall binding domain is essential for binding to peptidoglycan of cell membranes. Since designing and producing enzymes with strong catalytic activity is essential in future food industry, investigating the similarities of amylases of different species can be helpful in protein engineering of these enzymes. Homology alignment of bacterial and human amylase sequences shows that both sequences contain 511 amino acid that are highly conserved with several repeated sequences that seems to be necessary for catalysis activity and structural conformation of the protein. In this study, we aimed to characterize the most critical structural and catalytic domains of amylase using bioinformatics tools. In this study, it was shown that SLH and starch binding domain (SBD) are extremely conserved in both human and bacterial amylase. In addition, it was shown that similar domain with several repeated di- or tri-peptide sequences existed in almost all amylases indicating that these residues may have valuable functional, structural and evolutional information. The results of this study may provide new insights into biological aspects of proteins that have not yet been elucidated.

32-35

Authors: Seyed Javad Mowla , Ensieh Poursani, Majid Mehravar, James E. Trosko

Alternative splicing is an important mechanism that regulates gene expression and function in mammals, particularly in primates and human. Large-scale studies estimates a large number of human genes undergo alternative splicing. OCT4, a crucial stem cell marker, is one of the most important stemness markers that is not excluded in this regard. Up until now, it is known that OCT4 can produce several spliced variants in different cell types. In this study, we used RT-PCR and DNA sequencing approaches to verify OCT4 variant expression. Here, we reported a novel OCT4 spliced variant which is named as OCT4B3, for the first time. This variant is very similar to the OCT4B2 transcript, but a 207-bp fragment has been lost from Exon1b. Moreover, we investigated the expression pattern of OCT4B3 in several human cancer cell lines and found its expression in two cell lines of 1321N1 and 5637. Our experiments resulted in discovery of a novel OCT4 variant of OCT4B3 that is expressed in two human 5637 and 1321N1 cell lines.

3-15

Authors: Muhammad Irfan-maqsood, Hojjat Naderi-Meshkin, Asieh Heirani-Tabasi, Monireh Bahrami, Mahdi Mirahmadi, Halimeh Hassanzadeh, Mahmood Raeesolmohaddesin, Nasser Sanjar Moussavi, Hasnain Raza-Shah, Ahmad Reza Bahrami, Hamidreza Bidkhori, Maryam M. Matin

Keratinocytes are the main components of skin epidermis constituting more than 90% of it which are responsible to regulate skin regeneration during external or internal injury. These cells can be found in a heterogenic form containing proliferative, terminally differentiated and transit amplifying (TA) cells located in basal layer, outer keratinized layer and intermediate layers, respectively when isolated from an adult skin. Efforts are going on to characterize keratinocytes precisely as in comparison with widely used stem cells, mesenchymal stem cells (MSCs) and their application in cutaneous wound healing as a translational approach in regenerative medicine. In this work, we have applied a comprehensive approach to identify and characterize keratinocytes which are a valuable tool in wound healing. Keratinocytes have successfully been isolated from adult skin and the ratio of proliferative, terminally differentiated and TA cells based on the expression of α6-integrin and CD71 was studied using flow cytometry after 7, 14 and 21 days of their culture in vitro. RT and qRT-PCR was applied to study the change in genetic expression and relativity of cytokeratin markers with the passage of time. PopUp culturing and population doubling time was performed to study the proliferative potential of heterogenic population of cells. Their colonogenicity and wound healing potential were also studied to explain their healing behaviour during the time of injuries. 47% keratinocytes after 7 days, 62% after 14days and 93% keratinocytes after 21 days of culturing. Expression of CD71 was also observed in cells as, 5% after 7 days, 22% after 14 days and 66% of keratinocytes after 21 days were positive for CD71 expression. Gradual increased expression of genetic markers, K10, K14, INV and P63 was observed using qRT-PCR. Studying comparative data, it can be concluded that keratinocytes after 14 days of culturing are better to use for clinical purposes. This comprehensive protocol could be a valuable addition in cutaneous biology helping researchers to identify keratinocytes and to use them for their pre-clinical studies.

8-10

Authors: Naveed Akhtar , Kausar Saeed, Naseem Rafiq, Nasreen Khan

Dengue is one of the leading cause of death worldwide. The first outbreak of dengeu hemorrhagic fever was reported in 1994 in Pakistan and after that several outbreaks of the dengue were reoprted in Pakistan and in all these outbreaks the dengue serotype 2 (DENV2) was confirmed, which ensures that dengue serotype 2 (DENV2) is prevelant in Pakistan.

16-20

Authors: Afeefa Kiran Ch

Dengue fever is an epidemic killing millions of every year around the world especially in regions with moderate temperature primarily due to propagation of the vector, Aedes aegypti. Unknown genomic basis is the biggest challenge to compete with this challenge. A little information is available about the genotypic characterization of the virus infecting the population. Based on the genotype, dengue viruses are classified into four types named as DENV-1, DENV-2, DENV-3, DENV-4. All these serotypes are composed of several proteins know as NS proteins which are further categories into NS1, NS2A, NS2B, NS3, NS4A,NS4B, NS5 based on their functions. In this manuscript, detailed of serotypes classification, their components proteins, their structure and functions are discussed briefly which will be helpful to understand the basic of dengue viral genome.

51-56

Authors: Madjid Momeni-Moghaddam

Infectious Bronchitis Virus (IBV) is a highly infectious and contagious viral pathogen of chickens worldwide. IBV is a positive sense, single-stranded RNA virus that primarily targets the respiratory tract. IBV belongs to group III of the genus Coronavirus of the Coronaviridae family. In this study, viral RNA was extracted using phenol-chloroform based method. A pair of specific primers for RT-PCR and a pair of specific internal primers for Nested-PCR of the S1 region in the spike protein gene was designed and the tests were optimized for the detection of IBV. To determine the sensitivity of the test, 10 fold dilutions of virus stock (3 × 105 EID50 to 3 × 10-2 EID50/ml) were prepared in distilled water. A 531 bp fragment and a 284 bp of the spike protein gene (S1) were amplified in the RT-PCR and the Nested-PCR test, respectively. The PCR product was cloned in pTZ57R/T vector and sequenced. The sequence data confirmed the specificity of the test. The sensitivity of RT- PCR was determined to be 3×103 EID50 per ml. Nested-PCR was also carried out and sensitivity was increased to 3×10 EID50 per ml. Due to the high sensitivity of this test, this technique can be used as a robust and rapid diagnostic method for the detection of IBV.

67-70

Authors: Muhammad Irfan-Maqsood, Monireh Bahrami, Raheleh Amirkhah

Cancer is an abnormality of cells in which cells are proliferating in an uncontrolled manner due to some unwanted mutations. Any cells from any part of body can be cancerous and may form tumors later on. These tumors develop tumorsphere and their microenvironment modulate T cells as a survival mechanism and these immune cells failed to identify the cancerous cells, allowing them to metastasis. A number of therapeutic approaches such as radiotherapy, chemotherapy etc. are being applied in clinical practices to remove tumors or kill tumor cells. Cell therapy has shown a promising potential in last two decades in cancer treatment as some cells such as mesenchymal stem cells have tumor tropism and induce apoptosis in cancer cells. Recently, immunotherapeutic approaches have been considered the gold standard and it has been shown that T cells isolated from the cancer patient can be trained to target cancer cells. In this process, chimeric antibody receptor (CAR) and T-cell receptor (TCR) are introduced on the T cell surface to enhance T cells function and their transfer in the patient’s circulatory system result in the identification and killing of cancerous cells. This approach has been known as the adoptive cell transfer or adoptive cell therapy (T cells) and has shown 99% promising results in 243 clinical trials done so far. Clinical trials are going on to determine its efficacy in cancer treatment and a number of pharmaceutical firms are developing adoptive T cells as drugs to treat cancer in our daily routine clinical practice. Results obtained yet, have shown the perspective of adoptive cell therapy as the future approved cancer therapy.

73-75

Authors: Muhammad Ali

Prostate cancer is one of the deadliest male malignancy in the worldwide. It has been estimated that 1.1 million cases of prostate cancer were diagnosed in 2012 and within coming 10 years, the prostate cancer cases will be increased by 15 per cent in men. The multifaceted molecular anomaly of prostate cancer has confronted it from being dated owing to the orchestrated carcinogenic functions of negative regulators which work in a network. An as yet blurred view of therapeutic interventions of prostate cancer has been acknowledged, resulting from the addition of substantial fraction of information. Prostate-specific antigen (PSA) screening is currently known as a method for early detection of prostate cancer but its validity as prostate cancer biomarker is still controversial. Information obtained from the sequencing data of prostate cancer tissue biopsy, it has been described that non-coding RNAs such as microRNAs are good source of information to detect the prostate cancer. This information can be used to detect, predict metastatic state and even the state of prostate cancer progression. In this commentary, the role of microRNAs has been discussed in prostate cancer detection.

34-39

Authors: Asieh Heirani-Tabasi, Marzieh Hassanzadeh, Shabnam Hemmati-Sadeghi, Mina Shahriyari, Mahmood Raeesolmohaddeseen

Cell therapy has been considered as the third pillar of medicine. There are several kinds of cells attracted researchers attention whereas the growing interest in mesenchymal stem cells (MSCs) for regenerative purposes has enabled them to explore the versatile characteristics for regenerative purposes. So far, near about 5000 clinical trials using MSCs, authenticating the clinical applicability of these cells. Their multipotent differentiation and unique immunosuppressive properties have made them ideal candidate in cell therapeutic approaches. Recent advances have been observed in the homing of these cells to enhance their transplantation efficacy. These cells have been proposed as the ideal candidates for cancer therapy and have been engineered for targeted cancer therapy. Their successful applications and responses have termed them the ideal cells for the future of regenerative medicine.

46-48

Authors: Sohrab Boozarpour, Madjid Momeni-Moghaddam

Stem cell research is well known in clinics to treat diseases as huge research has resulted in successful isolation, maintenance and manipulation of stem cells in vitro. Much information regarding molecular biology of number of cells/stem cells is known and have shown a promising potential in biotechnology. Biotechnologist have used novel approaches in last decade for the production of human compatible recombinant proteins and recently, stem cells have been considered as potential biotechnological agents. Focus should be done to engineer cells for specific trait production such as germ cells. It also has been shown that maintenance of germ cells is not an easy task but successful trans-differentiation of other multipotent stem cells such as mesenchymal stem cells toward germ cell linages have opened the opportunities to engineer MSC and then trans-differentiate towards germ like-cells which can be used to develop transgenic animals. Focus should be done on cell based biotechnological productions to revolutionize health biotechnology.

3-12

Authors: Mahdi Ghadimi Hamzekolaii

Human diseases caused by mutations in extracellular matrix genes are often associated with an increased risk of cataract and lens capsular rupture. However, the underlying mechanisms of cataract pathogenesis in these conditions are still unknown. This review provides a brief overview of the function and structure of the extracellular matrix and reviews the progress that has been made in understanding the involvement of extracellular matrix in lens development and pathology.

22-28

Authors: Elahe Navaeian-kalat, Mohsen Jalali, Zohreh Fathi

Infertility is increasing rapidly in the world because of change in major lifestyle habits and it has been estimated that near about 75 million peoples in the world are facing infertility problems including both male and females. A number of therapeutic approaches like assisted reproductive techniques (ARTs), intra-uterine-insemination (IUI), hysteroscopic surgery, hormonal and chemotherapy have been applied in infertility clinics. Stem cell therapy has been proposed a promising candidate in this field where adult stem cells can be transdifferentiated to oocyte-like cells and SSC-like cells, which can be used to treat infertility like azoospermia etc. In this study, a number of research papers regarding infertility and stem cell therapy have been reviewed and hypothesized that trans-differentiation of stem cells towards SSC-like cells or OSC-like cells, and their clinical trials may authenticate the claims of stem cell therapeutic potential to treat infertility. Transdifferentiation is a process of lineage reprogramming where the fate of a (stem) cell has been determined. Increased research on transdifferentiation of multipotent and pluripotent stem cells (induced pluripotent stem cells [iPSCs], mesenchymal stem cells [MSCs] etc.) toward germ cell lineage in last decade hypothesized that infertility can be treated by using these kind of stem cells. Reliable successes have been achieved in the transdifferentiation of stem cells toward germ-like cells (oocyte-like cells and SSC-like cells). Clinical trials of transdifferentiated mesenchymal stem cells are the only way to answer this question. Clinical trials of trans-differentiated MSCs may give valuable results to combat increased infertility in the world. In conclusion, applications of MSCs in infertility clinics and start of their clinical trials may open new horizons of treatment in infertility.