Iranian Journal of Basic Medical Sciences
Volume:21 Issue: 9, Sep 2018

To present a brief overview of various natural sources of antimicrobials with the aim of highlighting invertebrates living in polluted environments as additional sources of antimicrobials.

A PubMed search using antibacterials, antimicrobials, invertebrates, and natural products as keywords was carried out. In addition, we consulted conference proceedings, original unpublished research undertaken in our laboratories, and discussions in specific forums.

Representative of a stupefying 95% of the fauna, invertebrates are fascinating organisms which have evolved strategies to survive germ-infested environments, yet they have largely been ignored. Since invertebrates such as cockroaches inhabit hazardous environments which are rampant with pathogens, they must have developed defense mechanisms to circumvent infections. This is corroborated by the presence of antimicrobial molecules in the nervous systems and hemolymph of cockroaches. Antimicrobial compounds have also been unraveled from the nervous, adipose, and salivary glandular tissues of locusts. Interestingly, the venoms of arthropods including ants, scorpions, and spiders harbor toxins, but also possess multiple antimicrobials.

These findings have rekindled the hopes for newer and enhanced therapeutic agents derived from a plentiful and diverse resource to combat fatal infectious diseases. Such antimicrobials from unusual sources can potentially be translated into clinical practice, however intensive research is needed over the next several years to realize these expectations.

Current therapeutic strategies for cancer are associated with side effects and lack of specificity in treatments. Biological therapies including monoclonal antibodies and immune effectors have been the subject of multiple research projects. Pore-forming proteins may become the other biological strategy to overcome the problems associated with current treatments. But detailed mechanisms of their action on target membranes remained to be elucidated. We aimed to study the cytotoxic effects of recombinant form of fragaceatoxin C on AML cell lines HL-60 and KG-1.
We cloned the FraC gene in pET-28a () bacterial expression vector and the expressed recombinant FraC protein was purified by affinity chromatography. Then, cytotoxic effects of the recombinant protein were examined on two AML cell lines, HL-60 and KG-1. Effects of serum and calcium ion were explored by hemolysis assay in more details.
Our results showed that the recombinant C-terminal polyhistidine-tagged FraC protein has potent cytotoxic effects on both AML cell lines, with IC50=5.6, and 4.6 µg.ml-1 for HL-60 and KG-1 cells, respectively. Serum showed dose-dependent and also time-dependent inhibitory effects on the hemolytic and cytotoxic activities of the FraC protein. Pre-incubation of the toxin with different concentrations of calcium ion also inhibited hemolytic activity of FraC toxin.
Results of the present study showed that FraC has potential anti-tumor effects. By detailed investigation of the inhibition mechanism of serum and calcium effects in the future, it can be possible to design target sites for clinical applications of the toxin.

Filamentous bacteriophage M13 was genetically engineered to specifically target mammalian cells for gene delivery purpose.
A vascular endothelial growth factor receptor 2 (VEGFR2)-specific nanobody was genetically fused to the capsid gene III of M13 bacteriophage (pHEN4/3VGR19).
A mammalian expression construct containing Cop-green fluorescent protein (Cop-GFP), as a reporter gene, was amplified by PCR and then sub-cloned in the pHEN4/3VGR19 phagemid. The resulting construct was transfected into 293KDR cell. The recombinant phage was extracted and confirmed and then transduced into VEGFR2 expressing cell (293KDR).
Seventy-two hr after transfection, green fluorescence was detected in 30% of the cells. About 1% of the cells which transduced by recombinant phages were able to express GFP.
It is hoped that the results from this study will help to find potential vectors to improve the efficiency of gene delivery. Taken together, we conclude that this newly-introduced vector can be used in cancer researches.

Fetal microchimerism is the persistence of allogeneic cell population that transfer from the fetus to the mother. The aim of this study was to evaluate the presence of fetal microchimerism in the pancreas of the mouse with acute pancreatitis (AP).
In this experimental study, female wild-type mice were mated with male EGFP. AP model was obtained by injection of caerulein two days after delivery. Sixty mice were divided into 3 groups: the virgin pancreatitis-induced animals, pregnant pancreatitis-induced animals mated with transgenic EGFP mice, and pregnant sham animals. To prove pancreatitis induction, the blood amylase and lipase were assessed; and pancreas was removed from a subpopulation of each group for histopathological examinations after 6 hr. The remaining mice were kept for 3 weeks and histopathological exanimation, immunohistochemistry, and PCR were performed.
EGFP cells were found in acini and around the blood vessels in the pancreas of pregnant pancreatitis-induced animals. They differentiated to acinar, adipocyte-like, and mesenchymal-like cells. PCR showed that 20% of the pregnant pancreatitis-induced animals were EGFP. The histopathological study showed improvement in pancreatitis scores in the mice with history of pregnancy.
It seems that pregnancy has a beneficial impact on caerulein-induced pancreatitis and improves the pancreatitis score in mouse.

Heat stress (HS) is a catastrophic stressor that dampens immunity. The current study investigates the effect of dietary administration with camel whey protein (CWP) on apoptotic pathway caused by HS.
Forty-five male mice were divided into three groups: a control group; HS group; and HS mice that were orally supplemented with CWP (CWP-HS group).
We found that reactive oxygen species (ROS), pro-inflammatory cytokines (IL-6), and C reactive protein (CRP) were elevated in the HS group along with a significant increase of caspase-9 and -3 and decrease of total antioxidant capacity (TAC). HS mice revealed impaired phosphorylation of Bcl-2 and Survivin, as well as increased expression of Bax, Bim and cytochrome C. Additionally, we observed an aberrant distribution of HSP-70 expressing lymphocytes in the spleen and thymus of HS mice. Moreover, histopathological examination showed alterations on the architectures of immune organs. In comparison with CWP-HS group, we found that CWP restored the levels of ROS, IL-6, TAC and CRP induced by HS. Furthermore, CWP restored the expression of Bcl-2/Bax, improved the histopathological changes in immune organs and HSP-70 distribution in the spleen and thymus.
Our findings revealed the possible ameliorative role of CWP supplementation against damages induced by exposure to HS.

Osteonecrosis of the jaw, as an exposed necrotic bone in the oral cavity, is one of the adverse effects of bisphosphonates, which have an affinity for bone minerals. This study investigates the cytotoxic effects of alendronate (ALN) as a nitrogen-containing bisphosphonate, on human dental pulp stem cells (hDPSCs).
The mesenchymal stem cells (MSCs), obtained from third molar tooth pulps were characterized by immunophenotyping assay in order to identify surface markers to evaluate their expression. To detect multipotency hDPSCs, they were differentiate into osteocytes and adipocytes. Cell proliferation was measured by MTT assay. PI staining of DNA fragmentation by flowcytometry (sub-G1 peak) was performed for determination of apoptotic cells and Bax, Bcl-2, and cleaved caspase 3 expressions. Protein expression was detected by Western blotting.
As the results revealed, ALN decreased viable cells (in 0.8–100 µM) after 72 hr and 168 hr (P Long-term effects of ALN on cell proliferation and apoptosis in hDPSCs can result in either initiation or potentiation of ALN-induced osteonecrosis.

This study aimed to investigate the effect of aerobic training on serum levels of Klotho, cardiac tissue levels of H2O2 and phosphorylation of ERK1/2 and P38 as well as left ventricular internal diameter (LVID), the left ventricle wall thickness (LVWT) and fibrosis in middle-aged rats.
Forty wistar rats, including young rats (n=10, 4 month-old) and middle-aged rats (n=30, 13-15 months-old) were enrolled in this experimental study. The all young and 10 middle-aged rats were sacrificed (randomly) under deep anesthesia without any exercise training as normal young control and normal middle-aged control respectively. The remaining 20 middle-aged rats participated in 4 (n=10) or 8-week (n=10) aerobic exercise training.
There were significant differences in the plasmatic Klotho levels and the heart tissue levels of phosphorylated-ERK1/2 (p-ERK1/2), P-P38 and H2O2, LVWT, LVID and fibrosis between young and middle-aged rats (P=0.01). Plasmatic Klotho level was significantly increased after eight weeks training (P=0.011). Also, p-ERK1/2 was significantly decreased after eight weeks and p-P38 was significantly decreased in the fourth (P=0.01) and eight weeks of training (P=0.01). A similar decrease was reported for aging-induced H2O2 in the fourth (P=0.016) and eighth weeks (P=0.001). LVID was significantly increased in eight weeks, but LVWT and fibrosis was significantly reduced in the eighth week (P=0.011, P=0.028, P=0.001 respectively).
Moderate aerobic training attenuates aging-induced pathological cardiac hypertrophy at least partially by restoring the Klotho levels, attenuating oxidative stress, and reduction in the phosphorylation of ERK1/2, P38 and fibrosis.

During type-1 diabetes treating by pancreatic islet transplantation, increasing oxidative stress and microbial contaminations are the main reasons of transplantation failure. In this study, we evaluated anti-apoptotic, antioxidant and antimicrobial potentials of phenolic compounds called ellagic acid (EA) and silybin on rat pancreatic islets.
By doing MTT assay, effective concentrations of EA and silybin were determined as 1500 and 2100 μM, respectively. Then, ELISA methods, flow cytometry and MIC were done to investigate antioxidant, anti-apoptotic and antibacterial effects of those compounds, respectively.
Results of FITC Annexin-V and PI staining via flow cytometry, and also caspase-3 and -9 activities performed that EA has anti-apoptotic effects on pancreatic cells. Both compounds significantly diminished reactive oxygen species, and enhanced antioxidant power and insulin secretion. Furthermore, the minimum inhibitory concentration test indicated that these two have antibacterial effects on both Gram-positive and Gram-negative bacteria which usually contaminate the pancreatic islets.
These findings support that use of EA and silybin can improve the function of islets which are used in transplantation, along with decreasing islets bacterial contamination.

In this study, effects of encapsulated umbilical cord stem cells (UCSCs)-derived hepatocyte-like cells (HLCs) in high mannuronic alginate scaffolds was investigated on CCl4-induced acute liver failure (ALF) in rats.
Material and UCSCs were encapsulated in high mannuronic alginate scaffolds. Then the UCSCs differentiated into HLCs for treatment of CCl4-induced ALF in rats. Thirty rats randomly divided into 5 groups: Intoxicated group received only CCl4 to induce ALF. In other groups including cell-free, UCSCs and HLCs, alginate scaffolds were transplanted into the liver 4 days after CCl4 injection. Biochemical markers including albumin (ALB), blood urea nitrogen (BUN), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) were evaluated. Histological changes and gene expression of ALB, alpha-fetoprotein (AFP), and cytokeratin 18 (CK-18) were also assessed.
Expression of CK-18 significantly increased in HLCs compared to the UCSCs in vitro. This indicates that UCSCs can effectively differentiate into the HLCs. In CCl4-intoxicated group, BUN, AST and ALT levels, and histological criteria, such as infiltration of inflammatory cells, accumulation of reticulocytes, nuclear pyknosis of hepatocyte and sinusoidal dilation, significantly increased. In this group, ALB secretion significantly decreased, while AFP expression significantly increased. Both UCSCs and HLCs encapsulated in alginate scaffolds effectively attenuated biochemical tests, improved liver cytoarchitecture, increased expression of ALB and reduced AFP expression.
Finding of the present study indicated that encapsulation of UCSCs or HLCs in alginate mannuronic scaffolds effectively improve CCl4-induced ALF.

The current investigation was undertaken to evaluate the effects of 17β- estradiol (17β-ED) on the potential of the mesenchymal stem cells (MSCs) for modulation of immunity responses in an animal model of multiple sclerosis (MS).
After isolation of MSCs, cells were cultured in presence of 100 nM 17β-ED for 24 hr. Modeling of experimental autoimmune encephalomyelitis (EAE) was achieved by using guinea pig spinal cord homogenate, in addition to complete Freund’s adjuvant in male Wistar rats. The processes of cell therapy were started following 12 days post-immunization. This duration allows all animals to develop a disability score. The achieved EAE clinical symptoms were regularly monitored every day until day 36, when all of examined rats were euthanized.
Cell therapy in the EAE rats with 17β-ED-primed MSCs exhibited more desirable consequences, which in turn lead to regression of the cumulative clinical score and neuropathological changes that are more than the therapy with untreated MSCs. The serum measures of myeloperoxidase (MPO), nitric oxide (NO) as well as splenocytes-originated pro-inflammatory interleukin-17 (IL-17) and tumor necrosis factor alpha (TNF-α) were significantly decreased in EAE rats treated by 17β-ED primed-MSCs compared to EAE rats that received untreated MScs.
Combination of 17β-ED and MSCs more effectively improved the signs and symptoms of EAE.

This study aimed to investigate the effect of Shogaol on dextran sodium sulfate (DSS)- induced ulcerative colitis (UC) in mice compared to an immune-suppressant chemotherapeutic medicine, known as 6-thioguanine (6-TG).
Thirty-six adult BALB/c mice were divided into six groups: group 1 (positive control): no DSS exposure and no treatment; group 2 (negative control): DSS exposure without treatment; group 3 (vehicle control): DSS exposure and olive oil treatment; group 4: DSS exposure and 0.3 mg/kg 6-TG treatment; group 5: DSS exposure and 20 mg/kg Shogaol treatment; and group 6: DSS exposure and 40 mg/kg Shogaol treatment. At day 16, the mice were euthanized and UC was evaluated according to colon length, histologically index score and expression scores of the epidermal growth factor receptor (EGFR).
The disease activity index (DAI) and histological index scores of mice treated with 40 mg/kg body weight (BW) Shogaol were approximately lower than the corresponding scores of mice treated with 6-TG. In addition, the rate of healing in the former mice was approximately 3 folds higher than that of the latter ones as indicated by the lack of EGFR expression in colonic glands and macrophages.
These findings showed that the therapeutic effect of 40 mg/kg BW Shogaol could be better than 6-TG in the treatment of UC, and it may draw the attention regarding the priority of using this cheap plant-derived substance for treatment of the inflammatory bowel diseases because treatment with 6-TG is usually associated with adverse side effects.

Mucopolysaccharidosis VI (MPS VI) or Maroteaux-Lamy syndrome is a rare metabolic disorder, resulting from the deficient activity of the lysosomal enzyme arylsulfatase B (ARSB). The enzymatic defect of ARSB leads to progressive lysosomal storage disorder and accumulation of glycosaminoglycan (GAG) dermatan sulfate (DS), which causes harmful effects on various organs and tissues and short stature. To date, more than 160 different mutations have been reported in the ARSB gene.
Here, we analyzed 4 Iranian and 2 Afghan patients, with dysmorphism indicating MPS VI from North-east Iran. To validate the patients’ type of MPS VI, urine mucopolysaccharide and leukocyte ARSB activity were determined. Meanwhile, genomic DNA was amplified for all 8 exons and flanking intron sequences of the ARSB gene to analyze the spectrum of mutations responsible for the disorder in all patients.
Abnormal excretion of DS and low leukocyte ARSB activity were observed in the urine samples of all 6 studied patients. In direct DNA sequencing, we detected four different homozygous mutations in different exons, three of which seem not to have been reported previously: p.H178N, p.H242R, and p.*534W. All three novel substitutions were found in patients with Iranian breed. We further detected the IVS5>C mutation in Afghan siblings and four different homozygous polymorphisms, which have all been observed in other populations.
results indicated that missense mutations were the most common mutations in the ARSB gene, most of them being distributed throughout the ARSB gene and restricted to individual families, reflecting consanguineous marriages.

Vaccination is one of the most effective means to protect humans and animals against brucellosis. Live attenuated Brucella vaccines are considered effective in animals but they may be potentially infectious to humans, so it is vital to improve the immunoprotective effects and safety of vaccines against Brucella. This study was designed to evaluate the immunogenicity of DNA vaccines encoding B. melitensis outer membrane proteins (Omp25 and Omp31) against B. melitensis Rev1 in a mouse model.
For this propose, Omp25 and Omp31 genes were cloned (individually and together) into the eukaryotic expression vector pcDNA3.1/Hygro (). Expressions of recombinant plasmids were confirmed by SDS-PAGE and Western blot analysis. Six groups of BALB/c mice (seven mice per group) were intramuscularly injected with three recombinant constructs, native pcDNA3.1/Hygro () and phosphate-buffered saline (PBS) as controls and subcutaneous injection of attenuated live vaccine Rev1.
Results indicated that DNA vaccine immunized BALB/c mice had a dominant immunoglobulin G response and elicited a T-cell-proliferative response and induced significant levels of interferon gamma (INF-γ) compared to the control groups.
Collectively, these finding suggested that the pcDNA3.1/Hygro DNA vaccines encoding Omp25 and Omp31 genes and divalent plasmid were able to induce both humoral and cellular immunity, and had the potential to be a vaccine candidate for prevention of B. melitensis infections.

Bacterial cellulose (BC) has applications in medical science, it is easily synthesized, economic and purer compared to plant cellulose. The present study aimed to evaluate BC, a biocompatible natural polymer, as a scaffold for the bone marrow mesenchymal stem cells (BMSCs) loaded with fisetin, a phytoestrogen.
BC hydrogel scaffold was prepared from Gluconaceter xylinus and characterized through scanning electron microscopy (SEM). Biocompatibility of BC was measured by MTT assay, BMSCs were obtained from femur of rat and the osteogenic potential of the BC scaffold cultured with BMSCs and loaded with fisetin, was investigated by measuring the alkaline phosphatase (ALP) activity, alizarin red staining (ARS) and real-time PCR in terms of osteoblast-specific marker, osteocalcin (OCN) and osteopontin (OPN).
Biocompatibility results did not show any toxic effects of BC scaffold on BMSCs, while it increased cell viability. The data showed that BC loaded fisetin differentiated BMSCs into osteoblasts as demonstrated by ALP activity assays and ARS in vitro. Moreover, results from gene expression assay showed the expression of OCN and OPN genes was increased in cells that were seeded on the BC scaffold loaded with fisetin.
According to the results of the present study, BC loaded with fisetin is an effective strategy to promote osteogenic differentiation and a proper localized delivery system, which could be a potential candidate in bone tissue engineering.

This study aims to evaluate the activity of mangosteen peels extract (MPE) as protection agent on induced-glucose mesangial cells (SV40 MES 13 cell line (Glomerular Mesangial Kidney, Mus Musculus)).
MPE was performed based on maceration method. Cytotoxic assay was performed based on MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) method, while the level of TGF-β1 (Transforming growth factor-β1) and fibronectin in glucose-induced mesangial cells were assayed and determined using ELISA KIT.
In viability assay, MPE 5 and 20 µg/ml has the highest activity to increase cells proliferation in glucose-induced mesangial cells at 5, 10, and 15 days of incubation in glucose concentration (5 and 25 mM) (P MPE can increase cell proliferation in glucose-induced mesangial cells and significantly reduce the level of TGF-β1 and fibronectin. MPE activity has correlates to inhibit the diabetic glomerulosclerosis condition and may increase mesangial cell proliferation.