Designing a Secretory form of RTX-A as an Anticancer Toxin: An In Silico Approach.

BACKGROUND: Cancer is a leading cause of death and a significant public health issue worldwide. Standard treatment methods such as chemotherapy, radiotherapy, and surgery are only sometimes effective. Therefore, new therapeutic approaches are needed for cancer treatment. Sea anemone actinoporins are pore-forming toxins (PFTs) with membranolytic activities. RTX-A is a type of PFT that interacts with membrane phospholipids, resulting in pore formation. The synthesis of recombinant proteins in a secretory form has several advantages, including protein solubility and easy purification. In this study, we aimed to discover suitable signal peptides for producing RTX-A in Bacillus subtilis in a secretory form. METHODS: Signal peptides were selected from the Signal Peptide Web Server. The probability and secretion pathways of the selected signal peptides were evaluated using the SignalP server. ProtParam and Protein-sol were used to predict the physico-chemical properties and solubility. AlgPred was used to predict the allergenicity of RTX-A linked to suitable signal peptides. Non-allergenic, stable, and soluble signal peptides fused to proteins were chosen, and their secondary and tertiary structures were predicted using GOR IV and I-TASSER, respectively. The PROCHECK server performed the validation of 3D structures. RESULTS: According to bioinformatics analysis, the fusion forms of OSMY_ECOLI and MALE_ECOLI linked to RTX-A were identified as suitable signal peptides. The final proteins with signal peptides were stable, soluble, and non-allergenic for the human body. Moreover, they had appropriate secondary and tertiary structures. CONCLUSION: The signal above peptides appears ideal for rationalizing secretory and soluble RTX-A. Therefore, the signal peptides found in this study should be further investigated through experimental researches and patents.

DNA binding properties and cytotoxic effects of two double rollover cycloplatinated (II) complexes on cancer cell lines.

In this study, the DNA binding capacity and cytotoxic effects of two double rollovers cycloplatinated complexes, [Pt2(µ-bpy-2H)(CF3COO)2(PPh3)2] and [Pt2(µ-bpy-2H)(I)2(PPh3)2] denoted as C1 and C2, respectively, were evaluated. By using UV-Visible spectroscopy the intrinsic binding constant (Kb) of C1 and C2 to DNA were determined as 2.9 × 105 M-1, and 5.4 × 105 M-1, respectively. Both the compounds were able to quench the fluorescence of ethidium bromide as a well known DNA intercalator. The calculated Stern-Volmer quenching constants (Ksv) for C1 and C2 were 3.5 × 103 M-1, and 1.2 × 104 M-1, respectively. Upon interaction of both the compounds with DNA, increase in viscosity of DNA solution were observed, further confiming the involvement of intercalative interactions between the complexes and DNA. The cytotoxic effects of complexes in compare to cisplatin were evaluated on different cancer cell lines by MTT assay. Interestingly, C2 showed the highest cytotoxicity on A2780R, a cisplatin resistant-cell line. Induction of apoptosis by the complexes was proved by flowcytometry. In all the studied cell lines, the extent of apoptosis induced by C2 was comparable or higher than cisplatin. Cisplatin induced more necrosis in all the cancer cell lines in the tested concentration.

Review of electrochemical and optical biosensors for testosterone measurement.

Testosterone is an anabolic steroid and a major sex hormone in males. It plays vital roles, including developing the testis, penis, and prostate, increasing muscle and bone, and sperm production. In both men and women, testosterone levels should be in normal ranges. Besides, testosterone and its analogs are major global contributors to doping in sport. Due to the importance of testosterone testing, novel, accurate biosensors have been developed. This review summarizes the various methods for testosterone measurement. Also, recent optical and electrochemical approaches for the detection of testosterone and its analogs have been discussed.

Non-coding RNAs in gynecologic cancer.

The term "gynecologic cancer" pertains to neoplasms impacting the reproductive tissues and organs of women encompassing the endometrium, vagina, cervix, uterus, vulva, and ovaries. The progression of gynecologic cancer is linked to various molecular mechanisms. Historically, cancer research primarily focused on protein-coding genes. However, recent years have unveiled the involvement of non-coding RNAs (ncRNAs), including microRNAs, long non-coding RNAs (LncRNAs), and circular RNAs, in modulating cellular functions within gynecological cancer. Substantial evidence suggests that ncRNAs may wield a dual role in gynecological cancer, acting as either oncogenic or tumor-suppressive agents. Numerous clinical trials are presently investigating the roles of ncRNAs as biomarkers and therapeutic agents. These endeavors may introduce a fresh perspective on the diagnosis and treatment of gynecological cancer. In this overview, we highlight some of the ncRNAs associated with gynecological cancers.

Exosomal noncoding RNAs in prostate cancer.

Prostate cancer (PCa) is the second most common cancer and the fifth leading cause of mortality among men. The recurrent reports of false-positive results of common PCa biomarkers have led to the introduction of some promising biomarkers for PCa, such as exosomal non-coding RNAs (ncRNAs). Exosomes contain various components, such as several ncRNAs (miRNAs and lncRNAs), which are important in the initiation and progression of PCa. These ncRNAs also reflect the state of the origin cell. In this article, we reviewed research on the importance and roles of ncRNAs in PCa, focusing on exosomal ncRNAs. We highlighted plasma exosomal miRNAs (8 miRNAs), urine exosomal miRNAs (19miRNAs), serum miRNAs (2 miRNAs), and five miRNAs in semen used for PCa diagnosis. Also, four exosomal lncRNAs in plasma and urine can be used as biomarkers for PCa diagnosis.

Aptamer-based biosensors for Pseudomonas aeruginosa detection.

Pseudomonas aeruginosa possesses innate antibiotic resistance mechanisms, and carbapenem-resistant Pseudomonas aeruginosa has been considered the number one priority in the 2017 WHO list of antimicrobial-resistant crucial hazards. Early detection of Pseudomonas aeruginosa can circumvent treatment challenges. Various techniques have been developed for the detection of P. aeruginosa detection. Biosensors have recently attracted unprecedented attention in the field of point-of-care diagnostics due to their easy operation, rapid, low cost, high sensitivity, and selectivity. Biosensors can convert the specific interaction between bioreceptors (antibodies, aptamers) and pathogens into optical, electrical, and other signal outputs. Aptamers are novel and promising alternatives to antibodies as biorecognition elements mainly synthesized by systematic evolution of ligands by exponential enrichment and have predictable secondary structures. They have comparable affinity and specificity for binding to their target to antibody recognition. Since 2015, there have been about 2000 journal articles published in the field of aptamer biosensors, of which 30 articles were on the detection of P. aeruginosa. Here, we have focused on outlining the recent progress in the field of aptamer-based biosensors for P. aeruginosa detection based on optical, electrochemical, and piezoelectric signal transduction methods.

Long non-coding RNAs (lncRNAs); roles in tumorigenesis and potentials as biomarkers in cancer diagnosis.

New research has indicated that long non-coding RNAs (lncRNAs) play critical roles in a broad range of biological processes, including the pathogenesis of many complex human diseases, including cancer. The detailed regulation mechanisms of many lncRNAs in cancer initiation and progression have yet to be discovered, even though a few of lncRNAs' functions in cancer have been characterized. In the present study, we summarize recent advances in the mechanisms and functions of lncRNAs in cancer. We focused on the roles of newly-identified lncRNAs as oncogenes and tumor suppressors, as well as the potential pathways these molecules could play. The paper also discusses their potential uses as biomarkers for the diagnosis and prognosis of cancer.

A Novel miRNA Located in the HER2 Gene Shows an Inhibitory Effect on Wnt Signaling and Cell Cycle Progression.

Human epidermal growth factor receptor 2 (HER2) is involved in the development of the majority of cancers. Therefore, it can be a potential target for cancer therapy. It was hypothesized that some of the broad effects of HER2 could be mediated by miRNAs that are probably embedded inside this gene. Here, we predicted and then empirically substantiated the processing and expression of a novel miRNA named HER2-miR1, located in the HER2 gene; transfection of a DNA fragment corresponding to HER2-miR1 precursor sequence (preHER2-miR1) resulted in ~4000-fold elevation of HER2-miR1 mature form in HEK293t cells. Also, the detection of HER2-miR1 in 5637, NT2, and HeLa cell lines confirmed its endogenous production. Following the HER2-miR1 overexpression, TOP/FOP flash assay and RT-qPCR results showed that Wnt signaling pathway was downregulated. Consistently, flow cytometry results revealed that overexpression of HER2-miR1 in Wnt+ cell lines (SW480 and HCT116) was ended in G1 arrest, unlike in Wnt- cells (HEK293t). Taking everything into account, our results report the discovery of a novel miRNA that is located within the HER2 gene sequence and has a repressive impact on the Wnt signaling pathway.

Mammalian target of rapamycin (mTOR) signaling pathway and traumatic brain injury: A novel insight into targeted therapy.

Traumatic brain injury (TBI) is one of the most concerning health issues in which the normal brain function may be disrupted as a result of a blow, bump, or jolt to the head. Loss of consciousness, amnesia, focal neurological defects, alteration in mental state, and destructive diseases of the nervous system such as cognitive impairment, Parkinson's, and Alzheimer's disease. Parkinson's disease is a chronic progressive neurodegenerative disorder, characterized by the early loss of striatal dopaminergic neurons. TBI is a major risk factor for Parkinson's disease. Existing therapeutic approaches have not been often effective, indicating the necessity of discovering more efficient therapeutic targets. The mammalian target of rapamycin (mTOR) signaling pathway responds to different environmental cues to modulate a large number of cellular processes such as cell proliferation, survival, protein synthesis, autophagy, and cell metabolism. Moreover, mTOR has been reported to affect the regeneration of the injured nerves throughout the central nervous system (CNS). In this context, recent evaluations have revealed that mTOR inhibitors could be potential targets to defeat a group of neurological disorders, and thus, a number of clinical trials are investigating their efficacy in treating dementia, autism, epilepsy, stroke, and brain injury, as irritating neurological defects. The current review describes the interplay between mTOR signaling and major CNS-related disorders (esp. neurodegenerative diseases), as well as the mTOR signaling-TBI relationship. It also aims to discuss the promising therapeutic capacities of mTOR inhibitors during the TBI.

Development of a recombinant nucleocapsid protein-based ELISA for the detection of IgM and IgG antibodies to SARS-CoV-2.

Coronavirus 2019 (COVID-19) is a global concern for public health. Thus, early and accurate diagnosis is a critical step in management of this infectious disease. Currently, RT-PCR is routine diagnosis test for COVID-19, but it has some limitations and false negative results. enzyme-linked immunosorbent assay (ELISA) against SARS-CoV-2 antigens seems to be an appropriate approach for serodiagnosis of COVID-19. In the current study, an ELISA system, using a recombinant nucleocapsid (N) protein, was developed for the detection of IgM and IgG antibodies to SARS-CoV-2. The related protein was expressed, purified, and used in an ELISA system. Sera samples (67) for COVID-19 patients, as well as sera samples from healthy volunteers (112), along with sera samples from non-COVID-19 patients were examined by the ELISA system. The expression and purity of the recombinant N protein were approved by SDS-PAGE and Western blotting. The sensitivity of ELISA system was 91.04 and 92.53% for the detection of IgG and IgM antibodies, respectively. Moreover, the specificity of the developed ELISA system for IgG and IgM were 98.21 and 97.32%, respectively. Our developed ELISA system showed satisfactory sensitivity and specificity for the detection of antiSARS-CoV-2 IgM and IgG antibodies and could be used as a complementary approach for proper diagnosis of COVID-19.

New Insights into the Biosensing of Parkinson's Disease Biomarkers: A Concise Review.

BACKGROUND: Parkinson's disease (PD) is a long-term, degenerative, and neurological disease in which a person loses control of certain body functions. The formulation of novel effective therapeutics for PD as a neurodegenerative disease requires accurate and efficient diagnosis at the early stages. OBJECTIVE: Analyzing data gathered by measurable signals converted from biological reactions allows for qualitative and quantitative evaluations. Among various approaches reported so far, biosensors are powerful analytical tools that have been used in detecting the biomarkers of PD. METHODS: Biosensor's biological recognition components include antibodies, receptors, microorganisms, nucleic acids, enzymes, cells and tissues, and biomimetic structures. This review introduces electrochemical, optical, and optochemical detection of PD biomarkers based on recent advances in nanotechnology and material science, which resulted in the development of high-performance biosensors in this field. RESULTS: PD biomarkers such as α-synuclein protein, dopamine (DA), urate, ascorbic acid, miRNAs, and their biological roles are summarized. Additionally, the advantages and disadvantages of the usual standard methods are reviewed. We compared electrochemical, optical, and optochemical biosensors' properties and novel strategies for higher sensitivity and selectivity. CONCLUSION: The development of novel biosensors is required for the early diagnosis of PD as sensitive, rapid, reliable, and cost-effective systems.

A brief overview on the application and sources of α-amylase and expression hosts properties in order to production of recombinant α-amylase.

By reducing the activation energy, enzymes accelerate the chemical reaction; therefore, they are good alternative for industrial catalysts. Amylase is a suitable enzyme as a catalyst for the chemical decomposition of starch. This enzyme is of great importance, and its production is highly profitable. α-Amylase is among the most important amylases produced naturally by animals, plants, and microorganisms. Still, the α-amylases produced by bacteria have a special place in industry and commerce. Moreover, a large volume of this enzyme can be produced by selecting an appropriate and optimized host to clone and express the α-amylase gene. The present study briefly reviews the structure, application, sources, and hosts used to produce recombinant α-amylase.

ß-Galactosidase: From its source and applications to its recombinant form.

Carbohydrate-active enzymes are a group of important enzymes playing a critical role in the degradation and synthesis of carbohydrates. Glycosidases can hydrolyze glycosides into oligosaccharides, polysaccharides, and glycoconjugates via a cost-effective approach. Lactase is an important member of ß-glycosidases found in higher plants, animals, and microorganisms. ß-Galactosidases can be used to degrade the milk lactose for making lactose-free milk, which is sweeter than regular milk and is suitable for lactose-intolerant people. ß-Galactosidase is employed by many food industries to degrade lactose and improve the digestibility, sweetness, solubility, and flavor of dairy products. ß-Galactosidase enzymes have various families and are applied in the food-processing industries such as hydrolyzed-milk products, whey, and galactooligosaccharides. Thus, this enzyme is a valuable protein which is now produced by recombinant technology. In this review, origins, structure, recombinant production, and critical modifications of ß-galactosidase for improving the production process are discussed. Since ß-galactosidase is a valuable enzyme in industry and health care, a study of its various aspects is important in industrial biotechnology and applied biochemistry.

Bispecific antibodies in colorectal cancer therapy: recent insights and emerging concepts.

Colorectal cancer (CRC) is identified as a life-threatening malignancy. Despite several efforts and proceedings available for CRC therapy, it is still a health concern. Among a vast array of novel therapeutic procedures, employing bispecific antibodies (BsAbs) is currently considered to be a promising approach for cancer therapy. BsAbs, as a large family of molecules designed to realize two distinct epitopes or antigens, can be beneficial microgadgets to target the tumor-associated antigen pairs. On the other hand, applying the immune system's capabilities to attack malignant cells has been proven as a tremendous development in cancer therapeutic projects. The current study has attempted to overview some of the approved BsAbs in CRC therapy and those under clinical trials. For this purpose, reputable scientific search engines and databases, such as PubMed, ScienceDirect, Google Scholar, Scopus, etc., were explored using the keywords 'bispecific antibodies', 'colorectal cancer', 'immunotherapy' and 'tumor markers'.

microRNAs in female infertility: An overview.

Infertility impacts a considerable number of women worldwide, and it affects different aspects of family life and society. Although female infertility is known as a multifactorial disorder, there are strong genetic and epigenetic bases. Studies revealed that miRNAs play critical roles in initiation and development of female infertility related disorders. Early diagnosis and control of these diseases is an essential key for improving disease prognosis and reducing the possibility of infertility and other side effects. Investigating the possible use of miRNAs as biomarkers and therapeutic options is valuable, and it merits attention. Thus, in this article, we reviewed research associated with female diseases and highlighted microRNAs that are related to the polycystic ovary syndrome (up to 30 miRNAs), premature ovarian failure (10 miRNAs), endometriosis (up to 15 miRNAs), uterine fibroids (up to 15 miRNAs), endometrial polyp (3 miRNAs), and pelvic inflammatory (6 miRNAs), which are involved in one or more ovarian or uterine disease-causing processes.

Therapeutic Effects of Resveratrol on Ischemia-Reperfusion Injury in the Nervous System.

Resveratrol is a phenol compound produced by some plants in response to pathogens, infection, or physical injury. It is well-known that resveratrol has antioxidant and protective roles in damages potentially caused by cancer or other serious disorders. Thus, it is considered as a candidate agent for the prevention and treatment of human diseases. Evidence has confirmed other bioactive impacts of resveratrol, including cardioprotective, anti-tumorigenic, anti-inflammatory, phytoestrogenic, and neuroprotective effects. Ischemia-reperfusion (IR) can result in various disorders, comprising myocardial infarction, stroke, and peripheral vascular disease, which may continue to induce debilitating conditions and even mortality. In virtue of chronic ischemia or hypoxia, cells switch to anaerobic metabolism, giving rise to some dysfunctions in mitochondria. As the result of lactate accumulation, adenosine triphosphate levels and pH decline in cells. This condition leads cells to apoptosis, necrosis, and autophagy. However, restoring oxygen level upon reperfusion after ischemia by producing reactive oxygen species is an outcome of mitochondrial dysfunction. Considering the neuroprotective effect of resveratrol and neuronal injury that comes from IR, we focused on the mechanism(s) involved in IR injury in the nervous system and also on the functions of resveratrol in the protection, inhibition, and treatment of this injury.

In Silico Design and Evaluation of PRAME+FliCΔD2D3 as a New Breast Cancer Vaccine Candidate.

Background: The most prevalent cancer in women over the world is breast cancer. Immunotherapy is a promising method to effectively treat cancer patients. Among various immunotherapy methods, tumor antigens stimulate the immune system to eradicate cancer cells. Preferentially expressed antigen in melanoma (PRAME) is mainly overexpressed in breast cancer cells, and has no expression in normal tissues. FliCΔD2D3, as truncated flagellin (FliC), is an effective toll-like receptor 5 (TLR5) agonist with lower inflammatory responses. The objective of the present study was to utilize bioinformatics methods to design a chimeric protein against breast cancer. Methods: The physicochemical properties, solubility, and secondary structures of PRAME+FliCΔD2D3 were predicted using the tools ProtParam, Protein-sol, and GOR IV, respectively. The 3D structure of the chimeric protein was built using I-TASSER and refined with GalaxyRefine, RAMPAGE, and PROCHECK. ANTIGENpro and VaxiJen were used to evaluate protein antigenicity, and allergenicity was checked using AlgPred and Allergen FP. Major histocompatibility complex )MHC( and cytotoxic T-lymphocytes )CTL( binding peptides were predicted using HLApred and CTLpred. Finally, B-cell continuous and discontinuous epitopes were predicted using ABCpred and ElliPro, respectively. Results: The stability and solubility of PRAME+FliCΔD2D3 were analyzed, and its secondary and tertiary structures were predicted. The results showed that the derived peptides could bind to MHCs and CTLs. The designed chimeric protein possessed both linear and conformational epitopes with a high binding affinity to B-cell epitopes. Conclusion: PRAME+FliCΔD2D3 is a stable and soluble chimeric protein that can stimulate humoral and cellular immunity. The obtained results can be utilized for the development of an experimental vaccine against breast cancer.

microRNA in inflammatory bowel disease at a glance.

Inflammatory bowel disease (IBD) as a chronic inflammation in colon and small intestine has two subtypes: ulcerative colitis (UC) and Crohn's disease (CD). Genome studies have shown that UC and CD are related to microRNAs (miRNAs) expression in addition to environmental factors. This article reviews important researches that have recently been done on miRNAs roles in CD and UC disease. First, miRNA is introduced and its biogenesis and function are discussed. Afterward, roles of miRNAs in inflammatory processes involved in IBD are showed. Finally, this review proposes some circulating and tissue-specific miRNAs, which are useful for CD and UC fast diagnosis and grade prediction. As a conclusion, miRNAs are efficient diagnostic molecules especially in IBD subtypes discrimination and can be used by microarray and real time PCR methods for disease detection and classification.

Design and evaluation of scFv-RTX-A as a novel immunotoxin for breast cancer treatment: an in silico approach.

Human epidermal growth factor receptor 2 (HER2) is overexpressed in breast cancer (BC) patients. Hence, immunotherapy is a proper treatment option for HER2-positive BC patients. Accumulating evidence has indicated that immunotoxin therapy is a novel approach to improve the potency of targeted therapy. Immunotoxins are antibodies or antibody fragments coupled with a toxin. We designed an immunotoxin. The physicochemical properties were evaluated using ProtParam servers and secondary structure was examined by PROSO II and GORV. Using I-TASSER, a 3D model was built and refined by GalaxyRefine. The model was validated using PROCHECK and RAMPAGE. To predict immunotoxin allergenicity and mRNA stability, AlgPred server and RNAfold were used. Furthermore, the immunotoxin and HER2 were docked by ZDOCK. The scFv+RTX-A could be a non-allergenic and stable chimeric protein, and the secondary structure of its components did not alter, and this protein had a proper 3D structure that might have stable mRNA structure which could bind to HER2. Given the fact that the designed immunotoxin was a non-allergenic and stable chimeric protein and that it could bind with high affinity to HER2 receptors, we proposed that this chimeric protein could be a useful candidate for HER-2 positive BC patients.

Suitable Signal Peptides for Secretory Production of Recombinant Granulocyte Colony Stimulating Factor in Escherichia coli.

BACKGROUND: Granulocyte colony-stimulating factor (G-CSF) expressed in engineered Escherichia coli (E. coli) as a recombinant protein is utilized as an adjunct to chemotherapy for improving neutropenia. Recombinant proteins overexpression may lead to the creation of inclusion bodies whose recovery is a tedious and costly process. To overcome the problem of inclusion bodies, secretory production might be used. To achieve a mature secretory protein product, suitable signal peptide (SP) selection is a vital step. OBJECTIVE: In the present study, we aimed at in silico evaluation of proper SPs for secretory production of recombinant G-CSF in E. coli. METHODS: Signal peptide website and UniProt were used to collect the SPs and G-CSF sequences. Then, SignalP were utilized in order to predict the SPs and location of their cleavage site. Physicochemical features and solubility were investigated by ProtParam and Protein-sol tools. Fusion proteins sub-cellular localization was predicted by ProtCompB. RESULTS: LPP, ELBP, TSH, HST3, ELBH, AIDA and PET were excluded according to SignalP. The highest aliphatic index belonged to OMPC, TORT and THIB and PPA. Also, the highest GRAVY belonged to OMPC, ELAP, TORT, BLAT, THIB, and PSPE. Furthermore, G-CSF fused with all SPs were predicted as soluble fusion proteins except three SPs. Finally, we found OMPT, OMPF, PHOE, LAMB, SAT, and OMPP can translocate G-CSF into extracellular space. CONCLUSION: Six SPs were suitable for translocating G-CSF into the extracellular media. Although growing data indicate that the bioinformatics approaches can improve the precision and accuracy of studies, further experimental investigations and recent patents explaining several inventions associated to the clinical aspects of SPs for secretory production of recombinant GCSF in E. coli are required for final validation.