Mycobacterium tuberculosis (M. tuberculosis) is the causative agent of tuberculosis in human. One of the major M. tuberculosis virulence factors is early secretory antigenic target of 6-kDa (ESAT-6), and EccB5 protein encoded by eccB5 is one of its components. EccB5 protein is a transmembrane protein in ESX-5 system. The aim of this study is to explore the characteristics of wild-type EccB5 and its mutant form N426I. We expressed the EccB5 protein by cloning the mutant and wild-type eccB5 gene in Escherichia coli (E. coli). We compared the protein structure of wild type and mutant form of EccB5 and found changes in structure around Asn426 (loop structure) in wild type and around Ile426 (β-strand) in the mutant. The truncated recombinant protein of EccB5 was successfully cloned and expressed using plasmid pCold I in E. coli DH5α and E. coli strain Rosetta-gami B (DE3) and purified as a 38.6 kDa protein by using the affinity column. There was no detectable adenosine triphosphatase activity in truncated forms of EccB5 and its mutant. In conclusion, our study reveals successful cloning and protein expression of truncated form of eccB5 gene of M. tuberculosis. EccB5 protein in ESX-5 system may be an important membrane component involved in the transport machinery of type VII secretion system, which is essential for growth and virulence.
Ovarian carcinogenesis can be induced by a large number of somatic gene mutations. Circulating tumor DNA (ctDNA) released into peripheral blood can provide insights into the genomic landscape of cancer cells and monitor their dynamics. Our aim was to detect and compare the genetic profiles in tumor tissue and plasma before and after tumor resection in ovarian cancer patients. All three samples were collected from each patient. In this study, we used a commercial cancer panel to identify somatic mutations in 26 genes in seven selected patients through next-generation sequencing on the Illumina platform. Overall, 16 variants with pathogenic effect were identified in the TP53, PIK3CA, PTEN, APC, NRAS, KRAS, GNAS, and MET genes involved in important signaling pathways. The genetic alterations found in the presurgical plasma in six of seven ovarian cancer patients were no longer present in the plasma after tumor surgical removal. Identical variants in formalin-fixed paraffin embedded (FFPE) tissues and preoperative plasma specimens were observed in only two cases. These findings suggest that the detected presurgical pathogenic variants absent in postsurgery plasma are associated with the primary ovarian tumor. Finally, the low-identified concordance between FFPE and plasma can be due to various factors, but most likely to high tumor heterogeneity and low ctDNA level.
Osteoarthritis (OA) of the knee causes disability, pain, and progressive destruction of cartilage in adult women. The objective of the study was to evaluate the concentrations of the urinary biomarker C-terminal telopeptide of type II collagen (CTX-II) and pain by radiographic grade in women with knee OA in northeastern Mexico: Cross-sectional study of 155 women with knee OA. Concentrations of biochemical parameters were evaluated and urine samples were collected to measure biomarker levels (uCTX-II) ng/mmol by competitive enzyme-linked immunoabsorbent assay (ELISA) technique and the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) scale was used for pain classification; median age of 49 years and 29.1 kg/m2 of body mass index (BMI). uCTX-II biomarker levels were grade 2 (210.7 ng/mmol), grade 3 (314.8 ng/mmol), and grade 4 (478.8 ng/mmol) relative to Kellgren and Lawrence, uCTX-II levels were compared with WOMAC scale and presented significant statistical difference (p = 0.0001). An association of the biomarker CTX-II and an increase in BMI was found in female patients with knee OA (odds ratio = 1.01; 95% confidence interval 1.001-1.005; p = 0.047).This study demonstrates an increase in the levels of the biomarker uCTX-II, the degree of pain, and radiographic grade in women with knee OA in northeastern Mexico.
Breast cancer is the major mortality cause of women worldwide. In the course of management of breast cancer, the identification of a biomarker is important in enhancing our knowledge on cancer pathology, predicting the response to treatment, and selecting the patients who are more favorable to receive certain treatments. These biomarkers have a prognostic value. In addition to traditional breast cancer prognosis factors such as the tumor size and grade, the axillary lymph node micrometastasis, and biomarkers such as HER2/neu, newly discovered biomarkers have been discovered. Some of these factors are genetic signature in tissue or in peripheral blood. Lipid profil, a simple and accessible biological examination, has been a novel path on the prediction of breast cancer risk of occurrence and recurrence in many studies. The main goal of our review is to evaluate lipid profile and breast cancer risk with an emphasis on the prognosis value of lipid profiles in breast cancer patient management.
Eight percent of women suffer from vulvodynia, a chronic pain condition with unknown etiology. Inflammation and dysregulation of estrogen signaling have been suggested to play a role in the pathogenesis of localized provoked vulvodynia (LPV). Therefore, the aim of the study was to analyze protein expression levels of estrogen-related receptors ERRα, ERRß, ERRγ, estrogen receptor (ERα), and progesterone receptor (PRα) and CD3-positive T cells in the vulvar vestibulum obtained from women suffering from LPV in comparison to healthy, unaffected controls. Vulvar vestibulum tissue specimens were obtained from LPV patients (n = 12) who had undergone modified posterior vestibulectomy and from 15 healthy controls. Protein expression of ERRα, ERRß, ERRγ, ERα, and PRα and CD3-positive T cells was analyzed by immunohistochemistry (IHC). Expression of ERRß was significantly more pronounced in samples from LPV compared to healthy controls (p = 0.006). No significant difference in the expression patterns of ERRα, ERRγ, ERα, PRα, or CD3 cells was detected. To our knowledge, this is the first study reporting ERR expression in normal vestibulum and in vestibulectomy samples from LPV patients. The higher level of ERRß expression detected by IHC may reflect dysregulation of estrogen signaling in LPV.
The generation of induced pluripotent stem cells (iPSCs) from differentiated mature cells is one of the most promising technologies in the field of regenerative medicine. The ability to generate patient-specific iPSCs offers an invaluable reservoir of pluripotent cells, which could be genetically engineered and differentiated into target cells to treat various genetic and degenerative diseases once transplanted, hence counteracting the risk of graft versus host disease. In this context, we review the scientific research streams that lead to the emergence of iPSCs, the roles of reprogramming factors in reprogramming to pluripotency, and the reprogramming strategies. As iPSCs serve tremendous correction potentials for various diseases, we highlight the successes and challenges of iPSCs in cell replacement therapy and the synergy of iPSCs and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing tools in therapeutics research.
In this review we outline a rationale for identifying neuroprotectants aimed at inducing endogenous Klotho activity and expression, which is epigenetic action, by definition. Such an approach should promote remyelination and/or stimulate myelin repair by acting on mitochondrial function, thereby heralding a life-saving path forward for patients suffering from neuroinflammatory diseases. Disorders of myelin in the nervous system damage the transmission of signals, resulting in loss of vision, motion, sensation, and other functions depending on the affected nerves, currently with no effective treatment. Klotho genes and their single-pass transmembrane Klotho proteins are powerful governors of the threads of life and death, true to the origin of their name, Fates, in Greek mythology. Among its many important functions, Klotho is an obligatory co-receptor that binds, activates, and/or potentiates critical fibroblast growth factor activity. Since the discovery of Klotho a little over two decades ago, it has become ever more apparent that when Klotho pathways go awry, oxidative stress and mitochondrial dysfunction take over, and age-related chronic disorders are likely to follow. The physiological consequences can be wide ranging, potentially wreaking havoc on the brain, eye, kidney, muscle, and more. Central nervous system disorders, neurodegenerative in nature, and especially those affecting the myelin sheath, represent worthy targets for advancing therapies that act upon Klotho pathways. Current drugs for these diseases, even therapeutics that are disease modifying rather than treating only the symptoms, leave much room for improvement. It is thus no wonder that this topic has caught the attention of biomedical researchers around the world.
Instead of the silicone implants previously used for repair and reconstruction of the auricle and nose lost due to accidents and disease, a new treatment method using tissue-engineered cartilage has been attracting attention. The quality of cultured cells is important in this method because it affects treatment outcomes. However, a marker of chondrocytes, particularly auricular chondrocytes, has not yet been established. The objective of this study was to establish an optimal marker to evaluate the quality of cultured auricular chondrocytes as a cell source of regenerative cartilage tissue. Gene expression levels were comprehensively compared using the microarray method between human undifferentiated and dedifferentiated auricular chondrocytes to investigate a candidate quality control index with an expression level that is high in differentiated cells, but markedly decreases in dedifferentiated cells. We identified glial fibrillary acidic protein (GFAP) as a marker that decreased with serial passages in auricular chondrocytes. GFAP was not detected in articular chondrocytes, costal chondrocytes, or fibroblasts, which need to be distinguished from auricular chondrocytes in cell cultures. GFAP mRNA expression was observed in cultured auricular chondrocytes, and GFAP protein levels were also measured in the cell lysates and culture supernatants of these cells. However, GFAP levels detected from mRNA and protein in cell lysates were significantly decreased by increases in the incubation period. In contrast, the amount of protein in the cell supernatant was not affected by the incubation period. Furthermore, the protein level of GFAP in the supernatants of cultured cells correlated with the in vitro and in vivo production of the cartilage matrix by these cells. The productivity of the cartilage matrix in cultured auricular chondrocytes may be predicted by measuring GFAP protein levels in the culture supernatants of these cells. Thus, GFAP is regarded as a marker of the purity and properties of cultured auricular chondrocytes.
Many studies have shown that mitochondrial metabolism has a fundamental role in induction of carcinogenesis due to the influence of increased levels of reactive oxygen species (ROS) generation in all steps of oncogene transformation and cancer progression. It is widely accepted that the anticancer effect of conventional anticancer drugs is due to induction of oxidative stress and elevated intracellular levels of ROS, which alter the redox homeostasis of cancer cells. On the other hand, the harmful side effects of conventional anticancer chemotherapeutics are also due to increased production of ROS and disruption of redox homeostasis of normal cells and tissues. Therefore, there is a growing interest toward the development of natural antioxidant compounds from various sources, which could impact the redox state of cancer and normal cells by different pathways and could prevent damage from oxidant-mediated reactions. It is known that chitosan exhibits versatile biological properties, including biodegradability, biocompatibility, and a less toxic nature. Because of its antioxidant, antibacterial, anticancer, anti-inflammatory, and immunostimulatory activities, the biopolymer has been used in a wide variety of pharmaceutical, biomedical, food industry, health, and agricultural applications and has been classified as a new physiologically bioactive material.
Mycobacterium tuberculosis (M. tuberculosis) is the causative agent of tuberculosis in human. One of the major M. tuberculosis virulence factors is early secretory antigenic target of 6-kDa (ESAT-6), and EccB5 protein encoded by eccB5 is one of its components. EccB5 protein is a transmembrane protein in ESX-5 system. The aim of this study is to explore the characteristics of wild-type EccB5 and its mutant form N426I. We expressed the EccB5 protein by cloning the mutant and wild-type eccB5 gene in Escherichia coli (E. coli). We compared the protein structure of wild type and mutant form of EccB5 and found changes in structure around Asn426 (loop structure) in wild type and around Ile426 (β-strand) in the mutant. The truncated recombinant protein of EccB5 was successfully cloned and expressed using plasmid pCold I in E. coli DH5α and E. coli strain Rosetta-gami B (DE3) and purified as a 38.6 kDa protein by using the affinity column. There was no detectable adenosine triphosphatase activity in truncated forms of EccB5 and its mutant. In conclusion, our study reveals successful cloning and protein expression of truncated form of eccB5 gene of M. tuberculosis. EccB5 protein in ESX-5 system may be an important membrane component involved in the transport machinery of type VII secretion system, which is essential for growth and virulence.
Crimean-Congo hemorrhagic fever (CCHF) is a severe human disease with mortality rates of up to 30%. The disease is widespread in Africa, Asia, the Middle East and Eastern Europe. The last few years have seen disease emergence in Spain for the first time and disease re-emergence in other regions of the world after periods of inactivity. Factors, such as climate change, movement of infected ticks, animals, and changes in human activity, are likely to broaden endemic foci. There are therefore concerns that CCHF might emerge in currently nonendemic regions. The absence of approved vaccines or therapies heightens these concerns; thus Crimean-Congo hemorrhagic fever virus (CCHFV) is listed by the World Health Organization as a priority organism. However, the current sporadic nature of CCHF cases may call for targeted vaccination of risk groups as opposed to mass vaccinations. CCHF vaccine development has accelerated in recent years, partly because of the discovery of CCHF animal models. In this review, we discuss CCHF risk groups who are most likely to benefit from vaccine development, the merits and demerits of available CCHF animal models, and the various approaches which have been explored for CCHF vaccine development. Lastly, we present concluding remarks and research areas which can be further explored to enhance the available CCHFV vaccine data.
A number of packaging systems are available for production of recombinant adeno-associated virus vectors (rAAVs). Among these, the use of a two-plasmid cotransfection system, in which Rep and Cap genes and Ad helper genes are on the same plasmid, has not been frequently employed for good manufacturing practices (GMP) production, even though it presents some practical advantages over the common three-plasmid (triple) transfection method. To confirm and expand the utility of the two-plasmid system, we generated GMP-compatible versions of this system and used those package reporter genes in multiple capsid variants in direct comparison with triple transfection. Vector yields, purity, and empty-to-full ratios were comparable between double and triple transfection methods for all capsid variants tested. We performed an in vivo side-by-side comparison of double and triple transfection vectors following both intravenous injection and intramuscular injection in mice. Expression and transduction were evaluated in muscle and liver 4 weeks after injection. Additional studies of bioactivity were conducted in vivo using packaged vectors carrying a variety of cargos, including the therapeutic transgene, microRNA, and single- or double-stranded vector. Results showed that cargos packaged using double transfection were equivalently bioactive to those packaged using a triple transfection system. In conclusion, these data suggest the utility of midrange (1E12-1E16) GMP-compatible packaging of adeno-associated virus (AAV) vectors for several AAV capsids.
The interest in extracting kafirins (KAF), the main storage protein from sorghum grain has recently increased due to its gluten-free content and the significant scientific evidence showing the health benefits of the bioactive peptides from cereal grains in human diets. The objectives were to obtain the highest percentage of KAF extraction using amyloglucosidase as pretreatment to increase the extraction yield and predict the bioactive peptides in the KAF. In this study, pretreatments with amyloglucosidase increased the extraction yield of KAF compared with extraction methods using only ethanol and sodium metabisulfite. Two protein fragment sequences were identified from KAF extract and were evaluated for potential bioactive peptide using the BIOPEP-UWM database, which suggest that KAF proteins from white sorghum may be considered as good precursors of dipeptidyl peptidase-inhibitor, angiotensin-converting enzyme inhibitor, antioxidant and hypotensive peptides following chymotrypsin, thermolysin, and subtilisin and their combination. Average scores aligned using PeptideRanker confirmed KAF proteins' potential sources of bioactive peptides with over 5 peptides scored over 0.8. In addition, 31 unexplored peptide sequences that could have biological activity were identified. Our results suggest that KAF can be used in the peptide productions with potential biological activity and beyond.
Effective graft technologies for bone repair have been a primary focus in the field of bone tissue engineering. We have previously fabricated and examined a nanocomposite composed of polyurethane, nano-hydroxyapatite, and decellularized bone particles, which demonstrated osteobiologic characteristics. To evaluate the underlying mechanisms of this biomaterial, human adipose-derived mesenchymal stem cell seeded scaffolds were assessed using a combinatorial approach of transcriptomic and metabolomic analyses. Data from osteogenic and signal transduction polymerase chain reaction arrays and small molecule abundances, measured through liquid chromatography-mass spectrometry, were cross-examined using Integrated Molecular Pathway Level Analysis, Database for Annotation, Visualization, and Integrated Discovery, and ConsensusPathDB online tools to generate a fundamental collection of scaffold-influenced pathways. Results demonstrated upregulation of key osteogenic, cellular adhesion cell signaling markers and indicated that Hedgehog and Wnt signaling pathways were primary candidates for the osteobiologic mechanisms of the scaffold design. The detection of complimentary metabolites, such as ascorbate, further indicates that scaffolds generate intricate cellular environments, promoting cell attachment and subsequent osteodifferentiation.
Dental pulp stem cells (DPSCs) have great potential for use in tissue engineering (TE)-based dental treatments. Electrical stimulation (EStim) has been shown to influence cellular functions that could play an important role in the success of TE treatments. Despite many recent studies focused on DPSCs, few have investigated the effect EStim has on these cells. The aim of this research was to investigate the effects of direct current (DC) EStim on osteo-/odontogenic differentiation of DPSCs. To do so cells were isolated from male Sprague Dawley rats (7-8 weeks old), and phenotype characterization and multilineage differentiation analysis were conducted to verify their "stemness." Different voltages of DC EStim were administrated 1 h/day for 7 days, and the effect of EStim on DPSC osteo-/odontogenic differentiation was assessed by measuring calcium and collagen deposition, alkaline phosphatase (ALP) activity, and expression of osteo- and odontogenic marker genes at days 7 and 14 of culture. We found that while 10 and 50 mV/mm of EStim had no effect on cell number or metabolic activity, 100 mV/mm caused a significant reduction in cell number, and 150 mV/mm resulted in cell death. Despite increased gene expression of osteo-/odontogenic gene markers,Osteocalcin,RunX2,BSP, andDMP1, at day 7 in EStim treated cells, 50 mV/mm of EStim decreased collagen deposition and ALP activity at both time points, and calcium deposition was found to be lower at day 14. In conclusion, under the conditions tested, EStim appears to impair DPSC osteo-/odontogenic differentiation. Additional studies are needed to further characterize and understand the mechanisms involved in DPSC response to EStim, with an eye toward its potential use in TE-based dental treatments.
The use of high concentrations of biotin as a dietary supplement to improve hair, skin, and nail quality has increased in the United States over the past few years. High concentrations of biotin have been shown to interfere with some diagnostic assays that use streptavidin-biotin interactions as one of the steps in the assay. The objective of this report is to evaluate potential biotin interference on the analytical and clinical sensitivity of a point of care (POC) antigen-antibody combo HIV-1 assay. We spiked biotin at concentrations ranging from 12.5 to 400 ng/mL into serum and plasma containing HIV-1 subtype B p24 antigen derived from culture supernatant. The p24 antigen was present in the matrices at 30 pg/mL. Fifty microliters of each sample was applied to Alere Determine HIV-1/2 Ag/Ab combo assay strips in duplicate and results were read by eye after 20 to 30 min. Biotin interfered with detection of HIV-1 p24 in serum and plasma. HIV-1 p24 was not detected at 30 pg/mL p24 when biotin was present at 200 ng/mL concentration. Our study demonstrated that elevated levels of biotin in samples may interfere with POC assays. It is important to consider biotin supplements as potential sources of falsely increased or decreased test results, especially in cases wherein supplementation cannot be ruled out.
The clinic of diabetes mellitus (DM) offers a number of hypotheses about the leading role of polymorphonuclear neutrophils (PMNs) in both oxidative stress and diabetic complications. However, the results of numerous studies are extremely controversial. Why is it so? We appreciated the clinical significance of simultaneous measurement data of several PMN parameters, which must complement each other. For this purpose, myeloperoxidase (MPO) and elastase (EL) were jointly analyzed in the blood plasma from 160 type 2 diabetes mellitus patients with high levels of HbA(1c). A weakly positive correlation (r similar to 0.56) was observed between MPO and EL analytical data, and any correlation between the concentrations of MPO/EL and HbA(1c)was absent. Medians of 160 measurements of MPO/EL concentrations were similar to 103/190 ng/mL, and 95% of all results were in the range below 320/1016 ng/mL, respectively. The share of DM patients whose concentrations of MPO, EL, or either of two parameters exceeded the corresponding reference values was 65%, 80%, and 82.5%, respectively. These findings-a high intensity of neutrophil degranulation process-indicated that some diabetic conditions promote the transfer of PMNs to an "arousal" or "subactivation" state, which is identical or similar to their activation, providingin vivoan almost inexhaustible source of extremely "aggressive" MPO and EL. Thus, the conjoint MPO/EL measurements confirm the leading role of PMNs in the development of various complications of diabetes. The paradox is that the diagnostic significance of MPO/EL as independent parameters in diabetic patients is unambiguous for a number of reasons.
The loss of estrogen with menopause is associated with an increase in central fat. The objective of this study was to evaluate the effects of menopause hormone therapy (HT) on body composition and metabolic parameters in postmenopausal women. A prospective study was conducted among postmenopausal women from the Climacteric clinic, Universidade Federal de São Paulo. Thirty-two participants, median age 51 years, were included. Sixteen women were eligible to receive a low-dose continuous combined HT, containing 1 mg of E2 plus 0.125 mg of trimegestone for 6 months. The other 16 women remained in the control group. In the HT group, significant decreases from baseline were evident for the total cholesterol (TC) (p < 0.05) and LDL levels (p < 0.05). The HDL significantly decreased (p < 0.05). However, the TC/HDL ratio also decreased (p = 0.05). The parameters of body composition, after 6 months of HT, were maintained. In the control group, body mass index levels increased from baseline, however, with nonstatistically significant differences (p = 0.06). Analyzing the body composition showed a significant increase in the trunk body fat (p = 0.04), trunk region fat (p = 0.04), and total region fat (p = 0.03) after 6 months. In conclusion, the present study provides evidence that HT can stunt the increase in total body fat and prevent the shift from a more central fat distribution observed in early postmenopausal period.
India is declared as the diabetic capital of the world. Clinically well-annotated blood samples will advance diabetes research for better diagnostic and treatment methods. Building a disease-specific biobank with high-quality peripheral blood mononuclear cells (PBMCs) and clinical follow-up data system will serve as a good platform for clinical research in diabetes. Processing and storage of high-quality biospecimen for translational research in diabetes demand the implementation of good clinical laboratory practices. “Certification or accreditation programs” that improve biorepository processes and frameworks are lacking in Indian context. To sustain and translate the research into clinical practice, good governance of the biobank and financial resources is required. For ethical issues related to health needs of the people and participants in the research, issues related to research process, translational research, and commercialization, data sharing should be addressed. For India to be an innovation and sustainable country Indian government is supporting translational research facilities, including biobanks. India has developed biobanks for various diseases; however, diabetes-specific research biorepository is lacking. Given the dangers of diabetic burden, India should set up a diabetes disease-specific repository learning from the global organizations and customize to the needs of Indian context. It is important to have private agencies get involved to develop biobanks and future research as there are commercial goals to translate research into practice. New technologies of specimen storing and preservation, data management, and data sharing should be adopted for developing cost-effective long-standing disease-specific population biobank in India.
The myeloid cells infiltrating the heart early after acute myocardial infarction elaborate a secretome that largely orchestrates subsequent ventricular wall repair. Regulating this innate immune response could be a means to improve infarct healing. To pilot this concept, we utilized (β1,3-d-) glucan-encapsulated small interfering RNA (siRNA)-containing particles (GeRPs), targeting mononuclear phagocytes, delivered to mice as a one-time intramyocardial injection immediately after acute infarction. Findings demonstrated that cardiac macrophages phagocytosed GeRPs in vivo and had little systemic dissemination, thus providing a means to deliver local therapeutics. Acute infarcts were then injected in vivo with phosphate-buffered saline (PBS; vehicle) or GeRPs loaded with siRNA to Map4k4, and excised hearts were examined at 3 and 7 days by quantitative polymerase chain reaction, flow cytometry, and histology. Compared with infarcted PBS-treated hearts, hearts with intrainfarct injections of siRNA-loaded GeRPs exhibited 69–89% reductions in transcripts for Map4k4 (mitogen-activated protein kinase kinase kinase kinase 4), interleukin (IL)-1β, and tumor necrosis factor α at 3 days. Expression of other factors relevant to matrix remodeling—monocyte chemoattractant protein-1 (MCP-1), matrix metalloproteinases, hyaluronan synthases, matricellular proteins, and profibrotic factors transforming growth factor beta (TGF-β), and connective tissue growth factor (CTGF)—were also decreased. Most effects peaked at 3 days, but, in some instances (Map4k4, IL-1β, TGF-β, CTGF, versican, and periostin), suppression persisted to 7 days. Thus, direct intramyocardial GeRP injection could serve as a novel and clinically translatable platform for in vivo RNA delivery to intracardiac macrophages for local and selective immunomodulation of the infarct microenvironment.
