Standard Isolation Protocol Research Articles

Ultrastructure and stability of cellular nanoparticles isolated from Phaeodactylum tricornutum and Dunaliella tertiolecta conditioned media

Background: Cells in general secrete nanoparticles (NPs) which are believed to mediate intercellular communication. Recently, great efforts have been made to utilize them as delivery vectors. We aimed to harvest and identify NPs from liquid cultures of two marine microalgae Dunaliella tertiolecta and Phaeodactyum tricornutum. Methods: NPs were isolated from the culture conditioned media by differential ultracentrifugation by the protocol used for the isolation of extracellular vesicles. Microalgae and isolated NPs were examined by scanning electron microscopy (SEM) while isolated NPs were examined also by cryogenic transmission electron microscopy (cryo-TEM). The Triton X-100 detergent and temperature sensitivity of NPs was assessed by dynamic light scattering (DLS) through monitoring the intensity of the scattered light (I) and the distribution of hydrodynamic radii of NPs (Rh). Results: Two mechanisms of formation of NPs with average Rh 200 nm were observed in the D. tertiolecta culture: a disintegration of tubular protrusions, and cell decay. A part of the imaged D. tertiolecta NPs were membrane-enclosed vesicles, but the isolates also contained electron-dense NPs and nanofilaments. P. tricornutum NPs in the culture and in the isolate were homogeneous in size and shape. Their average Rh was 104 nm. The addition of surfactant to isolates resulted in a change in Rh distribution and a decrease of I in samples from both species, indicating decay of a part of NPs. Changes in the width of the I(Rh) peaks were observed at temperatures above 45 °C. Conclusions: A part of NPs found in isolates from microalgae D. tertiolecta and P. tricornutum were membrane-enclosed vesicles. However, the isolates obtained by a standard protocol for extracellular vesicle isolation by ultracentrifugation contained also a significant amount of other similar-sized nanoparticles. The isolates were partly susceptible to the addition of detergent and to temperature up to 80 degrees.

Roles of Regulatory T Cell-Derived Extracellular Vesicles in Human Diseases.

Regulatory T (Treg) cells play crucial roles in maintaining immune self-tolerance and immune homeostasis, and closely associated with many human diseases. Recently, Treg cells-derived extracellular vesicles (Treg-EVs) have been demonstrated as a novel cell-contact independent inhibitory mechanism of Treg cells. Treg-EVs contain many specific biological molecules, which are delivered to target cells and modulate immune responses by inhibiting T cell proliferation, inducing T cell apoptosis, and changing the cytokine expression profiles of target cells. The abnormal quantity or function of Treg-EVs is associated with several types of human diseases or conditions, such as transplant rejection, inflammatory diseases, autoimmune diseases, and cancers. Treg-EVs are promising novel potential targets for disease diagnosis, therapy, and drug transport. Moreover, Treg-EVs possess distinct advantages over Treg cell-based immunotherapies. However, the therapeutic potential of Treg-EVs is limited by some factors, such as the standardized protocol for isolation and purification, large scale production, and drug loading efficiency. In this review, we systematically describe the structure, components, functions, and basic mechanisms of action of Treg-EVs and discuss the emerging roles in pathogenesis and the potential application of Treg-EVs in human diseases.

Characterization of primary canine Sertoli cells as a model to test male reproductive toxicant

Sertoli cells play critical roles in regulating spermatogenesis and testis development by providing structural and nutritional support. This study aimed to develop a standard protocol for canine Sertoli cell isolation and culture; and characterize its biological features, functionality, and application of compound toxicity testing. Canine testicles were received from the neuter clinic, and three-step of enzymatic digestion was applied to isolate Sertoli cells. We characterized the growth and purity of Sertoli cells with the expression of SOX9, GATA4, and Clusterin. In addition, we selected cadmium as a model toxicant to evaluate the toxic responses in the newly established Sertoli cells using High-content Analysis (HCA). With our optimized protocol, the purity of isolated Sertoli cells was above 95%, as determined with Sertoli cell-specific protein markers of SOX9 and GATA4. More importantly, primary Sertoli cell populations could be expanded rapidly in vitro, passaged (up to seven), and cryopreserved. The HCA-based assay revealed that cadmium at 1 μM induced both disruptions of cytoskeletal and DNA damage responses. Furthermore, we established an HCA assay with the newly isolated and optimized culture of canine Sertoli cells to evaluate the epigenetic markers of histone modification. We found cadmium-induced differential changes in histone modifications H3Me3K9, H3Me3K36, H4Me3K20, and H4acK5. In summary, we have established the standardized protocol to produce canine Sertoli cells with Sertoli cell-specific phenotype. The isolation and expansion of large quantities of canine Sertoli cells will provide broad applications in studying male infertility, reproductive toxicology, testicular cancer, and cell therapy.

Application of Bruce-Ladder Multiplex PCR for Identification of Brucella abortus Isolated from Cattle in Kachia Grazing Reserve and Jos Plateau

A study was carried out to isolate Brucella strains from cattle in Kachia Grazing Reserve (KGR) and some communities on Jos Plateau and to carry out phenotypic and molecular characterization of the isolates along with other isolates obtained from field submissions and those in the archive of National Veterinary Research Institute (NVRI), Vom. A total of 63 vaginal swabs, 36 milk samples, and 2 hygroma fluids were collected from KGR while 70 vaginal swabs, 50 milk samples and 2 hygroma fluids were collected on the Jos Plateau for Brucella isolation. They were cultured for Brucella isolation according to standard Brucella isolation protocol. Three Brucella abortus strains were isolated from KGR while 4 Brucella abortus strains were isolated from the Jos Plateau respectively. Eight isolates from field submissions and 5 from the archive were collected and resuscitated. Comprehensive characterization of the isolates in this study revealed that they were all Brucella abortus. Similarly, characterization of archived isolates and those from field submissions showed that they were Brucella abortus. The classical biotyping of all the isolates revealed that they were Brucella abortus biotype 3. Molecular characterization of all the isolates by Bruce-ladder multiplex Polymerase chain reaction (PCR) showed bands consistent with Brucella abortus. This is the first molecular characterization of Brucella isolates from Nigeria using the Bruce-ladder multiplex PCR and the first study that established that Brucella abortus biotype 3 is the predominant Brucella strain in Nigeria. The study established the endemicity of brucellosis due to Brucella abortus, in the two study areas. These findings have great veterinary and public health implications. There is therefore an urgent need for the institution and implementation of brucellosis control measures in these areas.

Effects of Vernonia amygdalina methanol leaf extract and fractions on Ascaridia galli in experimentally infected birds with regard to its pathological effect

BackgroundHelminthiasis, usually a chronic problem in poultry, is the major cause of economic losses in the poultry industry. This study was undertaken with the aim of evaluating the in vitro and in vivo anthelmintic effects of Vernonia amygdalina methanol leaf extract and fractions on Ascaridia galli in experimentally infected birds. Standard protocols for extraction, phytochemical screening, isolation and infection of parasite and histological examination were followed.ResultAt 50 mg/ml albendazole (ALB) caused the highest inhibition of embryonation (97.3%) followed by crude methanol extract (CME) (94.5%), ethyl acetate fraction (EAF) (81.3%) and butanol fraction (BF) (80.3%). The lowest faecal egg counts (FEC) and highest FEC at 21 days post-treatment were observed in the group treated with albendazole (99.2%) and CME at 800 mg/kg (96.9%). Deparasitization at 200 mg/kg caused by ALB was (93.8%), while at 200, 400 and 800 mg/kg, CME was (77.5%, 87.5% and 91.86%), BF was (75.51%, 79.61% and 85.73%) and EAF was (73.48%, 75.5% and 81.63%), respectively. At post-mortem, broiler chickens infected with A. galli and treated with CME, BF and EAF at 200 mg/kg had desquamation of the villi while at 400 and 800 mg/kg treatments, the intestines were intact.ConclusionThe extract and fractions of V. amygdalina were found to possess anthelmintic activity against A. galli due to the inhibition of embryonation of eggs and the reduction in faecal egg count; further research is required to fully ascertain their mechanism of action of the extract.

Microbiology and Epidemiology of Escherichia albertii-An Emerging Elusive Foodborne Pathogen.

Escherichia albertii, a close relative of E. coli, is an emerging zoonotic foodborne pathogen associated with watery diarrhea mainly in children and immunocompromised individuals. E. albertii was initially classified as eae-positive Hafnia alvei, however, as more genetic and biochemical information became available it was reassigned to its current novel taxonomy. Its infections are common under conditions of poor hygiene with confirmed transmission via contaminated water and food, mainly poultry-based products. This pathogen has been isolated from various domestic and wild animals, with most isolates being derived from birds, implying that birds among other wild animals might act as its reservoir. Due to the absence of standardized isolation and identification protocols, E. albertii can be misidentified as other Enterobacteriaceae. Exploiting phenotypes such as its inability to ferment rhamnose and xylose and PCR assays targeting E. albertii-specific genes such as the cytolethal distending toxin and the DNA-binding transcriptional activator of cysteine biosynthesis encoding genes can be used to accurately identify this pathogen. Several gaps exist in our knowledge of E. albertii and need to be bridged. A deeper understanding of E. albertii epidemiology and physiology is required to allow the development of effective measures to control its transmission and infections. Overall, current data suggest that E. albertii might play a more significant role in global infectious diarrhea cases than previously assumed and is often overlooked or misidentified. Therefore, simple, and efficient diagnostic tools that cover E. albertii biodiversity are required for effective isolation and identification of this elusive agent of diarrhea.

Abnormal neutrophil polarization in chronic obstructive pulmonary disease and how cigarette smoke extracts attract neutrophils

BackgroundAirway inflammation produced by neutrophils is a critical factor in the development of chronic obstructive pulmonary disease (COPD). Poor or excessive neutrophil polarization and chemotaxis may lead to pathogen accumulation and tissue damage. However, it is unclear how cigarette smoke extract (CSE) attracts neutrophils and to what extent COPD is affected by the improper polarization of these abnormal neutrophils. This study sought to assess the polarization and migration dynamics of neutrophils isolated from patients with different severities of COPD compared to healthy smoking and non-smoking control subjects, and to detect how CSE triggers the polarization of neutrophils.MethodsThe neutrophils were freshly isolated using standard isolation protocol. The polarization of the neutrophils was observed using a Zigmond chamber when stimulated by a linear concentration gradient of CSE or N-formyl-methionine-leucine-phenylalanine (fMLP). Confocal laser-scanning microscopy was used to observe the intracellular calcium of the neutrophils. The experimental data are presented as the mean ± standard deviation. SPSS 20.0 software was used for the statistical analysis. A P value <0.05 was considered statistically significant.ResultsThe neutrophils from the COPD patients showed a higher frequency of spontaneous polarization and a lower prevalence of directionality polarization than those from the healthy control (HC) and smoker subjects. The abnormal polarization of the neutrophils from the COPD patients was altered by the influence of store-operated calcium entry (SOCE) component matrix interaction molecules 1 and 2 and calcium release-activated calcium channel protein 1 [stromal interaction molecule 1 (STIM1), Stromal interaction molecule 2 (STIM2), and calcium release-activated calcium modulator 1 (ORAI1)].ConclusionsThe COPD neutrophils exhibited unique polarization and migration patterns compared to those of the cells examined from other populations. The attraction of CSEs to neutrophils was mediated by the SOCE/Akt/Src pathway.

First Report of Phytophthora nicotianae Causing Stem Canker of Catalpa bungei (Chinese Catalpa) in China.

Chinese catalpa, Catalpa bungei C.A. Mey is native to China and has been widely cultivated as an important tree species for timber and ornamental purposes (Tao et al. 2019). The properties and high durability of the wood can resist the damage caused by microorganisms and insects (Xiao Y et al. 2019). In September 2020, stem cankers were observed in 5-year-old and 3-year-old C. bungei in a pilot experiment field covering 16-hectare area in Shuyang city (Jiangsu province, China) and in a nursery in Binhai city (Jiangsu Province, China), respectively. The disease incidence in both locations was about 1% to 3%. The typical disease symptoms include small to large, dark-brown and irregular-sunken canker around and along the stem under 2 meters from the stem base. The phloem and xylem of the symptomatic stem were dark brown and the xylem had larger necrosis than the phloem. The cross section of the diseased stem was partially died. The symptomatic stem were collected in both locations for pathogen isolation. In total, seven purified isolates from the diseased samples were obtained using potato dextrose agar (PDA) following standard isolation protocol (Huang et al. 2019). In order to determine the pathogenicity, 3-year-old Chinese catalpa seedlings were artificially inoculated with each of the seven isolates in April 2021. After removing the bark of the stem by a sterilized punch (diameter 6mm), an agar plug (diameter 6mm) pre-colonized by the isolate was inoculated to the stem and the inoculation point was sealed with parafilm. The agar plug without pre-colonization was used as control. Six tree seedlings were inoculated for each isolate. Ten days after inoculation, only the treatment with isolate QS.1 showed obvious discoloration around the inoculation point. One month after inoculation, the canker around the inoculation point was formed (3.4 cm ± 1.0 cm) and spread to the xylem, similar to the symptoms observed in the field. Isolate QS.1 was re-isolated successfully from the inoculated stem based on morphological characters, confirming the Koch's postulates and QS.1 as the causal pathogen. The isolate QS.1 formed white colonies with abundant aerial mycelia on V8 juice agar and produced a large amount of persistent and papillary ovoid sporangia with size of 22 ~ 45μm (average 31μm) × 18 ~ 39μm (average 23μm) in 10% aqueous solution of V8. The spore was spherical with thick-wall and diameter of 24 ± 3.9μm. The morphology of QS.1 is similar to that of Phytophthora nicotianae. The genomic DNA of representative isolate QS.1 was extracted from mycelium by a modified CTAB method (Murray et al. 1980). The rDNA internal transcribed spacer (ITS) region, β-tubulin and EF1-α genes were amplified and sequenced with primers ITS1/ITS4 (White et al. 1990), BTub_F1/TUBUR1 (L. et al. 2004) and EF1A_for/EF1A_rev (Blair et al. 2008), respectively. The BLAST results of these sequences (Accession No. MZ646302, MZ672116, and MZ675589, respectively) showed 99%, 100% and 100% identity with sequences of P. nicotianae (Accession No. KJ494902, KY205750, and MH359041), respectively. Based on the morphological characteristics and DNA analysis, isolate QS.1 was identified as P. nicotianae. To our best knowledge, this is the first report of P. nicotianae causing stem canker on Chinese Catalpa. This disease may pose potential threat on Catalpa due to the increase in Catalpa planting for economic and ecological purposes in China.

Are Standard COVID-19 Isolation Protocols Appropriate in Haemodialysis Units? A Description of a Novel SARSCoV-2 Coronavirus Outbreak in a Haemodialysis Unit in Johannesburg, South Africa

Background: Recipients of outpatient haemodialysis are at an increased risk of acquiring severe SARS-CoV-2 infection. Isolation of infected patients reduces in-centre transmission, but protocols extrapolated from the general population may not be applicable in this setting. We describe the kinetics of an outbreak in a tertiary dialysis centre in Johannesburg, South Africa, to suggest an appropriate isolation strategy. Methods: Retrospective analysis of a clinical database employed to facilitate isolation of exposed and infected patients was undertaken. Modes of transmission, incubation and recovery periods in patients developing SARS-CoV-2 infection were assessed. The effects of factors modulating immune function on incubation and recovery periods were modelled using sigma-restricted partial least squares linear regression. Severity of infection and the outcomes thereof were described to assess the efficacy of the isolation protocols employed. Results: SARS-CoV-2 infection was diagnosed in 24.7% of patients receiving outpatient haemodialysis. Contact with an infected healthcare worker was the leading indication for surveillance swabbing in this cohort (49.12%). Forty per cent of all positive cases had antecedent contact with an infected healthcare worker, and possible patient-to-patient transmission occurred in one case. The median time to the diagnosis of infection following known exposure was 16.5 days. Comorbid diabetes and increasing dialysis vintage were associated with a shorter incubation period. The median time to clearance of infection was 33.5 days. The clinical disease severity prolonged the recovery period. No patient required mechanical ventilation, and there were no deaths during the study period. Conclusion: Haemodialysis patients manifest prolonged incubation and recovery periods. Serial monitoring with RT-PCR swabs may be required to ensure effective isolation.

Selection of DNA Aptamers for Differentiation of Human Adipose-Derived Mesenchymal Stem Cells from Fibroblasts.

In recent years, stem cell therapy has shown promise in regenerative medicine. The lack of standardized protocols for cell isolation and differentiation generates conflicting results in this field. Mesenchymal stem cells derived from adipose tissue (ASC) and fibroblasts (FIB) share very similar cell membrane markers. In this context, the distinction of mesenchymal stem cells from fibroblasts has been crucial for safe clinical application of these cells. In the present study, we developed aptamers capable of specifically recognize ASC using the Cell-SELEX technique. We tested the affinity of ASC aptamers compared to dermal FIB. Quantitative PCR was advantageous for the in vitro validation of four candidate aptamers. The binding capabilities of Apta 2 and Apta 42 could not distinguish both cell types. At the same time, Apta 21 and Apta 99 showed a better binding capacity to ASC with dissociation constants (Kd) of 50.46 ± 2.28 nM and 72.71 ± 10.3 nM, respectively. However, Apta 21 showed a Kd of 86.78 ± 9.14 nM when incubated with FIB. Therefore, only Apta 99 showed specificity to detect ASC by total internal reflection microscopy (TIRF). This aptamer is a promising tool for the in vitro identification of ASC. These results will help understand the differences between these two cell types for more specific and precise cell therapies.

Periodontal and Dental Pulp Cell-Derived Small Extracellular Vesicles: A Review of the Current Status.

Extracellular vesicles (EVs) are membrane-bound lipid particles that are secreted by all cell types and function as cell-to-cell communicators through their cargos of protein, nucleic acid, lipids, and metabolites, which are derived from their parent cells. There is limited information on the isolation and the emerging therapeutic role of periodontal and dental pulp cell-derived small EVs (sEVs, <200 nm, or exosome). In this review, we discuss the biogenesis of three EV subtypes (sEVs, microvesicles and apoptotic bodies) and the emerging role of sEVs from periodontal ligament (stem) cells, gingival fibroblasts (or gingival mesenchymal stem cells) and dental pulp cells, and their therapeutic potential in vitro and in vivo. A review of the relevant methodology found that precipitation-based kits and ultracentrifugation are the two most common methods to isolate periodontal (dental pulp) cell sEVs. Periodontal (and pulp) cell sEVs range in size, from 40 nm to 2 μm, due to a lack of standardized isolation protocols. Nevertheless, our review found that these EVs possess anti-inflammatory, osteo/odontogenic, angiogenic and immunomodulatory functions in vitro and in vivo, via reported EV cargos of EV–miRNAs, EV–circRNAs, EV–mRNAs and EV–lncRNAs. This review highlights the considerable therapeutic potential of periodontal and dental pulp cell-derived sEVs in various regenerative applications.

Isolation protocol of jumbo phage from winter grass soil

Jumbo phage can isolated by two different methods and protocols. It depends on size, phage species, substrate sources, environment, media and tools. Each method has positive and negative results within each factors. The research aimed to determine the suitable protocol to isolated the jumbo phages from winter grass soil during winter conditions, utilizing two main protocols, the enrichment protocol and non-enrichment protocol. In Sequent, research was conducted with several steps, including bacterial isolation, jumbo phage isolation, plaque test in Escherichia coli colony, bacterial genus identification, phage test in soil bacteria, protocol evaluation, DNA extraction and electrophoresis. Based on the plaque size and plaque total, each protocol did not significantly differ on plaque total among the E. coli or each isolated soil bacteria. On the E. coli test, the plaque total average was 65 plaque, nor 45 plaque emerge on each isolated soil bacteria. The significant difference on each protocol were on the clarity of plaque. Based on visual observation, the plaques with enrichment protocol are clearer. It was obvious that the isolation of jumbo phage from the winter grass during winter conditions can be done without the enrichment protocol. It can isolated with standard isolation protocol.

Drop-to-drop liquid-liquid extraction of DNA in an electrowetting-on-dielectric digital microfluidics.

Recent advancements in microfluidics and lab-on-a-chip technologies enabled miniaturization and automation of many downstream nucleic acid analysis steps such as PCR. However, DNA extraction/isolation protocol remains a stand-alone sample preparation step. For a quick sample-to-result solution, downstream protocols and sample preparation protocols need to be seamlessly integrated into a single lab-on-a-chip platform. As a step toward such integration, this paper introduces microfluidic DNA isolation using the liquid-liquid extraction (LLE) method in the drop-to-drop (DTD) format. The electrowetting-on-dielectric digital microfluidic platform is capable of handling a two-phase liquid system easily, which enables DTD LLE. In this study, the extraction of plasmid DNA (pDNA) from an aqueous sample to an ionic liquid is demonstrated. Prior to pDNA extraction study, the DTD LLE protocol was developed and optimized using organic dyes as solutes. The selective extraction of pDNA in the presence of proteins as interfering molecules is also demonstrated. This work implies that DTD LLE can substitute for magnetic beads steps in standard DNA isolation protocols.

Investigation of the role of polymorphic variants FTO gene in schizophrenia patients with metabolic syndrome

IntroductionMedication is primary tactics in schizophrenia treatment. First and second generation antipsychotics (FGA and SGA respectively) affect on core symptoms. Unfortunately, it causes side effects. Metabolic syndrome one of them and includes large number of affections like body mass index changes, lipidemias, hypertension and others.ObjectivesTo study the role of polymorphic variants FTO gene in metabolic syndrome in schizophrenia patients.MethodsWe were investigated 480 patients. Main criteria for inclusion in study was using antipsychotics, verified diagnosis of schizophrenia and metabolic syndrome. Mean age was 42,1±1,4 years. The metabolic syndrome was assessment based on clinical data. Standard phenol-chloroform protocol for DNA isolation was used. Genotyping was carried out on six SNP’s of FTO gene with real-time PCR. Statistical analysis was carried out with R 3.6.2 with basic functions and SNPassoc package.ResultsThe distribution of genotypes for variants rs7185735 and rs9939609 was not in according to Hardy-Weinberg equilibrium (a p-value less than 0.05) and excluded from further analysis. Patients with schizophrenia were divided into two groups: patients with metabolic syndrome and patients without it. We did not identify any statistically significant associations between genotypes and alleles of FTO gene and metabolic syndrome.ConclusionsWe did not find any associations of alleles and genotypes of FTO gene with metabolic syndrome in schizophrenia patients from Siberia region. Metabolic syndrome needs more further studies with larger number of samples and different populations. Conflict of interest. The authors declare no conflict of interests. Supported by Grant of RSF 19-75-10012.

MINIMALLY MANIPULATED BONE MARROW MESENCHYMAL STEM CELL PURIFICATION AND CHARACTERIZATION FROM PATIENTS WITH OSTEOARTHRITIS

<h3>Background</h3> Bone marrow mesenchymal stem cells (MSC) have been investigated due to its therapeutic effects as multipotent, immunoregulating and anti-apoptotic cells. However, MSC's standard isolation relies on its swiftly adherence to plastic culture flasks, lacking precise cell definition. Therefore, marked heterogeneity persists in the so-called MSC population hampering true multipotent MSC isolation and, consequently, setting back tissue engineering efficiency. The present study proposes a modified adhesion-based protocol to purify bone marrow MSC. <h3>Methods</h3> MSC were obtained from 24 patients undergoing total hip arthroplasty. We propose a simple additional step to the standard isolation protocol. Normally, marrow cells are cultivated for 3 days and the non-adherant cells are discarded. Instead, we used the non-adherant fraction to initiate a new culture, here named MSC-LA. Primary MSC obtained from standard (MSC-RA) or modified protocol (MSC-LA) were minimally manipulated and compared regarding classical MSC's properties, such as colony formation capacity, in vitro differentiation and cell surface markers. Vascular organization and maintenance capacity was also assessed through tube formation assay. <h3>Results</h3> MSC-LA corresponded to a 33% increase in yield, generated fewer but more populated primary colonies and more secondary colonies than MSC-RA. Moreover, MSC-LA displayed equivalent osteogenic but enhanced adipogenic differentiation than MSC-RA. In tube formation assay, MSC-LA demonstrated similar vessel organization capacity to MSC-RA, pruning disorganized tubule formation and assuming perivascular localization and morphology. In cytometry characterization, both MSC groups presented CD45-/CD31-/ CD90+/CD73+/CD44+/CD29+/CD10+/CD54+/CD34- phenotype. While MSC-RA showed greater CD45+, HLA+ and CD184+ cell contamination, MSC-LA displayed enhanced purity of CD24+, CD49d+ and CD146+ cells, markers associated to MSC phenotype. <h3>Conclusion</h3> Our adhesion-based isolation protocol promotes greater yield of MSC at very low cost, generating a purified MSC population that may hold greater stemness potential. MSC-LA can optimize tissue engineering therapies through lower contamination of undesired or totally differentiated cell populations supporting autologous MSC use in arthritis patients. Further analysis is necessary to confirm MSC-LA potential in in vivo settings.

Bovine Milk-Derived Exosomes as a Drug Delivery Vehicle for miRNA-Based Therapy

MicroRNAs (miRNAs) are small non-coding RNAs with a known role as mediators of gene expression in crucial biological processes, which converts them into high potential contenders in the ongoing search for effective therapeutic strategies. However, extracellular RNAs are unstable and rapidly degraded, reducing the possibility of successfully exerting a biological function in distant target cells. Strategies aimed at enhancing the therapeutic potential of miRNAs include the development of efficient, tissue-specific and nonimmunogenic delivery methods. Since miRNAs were discovered to be naturally transported within exosomes, a type of extracellular vesicle that confers protection against RNase degradation and increases miRNA stability have been proposed as ideal delivery vehicles for miRNA-based therapy. Although research in this field has grown rapidly in the last few years, a standard, reproducible and cost-effective protocol for exosome isolation and extracellular RNA delivery is lacking. We aimed to evaluate the use of milk-derived extracellular vesicles as vehicles for extracellular RNA drug delivery. With this purpose, exosomes were isolated from raw bovine milk, combining ultracentrifugation and size exclusion chromatography (SEC) methodology. Isolated exosomes were then loaded with exogenous hsa-miR148a-3p, a highly expressed miRNA in milk exosomes. The suitability of exosomes as delivery vehicles for extracellular RNAs was tested by evaluating the absorption of miR-148a-3p in hepatic (HepG2) and intestinal (Caco-2) cell lines. The potential exertion of a biological effect by miR-148a-3p was assessed by gene expression analysis, using microarrays. Results support that bovine milk is a cost-effective source of exosomes which can be used as nanocarriers of functional miRNAs with a potential use in RNA-based therapy. In addition, we show here that a combination of ultracentrifugation and SEC technics improve exosome enrichment, purity, and integrity for subsequent use.

Challenges and Limitations of Strategies to Promote Therapeutic Potential of Human Mesenchymal Stem Cells for Cell-Based Cardiac Repair.

Mesenchymal stem cells (MSCs) represent a population of adult stem cells residing in many tissues, mainly bone marrow, adipose tissue, and umbilical cord. Due to the safety and availability of standard procedures and protocols for isolation, culturing, and characterization of these cells, MSCs have emerged as one of the most promising sources for cell-based cardiac regenerative therapy. Once transplanted into a damaged heart, MSCs release paracrine factors that nurture the injured area, prevent further adverse cardiac remodeling, and mediate tissue repair along with vasculature. Numerous preclinical studies applying MSCs have provided significant benefits following myocardial infarction. Despite promising results from preclinical studies using animal models, MSCs are not up to the mark for human clinical trials. As a result, various approaches have been considered to promote the therapeutic potency of MSCs, such as genetic engineering, physical treatments, growth factor, and pharmacological agents. Each strategy has targeted one or multi-potentials of MSCs. In this review, we will describe diverse approaches that have been developed to promote the therapeutic potential of MSCs for cardiac regenerative therapy. Particularly, we will discuss major characteristics of individual strategy to enhance therapeutic efficacy of MSCs including scientific principles, advantages, limitations, and improving factors. This article also will briefly introduce recent novel approaches that MSCs enhanced therapeutic potentials of other cells for cardiac repair.

Insights Into the Proteomic Profiling of Extracellular Vesicles for the Identification of Early Biomarkers of Neurodegeneration.

Extracellular vesicles (EVs) are involved in the development and progression of neurodegenerative diseases, including Alzheimer's and Parkinson's disease. Moreover, EVs have the capacity to modify the physiology of neuronal circuits by transferring proteins, RNA, lipids, and metabolites. The proteomic characterization of EVs (exosomes and microvesicles) from preclinical models and patient samples has the potential to reveal new proteins and molecular networks that affect the normal physiology prior to the appearance of traditional biomarkers of neurodegeneration. Noteworthy, many of the genetic risks associated to the development of Alzheimer's and Parkinson's disease affect the crosstalk between mitochondria, endosomes, and lysosomes. Recent research has focused on determining the role of endolysosomal trafficking in the onset of neurodegenerative diseases. Proteomic studies indicate an alteration of biogenesis and molecular content of EVs as a result of endolysosomal and autophagic dysfunction. In this review, we discuss the status of EV proteomic characterization and their usefulness in discovering new biomarkers for the differential diagnosis of neurodegenerative diseases. Despite the challenges related to the failure to follow a standard isolation protocol and their implementation for a clinical setting, the analysis of EV proteomes has revealed the presence of key proteins with post-translational modifications that can be measured in peripheral fluids.

Comprehensive Characterization of Nanosized Extracellular Vesicles from Central and Peripheral Organs : Implications for Preclinical and Clinical Applications.

Extracellular vesicles (EV) are nano-sized vesicles that have been garnering a lot of attention for their valuable role as potential diagnostic markers and therapeutic vehicles for a plethora of pathologies. Whilst EV markers from biofluids such as plasma, serum, urine, cerebrospinal fluid and in vitro cell culture based platforms have been extensively studied, a significant knowledge gap that remains is the characterization of specific organ derived EVs (ODE). Here, we present a standardized protocol for isolation and characterization of purified EV isolated from brain, heart, lung, kidney and liver from rat and postmortem human tissue. Next, using quantitative mass spectrometry based proteomics, we characterized the respective tissue EV proteomes that identified synaptophysin (SYP), caveolin-3 (CAV3), solute carrier family 22 member 2 (SLC22A2), surfactant protein B (SP-B), and fatty acid-binding protein 1 (FABP1) as potential markers for the brain, heart, kidney, lung, and liver-EV, respectively. These respective tissue specific markers were further validated using both immunoblotting and a nanoplasmonic platform- single EV imaging analysis in the two species. To summarize, our study for the first time using traditional biochemical and high precision technology platforms provide a valuable proof of concept approach in defining specific ODE markers which further could be developed as potential therapeutic candidates for respective end-organ associated pathologies.

Molecular evidence of Francisella noatunensis sub sp. orientalis in Oreochromis niloticus in Yunnan Province, south-western China

Francisella sp. is an emerging bacterial pathogen affecting tilapia Oreochromis niloticus This study surveyed presence of Francisella sp. in tilapia raised in Yunnan Province, south-western China along with water quality parameters and management practices. A total of 224 tilapia specimens were sampled from 28 farms between June and September 2017. Necropsy of the sampled fish revealed no granulomatous lesions and the standard bacterial isolation protocols proved negative for Francisella sp. However, the presence of Francisella sp. was confirmed by polymerase chain reaction in 18 samples (8%) from 8 of the 28 farms (28.6%) sampled, though no increasing mortality was reported from these farms. The 16S ribosomal RNA gene sequences revealed 99.7% identity with F. noatunensis sub sp. orientalis. The presence of Francisella sp. infection showed significant association with water temperature.