Standard Isolation Methods Research Articles

Exosome-based drug delivery systems for enhanced neurological therapeutics.

Exosomes are small extracellular vesicles naturally secreted by cells into body fluids, enriched with bioactive molecules such as RNAs, proteins, and lipids. These nanosized vesicles play a crucial role in physiological and pathological processes by facilitating intercellular communication and modulating cellular responses, particularly within the central nervous system (CNS). Their ability to cross the blood-brain barrier and reflect the characteristics of their parent cells makes exosomal cargo a promising candidate for biomarkers in the early diagnosis and clinical assessment of neurological conditions. This review offers a comprehensive overview of current knowledge on the characterization of mammalian-derived exosomes, their application as drug delivery systems for neurological disorders, and ongoing clinical trials involving exosome-loaded cargo. Despite their promising attributes, a significant challenge remains the lack of standardized isolation methods, as current techniques are often complex, costly, and require sophisticated equipment, affecting the scalability and affordability of exosome-based therapies. The review highlights the engineering potential of exosomes, emphasizing their ability to be customized for targeted therapeutic delivery through surface modification or conjugation. Future advancements in addressing these challenges and leveraging the unique properties of exosomes could lead to innovative and effective therapeutic strategies in neurology.

Isolation, Characterisation and Identification of Bacteria from Three Different Unpasteurized Fruit Juices Sold at a Local Market in Guyana

Fruit juices are widely consumed for their nutritional value. However, fruit juices that are unpasteurised may be unsafe to drink because they are not subject to heat processing and much of their human handling results in contamination. The fruits used to make these juices have resident microflora, transient bacteria from handling and equipment, and bacteria from water used to wash fruits and in some cases make the juice. The objective of this research was to determine the bacteria present in unpasteurised passionfruit, cherry, and tamarind juices respectively. Through the use of a survey, and standard methods for isolation, identification and characterisation of bacteria, the results showed that temperature and pH did not significantly affect the diversity of bacteria. Cherry juice had the highest number of Colony Forming Units while passion fruit juice had the lowest. In total seven (7) species of bacteria were found including E.coli, S. aureus, Lactobacillus sp, Acetobacter sp, Bacillus subtilis, Bacillus cereus, and Micrococcus. The results for the Colony Forming Units were found to be above maximum acceptable levels for fruit juices. The results of this study demonstrate that unpasteurised juices may be a threat to public health and hazardous to humans if not processed in a sanitary manner.

P-290 Isolation and characterization of extracellular vesicles from human endometrial organoids

Abstract Study question Do human endometrial organoids produce extracellular vesicles (EVs)? Summary answer Endometrial organoids release EVs (ORG-EVs) that we successfully and comprehensively characterize according to guidelines. What is known already EVs play a crucial role in cellular communication, garnering increasing attention in physiological and pathological processes. Although the enormous potential of EVs has been extensively demonstrated across a large variety of research fields, the promise held by EVs derived from human endometrial organoids is still largely unexplored. Indeed, nowadays there is limited research available on the presence, role, and function of EVs derived from organoids of female reproductive tissue samples. This underscores the importance of adopting standardized methods for isolating, characterizing, and reporting findings, which is crucial for advancing in this promising and innovative, yet challenging, research field. Study design, size, duration This study involved establishing organoid cultures from endometrial biopsies of healthy women (n = 10) of reproductive age, who underwent ovarian surgery between January and October 2023. ORG-EVs were isolated using differential ultracentrifugation. Characterization of ORG-EVs, including assessment of their biophysical and biochemical properties, adhered to guidelines established by the International Society for Extracellular Vesicles (ISEV). Participants/materials, setting, methods Endometrial specimens from 10 women were employed to establish organoid cultures. ORG-EVs were isolated from spent medium through differential ultracentrifugation. Morphology, size distribution and concentration analysis were performed using Transmission Electron Microscopy (TEM) and Nanoparticle Tracking Analysis (NTA). Western blotting was used to confirm the presence of EVs markers. Main results and the role of chance TEM analysis confirmed the presence of isolated ORG-EVs, elucidating their morphology and structural integrity. NTA revealed that ORG-EVs had an average size of 160.4 ± 6.2 nm (mean ± SD) and a mean concentration of 5.2E+11 ± 2.1E+10 particles/ml (mean ± SD). Western blot analysis further confirmed the vesicular nature of the assessed particles, demonstrating positivity for standard EV markers such as tetraspanins (CD63 and CD9), and soluble proteins (ALIX and TSG101), while excluding the endoplasmic reticulum-resident protein calnexin (negative control). Additionally, ORG-EVs exhibited a positive signal for mucin1 (MUC1), a glycoprotein present on the surface of endometrial epithelial cells, thereby confirming their cellular origin. Limitations, reasons for caution This is a pilot study, characterized by a small sample size requiring larger cohorts to validate results and, at the same time, to undertake deeper exploration of ORG-EVs’ molecular cargo. Emphasizing the critical need for standardized isolation and characterization methods is paramount. Wider implications of the findings Endometrial organoids enhance EVs production, providing a powerful tool for reproductive aspects studies, and improving regenerative medicine and artificial reproductive technology. These initial findings propel further exploration, unveiling of ORG-EVs’ molecular cargo aiming to elucidate their potential role in human female reproductive tissues and mechanism related to embryo implantation process. Trial registration number not applicable

Comparison of morphology, protein concentration, and size distribution of bone marrow and Wharton's jelly-derived mesenchymal stem cells exosomes isolated by ultracentrifugation and polymer-based precipitation techniques

Exosomes which are tiny extracellular vesicles (30–150 nm), transport vital proteins and gene materials such as miRNA, mRNA, or DNA, whose role in cell communication and epithelia regulation is critical. Many techniques have been developed as a result of studying exosomes' biochemical and physicochemical properties, although there is still no standard method to isolate exosomes simply with high yield. Commercial kits have gained popularity for exosome extraction despite concerns about their effectiveness in scientific research. On the other hand, ultracentrifugation remains the gold standard isolation method. This study compares these two common exosome isolation methods to determine their impact on the quality and quantity of exosomes isolated from bone marrow (BM) and Wharton's jelly (WJ)-derived mesenchymal stem cells. Isolated exosomes from the two sources of the cell's conditioned medium by two methods (polymer kit and ultracentrifuge) were characterized using western blotting, scanning electron microscopy (SEM), dynamic light scattering (DLS), and the Bradford assay. Western blot analysis confirmed separation efficiency based on CD81 and CD63 markers, with the absence of calnexin serving as a negative control. The Morphology of exosomes studied by SEM image analysis revealed a similar round shape appearance and their sizes (30–150 nm) were the same in both isolation techniques. The DLS analysis of the sample results was consistent with the SEM ones, showing a similar size range and very low disparity. The exosome protein content concentration analysis revealed that exosomes isolated by the polymer-based kits contained higher protein concentration density and purity (p <0.001). In general, though the protein yield was higher when the polymer-based kits were used, there were no significant differences in morphology, or size between WJ-derived and BM-derived exosomes, regardless of the isolation method employed.

Advancing Diabetes Research: A Novel Islet Isolation Method from Living Donors.

Pancreatic islet isolation is critical for type 2 diabetes research. Although -omics approaches have shed light on islet molecular profiles, inconsistencies persist; on the other hand, functional studies are essential, but they require reliable and standardized isolation methods. Here, we propose a simplified protocol applied to very small-sized samples collected from partially pancreatectomized living donors. Islet isolation was performed by digesting tissue specimens collected during surgery within a collagenase P solution, followed by a Lympholyte density gradient separation; finally, functional assays and staining with dithizone were carried out. Isolated pancreatic islets exhibited functional responses to glucose and arginine stimulation mirroring donors' metabolic profiles, with insulin secretion significantly decreasing in diabetic islets compared to non-diabetic islets; conversely, proinsulin secretion showed an increasing trend from non-diabetic to diabetic islets. This novel islet isolation method from living patients undergoing partial pancreatectomy offers a valuable opportunity for targeted study of islet physiology, with the primary advantage of being time-effective and successfully preserving islet viability and functionality. It enables the generation of islet preparations that closely reflect donors' clinical profiles, simplifying the isolation process and eliminating the need for a Ricordi chamber. Thus, this method holds promises for advancing our understanding of diabetes and for new personalized pharmacological approaches.

Prevalence of Urinary Tract Infection (UTIs) Among HIV Sero-Positive Patients Attending Selected Hospitals at Otukpo Benue State, Nigeria

Prevalence of uropathogenic bacteria among HIV sero-positive Patients attending selected Hospitals at Otukpo Benue State Nigeria was investigated. A total of 392 mid-stream urine samples from Patients with HIV infection attending St. Daniel Hospital, Royal Specialist Hospital, General Hospital and St. Charles Hospital was collected and analyzed using standard Microbiological and biochemical methods for isolation and identification of organisms. Prevalence of the infection was also determined. Socio-demographic variables were obtained using a pre-designed structured questionnaire. Data were entered into a data base designed using SPSS Statistical software version 20 and analyzed using Chi square. Out of 392 samples, 115 were tested positive for urinary tract infections and five different types of bacteria were isolated. The bacteria isolated and their prevalence were Escherichia coli 42(36.5%), Proteus mirabilis 32(27.8%), Pseudomonas aeruginosa 17(14.8%), Staphylococcus aureus 12(10.4%) and Klebsiella pneumoniae 12(10.4%). The overall prevalence of urinary tract infection in HIV patients was 29.3%.The female had prevalence of 30.9%, while the male had 27.4%. The age group 16-25years had the highest occurrence 16(33.3%). Based on occupation, business people had the highest positivity rate of 38(31.1%). The married had 43(30.7%) positivity rate. The prevalence of urinary tract infections among the HIV negative individuals was 26.9%. The information provided in this report is useful in the clinical management of urinary tract infections among HIV sero-positive patients in the study area

Synthesis of Some New Amino Carbonyl Compounds by Mannich Reaction

Beta-aminocarbonyl compounds are important intermediates produced from Mannich condensation. In this research, benzaldehyde, para-hydroxy acetophenone or para-nitro acetophenone were reacted with a various of primary aromatic amines to give compounds (1-7) and (8-14), respectively. The chemical formulas of the synthesized compounds were confirmed by conducting some physical and spectroscopic measurements, such as, melting points, IR and H1-NMR spectrum. After testing the biological activity of some of the synthesized compounds (8,10,11,13) using Gram-positive (Streptococcus, and Staphylococcus aureus), Gram-negative (Klebsiella spp., and Escherichia coli) bacteria, as well as yeast strains, were chosen for this investigation (candidan albicans). Isolated bacteria were identified using standard methods of isolation and identification, including direct assays and culture on various media. Some theoretical tests have also been carried out such as SwissADME and cardioToxCSM prediction on all prepared compounds to predict their activity in terms of pharmacokinetic properties (ADME) and also predict their cardiotoxicity on some heart functions such as arrhythmia, cardiac failure, heart block, hERG toxicity, Hypertension (HT), and Myocardial Infarction (MI).&#x0D; &#x0D; Keywords: Secondary amines, aldehyde, Mannich condensation, antimicrobial activity.

Occurrence and Pathogenicity of Indigenous Entomopathogenic Fungi Isolates to Fall Armyworm (Spodoptera frugiperda J. E. Smith) in Western Amhara, Ethiopia

The fall armyworm (FAW) (Spodoptera frugiperda J. E. Smith) is widely recognized as an invasive lepidopteran pest in Africa. Adoption of synthetic pesticides remains an option for emergency control of the FAW, but their large-scale use by small-scale farmers is costly and environmentally unsafe. Management options based on entomopathogenic fungi could minimize risks for health and the environment. In our study, the occurrence of entomopathogenic fungi in different habitats along with their pathogenicity to fall armyworm was examined. A total of 56 soil samples were collected from three locations. A standard isolation method, baited with the wax moth larvae (Galleria mellonella), was used for the isolation of entomopathogenic fungi from soil. Twelve entomopathogenic fungi were isolated from the total soil samples collected. It was observed that Metarhizium spp. was more frequent and widespread than Beauveria spp. and their occurrence was high in the forest habitat. Pathogenicity of indigenous entomopathogenic fungi isolates on fall armyworm larvae was tested by applying conidial suspension containing 1 × 108 spores/ml. All of the isolates tested were pathogenic to larvae of fall armyworm, with mortalities varying from 30 to 80% at 10 days posttreatment. Our results suggest that native entomopathogenic fungi could be integrated for the management of Spodoptera frugiperda larvae.

Impact of Isolation Methods on Extracellular Vesicle Functionality In Vitro and In Vivo.

This study compares the impact of two isolation methods, ultracentrifugation (UC) and size exclusion chromatography (SEC), on small extracellular vesicles (sEVs) from primary human cardiac mesenchymal-derived progenitor cells (CPCs). sEV_UC and sEV_SEC exhibit similar size, marker expression, and miRNA cargo, but sEV_UC contains notably higher total protein levels. In vitro assays show that sEV_UC, despite an equal particle count, induces more robust ERK phosphorylation, cytoprotection, and proliferation in iPS-derived cardiomyocytes (iPS-CMs) compared to sEV_SEC. sEV_UC also contains elevated periostin (POSTN) protein levels, resulting in enhanced focal adhesion kinase (FAK) phosphorylation in iPS-CMs. Importantly, this effect persists with treatment with soluble free-sEV protein fraction from SEC (Prote_SEC), indicating that free proteins like POSTN in sEV_UC enhance FAK phosphorylation. In vivo, sEV contamination with soluble proteins doesn't affect cardiac targeting or FAK phosphorylation, underscoring the intrinsic tissue targeting properties of sEV. These findings emphasize the need for standardized sEV isolation methods, as the choice of method can impact experimental outcomes, particularly in vitro.

Assessment of microbial contamination of naira notes in Idah Local Government Area, Kogi State, Nigeria

Currency notes could become potential vehicles for the transmission of pathogenic microorganism to man due to repeated circulation for transactions as they change hands from one person to another. The study was carried out to investigate the microbial contamination of Nigeria Currency notes in circulation in Idah Local Government Area of Kogi State, Nigeria. A total of 400 hundred used bank notes of different denomination (N1000, N500, N200, N100, N50, N20, N10 and N5) were collected and analyzed for microbial contamination using standard microbiological methods of total viable count, isolation and identification microorganisms to determine the percentage prevalence of fungi and bacteria associated with the currency notes. The results revealed the presence of bacteria isolates which include Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Salmonella sp and Bacillus sp. The fungi isolated include Aspergillus niger, Mucor sp and Fusarium sp. The N100 banknotes among the higher denominations recorded the highest contamination by bacteria (Staphylococcus aureus) and fungi (Aspergillus niger) with percentage prevalence of 36.7% and 45.9% and least percentage prevalence of 8.6% and 17.4% for Bacillus sp and Fusarium sp respectively. The total bacterial and fungal count range from 1.0 x 104 cfu/ml to 7.2 x104 cfu/ml. The result of the investigation revealed the need for awareness sensitization and caution in the handling of the Nigeria Currency notes against transmission of infectious diseases as well as proper hygiene to mitigate possible cross- contamination between currency notes and food stuff.

The Prevalence of Aeromonas Species in Salad Vegetables Sourced from Four Local Government Areas of Plateau State

The current study was conducted to determine the prevalence of Aeromonas species in five salad vegetables, namely lettuce, cabbage, carrot, cucumber, and green pepper, in four local government areas of Plateau state. Aeromonas species have been linked to a number of gastroenteritis outbreaks connected to the consumption of contaminated fresh vegetables and fruits. In Nigeria, the use of untreated wastewater in irrigation is often regarded as an unavoidable alternative for compensating for water scarcity. This study was conducted to determine the prevalence of Aeromonas species in salad vegetables from four local government areas of Plateau State. 800 lettuce samples, including 160 lettuces, 160 cabbages, 160 carrots, 160 cucumbers and 160 green peppers, were analyzed for the presence of Aeromonas species using standard isolation, identification and characterization methods. Of the 800 samples, 202 samples were positive. Aeromonas spp. was isolated from 60(7.50%) lettuce samples, 52(6.50%) cabbage samples, 35(4.38%) carrot samples, 30(3.77%) cucumber samples and 25(3.12%) green pepper samples making lettuce the vegetable with the highest prevalence of the organism. According to the findings, samples of salad vegetables from Lamingo had the highest percentage prevalence of Aeromonas species, at 45 (6.25%), followed by samples from Miango, at 42 (5.25%), and samples from Vom, at 37 (4.64%) had the lowest percentage prevalence of the organism. According to the statistical analysis's findings, the prevalence of the organism in various vegetable types with relation to the study locations did not differ significantly (p&gt;0.05) at the 95% confidence level. The incidence of Aeromonas species isolated from the various study regions varied significantly depending on the season, with the organism being more prevalent during the wet season (3.7%) than during the dry season (1.35%).

Exosomes: Nomenclature, Isolation, and Biological Roles in Liver Diseases.

The biogenesis and biological roles of extracellular vesicles (EVs) in the progression of liver diseases have attracted considerable attention in recent years. EVs are membrane-bound nanosized vesicles found in different types of body fluids and contain various bioactive materials, including proteins, lipids, nucleic acids, and mitochondrial DNA. Based on their origin and biogenesis, EVs can be classified as apoptotic bodies, microvesicles, and exosomes. Among these, exosomes are the smallest EVs (30-150 nm in diameter), which play a significant role in cell-to-cell communication and epigenetic regulation. Moreover, exosomal content analysis can reveal the functional state of the parental cell. Therefore, exosomes can be applied to various purposes, including disease diagnosis and treatment, drug delivery, cell-free vaccines, and regenerative medicine. However, exosome-related research faces two major limitations: isolation of exosomes with high yield and purity and distinction of exosomes from other EVs (especially microvesicles). No standardized exosome isolation method has been established to date; however, various exosome isolation strategies have been proposed to investigate their biological roles. Exosome-mediated intercellular communications are known to be involved in alcoholic liver disease and nonalcoholic fatty liver disease development. Damaged hepatocytes or nonparenchymal cells release large numbers of exosomes that promote the progression of inflammation and fibrogenesis through interactions with neighboring cells. Exosomes are expected to provide insight on the progression of liver disease. Here, we review the biogenesis of exosomes, exosome isolation techniques, and biological roles of exosomes in alcoholic liver disease and nonalcoholic fatty liver disease.

Microplastic isolation method for wastewater and sludge samples by removal of excess organic and inorganic interferences

The isolation of microplastics from other organic and inorganic contents is a major hurdle to the effective quantification and characterization of microplastics in wastewater and biosolids samples. As a result, a well-established and standardized isolation method is vital for the analysis of microplastics. In this study, we evaluated biological hydrolysis, enzymatic hydrolysis, wet peroxidation and ethylenediaminetetraacetic acid treatment for microplastics isolation and demonstrated that the integration of these processes can effectively remove organic and inorganic contents to provide a clear microscope view for microplastics identification from wastewater and sludge samples. To the best of our knowledge, this study is the first to introduce biological hydrolysis and ethylenediaminetetraacetic acid treatment for the isolation of microplastics from environmental samples. The results reported could facilitate the establishment of a standardized procedure for microplastic isolation from wastewater and biosolid samples.

Stool or blood culture? A search for a gold standard for isolation of salmonella typhi from patients with clinical symptoms of enteric fever in Bauchi state tertiary hospital

Introduction: Salmonella typhi (S. typhi) infection is endemic in Nigeria. In recent time, blood culture and stool were the most common diagnostic means of confirming salmonella infection in humans, since it is based on culture for isolation, identification among others. Although, there is lack of reliable and standard method of culture for its isolation. This study aimed to determine the gold standard between blood and stool for the cultural isolation of S. typhi. Methods: The study used laboratory-based cross-sectional study; patients who presented symptoms of enteric fever visiting Abubakar Tafawa Balewa University Teaching Hospital (ATBUTH) during the period of the study were recruited. Informed consent of volunteers and guardians were obtained and in addition confidentially of the results were ensured. A total of 150 patients samples were collected and cultured including adults and children. The bacterial isolated were identified by gram’s reaction and biochemical characteristics. Result: Out of 150 samples examined, 7 (4.1%) tested positive for S. typhi comprising of 5(71.4%) culture from blood and 2(28.6%) culture from stool. The result of this study shows a significant mean difference (t-value = 2.95, p-value = 0.026) between Blood culture and stool culture at 5% level of significance. The culture from blood was found to be more sensitive than the culture from stool. It is therefore recommended gold standard for S. typhi isolation. Conclusion: The study revealed cultures from blood specimen’s yielded highest number of S.typhi isolates when related to culture from stool samples. Therefore, culture from blood sample should be of priority in the isolation of S. typhi for easy identification of the bacteria. Culture from blood sample remains the gold standard method for isolation of S. typhi when it comes to blood or stool sample.

Comparative analysis of magnetically activated cell sorting and ultracentrifugation methods for exosome isolation.

Mesenchymal stem cell-derived exosomes regulate cell migration, proliferation, differentiation, and synthesis of the extracellular matrix, giving great potential for the treatment of different diseases. The ultracentrifugation method is the gold standard method for exosome isolation due to the simple protocol, and high yield, but presents low purity and requires specialized equipment. Amelioration of technical optimization is required for quick and reliable confinement of exosomes to translate them to the clinic as cell therapeutics In this study, we hypothesized that magnetically activated cell sorting may provide, an effective, reliable, and rapid tool for exosome isolation when compared to ultracentrifugation. We, therefore, aimed to compare the efficiency of magnetically activated cell sorting and ultracentrifugation for human mesenchymal stem cell-derived exosome isolation from culture media by protein quantification, surface biomarker, size, number, and morphological analysis. Magnetically activated cell sorting provided a higher purity and amount of exosomes that carry visible magnetic beads when compared to ultracentrifugation. The particle number of the magnetically activated cell sorting group was higher than the ultracentrifugation. In conclusion, magnetically activated cell sorting presents a quick, and reliable method to collect and present human mesenchymal stem cell exosomes to clinics at high purity for potential cellular therapeutic approaches. The novel isolation and purification method may be extended to different clinical protocols using different autogenic or allogeneic cell sources.

Comparison of Two Culture-Based Detection Systems for the Isolation of Arcobacter butzleri, Arcobacter cryaerophilus, and Arcobacter skirrowii in Raw Ground Poultry

Arcobacters are emerging pathogens that have been underestimated due to a lack of a standardized isolation method. The aim of this research was to evaluate the ability to isolate Arcobacter butzleri, Arcobacter cryaerophilus, and Arcobacter skirrowii using two Arcobacter-specific culture detection systems: (i) the Houf broth and modified charcoal cefoperazone deoxycholate agar supplemented with cefoperazone, amphotericin B, and teicoplanin (HB/mCCDA+CAT), and (ii) the Nguyen-Restaino-Juárez Arcobacter enrichment broth and chromogenic agar (NRJ-B/M). Both detection systems were evaluated for productivity ratio, sensitivity, and specificity. As a result, the productivity ratio for both plating agars were >90%, which indicates that the selective agents used in the two plating agars did not inhibit Arcobacter growth. Moreover, sensitivity evaluations using artificially inoculated retail ground poultry (n = 780) determined that both detection systems were able to isolate A. butlzeri with >95% sensitivity at the 0.1 and 1.0–2.0 CFU/g detection level. The sensitivity in A. cryaerophilus isolation was higher for NRJ-B/M (78.0% at 0.1 CFU/g; 95.1% at 1.0–2.0 CFU/g) when compared with HB/mCCDA+CAT (34.1% at 0.1 CFU/g; 51.2% at 1.0–2.0 CFU/g). Both detection systems resulted in <50% sensitivity when isolating A. skirrowii at 0.1 and 1.0–2.0 CFU/g; however, the sensitivity for NRJ-B/M was significantly higher than HB/mCCDA+CAT. At the detection level of 5.0 CFU/g, both detection systems were able to isolate A. skirrowii with 100% sensitivity. Specificity comparisons using uninoculated ground poultry samples (n = 40) indicated the growth of background microbiota were significantly inhibited or could be easily differentiated on NRJ-B/M (90.0%, specificity) when compared with HB/mCCDA+CAT (30.0%, specificity). Overall, these results show that the NRJ-B/M detection system is a more sensitive and specific detection system when isolating Arcobacter spp. from ground chicken.

Optimization of Flow Cytometric Sorting Parameters for High-Throughput Isolation and Purification of Small Extracellular Vesicles.

Small extracellular vesicles (sEV) can be released from all cell types and carry protein, DNA, and RNA. Signaling molecules serve as indicators of the physiological and pathological state of a cell. However, there is no standard method for sEV isolation, which prevents downstream biomarker identification and drug intervention studies. In this article, we provide a detailed protocol for the isolation and purification of 50-200 nm sEV by a flow cell sorter. For this, a 50 µm nozzle and 80 psi sheath fluid pressure were selected to obtain a good sorting rate and stable side stream. Standard sized polystyrene microspheres were used to locate populations of 100, 200, and 300 nm particles. With additional optimization of the voltage, gain, and forward scatter (FSC) triggering threshold, the sEV signal could be separated from the background noise. These optimizations provide a panel of critical sort settings that enables one to obtain a representative population of sEV using FSC vs. side scatter (SSC) only. The flow cytometry-based isolation method not only allows for high-throughput analysis but also allows for synchronous classification or proteome analysis of sEV based on the biomarker expression, opening numerous downstream research applications.

Enzymatic isolation of articular chondrons: is it much different than that of chondrocytes?

In native articular cartilage, chondrocytes are completely capsulated by a pericellular matrix (PCM), together called the chondron. Due to its unique properties (w.r.t. territorial matrix) and importance in mechanotransduction, the PCM and chondron may be important in regenerative strategies. The current gold standard for the isolation of chondrons from cartilage dates from 1997. Although previous research already showed the low cell yield and the heterogeneity of the isolated populations, their compositions and properties have never been thoroughly characterized. This study aimed to compare enzymatic isolation methods for chondrocytes and chondrons and characterize the isolation efficiency and quality of the PCM. Bovine articular cartilage was digested according to the 5-hour gold standard chondron isolation method (0.3% dispase + 0.2% collagenase II), an overnight chondron isolation (0.15% dispase + 0.1% collagenase II), and an overnight chondrocyte isolation (0.15% collagenase II + 0.01% hyaluronidase). Type VI collagen staining, fluorescence-activated cell sorting (FACS) analysis, specific cell sorting and immunohistochemistry were performed using a type VI collagen staining, to study their isolation efficiency and quality of the PCM. These analyses showed a heterogeneous mixture of chondrocytes and chondrons for all three methods. Although the 5-hour chondron isolation resulted in the highest percentage of chondrons, the cell yield was significantly lower compared to the other isolation methods. FACS, based on the type VI collagen staining, successfully sorted the three identified cell populations. To maximize chondron yield and homogeneity, the overnight chondron enzymatic digestion method should be combined with type VI collagen staining and specific cell sorting.

PREVALENCE AND ANTI MICROBIAL SUSCEPTIBILITY OF CLINICAL ISOLATES OF ENTEROCOCCUS SPECIES WITH SPECIAL REFERENCE TO VANCOMYCIN RESISTANCE

Background: Among enterococcus species , 80 -90% and 5-10% of human infection are caused by enterococcus faecalis and faecium respectively .The rate of isolation of Enterococcus faecium and other species is increase at recent times from various clinical samples . Enterococcus faecium showing higher degree of drug resistance. Enterococcus gallinarum and Enterococcus casseliavus are intrinsically resistant to vancomycin thereby inappropriate treatment can be avoided. Materials and methods: The clinical samples included blood, urine and exudates (pus,tissues, sterile body uids) were collected aseptically and processed as per standard methods for isolation and identication of organism. Antimicrobial susceptibility testing was performed using Kirby Bauer disc diffusion method. A total of 64 Enterococcus strains were isolated from clinic Result: al samples during the study period.The maximum number of Enterococcus isolates were obtained from Exudates 37(57.8%) ,urine 23(35.9%) followed by blood 4(6.2%) .Among Enterococcus species ,E.faecalis 59 (92.2%) and E.faecium 5 (7.8%) was isolated. The isolates from urine and exudates were predominantly resistance to antimicrobials like ampicillin, high level aminoglycoside, ciprooxacin and sensitive to linezolid, vancomycin and nitrofurantoin for urine samples. Enterococcus faecalis isolates were uniformly sensitive to Ampicillin, Gentamicin, Ciprooxacin, Linezolid and Vancomycin. Enterococcus faecium isolates were sensitive to Linezolid and Vancomycin and resistant to Ampicillin, Gentamicin, Ciprooxacin. Conclusion: This study illustrates the prevalence and antibiotic susceptibility pattern of enterococcus species from various clinical samples . In our study Enterococci did not show resistant to vancomycin.

Why are brown bears protected against atherosclerosis even though their plasma cholesterol levels are twice that of humans?

Plasma cholesterol and triglyceride levels are twice as high in hibernating brown bears (Ursus arctos) than in healthy humans. Yet, bears display no sign of atherosclerosis development. To explore this apparent paradox, we analyzed lipoproteins from same ten individual bears plasma collected during winter (hibernation; February) and summer (active; June) in the same year. Plasma from fourteen healthy humans were analyzed as comparator. We used standard methods for lipoprotein isolation, composition and functional investigation. The results shows that in brown bears the absence of atherosclerosis despite elevated cholesterol is likely associated with two main athero-protective properties of circulating lipoproteins. First, a significant ten times lower affinity of low-density-lipoprotein (LDL) particles for arterial proteoglycans and secondly, an elevated plasma cholesterol efflux capacity. What does the brown bear data tell us? That elevated total cholesterol and apoB-containing lipoproteins not always associates with atherosclerosis disease. We need to look also at the lipoprotein biochemical features and functionality as they are relevant for arterial pathophysiology. What is the translatability into human of these results? We humans need to control our total and LDL-cholesterol levels. We are not brown bears!