Tissue Culture Samples Research Articles

Isolation of Mitochondria for Mitochondrial Supercomplex Analysis from Small Tissue and Cell Culture Samples.

Over the last decades, the evidence accumulated about the existence of respiratory supercomplexes (SCs) has changed our understanding of the mitochondrial electron transport chain organization, giving rise to the proposal of the "plasticity model." This model postulates the coexistence of different proportions of SCs and complexes depending on the tissue or the cellular metabolic status. The dynamic nature of the assembly in SCs would allow cells to optimize the use of available fuels and the efficiency of electron transfer, minimizing reactive oxygen species generation and favoring the ability of cells to adapt to environmental changes. More recently, abnormalities in SC assembly have been reported in different diseases such as neurodegenerative disorders (Alzheimer's and Parkinson's disease), Barth Syndrome, Leigh syndrome, or cancer. The role of SC assembly alterations in disease progression still needs to be confirmed. Nevertheless, the availability of enough amounts of samples to determine the SC assembly status is often a challenge. This happens with biopsy or tissue samples that are small or have to be divided for multiple analyses, with cell cultures that have slow growth or come from microfluidic devices, with some primary cultures or rare cells, or when the effect of particular costly treatments has to be analyzed (with nanoparticles, very expensive compounds, etc.). In these cases, an efficient and easy-to-apply method is required. This paper presents a method adapted to obtain enriched mitochondrial fractions from small amounts of cells or tissues to analyze the structure and function of mitochondrial SCs by native electrophoresis followed by in-gel activity assays or western blot.

Exosomal long non-coding RNA MALAT1: a candidate of liquid biopsy in monitoring of Wilms' tumor.

Wilms' tumor (WT) is a rare kidney cancer that primarily affects children. Exosomes are extracellular vesicles that cargo nucleic acids, proteins,etc. for cellular communication. Long non-coding RNAs (lncRNAs) have utility as biomarkers for cancer diagnosis, prognosis, and disease monitoring. We hypothesize that expression of lncRNA, metastasis-associated lung adenocarcinoma transcript-1(MALAT1), is dysregulated and possibly trafficked within exosomes to influence the tissue microenvironment for metastasis and recurrence of WT. We investigated the expression of MALAT1 in thirty WT samples by qPCR. Exosomes were isolated using a precipitated and affinity-binding-based kit, and characterized using TEM, NTA, and DLS. Mean number of exosomes was 9.01×108/mL in primary culture, 1.64×108/mL in urine, and 4.65×108/plasma:400µl. Average yield of total RNA was 1.28µg (primary-culture supernatant:1ml), 1.47µg (Urine:1ml), 1.65µg (Plasma:400 µL). We quantified MALAT1 in exosomes derived from these sources in patients of WT. Expression of MALAT1 was significantly downregulated (p=0.008) in WT samples. This is the first study that demonstrated the presence of lncRNA MALAT1 in various invasive and non-invasive samples of patients with WT(primary tissue culture, urine, and plasma samples).

Using Small Molecules for Targeting Heavy Metals in Neurotoxicity and Neuroinflammation.

Pharmaceutical drugs, natural toxins, industrial chemicals, and various environmental toxins negatively impact the nervous system. A significant cause of many neurodegenerative diseases is neurotoxicity. Although trace amounts of heavy metals are required for the proper functioning of several metabolic pathways, their dysregulation can cause many cellular and molecular alterations, which can enhance the risks associated with several neurodegenerative diseases. For example, high levels of heavy metals like manganese (Mn) affect the central nervous system with implications in both higher-order cognitive and motor functions. In addition, the buildup of amyloid aggregates and metal ions in the brain of patients with Alzheimer's disease is associated with disease pathogenesis. Small molecules capable of targeting neuroinflammation and neuroprotection pathways would be valuable to elucidate the pathological pathways associated with metal toxicity in neurogenerative disease. This chapter will summarize the necessary steps involved in (1) culturing of cell lines and maintenance of animal models, (2) design and preparation of samples of small molecules and treatment methodologies, (3) RNA and protein isolation and preparation of tissue and cell culture samples for quantitative studies, and (4) quantitative estimation of cellular products.

Sample preparation and data collection for serial block face scanning electron microscopy of mammalian cell monolayers.

Volume electron microscopy encompasses a set of electron microscopy techniques that can be used to examine the ultrastructure of biological tissues and cells in three dimensions. Two block face techniques, focused ion beam scanning electron microscopy (FIB-SEM) and serial block face scanning electron microscopy (SBF-SEM) have often been used to study biological tissue samples. More recently, these techniques have been adapted to in vitro tissue culture samples. Here we describe step-by-step protocols for two sample embedding methods for in vitro tissue culture cells intended to be studied using SBF-SEM. The first focuses on cell pellet embedding and the second on en face embedding. En face embedding can be combined with light microscopy, and this CLEM workflow can be used to identify specific biological events by light microscopy, which can then be imaged using SBF-SEM. We systematically outline the steps necessary to fix, stain, embed and image adherent tissue culture cell monolayers by SBF-SEM. In addition to sample preparation, we discuss optimization of parameters for data collection. We highlight the challenges and key steps of sample preparation, and the consideration of imaging variables.

Are Absorbable Plates More Resistant to Infection Than Titanium Implants? An Experimental Pre-Clinical Trial in Rabbits.

Background: Infection of orthopaedic implants after internal fixation of bone fractures remains a major complication with occasionally devastating consequences. Recent studies have reported that the use of absorbable materials, instead of metallic ones, may lead to a lower incidence of postoperative infection. In this experimental pre-clinical animal study, we compared the infection rate between absorbable implants consisting of copolymers composed from trimethylene carbonate, L-polylactic acid, and D, L-polylactic acid monomers, and titanium implants after the inoculation of a pathogenic microorganism. Material and Methods: We used an experimental implant-related infection model in rabbits. Sixty animals were randomly and equally divided into two groups. In all animals, the right femur was exposed via a lateral approach and a 2.5 mm two-hole titanium plate with screws (Group A), or a two-hole absorbable plate and screws (Group B), were applied in the femoral shaft. Afterwards, the implant surface was inoculated with Pseudomonas Aeruginosa at a concentration of 2 × 108 CFU/mL. The primary outcome was the comparison of the incidence of developed infection between the two groups. The wound condition was monitored on a daily basis and radiographies were obtained at 12 weeks postoperatively. Infection-related laboratory markers (white blood cell count, erythrocyte sedimentation rate, and C-reactive protein values) were assessed at 3, 6, and 16 weeks postoperatively. Histologic analysis and cultures of tissue samples were also performed to evaluate the presence of infection. Results: Clinical and laboratory signs of infection were evident in 11 rabbits in Group A (36.7%), and 4 in Group B (13.3%). The difference between the groups was statistically significant (p = 0.04). Five animals in Group B (16.7%) had clinical and histologic signs of a foreign-body reaction with significantly elevated CRP and ESR values but no simultaneous presence of infection was identified (p = 0.04). Bone remodelling with thickening of the periosteum and surrounding sclerosis was demonstrated radiologically in animals developing infection or foreign-body reactions. Conclusions: Absorbable plates and screws show lower susceptibility to infection compared to titanium ones. However, their application is associated with foreign-body reaction and the potential need for a second surgical intervention.

The value of sonication on orthopaedic implants in an everyday clinical setting – an exploratory study

BackgroundSonication of removed orthopaedic implants in suspected implant-associated infections (IAI) is widely applied internationally. However, evaluation of the utility of sonication on all implants removed in everyday standard practice is scarce. This exploratory study was performed to evaluate the application of sonication fluid (SF) culture on removed orthopaedic implants, irrespective of the reason for removal.MethodsOut of 100 removed orthopaedic implants collected between August 2019 and September 2020, 77 implants with availability of concurrent tissue culture samples were included in the study. Removed implants were categorized into a confirmed or suspected IAI group and a presumed aseptic group based on pre-operative diagnosis by the responsible surgeon. Implants were sonicated and SF culture performed under both aerobic and anaerobic conditions. The significance of all bacterial isolates was evaluated based on the CFU/mL cut-offs of the EBJIS guidelines, except for C. acnes where additional investigations were performed.ResultsThe results of SF culture in the two groups were compared with their corresponding tissue cultures. Out of the 12 cases in the confirmed/suspected IAI group, SF culture was positive in 11 cases and had increased diagnostic yield in two (17%) cases compared to tissue culture. Increased diagnostic yield of SF compared to tissue culture was seen in seven (11%) of the 65 implants in the presumed aseptic group. If growth of Cutibacterium species isolates were interpreted based on EBJIS cut-off for SF culture instead of the study-specific criteria, then two isolates considered to represent infection might have been missed while three other isolates considered contaminants would have fallen under the ‘infection confirmed’ category in the EBJIS guidelines.ConclusionSonication with SF culture has increased diagnostic yield compared to tissue cultures in all implants irrespective of reason for removal. However, positive SF cultures with Cutibacterium species should always be interpreted with extreme care.

Virus Inactivation by Formaldehyde and Common Lysis Buffers.

Numerous mammalian viruses are routinely analyzed in clinical diagnostic laboratories around the globe or serve as indispensable model systems in viral research. Potentially infectious viral entities are handled as blood, biopsies, or cell and tissue culture samples. Countless protocols describe methods for virus fixation and inactivation, yet for many, a formal proof of safety and completeness of inactivation remains to be shown. While modern nucleic acid extraction methods work quite effectively, data are largely lacking on possible residual viral infectivity, e.g., when assessed after extended culture times, which maximizes the sensitivity for low levels of residual infectiousness. Therefore, we examined the potency and completeness of inactivation procedures on virus-containing specimens when applying commonly used fixatives like formaldehyde or nucleic acid extraction/lysis buffers. Typical representatives of different virus classes, including RNA and DNA viruses, enveloped and non-enveloped, such as adenovirus, enterovirus, lentivirus, and coronavirus, were used, and the reduction in the in vitro infectiousness was assessed for standard protocols. Overall, a 30-minute incubation with formaldehyde at room temperature effectively inactivated all tested enveloped and non-enveloped viruses. Full inactivation of HIV-1 and ECHO-11 was also achieved with all buffers in the test, whereas for SARS-CoV-2 and AdV-5, only five of the seven lysis buffers were fully effective under the tested conditions.

Impact of antigen retrieval protocols on the immunohistochemical detection of epigenetic DNA modifications

This study compares three different pretreatment protocols for the immunohistochemical detection of 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) in nuclear DNA. The human biological samples analyzed included formalin-fixed and paraffin-embedded (FFPE) normal squamous epithelium, ethanol-fixed cultured cells, and metaphase chromosomes. The antigen retrieval methods included low pH Citrate and high pH Tris–ethylenediaminetetraacetic acid (EDTA) protocols, as well as a method using Pepsin pretreatment combined with HCl for DNA denaturation. A gradual increase in the detection levels of 5-mC and 5-hmC was observed when going from Citrate via Tris/EDTA to Pepsin/HCl retrieval. While the Citrate retrieval protocol was the least efficient for the detection of 5-mC and 5-hmC, it did preserve nuclear morphology and enabled visualization of differences in intra- and internuclear distribution patterns in tissue and cell culture samples by single- and double-fluorescence detection. Quantification of (hydroxy)methylation levels in FFPE material demonstrated a significant heterogeneity and differences in 5-mC and 5-hmC levels within and between nuclei in the different compartments of normal squamous epithelium. It was concluded that immunohistochemical detection of 5-mC and 5-hmC enables the correlation of these DNA modifications with histomorphological features in heterogeneous tissues, but this is influenced by different pretreatment protocols that must be carefully chosen to allow an appropriate interpretation of these epigenetic switches.

A Rapid Protocol for Preparing 8-Aminopyrene-1,3,6-Trisulfonate-Labeled Glycans for Capillary Electrophoresis-Based Enzyme Assays.

Purified glycan standards are required for glycan arrays, characterizing substrate specificities of glycan-active enzymes, and to serve as retention-time or mobility standards for various separation techniques. This chapter describes a method for the rapid separation, and subsequent desalting, of glycans labeled with the highly fluorescent fluorophore 8-aminopyrene-1,3,6-trisulfonate (APTS). By using fluorophore-assisted carbohydrate electrophoresis (FACE) on polyacrylamide gels, a technique amenable to equipment readily available in most molecular biology laboratories, many APTS-labeled glycans can be simultaneously resolved. Excising specific gel bands containing the desired APTS-labeled glycans, followed by glycan elution from the gel by simple diffusion and subsequent solid-phase extraction (SPE)-based desalting, affords a single glycan species free of excess labeling reagents and buffer components. The described protocol also offers a simple, rapid method for the simultaneous removal of excess APTS and unlabeled glycan material from reaction mixtures. This chapter describes a FACE/SPE procedure ideal for preparing glycans for capillary electrophoresis (CE)-based enzyme assays, as well as for the purification of rare, commercially unavailable glycans from tissue culture samples.

A retrospective analysis of blood culture-negative endocarditis at a tertiary care centre in Switzerland

Numerous studies from different countries have contributed to an improved understanding of blood culture-negative infective endocarditis. However, little is known about its epidemiology and microbiology in Switzerland. We aimed to assess the epidemiology and microbiology of blood culture-negative endocarditis at the University Hospital Zurich, Switzerland. We screened all patients hospitalised between 1997 and 2020 with possible or definite endocarditis at our institution. Thereof, we identified all cases with blood culture-negative endocarditis and retrospectively retrieved patient characteristics, microbiological, histopathological, radiographicand surgical data from medical records. Among 861 patients screened, 66 (7.7%) cases of blood culture-negative endocarditis were identified. Thereof, 31 cases could be microbiologically documented or not documented (n = 30), and in five cases a non-infectious aetiology was confirmed. Endocarditis predominantly affected men (77%) and the left heart (79%);predisposing factors were prosthetic valves (42%), congenital heart disease (35%) and prior endocarditis (14%). The most common reasons for negative blood cultures were antibiotic treatment prior to blood culture sampling (35%), fastidious and slow growing microorganisms (30%) and definite non-infective endocarditis (8%). Coxiella burnetii and Bartonella spp. were the most common fastidious bacteria identified. In addition to serology, identification of causative microorganisms was possible by microbiological and/or histopathological analysis of tissue samples, of which polymerasechain reaction testing (PCR) of the 16S ribosomal RNA proved to be most successful. The present study provides a detailed analysis of blood culture-negative endocarditis over a time span of more than 20 years in Zurich, Switzerland. Antibiotic treatment prior to blood collection, and fastidious and slow growing organisms were identified as main reasons for sterile blood cultures. Typical culture-negative bacteria were mainly found by PCR and/or culture of tissue samples.

The diagnostic potential of MicroDTTect compared to conventional culture of tissue samples in orthopedic infections

Objective. The evolution of new prosthetic and osteosynthesis devices has led to better outcomes and therefore more frequent surgical indications. As a consequence, an increased incidence of complications such as infections or aseptic loosening in orthopedic and trauma surgery are being recorded in general population. MicroDTTect is a quick and simple system that is useful to detect low grade or bio-film related infections. The aim of this study was to evaluate the reliability of MicroDTTect in detection of orthopedic infections compared to conventional culture of tissue samples. Methods. The population enrolled was composed of 13 patients undergoing surgery for prosthesis or osteosynthetic device failure or loosening. The MicroDTTect system and traditional culture of tissue samples were applied to identify the pathogens and compared with each other. Results. MicroDTTect had a higher sensitivity compared to conventional culture of tissue samples. Two cases resulted positive while the traditional culture sample showed a false negative result. In addition, with MicroDTTect a polymicrobial infection was identified, while with traditional methods was misdiagnosed.Conclusions. We showed that treatment of suspected implant infections using the MicroDTTect device improves microbiological diagnosis with more sensitive results, leading to a more accurate treatment.

Preparation of biological monolayers for producing high-resolution scanning electron micrographs.

Scanning electron microscopy (SEM) provides a technical platform for nanoscopic mapping of biological structures. Correct preparation of SEM samples can provide an unprecedented understanding of the nexus between cellular morphology and topography. This comparative study critically examines two coating methods for preparing biological samples for scanning electron microscopy, while also providing novel advice on how to prepare in vitro epithelial or endothelial samples for high-resolution scanning-electron microscopy (HR-SEM). Two obstacles often confront the biologist when investigating cellular structures grown under tissue culture conditions, namely., how to prepare and present the biological samples to the HR-SEM microscope without affecting topographical membrane and cellular structural alterations. Firstly, our use of the Millicell cellulose inserts on which to grow our cellular samples in preparation for HR-SEM is both novel and advantageous to comparing the permeability function of cells to their morphological function. Secondly, biological material is often non-conducting, thermally sensitive and fragile and, therefore, needs to be fixed correctly and coated with thin conducting metal to ensure high-resolution detail of samples. Immortalized mouse brain endothelial cells (bEnd5) was used as a basis for describing the preferences in the use of the protocol. We compare two biological sample coating modalities for the visualizing and analysis of texturized, topographical, membranous ultrastructures of brain endothelial cell (BEC) confluent monolayers, namely, carbon and gold:palladium (Au:Pd) sputter coating in preparation for HR-SEM. BEC monolayers sputter-coated with these two modalities produced three-dimensional micrographs which have distinctly different topographical detail from which the nanostructural cellular data can be examined. The two coating methods display differences in the amount of nanoscopic detail that could be resolved in the nanosized membrane cytoarchitecture of BEC monolayers. The micrographical data clearly showed that Au:Pd sputter-coated samples generate descript imagery, providing useful information for profiling membrane nanostructures compared to carbon-coated samples. The recommendations regarding the contrast in two modalities would provide the necessary guidance to biological microscopists in preparing tissue culture samples for HR-SEM.

A versatile new tool derived from a bacterial deubiquitylase to detect and purify ubiquitylated substrates and their interacting proteins.

Protein ubiquitylation is an important posttranslational modification affecting a wide range of cellular processes. Due to the low abundance of ubiquitylated species in biological samples, considerable effort has been spent on methods to purify and detect ubiquitylated proteins. We have developed and characterized a novel tool for ubiquitin detection and purification based on OtUBD, a high-affinity ubiquitin-binding domain (UBD) derived from an Orientia tsutsugamushi deubiquitylase (DUB). We demonstrate that OtUBD can be used to purify both monoubiquitylated and polyubiquitylated substrates from yeast and human tissue culture samples and compare their performance with existing methods. Importantly, we found conditions for either selective purification of covalently ubiquitylated proteins or co-isolation of both ubiquitylated proteins and their interacting proteins. As proof of principle for these newly developed methods, we profiled the ubiquitylome and ubiquitin-associated proteome of the budding yeast Saccharomyces cerevisiae. Combining OtUBD affinity purification with quantitative proteomics, we identified potential substrates for the E3 ligases Bre1 and Pib1. OtUBD provides a versatile, efficient, and economical tool for ubiquitin research with specific advantages over certain other methods, such as in efficiently detecting monoubiquitylation or ubiquitin linkages to noncanonical sites.

Fiabilité des ultrasons préopératoires à contraste amélioré (CEUS) dans les pseudarthroses des os longs pour le diagnostic microbiologique des biopsies tissulaires comparées aux sonications sur matériel

IntroductionBacterial infection in the context of fracture repair remains a severe complication in trauma surgery and may result in long bone nonunion. Since treatment options for aseptic and infected nonunions vary greatly, diagnostic methods should ideally differentiate between these two entities as accurately as possible. Recently, contrast-enhanced ultrasound (CEUS) has been introduced as a preoperative imaging technique to evaluate hypervascularity at the fracture site as sign of bacterial infection. HypothesisPreoperative CEUS predicts results of microbiological evaluation obtained either by culture of tissue samples or by analyzing the sonication fluid following removal and sonication of the implant. Patients and methodsOver the course of 6 months, 26 patients with long bone nonunions were included in this study. Patients’ clinical data were evaluated. Tissue samples were collected intraoperatively and examined by standard microbiological techniques. The sonication method was applied to removed implants. Additionally, 1–3 days before surgery, CEUS was performed to determine hypervascularity at the nonunion site as a possible parameter for infection. ResultsCulture of tissue samples indicated infection in 50% of cases and implant sonication in 57.7% of cases. However, there was merely a fair agreement (κ=0.231) between these two diagnostic methods. CEUS predicted results of tissue culture reliably (sensitivity 92.3% and specificity 100%), whereas implant sonication showed no significant correlations with results from CEUS. Hypertrophic and atrophic nonunions were evaluated separately to determine possible differences in vascularity. We found that contrast peak enhancement of CEUS was similar in atrophic and hypertrophic nonunions with positive culture of tissue samples. Both differed significantly from culture negative cases (p=0.0016 and 0.0062). Results of implant-sonication positive or negative cases in atrophic and hypertrophic nonunions, however, were less clear and could be misleading. DiscussionWe were able to confirm CEUS as a valuable preoperative diagnostic tool that reliably predicts microbiology of tissue culture samples, but not of implant sonication. Level of evidenceI; diagnostic study.

Preoperative contrast-enhanced ultrasound (CEUS) of long bone nonunions reliably predicts microbiology of tissue culture samples but not of implant-sonication

IntroductionBacterial infection in the context of fracture repair remains a severe complication in trauma surgery and may result in long bone nonunion. Since treatment options for aseptic and infected nonunions vary greatly, diagnostic methods should ideally differentiate between these two entities as accurately as possible. Recently, contrast-enhanced ultrasound (CEUS) has been introduced as a preoperative imaging technique to evaluate hypervascularity at the fracture site as sign of bacterial infection. HypothesisPreoperative CEUS predicts results of microbiological evaluation obtained either by culture of tissue samples or by analyzing the sonication fluid following removal and sonication of the implant. Patients and methodsOver the course of 6 months, 26 patients with long bone nonunions were included in this study. Patients’ clinical data were evaluated. Tissue samples were collected intraoperatively and examined by standard microbiological techniques. The sonication method was applied to removed implants. Additionally, 1–3 days before surgery, CEUS was performed to determine hypervascularity at the nonunion site as a possible parameter for infection. ResultsCulture of tissue samples indicated infection in 50% of cases and implant sonication in 57.7% of cases. However, there was merely a fair agreement (κ=0.231) between these two diagnostic methods. CEUS predicted results of tissue culture reliably (sensitivity 92.3% and specificity 100%), whereas implant sonication showed no significant correlations with results from CEUS. Hypertrophic and atrophic nonunions were evaluated separately to determine possible differences in vascularity. We found that contrast peak enhancement of CEUS was similar in atrophic and hypertrophic nonunions with positive culture of tissue samples. Both differed significantly from culture negative cases (p=0.0016 and 0.0062). Results of implant-sonication positive or negative cases in atrophic and hypertrophic nonunions, however, were less clear and could be misleading. DiscussionWe were able to confirm CEUS as a valuable preoperative diagnostic tool that reliably predicts microbiology of tissue culture samples, but not of implant sonication. Level of evidenceI; diagnostic study.

Low Pathogenic Strain of Infectious Pancreatic Necrosis Virus (IPNV) Associated with Recent Outbreaks in Iranian Trout Farms.

Infectious pancreatic necrosis (IPN), first described as acute viral catarrhal enteritis, is a highly contagious disease with variable pathogenicity that has been linked to genetic variation in the viral VP2 gene encoding the capsid protein. In this study, the IPN virus (IPNV) is isolated from the moribund fish from five of fourteen Iranian trout farms from 2015 to 2017. The affected fish showed mortality rates ranging from 20% to 60%, with the main clinical signs of exophthalmia, darkened skin, and mild abdominal distension, as well as yellow mucoid fluid in the intestine. Histopathological examination of intestinal sections confirmed acute catarrhal enteritis in all samples. RT-PCR assay of the kidney tissue and cell culture (CHSE-214) samples consistently confirmed the presence of the virus. The phylogenetic analysis of the partial VP2 sequence revealed that the detected isolates belong to genogroup 5, and are closely related to the Sp serotype strains of European origin. Characterization of VP2 of all isolates revealed the P217T221 motif that previously was associated with avirulence or low virulence, while all IPNV-positive fish in this study were clinically affected with moderate mortality. The IPNV isolates from Iran are associated with two lineages that appear to have originated from Europe, possibly via imported eggs.

Escherichia coli, a common constituent of benign prostate hyperplasia‐associated microbiota induces inflammation and DNA damage in prostate epithelial cells

The role of microbiota in the pathophysiology of benign prostate hyperplasia (BPH), especially in creating an inflammatory milieu may not be avoided. The major objectives of this study were to investigate the microbial composition of BPH tissues, its association with inflammation and check the effect of clinically isolated bacteria on prostate epithelial cells. The study includes 36 patients with a pathological diagnosis of BPH. Following strict aseptic measures, tissues were collected after transurethral resection of prostate, multiple pieces of the resected tissues were subjected to histopathological analysis, bacterial culture and genomic DNA extraction. Microbial composition was analyzed by culture and/or next-generation sequencing methods. Annotation of operational taxonomy unit has been done with an in-house algorithm. The extent of inflammation was scored through histological evaluation of tissue sections. The effect of clinical isolates on nuclear factor-κB (NF-κB) activity and induction of DNA-damage in the prostate epithelial cells were evaluated. Histopathological analysis of the BPH tissues showed the presence of inflammation in almost all the tissues with a varied level at different regions of the same tissue section and the level of overall inflammation was different from patients to patients. Microbial culture of tissue samples showed the presence of live bacteria in 55.5% (20 out of 36) of the patient tissues. Majority of the isolates were coagulase-positive Staphylococcus, E. coli and Micrococcus spp. Further, V3 16S rRNA sequencing of the DNA isolated from BPH tissues showed the presence of multiple bacteria and the most common phylum in the BPH tissues were found to be Proteobacteria, Actinobacteria, Firmicutes, and Bacteroidetes. The E. coli, isolated from one of the tissue was able to activate NF-κB and induce DNA damage in prostate epithelial cells. Phospho-histone γH2A.X staining confirmed the presence of cells with damaged DNA lesion in BPH tissues and also correlated with the severity of inflammation. Our study has shown that the BPH tissues do have a divergent microbial composition including the commonly found E. coli (phylum Proteobacteria), and these bacteria might contribute to the BPH-associated inflammation and/or tissue damage. The BPH-associated E. coli induced NF-κB signaling and DNA damage in prostate epithelial cells in vitro.

Microbiological diagnosis of intramedullary nailing infection: comparison of bacterial growth between tissue sampling and sonication fluid cultures.

Intramedullary nailing (IMN) has been frequently indicated to treat long bone open and closed fractures, but IMN infection (IMNI) may have devastating consequences. Sonication has been regarded as an important add-on for microbial identification on a variety of orthopaedic implant-associated infections, but its role in the IMNI is poorly studied. We aim at evaluating the accuracy obtained by conventional peri-implant tissue culture (TC) samples with sonication fluid cultures (SCs) of IMNI. Longitudinal prospective cohort study ongoing since June 2014, which included patients with indication for IMN removal due to any reason. Clinical diagnosis of INMI was defined according to publication addressing fracture-related infections. Minimal of two samples from TC were cultured. SCs followed the protocol previously published. Statistical analysis was performed using McNemar's test for related proportions. We included 54 patients submitted to IMN retrieval, of whom 47 presenting clinical signs of IMNI. Sensitivity for detecting microorganisms using TC and SC was 89.4% (42/47) and 97.6% (40/41), and specificity was 71.4% (5/7) for both TC and SC (p = 1.00). Positive and negative predictive values for TC and SC were 95.5% (42/44), 95.2% (40/42), 50% (5/10), and 83.3% (5/6), respectively. The most frequent organisms isolated in both TC and SC were Staphylococcus aureus, S. epidermidis, and Enterococcus sp. Polymicrobial infection was diagnosed in 14.8% (8/54) and 25% (12/48) by TC and SC, respectively (p = 0.19). Sonication fluid and tissue samples presented similar accuracy on the diagnosis of IMNI, but SC was advantageous of detecting polymicrobial infection.

Diabetes-related sex differences in the brain endothelin system following ischemia in vivo and in human brain endothelial cells in vitro.

The endothelin (ET) system has been implicated to contribute to the pathophysiology of cognitive impairment and stroke in experimental diabetes. Our goals were to test the hypotheses that (1) circulating and (or) periinfarct ET-1 levels are elevated after stroke in both sexes and this increase is greater in diabetes, (2) ET receptors are differentially regulated in the diabetic brain, (3) brain microvascular endothelial cells (BMVEC) of female and male origin express the ETA receptor subtype, and (4) diabetes- and stroke-mimicking conditions increase ET-1 levels in BMVECs of both sexes. Control and diabetic rats were randomized to sham or stroke surgery. BMVECs of male (hBEC5i) and female (hCMEC/D3) origin, cultured under normal and diabetes-mimicking conditions, were exposed to normoxia or hypoxia. Circulating ET-1 levels were higher in diabetic animals and this was more pronounced in the male cohort. Stroke did not further increase plasma ET-1. Tissue ET-1 levels were increased after stroke only in males, whereas periinfarct ET-1 increased in both control and diabetic females. Male BMVECs secreted more ET-1 than female cells and hypoxia increased ET-1 levels in both cell types. There was sexually dimorphic regulation of ET receptors in both tissue and cell culture samples. There are sex differences in the stroke- and diabetes-mediated changes in the brain ET system at the endothelial and tissue levels.

Comparison of different human tissue processing methods for maximization of bacterial recovery

Tissues are valuable microbiological samples that have proved superiority over swabs. Culture of tissue samples is used in the diagnosis of a variety of infections. However, as well as factors such as the site of obtaining the sample, the number of samples, and previous antibiotic use, the method of tissue processing may have an important effect on sensitivity. Data from the literature comparing different tissue processing methods is very limited. This study aimed to compare different mechanical and chemical methods of tissue processing in terms of efficacy and retaining the viability of the bacteria in the tissues. Standard suspensions of Staphylococcus aureus and Escherichia coli were prepared and treated differently to test the effect of that treatment on bacterial viability. Artificially inoculated pork tissue and known infected human tissue samples were then processed by different methods prior to culture, and results were compared. Percentages of reduction in the number of viable bacteria compared to the control by homogenization was similar to 5-min dithiothreitol treatment but significantly lower than bead beating. Bacterial recovery from homogenized human tissues was significantly higher than from any other method of treatment. Although bead beating could be the most efficient method in obtaining a homogeneous tissue product, it significantly reduces the number of viable bacteria within tissues. Homogenization offers the most effective easily controllable retrieval of bacteria from tissue and retains their viability. Guidelines for diagnosing infections using tissue samples should include a standardized processing method.