Fluorescence Spectroscopy Based Identification of Pseudomonas Aeruginosa and Escherichia Coli Suspensions.

In this article, Fluorescence spectroscopy has been employed for the identification of Pseudomonas aeruginosa (PA) and Escherichia coli (E. coli) in water suspension. Emission spectra of PA and E. coli suspensions have been acquired by using excitation wavelengths from 270 to 420 nm with steps of 10 nm to explore their spectral features. It has been found that the emission spectra of tryptophan, tyrosine, NADH and FAD, being the intracellular biomolecules present in both bacteria, can be used as fingerprints for their identification, differentiation and quantification. Both bacterial strains can clearly be differentiated from water and from each other by using λex 270-290 nm through spectral analysis and from λex: 300-500 nm by applying statistical analysis. Furthermore, calibration curves for different bacterial loads of PA and E. coli suspensions have been produced between colonies forming units per ml (CFUs/ml) the integrated intensities of their emission spectra. CFUs/ml of both bacterial suspensions have been determined through plate count method which was used as cross-reference for the analysis of emission spectra of both bacterial suspensions. These curves may be used to estimate CFU/ml of both PA and E. coli in unknown water suspensions by determining the integrating intensity of their emission spectra.

Overexpression of miR-199b-5p in Colony Forming Unit-Hill's Colonies Positively Mediates the Inflammatory Response in Subclinical Cardiovascular Disease Model: Metformin Therapy Attenuates Its Expression.

Well-controlled type 1 diabetes (T1DM) is characterized by inflammation and endothelial dysfunction, thus constituting a suitable model of subclinical cardiovascular disease (CVD). miR-199b-5p overexpression in murine CVD has shown proatherosclerotic effects. We hypothesized that miR-199b-5p would be overexpressed in subclinical CVD yet downregulated following metformin therapy. Inflammatory and vascular markers were measured in 29 individuals with T1DM and 20 matched healthy controls (HCs). miR-199b-5p expression in CFU-Hill's colonies was analyzed from each study group, and correlations with inflammatory/vascular health indices were evaluated. Significant upregulation of miR-199b-5p was observed in T1DM, which was significantly downregulated by metformin. miR-199b-5p correlated positively with vascular endothelial growth factor-D and c-reactive protein (CRP: nonsignificant). ROC analysis determined miR-199b-5p to define subclinical CVD by discriminating between HCs and T1DM individuals. ROC analyses of HbA1c and CRP showed that the upregulation of miR-199b-5p in T1DM individuals defined subclinical CVD at HbA1c > 44.25 mmol and CRP > 4.35 × 106 pg/mL. Ingenuity pathway analysis predicted miR-199b-5p to inhibit the target genes SIRT1, ETS1, and JAG1. Metformin was predicted to downregulate miR-199b-5p via NFATC2 and STAT3 and reverse its downstream effects. This study validated the antiangiogenic properties of miR-199b-5p and substantiated miR-199b-5p overexpression as a biomarker of subclinical CVD. The downregulation of miR-199b-5p by metformin confirmed its cardio-protective effect.

Fluorescence Spectroscopy Based Characterization of Pseudomonas Aeruginosa Suspension.

In this article, optical characterization of Pseudomonas aeruginosa (PA) suspension has been performed by using Fluorescence spectroscopy. Optical density (OD) and plate count methods have been employed as a reference for the analysis of emission spectra of Pseudomonas aeruginosa in water suspension. Emission spectra of PA suspension has been acquired by using excitation wavelengths from 270 to 420 nm with step of 10 nm to explore its spectral behavior. It has been found that emission spectra of tryptophan, tyrosine, NADH and FAD, the intracellular biomolecules of bacteria, can be used as finger prints for the detection of Pseudomonas aeruginosa. Furthermore, the effect of water matrix on the spectral emission of Pseudomonas aeruginosa has been investigated that might be one of the limitation of Fluorescence spectroscopy for complex water matrices. Moreover, a calibration curve has been produced between ODs600 of Pseudomonas aeruginosa suspensions of different bacterial load and integrated intensities of the emission spectra of same samples. These ODs600 and integrating intensities have been further vetted through plate count method by determining their corresponding colony forming units per ml (CFU/ml). This calibration curve may be used to determine CFU/ml of Pseudomonas aeruginosa in water sample by determining integrating intensity of its emission spectrum.

The effect of caffeine intake and passive smoking on umbilical cord blood unit's quality parameters.

Today cord blood (CB) is a valuable source of hematopoietic stem cells to treat many hematological disorders. One of the limitations of CB utilization is the reduced number of nucleated cells including stem cells. Therefore, CB banks around the world have developed strategies in an attempt to improve donor selection and the quality of the CB inventory. This study aimed to determine the impact of passive smoking and caffeine consumption on CB quality. CBs were obtained from mothers who gave birth at King Abdulaziz Medical City. All mothers gave their informed consent. Personal interviews about the mother's demographics, smoking status and exposure, and caffeine consumption executed, followed by a chart review to analyze maternal and neonatal factors. Laboratory testing was performed on all collected CB units. Using descriptive statistics, maternal and newborn factors were analyzed. T-test or Mann-Whitney U Test, as appropriate, for continuous variables analysis to study the effect of second hand smoking and coffee consumption for the primary outcome. Our study demonstrated a reduction in CB MNC, including lymphocytes, in caffeine consumers among pregnant donors, as well as a reduction in cell potency activities, including total CFU and BFU-E. The effect of passive cigarette smoking on the same cohort was insignificant. Outcome of this study will help in optimizing the quality and quantity of stem cell harvesting from CB to get the maximum benefit and such knowledge will raise the awareness among pregnant women.

Effect of different finishing and polishing procedures on surface roughness and microbial adhesion on highly-filled composites for injectable mold technique.

OBJECTIVE: To evaluate the effect of different finishing and polishing procedures on surface roughness and microbial adhesion and viability of Streptococcus mutans on novel highly-filled composites for injectable mold technique. MATERIALS AND METHODS: One hundred sixty specimens were divided into four material groups: FSF (Filtek Supreme Flowable Restorative), TE (Tetric EvoFlow), GUI (G-aenial Universal Injectable) and GUF (G-aenial Universal Flo). Within each group, specimens were split regarding finishing and polishing (F/P) procedures into: SLD (Sof-Lex Discs), SLS (Sof-Lex Spirals), OG (OneGloss) and PG (PoGo). Surface roughness was analyzed using profilometer (Ra and Rz) and scanning electron microscopy (SEM) and Strep. mutans biofilm formation was analyzed using colony forming unit (CFU) and cell viability assay. Two-way analysis of variance (ANOVA) followed by Tukey's post hoc test were used for comparison among groups, Pearson's coefficient was applied for the correlation between Ra and CFU/ml and all data were presented as mean ± SD. RESULTS: Both materials and F/P procedures affect Ra, Rz and Strep. mutans CFU/ml values (p ≤ 0.05). Considering the Ra and Rz, GUI and GUF revealed lower values, compared to FSF and TE and SLD and SLS revealed lower values, compared to OG and PG. Considering the Strep. mutans CFU/ml and viability, GUI and TE presented lower values, compared to GUF and FSF and SLD and SLS presented lower values, compared to OG and PG. Moderate positive correlation was found between Ra and CFU/ml (r = 0.552). CONCLUSIONS: The smoothest surfaces possess GUI and GUF, among materials and SLD and SLS, among F/P procedures. GUI adhered the lowest amount of Strep. mutans, due to the smoothest surfaces. FSF and GUF revealed the highest amount of Strep. mutans, due to bis-GMA, bis-MEPP and TEGDMA in their composition. CLINICAL SIGNIFICANCE: The findings of the present study may be beneficial for the proper selection of highly-filled composites and an adequate finishing and polishing procedure when performing the injectable mold composite resin veneer technique.

Stromal Lineage Precursors from Rodent Femur and Tibia Bone Marrows after Hindlimb Unloading: Functional Ex Vivo Analysis.

Rodent hindlimb unloading (HU) model was developed to elucidate responses/mechanisms of adverse consequences of space weightlessness. Multipotent mesenchymal stromal cells (MMSCs) were isolated from rat femur and tibia bone marrows and examined ex vivo after 2 weeks of HU and subsequent 2 weeks of restoration of load (HU + RL). In both bones, decrease of fibroblast colony forming units (CFU-f) after HU with restoration after HU + RL detected. In CFU-f and MMSCs, levels of spontaneous/induced osteocommitment were similar. MMSCs from tibia initially had greater spontaneous mineralization of extracellular matrix but were less sensitive to osteoinduction. There was no recovery of initial levels of mineralization in MMSCs from both bones during HU + RL. After HU, most bone-related genes were downregulated in tibia or femur MMSCs. After HU + RL, the initial level of transcription was restored in femur, while downregulation persisted in tibia MMSCs. Therefore, HU provoked a decrease of osteogenic activity of BM stromal precursors at transcriptomic and functional levels. Despite unidirectionality of changes, the negative effects of HU were more pronounced in stromal precursors from distal limb-tibia. These observations appear to be on demand for elucidation of mechanisms of skeletal disorders in astronauts in prospect of long-term space missions.

Combination of Mycobacterium tuberculosis RS Ratio and CFU Improves the Ability of Murine Efficacy Experiments to Distinguish between Drug Treatments.

Murine tuberculosis drug efficacy studies have historically monitored bacterial burden based on CFU of Mycobacterium tuberculosis in lung homogenate. In an alternative approach, a recently described molecular pharmacodynamic marker called the RS ratio quantifies drug effect on a fundamental cellular process, ongoing rRNA synthesis. Here, we evaluated the ability of different pharmacodynamic markers to distinguish between treatments in three BALB/c mouse experiments at two institutions. We confirmed that different pharmacodynamic markers measure distinct biological responses. We found that a combination of pharmacodynamic markers distinguishes between treatments better than any single marker. The combination of the RS ratio with CFU showed the greatest ability to recapitulate the rank order of regimen treatment-shortening activity, providing proof of concept that simultaneous assessment of pharmacodynamic markers measuring different properties will enhance insight gained from animal models and accelerate development of new combination regimens. These results suggest potential for a new era in which antimicrobial therapies are evaluated not only on culture-based measures of bacterial burden but also on molecular assays that indicate how drugs impact the physiological state of the pathogen.

Percutaneous autologous bone marrow concentrate for knee osteoarthritis: patient-reported outcomes and progenitor cell content.

PURPOSE: Knee osteoarthritis (OA) is a common, progressively debilitating joint disease, and the intra-articular injection of autologous bone marrow concentrate (BMC) may offer a minimally invasive method of harnessing the body's own connective tissue progenitor cells to counteract accompanying degenerative effects of the disease. However, the extent to which the progenitor cell content of BMC influences treatment outcomes is unclear. We sought to determine whether patient-reported outcome measures associated with BMC treatment for knee OA are related to the concentration of progenitor cells provided. METHODS: In the present study, 65 patients (72 knees) underwent treatment for knee OA with autologous BMC and self-reported their outcomes for up to one year using follow-up questionnaires tracking function, pain, and percent improvement. A small fraction of each patient's BMC sample was reserved for quantification with a haematological analyzer and cryopreserved for subsequent analysis of potential connective tissue progenitor cells using a colony-forming unit fibroblast (CFU-F) assay. RESULTS: Patients reported significant increases in function and overall percent improvement in addition to decreases in pain relative to baseline levels following treatment with autologous BMC that persisted through 12 months. Patients reporting improved outcomes (46 of 72 knees) received BMC injections having higher CFU-F concentrations than non-responding patients (21.1×103 ± 12.4×103 vs 14.3×103 ± 7.0 x103 CFU-F per mL). A progenitor cell concentration of 18×103 CFU-F per mL of BMC was found to best differentiate responders from non-responders. CONCLUSION: This study provides supportive evidence for using autologous BMC in the minimally invasive treatment of knee OA and suggests that increased progenitor cell content leads to improved treatment outcomes. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT03011398, 1/7/17.

Investigation of indoor and outdoor air quality in a university campus during COVID-19 lock down period.

The pandemic of COVID-19 currently shadows the world; the whole earth has been on an unprecedented lockdown. Social distancing among people interrupted domestic and international air traffic, suspended industrial productions and economic activities, and had various far-reaching and undetermined implications on air quality. Improvement in air quality has been reported in many cities during the lockdown. On March 22, 2020, the Turkish government enforced strict lockdown measures to reduce coronavirus disease transmission. This lockdown had a significant impact on the movement of people within the country, which resulted in a major drop in worldwide commercial activities. During this period, university campuses were emptied due to the transition to distance education. In this study, various air pollutants sulfur dioxide (SO2), nitrogen dioxide (NO2), ozone (O3), fine particulate matter (PM2.5), total bacteria, and total fungi were measured in different indoor environments at Eskisehir Technical University Campus in Eskisehir, Turkey during COVID-19 lock down period. Also, to calculate the indoor and outdoor ratios (I/O) of the pollutants, simultaneous outdoor measurements were also carried out. The average indoor SO2, NO2, O3, and PM2.5 concentrations in different indoor environments ranged between 2.10 and 54.58, 1.36-30.89, 12.01-39.05, and 21-94 µg/m3, respectively. The total number of bacteria and fungi ranged between 21.83-514.15 and 13.10-83.36 CFU/m3, respectively. Our study intends to give a glimpse to quantify the impact of a pandemic on air quality in different indoor environments in a university campus in Eskisehir, Turkey and calls for follow-up studies. Indoor concentrations were evaluated together with outdoor concentrations. In general, it can be said that the calculated I/O ratios for SO2, NO2, O3, bacteria, and fungi were less than 1 in most indoor environments.

Ways to reduce the labor intensiveness of applying the method of optical estimation of microbial cell concentration in suspension.

The labor intensity (in hours) of the optical method of microbial cell counting in suspension compared to the method of microbial cell counting using a Goryaev chamber is evaluated. The relevance of assessing the production labor intensity of microbial cell counting methods in suspension is related to the need to use them in many studies. Often the commonly used methods are too labour-intensive, time-consuming, or require expensive equipment. A comparative experiment was carried out with our previously developed "Method for optical estimation of microbial cell concentration in suspension" (Priority certificate No. 2016141859 dated 25.10.2016) and the method of microbial cell counting using a Goryaev chamber. Production labor intensity of the measurements performed was calculated in hours according to the formula: Tp=Tt+Tob, where Tp is production labour input, Tt is technological labour input, Tob is maintenance labour input. Technological labour input of measurements with use of Goryaev's chamber made up 32,18 ± 0,95, whereas with optical method - 1,03±0,06 (reliability of differences at p<0,01) at amount of measurements n = 100. Labour input of service at optical method 0,24 ± 0,03, at application of method with use of Goryaev chamber 0,15±0,01 hours. Labour input of measurements of concentration of microbial cells in suspension at application of method of measurement with Goryaev chamber remains (p<0,01) higher than at an optical method of estimation, 32,33±0,96 and 1,27±0,05 hours accordingly. When using the optical method of concentration estimation in the suspension it is necessary to carry out not a small amount of necessary mathematical calculations, which in the future, probably, corrected by creating a special program for a personal computer. The labour input of results obtained by measuring by optical evaluation of the concentration of microbial cells in suspension is lower than that obtained by using a measurement method using a Goryaev chamber. Taking into consideration that its implementation does not require purchase of special equipment as in turbidimetry, its cost-effectiveness compared to existing ones is obvious.

Expression of gilt acts as a positive regulator of mouse hematopoietic progenitor cells.

Gamma interferon inducible lysosomal thiol reductase (GILT), is known to be involved in immunity, but its role in hematopoiesis has not been previously reported. Herein, we demonstrate using gilt knockout (-/-) mice that loss of gilt associates with decreased numbers and cycling status of femoral hematopoietic progenitor cells (CFU-GM, BFU-E, and CFU-GEMM) with more modest effects on splenic progenitor cells. Thus, GILT is associated with positive regulation of hematopoietic progenitor cells in mice, mainly in bone marrow.

Cu(II) biosorption by living biofilms: Isothermal, chemical, physical and biological evaluation.

Dissolved copper in stormwater runoff is a significant environmental problem. Biosorption of dissolved metals using microorganisms is known as a green, low-cost and efficient method. However, the role of live biological agents in the remediation of dissolved copper in Sustainable Drainage (SuDS) has not been reported. In this study, the effect of pH, initial concentration and temperature, on bacteria in different stages of biofilm development on a geotextile, along with Cu(II) removal efficiencies, were evaluated. Maximum Cu(II) removal efficiency (92%) was observed at pH 6. By decreasing the pH from 6 to 2, a log 5 reduction in bacteria was observed and Carboxyl groups transformed from -COO- to -COOH. The maximum biosorption capacity (119 mg g-1) was detected on day 1 of biofilm development, however, maximum removal efficiency (97%) was measured on day 21 of biofilm incubation. Exteracellular Polymeric Substance (EPS) showed a better protection of CFUs in more mature biofilms (day 21) with less than 0.1 log decrease when exposed to 200 mL-1 Cu(II), whereas, biofilm on day 1 of incubation showed a 2 log reduction in CFUs number. Thermodynamic studies showed that the maximum Cu(II) biosorption capacity of biofilms, incubated for 7 days (117 mg g-1) occurred at 35 °C. Thermodynamic and kinetic modelling of data revealed that a physical, feasible, spontaneous and exothermic process controlled the biosorption, with a diffusion process observed in external layers of the biofilm, fitting a pseudo-second order model. Equilibrium data modelling and high R2 values of Langmuir model indicated that the biosorption took place by a monolayer on the living biofilm surface in all stages of biofilm development.

Cryopreserved bone marrow aspirate concentrate as a cell source for the colony-forming unit fibroblast assay.

PURPOSE: The prevalence of connective tissue progenitor cells within a cell-based therapy is often quantified using the colony-forming unit fibroblast (CFU-F) assay. The present study investigates the feasibility of using cryopreserved bone marrow aspirate concentrate (BMAC) as an alternative cell source to fresh BMAC for CFU-F quantification. METHODS: Freshly prepared and corresponding cryopreserved BMAC samples from patients receiving autologous cell therapy (n = 98) were analyzed using the CFU-F assay for comparison. Cultures were established by directly plating BMAC at low cell densities and maintained for a 2-week growth period. Colonies were enumerated to determine CFU-F frequency, and a subset of cultures was imaged and analyzed to quantify colony area and density. RESULTS: A nonlinear relationship was observed between plating density and CFU-F frequency over a wide range in plating densities (~30-fold). Cryopreserved BMAC yielded recoverable (77 ± 23%) and viable (73 ± 9%) nucleated cells upon thawing. After cryopreservation, CFU-F frequencies were found to be significantly lower (56.6 ± 34.8 vs. 50.3 ± 31.7 colonies per million nucleated cells). Yet the number of CFU-F in fresh and cryopreserved BMAC were strongly correlated (r = 0.87) and had similar area and densities. Further, moderate correlations were observed between the number of CFU-F and nucleated cells, and both the mean colony area and density were negatively correlated with patient age. Notably, no relationship was found between CFU-F frequency and age, regardless of whether fresh or cryopreserved BMAC was used. CONCLUSIONS: Freshly prepared and cryopreserved BMAC yielded correlated results when analyzed using the CFU-F assay. Our findings support the cryogenic storage of patient BMAC samples for retrospective CFU-F analyses, offering a potential alternative for characterizing BMAC and furthering our understanding of progenitor cells in relation to clinical outcome.

Variations in the C-unit of bedaquiline provides analogues with improved biology and pharmacology.

Analogues of the anti-tuberculosis drug bedaquiline, bearing a 3,5-dimethoxy-4-pyridyl C-unit, retain high anti-bacterial potency yet exert less inhibition of the hERG potassium channel, in vitro, than the parent compound. Two of these analogues (TBAJ-587 and TBAJ-876) are now in preclinical development. The present study further explores structure-activity relationships across a range of related 3,5-disubstituted-4-pyridyl C-unit bedaquiline analogues of greatly varying lipophilicity (clogP from 8.16 to 1.89). This broader class shows similar properties to the 3,5-dimethoxy-4-pyridyl series, being substantially more potent in vitro and equally active in an in vivo (mouse) model than bedaquiline, while retaining a lower cardiovascular risk profile through greatly attenuated hERG inhibition.

Preeclampsia in pregnancy affecting the stemness and differentiation potency of haematopoietic stem cell of the umbilical cord blood.

BACKGROUND: Umbilical cord blood (UCB) has been proposed as the potential source of haematopoietic stem cells (HSC) for allogeneic transplantation. However, few studies have shown that a common disease in pregnancy such as preeclampsia would affect the quality of UCB-HSC. Total nucleated cell count (TNC) is an important parameter that can be used to predict engraftment including UCB banking. Colony forming unit (CFU) assay is widely used as an indicator to predict the success of engraftment, since direct quantitative assay for HSC proliferation is unavailable. The aim of this study is to investigate the effects of preeclampsia in pregnancy on the stemness and differentiation potency of UCB-HSC. METHODS: Mononuclear cells (MNC) were isolated from UCB and further enriched for CD34+ cells using immune-magnetic method followed by CFU assay. A panel of HSC markers including differentiated haematopoietic markers were used to confirm the differentiation ability of UCB-HSC by flow cytometry analysis. RESULTS/ DISCUSSION: The HSC progenitor's colonies from the preeclampsia group were significantly lower compared to the control. This correlates with the low UCB volume, TNC and CD34+ cells count. In addition, the UCB-enriched CD34+ population were lymphoid progenitors and capable to differentiate into natural killer cells and T-lymphocytes. CONCLUSION: These findings should be taken into consideration when selecting UCB from preeclamptic mothers for banking and predicting successful treatment related to UCB transplant.

Effect of a novel quaternary ammonium silane cavity disinfectant on cariogenic biofilm formation.

OBJECTIVE: Evaluate effect of quaternary ammonium silane (QAS) cavity disinfectant on cariogenic biofilm. MATERIALS AND METHODS: Single- (Streptococcus mutans or Lactobacillus acidophilus), dual- (Streptococcus mutans/Lactobacillus Acidophilus), and multi-species (Streptococcus mutans, Actinomyces naeslundii, and Streptococcus sanguis) biofilms were grown on acid-etched dentine discs. Biofilms were incubated (120 min/37 °C) and allowed to grow for 3 days anaerobically. Discs (no treatment) served as control (group 1). Groups II, III, IV, and V were then treated with 2% chlorhexidine, and 2%, 5%, and 10% QAS (20 s). Discs were returned to well plates with 300 µL of bacterial suspension and placed in anaerobic incubator at 37 °C and biofilms redeveloped for 4 days. Confocal microscopy, Raman, CFU, and MTT assay were performed. RESULTS: Raman peaks show shifts at 1450 cm-1, 1453 cm-1, 1457 cm-1, 1460 cm-1, and 1462 cm-1 for control, 2% CHX, 2%, 5%, and 10% QAS groups in multi-species biofilms. There was reduction of 484 cm-1 band in 10% QAS group. CLSM revealed densely clustered green colonies in control group and red confluent QAS-treated biofilms with significantly lower log CFU for single/dual species. Metabolic activities of Streptococcus mutans and Lactobacillus acidophilus decreased with increasing QAS exposure time. CONCLUSION: Quaternary ammonium silanes possess antimicrobial activities and inhibit growth of cariogenic biofilms. CLINICAL SIGNIFICANCE: Available data demonstrated use of QAS as potential antibacterial cavity disinfectant in adhesive dentistry. Experimental QAS can effectively eliminate caries-forming bacteria, when used inside a prepared cavity, and can definitely overcome problems associated with present available cavity disinfectants.

Deferoxamine Protects Stromal/Stem Cells of "Lull pgm System"-Processed Lipoaspirates Against Damages Induced by Mitochondrial Respiration Inhibition.

BACKGROUND: The ischemic environment of the receiving area compromises the outcome of autologous fat grafts. The aim of this study was to isolate and expand the stromal vascular fraction from patient lipoaspirates and investigate the gain in cell viability exerted by some protective agents against the blockage of mitochondrial respiration. METHODS: The aspirates were (1) washed, using the "Lull pgm system," (2) centrifuged and (3) decanted. The corresponding stromal vascular fractions were isolated, and after cell adherence selection, the stromal/stem cell subpopulations were exposed to Antimycin A for 1 h. Then, the protection induced by cell pretreatment with deferoxamine, diazoxide and IGF-1 was evaluated. RESULTS: The residual cell viability of the "Lull pgm system"-washed samples was greater than that of the centrifuged samples (p < 0.05), and this advantage was maintained during the following 12 days of culture. The administration of 400 µM deferoxamine before Antimycin A treatment increased the number of viable cells from 56.5 to 80.8% (p < 0.05). On the contrary, the pretreatment with 250 µM diazoxide or 0.1 µg/ml IGF-1 did not exert any significant pro-survival action. Echinomycin abolished the positive effect of deferoxamine, suggesting that its protection involved HIF-1α. CONCLUSIONS: Adipose-derived stromal-stem cells survive the inhibition of mitochondrial respiration better if the lipoaspirate is washed using the "Lull pgm system" rather than centrifuged. Moreover, a significant contribution to cell survival can be obtained by preconditioning stromal-stem cells with deferoxamine. In a clinical perspective, this drug could be safely administered before surgery to patients undergoing autologous fat transfer. NO LEVEL ASSIGNED: This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266.

Correlation of RAS-Pathway Mutations and Spontaneous Myeloid Colony Growth with Progression and Transformation in Chronic Myelomonocytic Leukemia-A Retrospective Analysis in 337 Patients.

Although the RAS-pathway has been implicated as an important driver in the pathogenesis of chronic myelomonocytic leukemia (CMML) a comprehensive study including molecular and functional analyses in patients with progression and transformation has not been performed. A close correlation between RASopathy gene mutations and spontaneous in vitro myeloid colony (CFU-GM) growth in CMML has been described. Molecular and/or functional analyses were performed in three cohorts of 337 CMML patients: in patients without (A, n = 236) and with (B, n = 61) progression/transformation during follow-up, and in patients already transformed at the time of sampling (C, n = 40 + 26 who were before in B). The frequencies of RAS-pathway mutations (variant allele frequency ≥ 20%) in cohorts A, B, and C were 30%, 47%, and 71% (p < 0.0001), and of high colony growth (≥20/105 peripheral blood mononuclear cells) 31%, 44%, and 80% (p < 0.0001), respectively. Increases in allele burden of RAS-pathway mutations and in numbers of spontaneously formed CFU-GM before and after transformation could be shown in individual patients. Finally, the presence of mutations in RASopathy genes as well as the presence of high colony growth prior to transformation was significantly associated with an increased risk of acute myeloid leukemia (AML) development. Together, RAS-pathway mutations in CMML correlate with an augmented autonomous expansion of neoplastic precursor cells and indicate an increased risk of AML development which may be relevant for targeted treatment strategies.

Molecular Basis and Clinical Application of Growth-Factor-Independent In Vitro Myeloid Colony Formation in Chronic Myelomonocytic Leukemia.

We have originally reported that colony-forming units granulocyte/macrophage (CFU-GM) formation is an in vitro feature of chronic myelomonocytic leukemia (CMML) and a strong predictor for short survival. Elucidation of the molecular basis underlying this in vitro phenomenon could be helpful to define molecular features that predict inferior outcome in patients. We studied the correlation between the mutational landscape and spontaneous colony formation in 164 samples from 125 CMML patients. As compared to wildtype samples, spontaneous in vitro CFU-GM formation was significantly increased in samples containing mutations in NRAS, CBL and EZH2 that were confirmed as independent stimulatory factors by multiple regression analysis. Inducible expression of mutated RAS but not JAK2 was able to induce growth factor independence of Ba/F3 cells. Whereas high colony CFU-GM growth was a strong unfavorable parameter for survival (p < 0.00001) and time to transformation (p = 0.01390), no single mutated gene had the power to significantly predict for both outcome parameters. A composite molecular parameter including NRAS/CBL/EZH2, however, was predictive for inferior survival (p = 0.00059) as well as for increased risk of transformation (p = 0.01429). In conclusion, we show that the composite molecular profile NRAS/CBL/EZH2 derived from its impact on spontaneous in vitro myeloid colony formation improves the predictive power over single molecular parameters in patients with CMML.

The application of the loop-mediated isothermal amplification (LAMP) method for diagnosing Enterococcus hirae-associated endocarditis outbreaks in chickens.

BACKGROUND: Enterococcus hirae is considered a part of the normal intestinal biota of several domestic animals, including poultry. However, this species is also associated with infective endocarditis in chickens, a disease that leads to unexpected deaths and serious economical losses. Enterococcus hirae is identified predominantly with the use of conventional bacteriological methods, biochemical tests and PCR. Rapid, sensitive and specific methods for detecting E. hirae in clinical samples are required in poultry production. The aim of this study was to use the Loop-Mediated Isothermal Amplification (LAMP) for the identification and quantification of E. hirae in heart samples from broiler chickens. RESULTS: The specificity of the LAMP method was confirmed for 7 enterococcal strains and 3 non-enterococcal strains. E. hirae was detected in all of the 22 analyzed clinical bacterial isolates and in all of the 9 heart samples. Three sets of primers supported the detection of E. hirae with high sensitivity and specificity within one hour. The highest detection rate of a LAMP product was approximately 7 min for an E. hirae strain and 12 min for a positive heart sample. The detection limit for the E. hirae ATCC 10541 standard was 1.3 × 102 CFU (43.4 fg) or 13.8 copies of the E. hirae genome equivalent per reaction. The reaction was 10-fold more sensitive than conventional species-specific PCR. The LAMP assay supported the determination of the E. hirae load in chicken hearts with endocarditis in field cases. The average number of E. hirae cells in hearts was 5.19 × 107 CFU/g of tissue, and the average number of E. hirae genome equivalents in hearts was 5.51× 106 copies/g of tissue. Bacterial counts were significantly higher in the LAMP assay than in the standard plate count. CONCLUSIONS: The LAMP assay is a useful diagnostic tool and an effective alternative to conventional methods for the detection of this enterococcal species. The sodA-based LAMP assay supported direct identification of E. hirae from pure cultures and heart samples without previous bacterial cultivation. This is the first study to apply the LAMP method for the purpose of diagnosing E. hirae-associated endocarditis in poultry.