Increase In Colony-forming Units Research Articles

Evaluation of ATP bioluminescence for rapid determination of cleanliness of livestock trailers after a commercial wash.

Pathogens such as porcine epidemic diarrhea virus (PEDV), porcine reproductive and respiratory syndrome (PRRSV), and E. coli are known to spread by contaminated vehicles and equipment. Pork producers have adopted trailer wash policies where each trailer is washed, disinfected, and dried before it can return to a farm. Cleanliness of livestock trailers after washing is determined by visual inspection rather than any objective method. Adenosine triphosphate (ATP) bioluminescence is used in many industries to provide real-time feedback on surface cleanliness through the detection of ATP from organic sources. That same technology may provide trailer wash facilities a way of objectively characterizing a livestock trailer's suitability to return to a farm after washing. Two ATP luminometers (3M Clean-Trace and Neogen AccuPoint) were used to estimate the correlation between ATP bioluminescence readings and aerobic bacterial plate counts (APCs) from sampled surfaces and to determine locations within a livestock trailer that can accurately estimate surface cleanliness. Five locations in livestock trailers were evaluated. Those locations included the nose access door (NAD), back door flush gate, rear side access door (RSAD), belly flush gate (BFG), and belly side access door (BSAD). There was a positive log-log association between the two luminometers (r = 0.59, P < 0.01). Every log unit increase in one unit, resulted in a 0.42 log increase (P < 0.01) in the other unit. ATP can come from bacteria, yeasts, molds, and manure. There was a poor association (r ≥ 0.10, P ≥ 0.02) between APCs and the ATP luminometers. Still, an increase in relative light units (RLUs) resulted in a corresponding increase in colony-forming units. The greatest area of surface contamination measured by APC was the NAD. RLUs were also greater in the NAD compared to the RSAD, the BFG, and the BSAD (P ≤ 0.01). Because APCs and luminometer RLUs provided similar outcomes, statistical process control charts were developed to determine control limits for RLUs. This provides real-time feedback to trailer wash workers in determining cleanliness outcomes for livestock trailers. These data suggest that ATP bioluminescence can be a reliable method to monitor cleaning effectiveness in livestock trailers. Bioluminescence is a monitoring tool that should be used in conjunction with microbial methods to monitor procedures for cleaning and disinfection.

Degradation features of bioplastics-based disposable straws in soil and aerobic mineralization: Insight into final properties and chemical pathways.

The transition to a sustainable economy has led industries to seek new raw materials for the production of renewable-based plastics. The present work shows the confirmation of formulation and initial degradation features in non-controlled soil of five types of straws including bio- and fossil-based polymers demonstrating that those based on starch could contain bio-based polymers; bio-based-polyethylene (PE) and -poly(propylene) (PP). Biodegradation percentage, structural, thermal, morphological and visual analysis are discussed indicating that after twelve weeks there is low weight loss percentage which is higher for cellulose, followed by starch/low density poly(ethylene) (LDPE) (S/LDPE), corn starch/poly(propylene) (S/PP), poly(lactic acid) (PLA) and PP. Selected avocado and corn-based straws (S/LDPE and S/PP) were subjected to controlled CO2 evolution test through ASTM D5338-15 essay to stablish the biodegradation percentage. Derived from CO2 evolution test of selected S/LDPE and –S/PP, it is established that the carbon present in the sample follows two possible routes; 1) Biomass growth, corroborated by the increase in colony-forming units in fungi and aerobic mesophilic bacteria, and 2) metabolization expressed in CO2 production. The S/LDPE straw presented higher biodegradability in both tests than S/PP. Insights of chemical pathways and final monitoring properties are discussed. These findings provide information to be used to support policies around the world to consider more sustainable materials that are not just bio-based materials but ensure their biodegradation into bioplastic products.

Survival of bovine-associated serotypes of Salmonella enterica in bedding sand

Cattle persistently infected with bovine-adapted serotypes of Salmonella enterica are an important animal health and food safety issue. One possible mechanism by which infection is sustained in a dairy herd is by survival of Salmonella in sand used as bedding material. In this study we assessed the survival of 107 to 108 cfu bovine-associated serotypes of Salmonella enterica (sv. Cerro, Dublin, and Heidelberg) in sterile sand, recycled bedding sand, and gray water collected from a Wisconsin dairy farm. All 3 serotypes persisted at relatively high numbers (>106 cfu/g) for at least 28 d in sterile sand, with Salmonella sv. Dublin decreasing less than 1 log10 over 70 d. To our surprise, when low numbers of Salmonella sv. Dublin (103 cfu) were inoculated into sterile sand, the organism multiplied within 3 d to approximately 106 cfu/g sand and persisted at that level for 28 d. When we inoculated Salmonella sv. Dublin into recycled bedding sand or sand taken directly from cow pens, we observed a significant decrease in colony-forming units by d 7. In contrast, we observed a significant increase in colony-forming units when Salmonella sv. Dublin was inoculated into gray water from the sand recycling system. These data demonstrate that Salmonella can persist for extended periods of time in bedding sand, although this is limited to some extent by the native microbiota in recycled bedding sand.

Mycobacterium tuberculosis ketol-acid reductoisomerase down-regulation affects its ability to persist, and its survival in macrophages and in mice

Branched-chain amino acids (BCAAs) leucine, isoleucine and valine biosynthetic pathways have been reported from plants, fungi and bacteria including Mycobacterium tuberculosis (Mtb) but are absent in animals. This makes interventions with BCAAs biosynthesis an attractive proposition for antimycobacterial drug discovery. In the present study, Mycobacterium tuberculosis H37Ra (Mtb-Ra) ketol-acid reductoisomerase encoding ORF MRA_3031 was studied to establish its role in Mtb-Ra growth and survival. Recombinant knockdown (KD) and complemented (KDC) strains along with wild-type (WT) Mtb-Ra were studied under in-vitro and ex-vivo conditions. KD was defective for survival inside macrophages and showed time dependent decrease in its colony forming unit (CFU) counts, while, WT and KDC showed time dependent increase in CFUs, after macrophage infection. Also, KD showed reduced ability to form persister cells, had altered membrane permeability against ethidium bromide and nile red dyes, and had reduced biofilm maturation, compared to WT and KDC. The in-vivo studies showed that KD infected mice had lower CFU counts in lungs, compared to WT. In summary Mtb shows survival deficit in macrophages and in mice after ketol-acid reductoisomerase down-regulation.

PROLIFERATION OF SACCHAROMYCES CEREVISIAE EXPOSED TO PULSED MAGNETIC FIELDS OF LOW INTENSITY

Yeast cultures of Saccharomyces cerevisiae were exposed to pulsed magnetic fields of low intensity. Field intensities from 0.92 mT to 2.73[Formula: see text]mT and frequencies of 20, 77.5, 135, 192.5 and 250[Formula: see text]Hz were employed. Proliferation and cell viability measurements were made in exposed and control cultures. For the frequency of 20[Formula: see text]Hz, statistically significant differences were found ([Formula: see text]) in cell proliferation using field intensities of 1.81 and 2.71[Formula: see text]mT. No changes were observed at this frequency for lower field strengths. At 77.5[Formula: see text]Hz, only changes in proliferation were observed for 0.92[Formula: see text]mT ([Formula: see text]). No changes were observed to any other frequency or intensity of the magnetic field. The cell viability studies were performed at different times, from 4 to 12[Formula: see text]h of exposure. At 1.81[Formula: see text]mT and 20[Formula: see text]Hz, an increase in colony-forming units (CFUs) of 26.1% was observed ([Formula: see text]) at 4[Formula: see text]h, and after 10[Formula: see text]h of exposure the increase in CFUs was 51.5% ([Formula: see text]). But when exposing the cells for 9[Formula: see text]h to a magnetic field of 2.71[Formula: see text]mT at 20[Formula: see text]Hz, the CFUs decreased by 25.9% ([Formula: see text]), and at 0.92[Formula: see text]mT and 77.5[Formula: see text]Hz, a decrease in CFUs of 18.6% was observed ([Formula: see text]) at 4[Formula: see text]h, while after 12 h of exposure an increase in CFU of 23.5% ([Formula: see text]) was determined. These results suggest the presence of a frequency window between 20[Formula: see text]Hz and 77.5[Formula: see text]Hz, where the effects of the magnetic field are significant.

Long filamentous state of Listeria monocytogenes induced by sublethal sodium chloride stress poses risk of rapid increase in colony-forming units

Bacterial cell filamentation is induced in response to sublethal stress in Listeria monocytogenes. The formation of filamentous cells, which have plural nucleoids and are connected in rows, is thought to result in the underestimation of viable cell counting using the agar plating method. Filamentous cells are able to return to their initial normal-sized morphology under stress-free conditions. However, the differences in the growth behavior of filamentous cells remain poorly understood. In this study, we established a long filamentous population induced by sublethal sodium chloride stress and compared the growth characteristics of normal-sized cells and filamentous cells in a stress-free environment. Sodium chloride (NaCl) (10%) was selected as the sublethal stress for L. monocytogenes IID 579 filamentation, subsequently inducing high levels of filamentation in L. monocytogenes IID 579. Fluorescent microscopy showed that single filamentous cells have multi nucleoids with or without membrane septa, while time-lapse imaging revealed that the formation of membrane septa and cell division of filamentous cells occurred simultaneously at multiple sites, unlike in normal-sized cells. The viable cell count of filamentous L. monocytogenes increased faster than that of normal-sized cells only in the early exponential phase, caused by reverting to the normal morphology from the filamentous state. Moreover, in the limited nutrient media in which normal cells did not proliferate, the colony-forming units of filamentous cells increased rapidly depending with a decrease in the filamentous cell rates. The thermal resistance of filamentous cells induced by NaCl was found to increase, suggesting that stress adaptation protects filamentous L. monocytogenes from various food-related stresses. These findings demonstrate that filamentous cells may have a higher stress tolerance than non-stressed cells, with a risk of a rapid increase in colony-forming units after the release of stress factors. The contamination by filamentous cells should be avoided, which will require conditions suitable for the prevention of filamentation in food safety.

196. Reducing bioburden in the operating room: manual cleaning and pulsed-xenon ultraviolet light disinfection produces significant reduction in colony forming units

BACKGROUND CONTEXT Though techniques for sterilizing surgical tools and supplies have steadily improved, current standards for operating room (OR) disinfection have become antiquated – providing continued convenience for pathogenic organisms and surgical site infections. Pulsed-xenon ultraviolet (PX-UV) disinfection is an opportunity for standardized, touch-free disinfection that may serve as a supplement to manual terminal cleaning. The combination of manual and PX-UV disinfection may provide the greatest reduction in OR bioburden, as measured by colony-forming units (CFUs), and frequency of site contamination. Here, the bioburden and site contamination of ORs following manual cleaning and PX-UV disinfection are explored. PURPOSE Determine impact of manual vs manual and PX-UV disinfection on CFUs in spine operating suites. STUDY DESIGN/SETTING N/A PATIENT SAMPLE N/A OUTCOME MEASURES N/A METHODS Fifteen ORs were sampled at three different time points – before terminal manual cleaning, after terminal manual cleaning, and after PX-UV. For each OR, at each time point, five high-touch surfaces were cultured using a Tryptone Soy Agar touch plate. A total of 225 samples were acquired. Samples were incubated for 24 hours and the number of colony-forming units reported. Distinct colonies were identified. Descriptive statistics and Rank Sum Testing with a Bonferroni correction were used to analyze results. RESULTS There was a 26.8% reduction of CFUs after manual cleaning (p>0.8014) and an 81.0% reduction of CFUs after PX-UV (p = 0.0086). Overall, the combination of manual and PX-UV disinfection resulted in an 86.10% reduction in CFUs (p = 0.004). The frequency of sites with CFUs prior to cleaning was 26.7%. There was no change in frequency of sites with CFUs after manual cleaning. Following PX-UV cleaning, the frequency of sites with CFUs reduced to 8.0%, which is a 70.0% reduction in sites with CFUs. Interestingly, the frequency of sites with an increase in CFUs after manual cleaning was 20.0%. There was no occurrence of an increase in CFUs after PX-UV disinfection. CONCLUSIONS The combination of PX-UV with manual cleaning yields the greatest reduction in OR bioburden as measured by CFUs. Manual cleaning alone resulted in a low reduction of CFUs and no change in the frequency of site contamination. Though some individual sites demonstrated elimination of CFUs following manual cleaning, there were a number of sites that had an increase in CFUs. This may represent cross contamination due to the inconsistent nature of manual terminal cleaning. The use of PX-UV disinfection resulted in a significant reduction in CFUs when compared to both the pre- and postmanual cleaning time points. PX-UV disinfection also yielded a reduction in the frequency of site contamination. This reduction in both bioburden and contaminated sites may contribute to a decreased risk of surgical site infection. FDA DEVICE/DRUG STATUS This abstract does not discuss or include any applicable devices or drugs.

Extrahepatic deficiency of transferrin receptor 2 is associated with increased erythropoiesis independent of iron overload

Transferrin receptor 2 (TFR2) is a transmembrane protein expressed mainly in hepatocytes and in developing erythroid cells and is an important focal point in systemic iron regulation. Loss of TFR2 function results in a rare form of the iron-overload disease hereditary hemochromatosis. Although TFR2 in the liver has been shown to be important for regulating iron homeostasis in the body, TFR2's function in erythroid progenitors remains controversial. In this report, we analyzed TFR2-deficient mice in the presence or absence of iron overload to distinguish between the effects caused by a high iron load and those caused by loss of TFR2 function. Analysis of bone marrow from TFR2-deficient mice revealed a reduction in the early burst-forming unit-erythroid and an expansion of late-stage erythroblasts that was independent of iron overload. Spleens of TFR2-deficient mice displayed an increase in colony-forming unit-erythroid progenitors and in all erythroblast populations regardless of iron overload. This expansion of the erythroid compartment coincided with increased erythroferrone (ERFE) expression and serum erythropoietin (EPO) levels. Rescue of hepatic TFR2 expression normalized hepcidin expression and the total cell count of the bone marrow and spleen, but it had no effect on erythroid progenitor frequency. On the basis of these results, we propose a model of TFR2's function in murine erythropoiesis, indicating that deficiency in this receptor is associated with increased erythroid development and expression of EPO and ERFE in extrahepatic tissues independent of TFR's role in the liver.

Mycobacterial antigens accumulation in foamy macrophages in murine pulmonary tuberculosis lesions: Association with necrosis and making of cavities.

Understanding mechanisms of cavitation in tuberculosis (TB) is the missing link that could advance the field towards better control of the infection. Descriptions of human TB suggest that postprimary TB begins as lipid pneumonia of foamy macrophages that undergoes caseating necrosis and fragmentation to produce cavities. This study aimed to investigate the various mycobacterial antigens accumulating in foamy macrophages and their relation to tissue destruction and necrosis. Pulmonary tissues from mice with slowly progressive TB were studied for histopathology, acid-fast bacilli (AFB) and presence of mycobacterial antigens. Digital quantification using Aperio ImageScope was done. Until week 12 postinfection, mice were healthy, and lesions were small with scarce AFB and mycobacterial antigens. Colony-forming units (CFUs) increased exponentially. At week 16-33, mice were sick, macrophages attained foamy appearance with an increase in antigens (P<.05), 1.5 log increase in CFUs and an approximately onefold increase in AFB. At week 37-41, mice started dying with a shift in morphology towards necrosis. A >20-fold increase in mycobacterial antigens was observed with only less than one log increase in CFUs and sevenfold increase in AFB. Secreted antigens were significantly (P<.05) higher compared to cell-wall antigens throughout infection. Focal areas of necrosis were associated with an approximately 40-fold increase in antigen MPT46, functionally active thioredoxin, and a significant increase in all secreted antigens. In conclusion, mycobacterial antigens accumulate in the foamy macrophages in TB lesions during slowly progressive murine pulmonary TB. Secreted antigens and MPT46 correlated with necrosis, thereby implying that they might trigger the formation of cavities.

Serum-Associated Antibiotic Tolerance in Pediatric Clinical Isolates of Pseudomonas aeruginosa.

When grown in human serum, laboratory isolates of Pseudomonas aeruginosa exhibit tolerance to antibiotics at inhibitory concentrations. This phenomenon, known as serum-associated antibiotic tolerance (SAT), could lead to clinical treatment failure of pseudomonal infections. Our purpose in this study was to determine the prevalence and clinical impact of SAT in Pseudomonas isolates in hospitalized children. The SAT phenotype was assessed in patients aged <18 years admitted with respiratory or blood cultures positive for P. aeruginosa. The SAT phenotype was a priori defined as a ≥2-log increase in colony-forming units when grown in human serum compared with Luria-Bertani medium in the presence of minocycline or tobramycin. SAT was detected in 29 (64%) patients. Fourteen patients each (34%) had cystic fibrosis (CF) and tracheostomies. Patient demographics and comorbidities did not differ by SAT status. Among CF patients, SAT was associated with longer duration of intravenous antibiotics (10 days vs 5 days; P < .01). This study establishes that SAT exists in P. aeruginosa from human serum and may be a novel factor that contributes to differences in clinical outcomes. Future research should investigate the mechanisms that contribute to SAT in order to identify novel targets for adjunctive antimicrobial therapies.

Role of homologous recombination genesRAD51,RAD52, andRAD59in the repair of lesions caused by γ-radiation to cycling and G2/M-arrested cells ofCandida albicans

We have analysed the role of homologous recombination (HR) genes on the repair of double-strand breaks induced by γ-ionising radiation in Candida albicans. Depletion of either CaRad51 or CaRad52 caused a dramatic drop in the number of survivors compared with wild type, whereas depletion of CaRad59 caused a moderate decrease. Besides, compared with Saccharomyces cerevisiae, C.albicans relies more on HR proteins for repair of ionising radiation lesions. Pulse-field electrokaryotypes of survivors identified genetic alterations mainly in the form of aneuploidy in HR mutants and chromosome length polymorphism and ectopic translocation in wild type. Increasing irradiation (4 to 80krad) of both cycling and nocodazole-treated (G2/M-arrested) cells revealed a gradual loss of chromosomes, larger chromosomes being more affected than smaller ones. For cycling wild-type cells, shattered chromosomes were progressively restored following incubation in yeast extract, peptone, dextrose medium, but not in phosphate-buffered saline, and this accompanied by a moderate increase in colony-forming units, suggesting that repair was followed by replication of survivors. Irradiated G2/M arrested cells from wild type but not from HR mutants partially restored the chromosome ladder following incubation (4-8hr) in yeast peptone dextrose-nocodazole. However, HR mutants showed a chromosome shattering pattern similar to wild type, an indication that lesions other than double-strand breaks, likely single-strand break, are responsible for their drastically reduced survivability.

Suppression of m6A reader Ythdf2 promotes hematopoietic stem cell expansion.

Transplantation of hematopoietic stem cells (HSCs) from human umbilical cord blood (hUCB) holds great promise for treating a broad spectrum of hematological disorders including cancer. However, the limited number of HSCs in a single hUCB unit restricts its widespread use. Although extensive efforts have led to multiple methods for ex vivo expansion of human HSCs by targeting single molecules or pathways, it remains unknown whether it is possible to simultaneously manipulate the large number of targets essential for stem cell self-renewal. Recent studies indicate that N6-methyladenosine (m6A) modulates the expression of a group of mRNAs critical for stem cell-fate determination by influencing their stability. Among several m6A readers, YTHDF2 is recognized as promoting targeted mRNA decay. However, the physiological functions of YTHDF2 in adult stem cells are unknown. Here we show that following the conditional knockout (KO) of mouse Ythdf2 the numbers of functional HSC were increased without skewing lineage differentiation or leading to hematopoietic malignancies. Furthermore, knockdown (KD) of human YTHDF2 led to more than a 10-fold increase in the ex vivo expansion of hUCB HSCs, a fivefold increase in colony-forming units (CFUs), and more than an eightfold increase in functional hUCB HSCs in the secondary serial of a limiting dilution transplantation assay. Mapping of m6A in RNAs from mouse hematopoietic stem and progenitor cells (HSPCs) as well as from hUCB HSCs revealed its enrichment in mRNAs encoding transcription factors critical for stem cell self-renewal. These m6A-marked mRNAs were recognized by Ythdf2 and underwent decay. In Ythdf2 KO HSPCs and YTHDF2 KD hUCB HSCs, these mRNAs were stabilized, facilitating HSC expansion. Knockingdown one of YTHDF2's key targets, Tal1 mRNA, partially rescued the phenotype. Our study provides the first demonstration of the function of YTHDF2 in adult stem cell maintenance and identifies its important role in regulating HSC ex vivo expansion by regulating the stability of multiple mRNAs critical for HSC self-renewal, thus identifying potential for future clinical applications.

Megakaryocyte and Osteoblast Interactions Modulate Bone Mass and Hematopoiesis.

Emerging evidence demonstrates that megakaryocytes (MK) play key roles in regulating skeletal homeostasis and hematopoiesis. To test if the loss of MK negatively impacts osteoblastogenesis and hematopoiesis, we generated conditional knockout mice where Mpl, the receptor for the main MK growth factor, thrombopoietin, was deleted specifically in MK (Mplf/f;PF4cre). Unexpectedly, at 12 weeks of age, these mice exhibited a 10-fold increase in platelets, a significant expansion of hematopoietic/mesenchymal precursors, and a remarkable 20-fold increase in femoral midshaft bone volume. We then investigated whether MK support hematopoietic stem cell (HSC) function through the interaction of MK with osteoblasts (OB). LSK cells (Lin-Sca1+CD117+, enriched HSC population) were co-cultured with OB+MK for 1 week (1wk OB+MK+LSK) or OB alone (1wk OB+LSK). A significant increase in colony-forming units was observed with cells from 1wk OB+MK cultures. Competitive repopulation studies demonstrated significantly higher engraftment in mice transplanted with cells from 1wk OB+MK+LSK cultures compared to 1wk OB+LSK or LSK cultured alone for 1 week. Furthermore, single-cell expression analysis of OB cultured±MK revealed adiponectin as the most significantly upregulated MK-induced gene, which is required for optimal long-term hematopoietic reconstitution. Understanding the interactions between MK, OB, and HSC can inform the development of novel treatments to enhance both HSC recovery following myelosuppressive injuries, as well as bone loss diseases, such as osteoporosis.

Changes of the skin barrier and bacterial colonization after hair removal by clipper and by razor

Background: Inappropriate hair removal increases the risk of surgical site infections which are associated with a higher morbidity and mortality of surgical patients. Here, the effects of a clipping device and a disposable razor on the skin barrier, microbial burden and surface structure were compared. Methods: Changes in bacterial colonization, transepidermal water loss, antioxidant status and the skin surface structure were investigated on the calves of 12 healthy volunteers. Measurement time points were at baseline (t base ) and 24 hours after hair removal (t 24 ). Results: Both, the disposable razor and the clipper showed a decrease in log colony-forming units count from t base (mean(t base ) ± standard deviation = 2.6 ± 1.27, median ± standard error = 2.6 ± 0.37) to t 24 at p razo r = 0.05 and p clipper = 0.06 respectively. At t 24 clipping resulted in a higher reduction of log colony-forming units (mean(t 24 ) = 1.76 ± 0.8, median = 1.69 ± 0.23) compared to the use of the disposable razor (mean(t 24 ) = 1.84 ± 0.85, median = 1.91 ± 0.24). Furthermore, the razor-treated group showed an increase in colony-forming units from t 0 to t 24 , whereas clipping lead to a continuous decrease in colony-forming units from t 0 to t 24 . An enhanced appearance of microlesions and a significant increase of transepidermal water loss after shaving using the disposable razor (p = 0.005) were found indicating skin barrier disruptions. Clipping showed no significant effect on transepidermal water loss. Conclusion: Hair removal using the clipping device results in less disruption of the skin barrier compared to the razor, avoiding the development of microlesions. This could be favorable for the prevention of surgical side infections and postoperative wound management.

Persistence of Beauveria bassiana sensu lato and Metarhizium anisopliae sensu lato in Vermont (USA) forest soil

Persistence of granular formulations of Beauveria bassiana sensu lato, strains GHA726 and ERL1170, and Metarhizium anisopliae sensu lato, strains ERL824 and ERL701, applied to forest soil was documented. One formulation was prepared by producing fungi on millet and at application adding cooked, dried millet to provide additional nutrients for fungal growth. The other was made by producing fungi on corn starch ‘packing peanuts’, a biodegradable packaging material, adding additional packing peanuts as a nutrient base. The trial was conducted in three sites in a forest predominating in sugar maple (Acer saccharum) in Underhill, VT, USA. In each site, 33 dominant sugar maples were randomly selected. A 1-m2 plot located 1 m from the bole of the tree was established. Soil samples were collected before application, and 1 h, 2, 4, 8, 12 and 16 wk post-application. Fungal levels were estimated based on number of colony-forming units (CFUs). A significant increase in CFUs was observed 2 wk post-application in plots treated with isolates GHA726, ERL1170 and ERL824 as a result of fungal reproduction on nutrients in the formulations. Evidence of greater persistence for the B. bassiana than the M. anisopliae isolates was observed, based on CFU levels over time. Isolates formulated with millet proliferated more than those on packing peanuts. Use of entomopathogenic fungi in forest soil has potential for managing soil-dwelling insect pests such as pear thrips, Taeniothrips inconsequens, and the black vine weevil, Otiorhynchus sulcatus, though the type of formulation and the specific isolate used must be selected carefully.

Salmonella-induced Diarrhea Occurs in the Absence of IL-8 Receptor (CXCR2)-Dependent Neutrophilic Inflammation.

Gastroenteritis is the most common manifestation of nontyphoidal Salmonella enterica infections, but little is known about the pathogenesis of diarrhea in this infection To determine whether polymorphonuclear neutrophils (PMNs) are required for diarrhea for Salmonella colitis, we infected kanamycin-pretreated interleukin 8R (IL-8R) mutant mice and controls, both with nonmutant Slc11a1 (Nramp1, ItyR). We compared the 2 mouse strains for increases in fecal water content (diarrhea) 3 days after infection, changes in expression of ion transporters in colonic epithelial cells, proliferation of epithelial cells, and severity of infection as measured by colony-forming units (CFUs). The IL-8R knockout mice had fewer PMNs in the colon but the other variables we measured were unaffected except for an increase in CFUs in the colon. The pathologic changes in the cecum were similar in both groups except for the lack of PMNs in the IL-8R knockout mice. There was minimal damage to the colon more distally. In the early stage of Salmonella colitis, PMNs are not required for diarrhea or for the decrease in expression of colonic epithelial cell apical ion transporters. They contribute to defense against infection in the cecum but not extracolonically at this stage of Salmonella colitis.

Polysaccharide isolated from Parmelia tinctorum ameliorates ionizing irradiation-induced damage in mice.

In this study, WPT-A, a type of water-soluble homogeneous lichen polysaccharide, was isolated and purified from Parmelia tinctorum. We investigated whether WPT-A has radioprotective effects when administered before total-body irradiation (TBI). Mice were treated with WPT-A via intraperitoneal injection (i.p.) once per day for three consecutive days prior to 7, 7.5, 8.5, 10 or 10.5-Gy TBI. Our results indicated that the survival rate was enhanced at a range of levels of TBI. The calculated dose reduction factor (DRF) was 1.2. White blood cell (WBC) counts, spleen colony forming units (CFU-S) and bone marrow nucleated cell (BMNC) counts were used to investigate the radioprotective effects of WPT-A on the hematopoietic system. The treatment groups received WPT-A at 20, 50 and 80 mg/kg b.w. doses before 6.5-Gy TBI and showed significantly higher BMNC and WBC counts compared with the radiation-only group. The groups administered 50 and 80 mg/kg b.w. WPT-A showed a significant increase in CFU-S compared with the radiation-only group. We also carried out a single cell gel electrophoresis assay to explore the radioprotective effects of WPT-A on DNA damage. The results from single-cell gel electrophoresis of peripheral blood leukocytes showed that WPT-A attenuated radiation-induced DNA damage. These results indicate a potential use for WPT-A as a radioprotector.

Salutary Effect of Pegylated Interferona in PV and ET As Evaluated by Quantitation of Pre-JAK2V617F and JAK2V617F-Bearing Stem Cells and Granulocytes and Correlation with Circulating Regulatory T Cells and HSC Cell Cycle Status

AbstractAbstract ▪807▪This icon denotes a clinically relevant abstractThe discovery of JAK2 (JAK2V617F) mutation in polycythemia vera (PV) and essential thrombocythemia (ET) has revolutionized our understanding of PV and ET. However, JAK2V617F is not the disease-initiating mutation and constitutes only part of the clone. Pegylated interferona (PegInfα), a form of interferon-alpha with a longer half-life and more tolerable toxicity, can induce not only hematological remission but also reduction of JAK2V617F allelic burden and suppression of the pre-JAK2V617F PV clone, as demonstrated by the return of polyclonal hematopoiesis (Liu Blood, 03). It is not clear whether this salutary effect is achieved by stimulation of an immune response against the PV clone, preferential activation of normal dormant stem cells (HSCs), or suppression of the PV clone by PegInfα. Using X-chromosome based clonality (qTCA) and JAK2V617F allelic burden quantitative assays, we analyzed selected biomarkers in HSCs, granulocytes and platelets of 18 patients treated with PegInfα, and evaluated cell cycle activity in HSCs. Previous data have indicated that PegInfα modulates the number of CD4+FOXP3+ regulatory T cells (Tregs) in the periphery. This is perhaps an indicator that PegInfα alters immunity against the JAK2V617F-bearing clone. We hypothesized that PegInfα mobilizes marrow Tregs to the periphery, thus decreasing their immunosuppressive and tumor promoting influence on the marrow microenvironment. Therefore, we correlated the number of Tregs before, during and after treatment with JAK2 allelic burden in peripheral blood and marrow samples in ongoing studies.In ongoing studies, 18 patients (7 females and 11 males) with PV and ET were treated with PegInfα, with a median follow-up of 6 months. There were variable JAK2V617F allelic burdens at enrollment (1.2% – 99.8%). Following treatment, 7 of 18 patients demonstrated a decrease in JAK2V617F allelic burden, indicating that PegInfα had an effect on the JAK2V617F-positive clone, while 11 had no response or had an increase in JAK2V617F levels. Six out of 7 female patients were informative for at least one exonic polymorphism out of 5 X-chromosome genes, and all 6 had clonal hematopoiesis as determined by qTCA at the start of treatment. Two of these 6 patients developed polyclonal hematopoiesis during therapy, but in one the JAK2V617F allelic burden was unchanged, indicating that, in this patient, PegInfα had a selective salutary effect on a pre-JAK2V617F clone, while in other patients PegInfα clearly decreased the JAK2V617F-positive clone. In the 7 patients who demonstrated decreased JAK2V617F levels, there was an inverse relationship of JAK2V617F level with the number of circulating peripheral blood Tregs (examples shown in Figs 1 and 2). Statistical analysis of 18 patients showed a significant negative relationship between the number of circulating Tregs and JAK2V617F allelic burden (Spearman’s correlation coefficient -0.4308, p<0.001). This suggests that an immunomodulatory effect by PegInfα involving Tregs may, at least in part, be associated with suppression of pre-JAK2V617F and JAK2V617F-positive clonal hematopoiesis.We assessed total colony forming ability in methylcellulose culture media as a measure of hematopoietic progenitor abundance. In 5 patients treated with Pegasys, all 5 demonstrated an increase in colony-forming units. Furthermore, when cell cycle status of CD34+ HSCs was ascertained, we found a decrease in the percentage of HSCs in G0 (quiescence) in 4 out of 6 samples. These findings suggest that PegInfα therapy promotes normal HSC proliferation.For the first time we directly demonstrate that PegInfα therapy in some patients recruits dormant pre-clonal PV HSCs to active hematopoiesis, thereby resulting in production of polyclonal granulocytes and platelets. PegInfα also appears to mobilize Tregs to the circulation, an effect that may be associated with changes in the marrow microenvironment. We conclude that PegInfα can suppress both pre-JAK2V617F and JAK2V617F PV clones in some PV patients, as demonstrated by evaluation of HSCs, granulocytes and platelets, but this often occurs asynchronously. Our preliminary data suggest that PegInfα therapy is associated with an increase in cell cycle activity in HSCs and mobilization of Tregs to the circulation. This provides a possible mechanism by which polyclonal hematopoiesis is restored after PegInfα treatment. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Diabetes and aging: a different phenotypic commitment of circulating and resident stem cells?

Atherosclerosis is a vascular disease largely attributed to chronic vascular injury, and its clinical manifestations appear more frequently in aged subjects. Accumulation of vascular smooth muscle cells in the tunica intima plays a major role in the pathogenesis of atherosclerosis. Arterial smooth muscle cells are heterogeneous even in the normal vessel wall and display more marked different phenotypes in pathological conditions. Smooth muscle cells in atherosclerotic plaques display a de-differentiated or ‘‘synthetic’’ phenotype compared to a ‘‘contractile’’ phenotype in the normal media. Aorta stiffens with age and other cardiovascular risk factors. In particular, diabetes-induced activation of the renin–angiotensin system increases the expression of angiotensin II, further increasing aortic calcification and stiffness. Thus, alterations of aortic and carotid walls in patients with diabetes were traditionally considered a sort of ‘‘accelerated aging.’’ In the last years, the contribution of stem cells to atherosclerosis has been highlighted. Bone marrow and peripheral blood-derived endothelial and vascular smooth muscle cell resident progenitors both contribute to vascular remodeling during atherogenetic process and aging [1]. Both circulating and resident progenitor cells have been evocated to contribute to the response of the adult arterial wall to damage. Chronic treatment with bone marrow-derived progenitor cells from young non-atherosclerotic ApoE/ mice prevents atherosclerosis progression in ApoE/ recipients despite persistent hypercholesterolemia, whereas bone marrow-derived progenitor cells from older ApoE/ mice with atherosclerosis were much less effective [2]. These findings suggest that the progressive bone marrow-derived progenitor cells deficit may contribute to the development of atherosclerosis. Nevertheless, atherosclerotic lesions are characterized from the increase of stem cell markerexpressing cells, and macroscopically normal aortas from human and rat aged donors show an increased number of VEGFR-1 and c-kit cells in the thickened intima [3]. Also, diabetes alters the function of circulating progenitor cells. Depletion of bone marrow-derived angiogenic cell populations may further promote atherogenesis and aortic calcification in patients with diabetes mellitus [4]. In multivariable analyses, the increase in colony-forming units from endothelial progenitor cells was associated with the decrease in coronary artery and abdominal aortic calcification [5]. These changes were not associated with changes in CD34 expression, suggesting that a decreased angiogenic potential contributes to the development of human atherosclerosis. Moreover, decreasing colonyforming capacity associated with the progressive increase of calcification scores [5]. Recently, it has been reported that diabetes mellitus patients had significantly higher expression of osteocalcin and bone alkaline phosphatase on circulating VEGFR-2/CD34 progenitor cells than control subjects [6]. Moreover, cultured VEGFR-2/CD34 cells from diabetes mellitus patients formed structures highly suggestive of calcified nodules, strongly suggesting that circulating progenitor cells from diabetic patients show a drift toward a pro-calcific phenotype that may be driven by inflammatory signals in response to injury [6], similarly to that observed in non-vascular tissues [7]. Monocyte– macrophage recruitment is a crucial step for a correct angiogenesis, and this mechanism is mainly mediated by VEGFR-1, that favors the increase of vessel lumen, vessel Communicated by Massimo Federici.

Antimicrobial Efficacy of Simarouba glauca (Lakshmi Taru) Plant Extract against Enterococcus faecalis Biofilm: An in vitro Study

ABSTRACT Aim The aim of this study was to assess the antimicrobial activity of Simarouba glauca plant extract as intracanal medicament against Enterococcus faecalis and to compare its effect with that of metapex (calcium hydroxide with iodoform paste). Materials and methods In the study, 30 human premolar teeth infected with E. faecalis were used. Contaminated teeth were randomly separated into three groups (n = 10) and treated as follows: Group I, calcium hydroxide-based medication; group II, S. glauca-based medication; and group III, contaminated teeth without medication. After 7 days of treatment, teeth from each group were assessed for the level of microbial growth after a period of treatment. Results Metapex and S. glauca were associated with a reduction in E. faecalis after removal of the dressing; however, saline is associated with an increase in colony-forming units (CFU) per milliliter. Conclusion In this laboratory study using extracted teeth, S. glauca (Lakshmi Taru) was able to significantly reduce the CFU per milliliter values of E. faecalis in the root canal system similar to metapex. How to cite this article Varada SL, Veetil JE, Nair GC. Antimicrobial Efficacy of Simarouba glauca (Lakshmi Taru) Plant Extract against Enterococcus faecalis Biofilm: An in vitro Study. Cons Dent Endod J 2017;2(2):43-47.