Mean Number Of Colonies Research Articles

Development of bacteriostatic central venous port using photobiomodulation: a comparative in vitro study

Photobiomodulation (PBM) occurs when a cell is exposed to low energy intensities. A novel central venous port (CVP) with light-emitting diodes (LEDs) that emits red light with a wavelength of 680 nm via wireless energy transmission technology has been established. This comparative in vitro study examined whether PBM can reduce the growth of methicillin-resistant Staphylococcus aureus (MRSA), a common cause of central venous (CV) infections, in vitro. In this comparative in vitro study, the red light with a wavelength of 680 nm was used to irradiate an MRSA suspension in phosphate-buffered saline for 7.5, 15, 30, or 60 min in a 3.5 cm Petri dish with an area of 8.5 cm2. The total energy was 85 J at 7.5 min, 170 J at 15 min, 340 J at 30 min, and 680 J at 60 min. Six dishes for each time and 6 temperature-controlled samples were prepared. Each sample was incubated overnight at 37℃. The Shapiro-wilk test was used to determine whether the data were normally distributed. The numbers of colonies were counted and compared using one-factor ANOVA and Bonferroni’s post-hoc test. The mean numbers of colonies in the control group were 60.3, where the numbers of colonies in the irradiated group were 51.4 at 7.5 min, 53.5 at 15 min, 44.6 at 30 min, 34.3 at 60 min. The mean number of colonies in the 60 min irradiated group differed significantly from that in the control, 7.5 min, and 15 min groups. The Bonferroni’s post-hoc test showed significant difference in the number of colonies between control vs. 30 min control vs. 60 min, 7.5 min vs. 60 min, 15 min vs. 60 min. PBM with 680 nm LEDs on MRSA for 340 J at 30 min and 680 J at 60 min inhibited the growth of cell colonies. These findings support the use of photobiomodulation in Central venous port to prevent CV access port-Blood stream infection.

Combination of 70% ethanol and 2% potassium aluminum sulfate as a modified antibacterial disinfectant

Background: Mobile phones used by health-care workers can harbor pathogenic bacteria. Effective techniques are therefore needed to prevent bacterial transmission through mobile phones, including the development of new or modified disinfectants. Methods: In total, 100 swabs were collected from the mobile phones of health-care workers. All samples were directly cultured and incubated for 24 h at 37°C. The samples were divided into two groups (n = 50 each): one disinfected with 70% ethanol alone and the other with 70% ethanol containing 2% potassium aluminum sulfate (alum). Results: Bacterial growth before disinfection with 70% ethanol alone was observed in 92% of samples, which decreased to 66% after disinfection. With 70% ethanol plus 2% alum, bacterial growth before disinfection was 88%, decreasing to 16% after disinfection. The mean number of colonies before disinfection with 70% ethanol alone was 22.80 ± 10.38, while after disinfection, it was 3.48 ± 1.40 (P < 0.00001). Similarly, the number of colonies before disinfection with 70% ethanol plus 2% alum was 19.84 ± 9.34, while that after disinfection was 0.68 ± 0.33 (P < 0.00001). Conclusion: To the best of our knowledge, this is the first study to evaluate the combination of 70% ethanol with 2% alum to enhance the antimicrobial efficacy of ethanol as a new modified disinfectant.

Prevalence and identification of oral candida species in patients with type 2 diabetes in Yogyakarta

Patients with diabetes are prone to recurring and even resistant Candidiasis, making treatment challenging. Many hypotheses proposed related to susceptibility of diabetic patients to Candida. The prevalence and species of Candida in a particular diabetic community might be different compared to other diabetic community. This study aimed to determine the prevalence of Candida colony and its species in the oral cavity of diabetic patients included in the CDM (Chronic Disease Management) program in Yogyakarta. One hundred patients with type 2 diabetes (n= 100) were recruited as the subjects of this study. The subjects were classified into controlled and uncontrolled Diabetes mellitus (DM). Samples of oral rinse solution were collected to determine the species of Candida and number of Candida colonies using CHROMagar Candida medium. There were 47 and 53 of subjects with controlled and uncontrolled DM, respectively. The mean number of Candida colony in the subjects with controlled diabetes (1003.13) was higher than that in the subjects with uncontrolled diabetes (478.43). The Candida colony most commonly identified in the subjects with controlled and uncontrolled diabetes were C. albicans and C. glabrata, respectively. Female patients had higher mean number of Candida colony (859.51) compared to male (299.21). The Candida colony most often identified in both genders was C. albicans. In addition, the subjects of this study consisted of 83 geriatric subjects and 17 non geriatric subjects, in which the mean number of Candida colony in the geriatric subjects (761.77) was higher than that in the non-geriatric subjects (545.71). The Candida colony most often identified in the geriatric subjects and non-geriatric subjects was C. albicans and C. glabrata, respectively. The Mann Whitney test demonstrated that there was a significant difference (p = 0.009) of the mean number of Candida colony between male and female. However, there was no significant difference of the mean number of Candida colony between ages (p = 0.060) and diabetic status (p=0.175). It can be concluded that the Candida species most commonly identified in all the subjects was Candida albicans with the mean colony number of 349.96, followed by C. glabrata (225.97), C. krusei (144.91), C. tropicalis (3.67), and other species (2.02).

Türkiye’nin kuzeyinde ova ve kentsel alanlardan geçen bir akarsuyun mikrobiyal kirlilik göstergelerindeki mekânsal-zamansal değişimlerin değerlendirilmesi

The degradation of watersheds due to urban development increases the vulnerability of surface waters to pollutants of anthropogenic origin. Urban streams draining near swimming waters can contribute significantly to the deterioration of water quality. In the study, the microbial quality of the Terme River was investigated seasonally by collecting samples from four different stream sites. Microbiological analyzes were performed using standard methods with membrane filtration. The colony-forming unit (CFU) method determined fecal contamination in samples. The mean number of colonies in Terme River water samples was determined as 712 CFU/100 mL, 278 CFU/100 mL, and 64 CFU/100 mL for total coliform, Escherichia coli, and Clostridium perfringens, respectively. This situation indicates that there was fecal contamination in the stream. According to the bacteriological parameters in the Water Pollution Control Regulation, Terme River was found to be of class II water quality in terms of total coliform bacteria count. The Research results determined that the Terme River is not much polluted in terms of microbiological quality but has values under the pressure of pollution. To not increase the pollution level, the direct mixing of domestic wastes/wastewater, animal wastes, and drainage channels from the settlements and agricultural areas around the river should be prevented. The public should be made aware of the necessary precautions.

Bacterial Reverse Mutation Test of Verbenalin.

Verbenalin is a compound found in herbs such as Cornus officinalis and Verbena officinalis. This study investigated whether verbenalin is safe by analyzing its mutagenicity. To examine the mutagenic potential of verbenalin, a bacterial reverse mutation test (Ames test) was conducted with Salmonella typhimurium and Escherichia coli strains. Experiments with and without metabolic activity were performed. The mean colony number was less than double that of the control. Growth inhibition and precipitation of verbenalin were not apparent in all strains at different concentrations regardless of metabolic activity. Verbenalin did not show any signs of mutagenicity in this study. Additional toxicity studies including repeated oral toxicity, reproductive toxicity, and carcinogenicity tests are needed.

Pre-Clinical Investigation of a Novel Small Molecule Inhibitor Targeting YBX1 in AML

Recently, our group identified the pleiotropic DNA and RNA binding protein YBX1 as a specific dependency and therapeutic target in acute myeloid leukemia (AML) that is essential for stabilizing oncogenic transcripts and thereby maintains oncogenic protein expression (Perner et al., 2021). Selective pharmacologic targeting of YBX1 in AML, however, has not been achieved so far. Here, we investigate the functional consequences of a novel small molecule inhibitor (SU056) of YBX1 in AML. We examined the viability of several human AML cell lines (MOLM13, HL60, EOL1, THP1, Kasumi1, ML2, Mv4;11, OCI-AML3, NOMO1) upon SU056 treatment and found significant reduction of cell growth in a concentration dependent manner. Inhibition of cell growth could be mainly attributed to impaired proliferative capacity at nanomolar concentrations. Pharmacologic inhibition of YBX1 resulted in impaired colony formation (mean colony number DMSO: 231.3±13.88 vs. SU056 500nM: 154.7±8.29; p<0.0001), cell cycle arrest (cells in S phase: DMSO: 37.3±2.4 vs. SU056 500nM: 27.2±1.67; p=0.0127) and induction of myeloid differentiation. Consistently, in a xenograft model of human AML, SU056 treatment resulted in prolonged disease latency (MOLM13 diluent: 62 days OS vs. SU056: not reached, p=0.0013; OCI-AML3 diluent: 79 days OS vs. not reached, p=0.0001) and significant reduction of disease penetrance (MOLM13 diluent: 100% vs. SU056: 24,9%; OCI-AML3 diluent: 100% vs. SU056: 9,1%). Consistently, percentage of SU056 treated human leukemic cells in the bone marrow of recipient mice was decreased for both, MOLM13 (mean diluent: 41,3% vs. SU056: 7,6%) and OCI-AML3 (mean diluent: 28,9% vs. SU056: 2,9%). Likewise, pharmacologic inhibition of YBX1 reduced colony formation and total cell numbers of primary human AML cells in a dose-dependent manner. To be clinically relevant, such treatment must have a suitable therapeutic index between human hematopoietic stem- and progenitor cells (HSPCs) and their malignant counterpart. This is particularly important, considering the role of YBX1 in mRNA splicing and processing. Of note, pharmacologic inhibition of YBX1 did not perturb colony formation of normal HSPCs derived from apheresis donors. These findings were confirmed by investigating primary human leukemic cells in a patient-derived xenograft (PDX) model of AML. We aimed to assess whether loss in leukemia cell competition induced by pharmacologic targeting of YBX1 is sufficient for normal hematopoietic cells to outcompete leukemic cells in vivo. 1x106 normal human HSPCs were transplanted along with 2x104 leukemic cells to assess for efficacy of SU056-treatment versus diluent control. In vivo treatment with SU056 (20mg/kg, i.p.) for 3 weeks resulted in a significant decrease in the AML blast number in the PB (mean blast %: diluent: 38.5%; SU056: 2.25%; p<0.0001). This resulted in increased survival of SU056 treated recipients (OS diluent: 83 days vs. SU056: not reached). To confirm the proposed mechanism by which SU056 impairs leukemia growth we performed global transcriptome and proteome analysis following treatment with SU056 (500nM, 96h). Proteome profiling revealed a total of 2641 deregulated proteins. Gene-ontology analysis indicated deregulation of YBX1 targets and proteins associated with cell cycle, DNA repair, mRNA splicing/processing, and transcriptional regulation. Of note, overlap between transcriptional regulation (as determined by mRNA-Seq) and changes in proteome abundance revealed less than 5% target genes/proteins, indicating that SU056 may in fact interfere with binding of oncogenic mRNAs to YBX1 and thereby may reduce translation of oncogenic transcripts. In order to define functional consequences of pharmacologic YBX1 inhibition and explore potential vulnerabilities of drug combinations, we performed a genome-wide CRISPR/Cas9 screen in human AML cells treated with SU056 versus diluent control. Validation experiments are currently ongoing. Taken together, our data confirm the therapeutic potential to target YBX1 using the novel small molecule SU056. Given its broad applicability across genetic subtypes of AML and lack of toxicity on normal HSPCs, SU056 as a single agent or in combination with established targeted therapies may efficiently help to target and outcompete leukemic cells. Experiments to assess for potential synergy with already established targeted therapies will be presented.

Functional Role of Histone Variant macroH2A1 (H2AFY) in Acute Myeloid Leukemia (AML)

Histone variants can replace replication-coupled histones in the nucleosome. Among core histones, the H2A family exhibits highest sequence divergence resulting in the largest number of variants. Dysregulated expression of macrohistone (macroH2A) variants has been reported in different cancers. Their role in normal hematopoiesis and hematopoietic cancers remained so far poorly understood. We found high expression of macrohistone H2AFY (macroH2A1) in hematopoietic stem and progenitor cells (HSPCs) and primary AML as compared to differentiated hematopoietic cells. To assess for the functional dependency of human AML cells on macroH2A1, we performed in vitro CRISPR-Cas9 negative selection competition assays. Genetic inactivation of H2AFY in 4/9 AML cell lines (Molm-13, MV4;11, THP-1, OCI-AML3) resulted in outcompetition of H2AFY-deleted cells against non-deleted competitors. To confirm these findings in vivo, we performed xenotransplantation experiments. Genetic inactivation of H2AFY in human AML cells resulted in delayed leukemia development in recipient mice (median survival for NT-control: 35.5 days vs. H2AFY-KO: 44.0 days; p=0.002). To validate the functional impact of macroH2A1 on leukemic stem cells (LSCs), we generated a novel conditional knockout mouse model for H2AFY [B6(Cg)-H2afytm1Flh/J (H2afy flox/flox)]. Here, isolated lineage-depleted H2afy+/+, Mx1-Cre+ and H2afyfl/fl, Mx1-Cre+ bone marrow cells (Lin-BMCs) were retrovirally infected with MLL-AF9-GFP (MA9) and transplanted into sub-lethally irradiated recipient mice. Following establishment of leukemia, we transplanted 1x105 leukemic cells and inactivated H2afy in the leukemic cells by pIpC injection on day 7, 9 and 11 post-transplantation. Genetic deletion of H2afy resulted in delayed disease progression (median survival +/+: 83.5 days vs. -/-: 162.0 days, p=0.0013) and reduction of disease penetrance to 61%. Organ infiltration by leukemic cells led to disease development in a limited number of H2afy-/- recipient mice. Isolated H2afy-/- leukemic cells showed a decreased in proliferation and an increased in differentiation. Importantly, 37% of H2afy-/- recipient mice that developed AML showed only partial excision of H2afy, indicating selection of partially excised clones. Long-term survivors without leukemia development showed no molecular evidence for counterselection. Irrespective of the time of disease development and survival, immunophenotypic analysis of (Lin-cKit+Sca-1-FcγR+CD34+GFP+) LGMPs revealed decreased numbers of LSCs following H2afy-deletion (+/+: 2631/106 BMC; -/-: 727/106 BMC; p=0.0002). To assess for the functional consequences of H2afy-deletion on the LSC pool, we performed limiting dilution assays. When transplanting 1x105, 1x104, 1x103 and 1x102 H2afy+/+ or -/- leukemic cells into sub-lethally irradiated recipients, LGMP frequency in H2afy-deficient cells was significantly reduced (1/1 vs. 1/29928; p=1.25e-19). The reduction of LGMP frequency correlated with reduced plating efficiency and increased differentiation. Consistently, primary human AML cells showed impaired colony forming capacity following H2afy deletion (mean colony number NT-control: 60.0±17.1 vs. H2afy-KD: 30.1±8.9; p=0.0023). To assess for the impact of H2afy deletion on normal HSPCs, we performed competitive repopulation assays. Immunophenotypic abundance of HSPC in H2afy+/+ vs. -/- animals revealed comparable numbers in all relevant subpopulations. Serial competitive transplantation of H2afy+/+ and -/- BMC into primary and secondary recipient animals showed no competitive disadvantage or lack of self-renewal capacity of H2afy-/- cells. Likewise, colony formation of healthy human donor CD34+ cells was not impaired following depletion of H2AFY by RNAi. Transcriptome analysis of LGMP populations indicated increased of myeloid cell development and loss of LSC signatures in H2afy-deficient LGMPs. Likewise, ATAC-seq of H2afy+/+ and -/- LGMP population revealed profound changes in the chromatin structure and a strong correlation between chromatin accessibility and mRNA expression. Together, our results provide first evidence for a critical function of H2afy (macroH2A1) in models of AML while being dispensable for normal HSPC function. Experiments to assess for the mechanistic consequences of H2afy deletion on chromatin are currently under way.

Robotic endoscopic cooperative surgery for colorectal tumors: a feasibility study (with video).

Laparoscopic endoscopic cooperative colorectal surgery (LECS-CR) is a promising technique to achieve full-thickness resection of colorectal tumors. This approach has shown good rates of complete resection and low local recurrence, especially for large laterally spreading tumors, which are difficult to remove via endoscopy alone. However, it is often difficult to prevent peritoneal leakage of intestinal content, causing infections and risks of cancer spreading. It was hypothesized that a robotic assistance could make the procedure easier and decrease intestinal fluid leakage. This preclinical trial aims to assess the feasibility of robotic and endoscopic cooperative colorectal surgery (RECS-CR). LECS-CR was performed in five female pigs and RECS-CR was also performed in five female pigs. With the animal under general anesthesia, pseudotumors were created on the colonic mucosa at a distance comprised between 20 and 25cm from the anal verge. Desired resection margins were marked endoscopically and two stay sutures were placed either robotically or laparoscopically. A mucosa-to-submucosa dissection was performed endoscopically along the markings. Complete full-thickness dissection was performed cooperatively. The specimen was withdrawn endoscopically. The colon was closed using a self-fixating running suture. Abdominal contaminations, operating times, complications, and complete resections were evaluated and compared between LECS-CR and RECS-CR. The mean number of colonies of Escherichia coli in the RECS group was significantly lower than in the LECS group (36.7 ± 30.2 vs. 142.2 ± 78.4, respectively, p < 0.05). Operating time was comparable (118 ± 11.2 vs. 98.6 ± 25.7, respectively, p = 0.22). Two stenoses occurred in the LECS group. R0 resection was achieved in all cases. This study suggests that RECS-CR is feasible and has the potential to reduce intestinal content leakage, potentially preventing postoperative infections.

The role of quercetin in primary culture of ovine spermatogonial stem cells

The aim of the present study was to examine the effect of quercetin on the survival and primary culture of ovine spermatogonial stem cells (SSCs). The two-time enzymatic digestion process was employed to obtain SSCs from lamb testes. In the next step, the use of filtration and differential plating methods caused an increase in the number of SSCs in the cell suspension resulting from enzymatic and mechanical digestions. Mitomycin-C-treated Sertoli cells were used to prepare the feeder layer. The stem cells were then cultured on the Sertoli cell feeder layer. The identification of the colonies was done through alkaline phosphatase staining methods and specific gene expression of ram’s SSCs (nanog and Plzf). The results of methylthiazolyldiphenyl-tetrazolium bromide assay on SSCs 72 h after culture with different treatments of quercetin demonstrated that the highest percentage of survival was for 5 μM and 10 μM concentrations, respectively; however, compared to the control, no significant difference was observed. In comparison with the control, the concentration equal to and greater than 20 μM quercetin caused a significant decrease in the survival of SSCs (P &amp;lt; 0.05). Seven days after culture, 40 μM quercetin caused a substantial reduction in the mean number of colonies, compared to the control (P &amp;lt; 0.05). The results demonstrated that compared to the control, 5 μM to 40 μM of quercetin significantly reduced Plzf gene expression. Furthermore, the concentration equal to and higher than 10 μM quercetin significantly decreased bcl-2 gene expression in the cells under study (P &amp;lt; 0.05). Based on the findings of the present study, the use of quercetin for the primary culture of ovine SSCs is not recommended. It is suggested that the function of this antioxidant should be investigated on the differentiation of SSCs.

Abstract 3907: Synergistic effect of targeting both CDK4/6 and mTORin sarcoma cell lines

Abstract Background: The phosphatidylinositide-3-kinase/Protein kinase B (AKT)/ mammalian target of rapamycin (mTOR) signaling is central for cancer growth, survival and motility. One of the key restraints on cell growth downstream is retinoblastoma (Rb) tumour suppressor, which is negatively regulated by the cyclin-dependent protein kinases (CDK) 4/6 protein. We hypothesize that blocking both the CDK4/6 and the mTOR allows the double brakes to tumour growth. Aim: Our aim is to investigate whether combination therapy using mTOR and CDK4/6 inhibitors (palbociclib and ridaforolimus) would have a synergistic growth inhibitory effect in sarcoma. Methods: The effect and mechanism of palbociclib and ridaforolimus was investigated in a panel of twelve sarcoma cell lines by Crystal-violet colorimetric and Western blot assays. HT1080 fibrosarcoma metastatic mouse model was investigated for in vivo therapeutic study. Results: Palbociclib showed anti-proliferation in all cell lines (IC50s: 0.1-1.1µM), while ridaforolimus had growth inhibition in 10/12 cell lines (IC50s: 0.4-26nM). The palbociclib-ridaforolimus combination achieved synergistic effect (CIs: 0.1-0.9) in 9/12 cell lines, including applying the drugs in different sequence (together or pre-treatment with one drug for 24-48 hours) and ratio (1:1, 1:2, 1:4, 2:1 or 4:1). The Western blot demonstrated that the 2-drug combination further blocked both CDK/Rb/E2F and AKT/mTOR pathways, via promoting apoptosis. In the experimental lung metastatic HT1080 mouse model, using palbocilib (10 and 30mg/kg, every other day) and ridaforolimus (0.1 and 0.3mg/kg, daily for 5 days per week) for 4 weeks synergistically (CI = 0.7) inhibited the development of lung metastases (mean number of colonies: 132 in low combination dose and 38 for high combination dose) compared to monotherapy [243 (palbociclib) and 255 (ridaforolimus) in low dose, 113 and 97 for high dose) and vehicle control (286). In the orthotopic fibrosarcoma HT1080 mouse model, the palbociclib-ridaforolimus combination therapies achieved significant (p&amp;lt;0.001) and synergistic (CI=0.00375) growth inhibitory effect compared to monotherapies and control groups. Conclusions: This study demonstrated that palbociclib-ridaforolimus combination is active in a variety of sarcoma subtypes and worthy of further development towards a clinical trial. Citation Format: Xiaochun Wang, David Goldstein, Philip Crowe, Jia-Lin Yang. Synergistic effect of targeting both CDK4/6 and mTORin sarcoma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3907.

Forest Fragmentation Shapes Resource Partitioning for Endemic Pollinators (Hymenoptera: Meliponini)

A field study was undertaken along six diverse habitat gradients of Kenya to evaluate the occurrence and underlining drivers that shape meliponine bee species diversity. These gradients ranged from continuous forest habitats (Indigenous forests, mixed forests and exotic forest patch) representing unfragmented habitats to dispersed habitats (Mixed deciduous woodlands, Grasslands dominated by perennial grasses such as Cenchrus ciliaris, Chloris roxburghiana and Acacia dominated bush lands) representing fragmented habitats respectively. A total of four different species of meliponine bees were encountered with Hypotrigona gribodoi being the most abundant species, followed by Meliponula ferruginea (black), Plebeina hildebrandti and Hypotrigona ruspolii in descending order. Occurrence and diversity of Hypotrigona gribodoi, Melipona ferruginea (black), Plebeina hildebrandti and Hypotrigonaruspolii was significantly influenced by the degree of fragmentation (vegetation type) and floral diversity (P = 0.0056); the mean number of colonies recorded per study site ranged from 2.3 in dispersed habitats in the lowlands to 1.0 in continuous forest landscapes in the highlands, whereas the mean population density ranged from 2.0 to 172 colonies/25 ha-transects. The results provide substantial support to better understand interactions between habitat structure and the degree of fragmentation in linking species diversity to habitat loss.

Miniaturization of the Clonogenic Assay Using Confluence Measurement

The clonogenic assay is a widely used method to study the ability of cells to ‘infinitely’ produce progeny and is, therefore, used as a tool in tumor biology to measure tumor-initiating capacity and stem cell status. However, the standard protocol of using 6-well plates has several disadvantages. By miniaturizing the assay to a 96-well microplate format, as well as by utilizing the confluence detection function of a multimode reader, we here describe a new and modified protocol that allows comprehensive experimental setups and a non-endpoint, label-free semi-automatic analysis. Comparison of bright field images with confluence images demonstrated robust and reproducible detection of clones by the confluence detection function. Moreover, time-resolved non-endpoint confluence measurement of the same well showed that semi-automatic analysis was suitable for determining the mean size and colony number. By treating cells with an inhibitor of clonogenic growth (PTC-209), we show that our modified protocol is suitable for comprehensive (broad concentration range, addition of technical replicates) concentration- and time-resolved analysis of the effect of substances or treatments on clonogenic growth. In summary, this protocol represents a time- and cost-effective alternative to the commonly used 6-well protocol (with endpoint staining) and also provides additional information about the kinetics of clonogenic growth.

Factors influencing microbial colonies in the air of operating rooms

BackgroundThe operating room (OR) of the hospital is a special unit that requires a relatively clean environment. The microbial concentration of an indoor OR extrinsically influences surgical site infection rates. The aim of this study was to use active sampling methods to assess microbial colony counts in working ORs and to determine the factors affecting air contamination in a tertiary referral medical center.MethodsThis study was conducted in 28 operating rooms located in a 3000-bed medical center in northern Taiwan. The microbiologic air counts were measured using an impactor air sampler from May to August 2015. Information about the procedure-related operative characteristics and surgical environment (environmental- and personnel-related factors) characteristics was collected.ResultsA total of 250 air samples were collected during surgical procedures. The overall mean number of bacterial colonies in the ORs was 78 ± 47 cfu/m3. The mean number of colonies was the highest for transplant surgery (123 ± 60 cfu/m3), followed by pediatric surgery (115 ± 30.3 cfu/m3). A total of 25 samples (10%) contained pathogens; Coagulase-negative staphylococcus (n = 12, 4.8%) was the most common pathogen. After controlling for potentially confounding factors by a multiple regression analysis, the surgical stage had the significantly highest correlation with bacterial counts (r = 0.346, p < 0.001). Otherwise, independent factors influencing bacterial counts were the type of surgery (29.85 cfu/m3, 95% CI 1.28–58.42, p = 0.041), site of procedure (20.19 cfu/m3, 95% CI 8.24–32.14, p = 0.001), number of indoor staff (4.93 cfu/m3, 95% CI 1.47–8.38, p = 0.005), surgical staging (36.5 cfu/m3, 95% CI 24.76–48.25, p < 0.001), and indoor air temperature (9.4 cfu/m3, 95% CI 1.61–17.18, p = 0.018).ConclusionsUnder the well-controlled ventilation system, the mean microbial colony counts obtained by active sampling in different working ORs were low. The number of personnel and their activities critically influence the microbe concentration in the air of the OR. We suggest that ORs doing complex surgeries with more surgical personnel present should increase the frequency of air exchanges. A well-controlled ventilation system and infection control procedures related to environmental and surgical procedures are of paramount importance for reducing microbial colonies in the air.

Effectiveness of isopropyl alcohol and ultraviolet-based sanitiser on decontamination of mobile phones used by dental personnel

Introduction: Mobile phones have become an inevitable mode of communication. Dental office and the dental operators along with their mobile phones are exposed to numerous pathogens as a part of their profession, leading to nosocomial infection. This study aimed at assessing the effectiveness of isopropyl alcohol and a customised ultraviolet-based (UV) sanitiser in decontamination of mobile phones. Methods: A cross-sectional study was carried out on 30 touch screen mobile phones belonging to dental professionals in a college setting. Swabs were collected along the screen, camera lens and on/off buttons of mobile phones. Swabs were streaked onto nutrient agar and incubated at 37°C for 24 h for the assessment of microbial load before and after the disinfection procedures. The disinfection process was performed using 70% isopropyl alcohol and an UV-based sanitiser. Mann—Whitney U-test was used to compare the values between the two groups. Wilcoxon signed-ranks test was used to compare values within each group Results: There was a statistically significant reduction in the mean number of colonies (P = 0.001) after decontamination by the two groups (isopropyl alcohol and UV-based sanitiser) indicating that both agents were effective in disinfection. Conclusion: The study concluded that the percentage reduction in microbial load of the mobile phones was better with isopropyl alcohol compared to UV-based sanitiser. It is recommended that mobile phones in the dental setup be regularly decontaminated and dentists must adhere to strict infection control protocols specifically in relation to hand hygiene.

Abstract MIP-051: EFFICACY OF MTORC1/2 INHIBITION ON OVARIAN CANCER STEM CELLS

Abstract INTRODUCTION: Ovarian cancer is the most lethal gynecologic malignancy. Initial treatment with platinum and taxane drugs are effective, however inevitably these patients will recur and become resistant to cytotoxic therapies. Thus, new alternatives for durable treatments need to be identified. One approach is to explore the heterogeneity of epithelial ovarian cancer and identify the subpopulation of cells that are resistant to treatment. It has been hypothesized, that cancer stem cells (CSCs) can survive chemotherapy and have enhanced tumor-initiating capabilities. The AKT-PI3K-mTOR pathway is well studied and has been shown to be critical for tumor cell survival. This pathway closely regulates both mTOR complexes (mTORC1 and mTORC2). mTORC1 regulates protein synthesis and autophagy whereas mTORC2 regulates cell motility. mTORC1 and 2 ultimately affect cell proliferation, survival and angiogenesis, all inherently important in tumorgenesis. OBJECTIVES: Rapalogs are specific and potent inhibitors of mTORC1 but have no effect of mTORC2. We investigate the efficacy of dual mTORC1/2 inhibitors and its effect on the CSC population. METHODS: Carboplatin resistant ovarian cancer cell line OVCAR3 were cultured and treated with dimethyl sulfoxide (DMSO), carboplatin, or INK128, a potent dual inhibitor of mTORC1/2. Using cell surface markers CD133 and CD44, CSCs were quantified by flow cytometry. OVCAR3 spheroids were cultured as a surrogate for CSCs. Colony formation-survival assays were performed with spheroids treated with the above mentioned conditions. Analysis was performed using ArrayScan technology. Doxycyline inducible silencing cell lines derived from OVCAR3 were generated; Non-silenced (Nsi), Raptor (Sh-mTORC1) and Rictor (Sh-mTORC2). Colony formation assays using spheroids from silenced lineages were performed to evaluate growth and survival. Western blot analysis was performed to confirm silencing of targets, and to evaluate protein expression in CSCs. RESULTS: 4.87% of the untreated OVCAR3 population were CSCs. INK128 treatment of the cells confered a 2 fold increase of CD44+/CD133+ cells, presumably CSCs, P&amp;lt;0.005 and no difference between DMSO and carboplatin treated cells (NS). INK128 treated cells formed fewer colonies in comparison to DMSO (mean number of colonies 36.3 + 23.8 vs. 83.3 + 32.1, P&amp;lt;0.001) and smaller colonies (mean number of cells in a colony 5260 + 835.8 vs 6907 + 702.4 P &amp;lt;0.0002). Similar populations of CD44+/CD133+ were isolated in Nsi and Raptor (NS), and there were no difference in the number or size of colonies. In comparison to Nsi, Rictor resulted in 50% decrease in CD44+/CD133+ cells, (P &amp;lt;0.002) and fewer colonies (1.6+1.3 vs 10+7.2, p&amp;lt; 0.03). There was no difference in colony size. CSCs expressed Oct4, Sox2 and phosphorylated 4E-BP1 confirms activation of AKT. CONCLUSIONS: The PI3K/AKT/mTOR pathway appears to have a role in the biogenesis of platinum resistant ovarian cancer cells. The inhibition of mTORC1/2 delays growth of OVCAR3 cells, and may have a role in activating in stem like CSCs. However, it does not appear to selectively inhibit CD133+/CD44+ cells. These findings suggest that targeting CSCs may lead to advances in ovarian cancer treatment. Citation Format: Jing-Yi Chern MD, Melissa Frey MD, Fernanda Musa MD, Amandine Alard PhD, Stephanie V. Blank, MD, Robert Schneider, PhD. EFFICACY OF MTORC1/2 INHIBITION ON OVARIAN CANCER STEM CELLS [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr MIP-051.

Mechanism of Soil Eco-Functional Stability Under Pyrene/Cadmium Simplex and Combined Pollution Stress

In current scenario,the soil pollution has become very severe and its effects on agricultural and ecological security issues cannot be ignored as various contaminants are discharged into soil.Thus,the soil pollution is exigent and has to be solved.This research took soil resistance (Rt),resilience (Rl) and stability (Sb) as evaluation indexes for judging soil quality by exerting different concentration (concentration ratio) gradient of pyrene (PYR),cadmium (Cd) and pyrene/cadmium (PYR/Cd) combined pollutants.A sympathetic description was showed from the aspects of microbial activity,diversity and abundance of soil ecosystem,and the models were constructed to describe the dose-response relationship between PYR-Sb and Cd-Rt.The research showed that different types of pollutants had certain inhibition on soil DOC content.In Cd and PYR simplex pollution,soil microbial mean biomass and colony number decreased with increasing concentration of pollutants.In PYR/Cd combined pollution,the ratio of PYR and Cd had a negative correlation with the decreasing rate of DOC and resistance,meanwhile Cd had a prominent influence on the above-mentioned correlations,in other words,the soil with higher concentration of Cd had lower DOC decrease rate and resistance,and Cd would have dominant inhibition effect on microorganisms under PYR/Cd combined pollution.In addition,this study found the significant correlation of cPYR-Sb and cCd-Rt,and built the binomial forecasting model to describe the dose-response relationship of cPYR-Sb and cCd-Rt.

CANDIDAL COLONIZATION AND PALATAL DENTURE BASE ADAPTATION IN RELATION TO DENTURE BASE MATERIALS AND THEIR PROCESSING TECHNIQUES

Purpose: The purpose of this research was to compare between the effect of denture base materials and their processing techniques on palatal denture base adaptation and candidal colonization. Materials and methods: Thirty identical denture bases was constructed by silicon mold which was made from a educational edentulous maxillary cast . Thirty maxillary stone casts were obtained by pouring hard dental stone in the previous silicon mold . Sheets of wax with 2mm thickness, each were adapted to their corresponding stone casts then divided equally into three groups. Group I: Ten maxillary heat cured acrylic resin denture bases processed by long cycle hot water bath curing technique. Group II: ten maxillary acrylic resin denture bases processed by microwave energy. Group III: Ten maxillary monomer free thermoplastic resin denture bases processed by sabelix injecting system. Palatal denture base adaptation and candidal colonization were evaluated. The data was recorded and statistically analyzed. Results: The results of this study showed that statistically there was significant difference in palatal denture base adaptation between the tested groups as(p=0.024). The least mean gap (stone thickness) at the posterior palatal seal area was found in Acrifast denture base resin (G III) followed by GI. The least mean numbers of candidal colonies for the three groups was found in Acrifast denture base resin (G III) followed by GI . Statistically there was highly significant difference of candidal colonization between the three tested groups as(p<0.001). Conclusion: Based on the limits of this study, it could be concluded that injectable monomer free thermoplastic resin denture base material is preferred to minimize candidal colonization, enhance the accuracy of denture base adaptation and increase the opportunity for obtaining successful complete denture

Sodium Nitroprusside Changed The Metabolism of Mesenchymal Stem Cells to An Anaerobic State while Viability and Proliferation Remained Intact.

We used sodium nitroprusside (SNP), a nitric oxide (NO) releasing molecule, to understand its effect on viability and proliferation of rat bone marrow mesenchymal stem cells (BM-MSCs). This experimental study evaluated the viability and morphology of MSCs in the presence of SNP (100 to 2000 µM) at 1, 5, and 15 hours. We chose the 100, 1000, and 2000 µM concentrations of SNP for one hour exposure for further analyses. Cell proliferation was investigated by the colony forming assay and population doubling number (PDN). Na+, K+, and Ca2+ levels as well as activities of lactate dehydrogenase (LDH), alkaline phosphatase (ALP), aspartate transaminase (AST), and alanine transaminase (ALT) were measured. The viability of MSCs dose-dependently reduced from 750 µM at one hour and 250 µM at 5 and 15 hours. The 100 µM caused no change in viability, however we observed a reduction in the cytoplasmic area at 5 and 15 hours. This change was not observed at one hour. The one hour treatment with 100 µM of SNP reduced the mean colony numbers but not the diameter when the cells were incubated for 7 and 14 days. In addition, one hour treatment with 100 µM of SNP significantly reduced ALT, AST, and ALP activities whereas the activity of LDH increased when incubated for 24 hours. The same treatment caused an increase in Ca2+ and reduction in Na+ content. The 1000 and 2000 µM concentrations reduced all the factors except Ca2+ and LDH which increased. The high dose of SNP, even for a short time, was toxic. The low dose was safe with respect to viability and proliferation, especially over a short time. However elevated LDH activity might increase anaerobic metabolism.

Low-molecular-weight carbohydrate Pentaisomaltose may replace dimethyl sulfoxide as a safer cryoprotectant for cryopreservation of peripheral blood stem cells.

Cryopreserved hematopoietic stem cell products are widely used for certain hematologic malignancies. Dimethyl sulfoxide (DMSO) is the most widely used cryoprotective agent (CPA) today, but due to indications of cellular toxicity, changes of the cellular epigenetic state, and patient-related side effects, there is an increasing demand for DMSO-free alternatives. We therefore investigated whether Pentaisomaltose (PIM), a low-molecular-weight carbohydrate (1 kDa), can be used for cryopreservation of peripheral blood stem cells, more specifically hematopoietic progenitor cell apheresis (HPC(A)) product. We cryopreserved patient or donor HPC(A) products using 10% DMSO or 16% PIM and quantified the recovery of CD34+ cells and CD34+ subpopulations by multicolor flow cytometry. In addition, we compared the frequency of HPCs after DMSO and PIM cryopreservation using the colony-forming cells (CFCs) assay. The mean CD34+ cell recovery was 56.3 ± 23.7% (11.4%-97.3%) and 58.2 ± 10.0% (45.7%-76.9%) for 10% DMSO and 16% PIM, respectively. The distribution of CD34+ cell subpopulations was similar when comparing DMSO or PIM as CPA. CFC assay showed mean colony numbers of 70.7 ± 25.4 (range, 37.8-115.5) and 67.7 ± 15.7 (range, 48-86) for 10% DMSO and 16% PIM, respectively. Our findings demonstrate that PIM cryopreservation of HPC(A) products provides recovery of CD34+ cells, CD34+ subpopulations, and CFCs similar to that of DMSO cryopreservation and therefore may have the potential to be used for cryopreservation of peripheral blood stem cells.

Candida concentrations determined following concentrated oral rinse culture reflect clinical oral signs.

BackgroundOral candidiasis is an infection caused by a yeast-like fungus called Candida. Various methods can be used to isolate Candida from the oral cavity. However, it is difficult to correctly and satisfactorily diagnose oral candidiasis because currently no microbiological or laboratory standards based on samples from the oral cavity are available. The aim of this study is to establish a reliable laboratory test for diagnosing oral candidiasis.MethodsOral swab, rinse and concentrated rinse samples were obtained from 200 consecutive outpatients (103 male patients and 97 female patients; mean age, 47.2 years; age range, 9–89 years). Candida colonies from cultured samples were enumerated to compare the sensitivities and specificities of the above sampling methods, and the associations between Candida detection or concentration and the clinical oral signs were examined.ResultsThe mean colony numbers were 263 ± 590 CFU/swab for the swab method, 2894 ± 6705 CFU/100 μL for the rinse method, and 9245 ± 19,030 CFU/100 μL for the concentrated rinse method. The median numbers were 23 CFU/swab for the swab method, 56 CFU/100 μL for the rinse method, and 485 CFU/100 μL for the concentrated rinse method. Candida was detected in the oral cavity of 33.5 % and 52.0 % of the outpatients by the swab method and concentrated rinse, respectively. Candida concentrations determined by the concentrated rinse were closely related to the severity of the clinical oral signs. The positive predictive values of residual root, redness of the oral mucosa, denture, glossalgia, dry mouth, and taste disorder were useful predictors of oral candidiasis.ConclusionsConcentrated rinse sampling is suitable for evaluating oral candidiasis, and Candida concentrations examined using this method strongly associated with the oral signs associated with Candida infection.