Cell Counting Method Research Articles

Pilot implementation of a cell culture‐based educational module in a gateway cellular and molecular biology course at a Liberal Arts PUI.

Undergraduate biology students are required to learn, understand and apply a variety of cellular and molecular biology concepts and techniques in preparation for biomedical, graduate and professional programs and careers in science. In 2015, we developed and published a simple lab module to teach the concepts of cell division, cellular communication and cancer, through the application of animal cell culture techniques, to sophomore level biology majors. This lab module exploits the mouse mammary tumor cell line (MMT) as a model for breast cancer. Students learn to grow and characterize animal cells in culture and test the effects of traditional and non‐traditional chemotherapy agents on cell proliferation. Specifically, students determine the optimal cell concentration for growing cells, learn how to prepare and dilute drug solutions, identify the best dosage and treatment time course of the antiproliferative agents, and ascertain the rate of cell death in response to various treatments. The module employs both a standard cell counting technique using a hemocytometer and a novel cell counting method using microscopy software. The experimental procedure lends to open‐ended inquiry as students can modify critical steps of the protocol, including testing homeopathic agents and over‐the‐counter drugs. Here we report the results of the initial implementation of this teaching module into a gateway cellular and molecular biology course at a liberal arts college in NYC. The application of the module was modified to fit into the last two weeks of the fall academic term, when students were learning about the core concepts of cell signaling, cell cycle control and cancer. This restricted time frame allowed for the study and application of core biology concepts and principles of lab methods but necessitated the elimination of hands‐on lab work. The students first read the published article describing the lab module and expanded their knowledge by selecting additional primary literature articles. Student learning was assessed through oral discussion, written instruments and a performance activity. Assessments posed questions about the lab module, specifically addressing core concepts, principles behind the methodology and data analysis. Students also expanded the experimental strategy and proposed additional experiments and predicted the results. Student knowledge base was initially tested by a pre‐survey at the start of this project, followed by a post‐survey at the end of the semester. Our results indicate that this teaching module helped students use the scientific process to understand the cell cycle, cellular signaling pathways, cancer and modes of treatment, while honing their ability to analyze experimental data in a way not routinely taught in the undergraduate classroom.Support or Funding InformationMarymount Manhattan College Faculty Research Award Sister Collette Mahoney Science Research Grant Badgeley Foundation Joseph A. Alexander FoundationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Using a Laboratory Teaching Module to Train Undergraduate Students in Cell Culture Based Research

Undergraduate biology students routinely conduct authentic scientific research. At PUI institutions lacking graduate programs like ours, undergraduates often participate in laboratory‐based research early in their academic career, well before they have acquired the requisite lab skills and foundational concepts necessary for success at the bench. To address this, in 2013 our lab developed and published a research training program for undergraduates that relies on structured, skills‐based activities and peer mentoring. Recently we developed and published a laboratory teaching module for use in sophomore‐level cellular and molecular biology course, that uses cell culture‐based experiments to teach core concepts in cell signaling and cell cycle control. The ease and success of this teaching module led our research group to modify our traditional training program and use this teaching module instead to train our newest undergraduate researchers. This teaching module employs the mouse mammary tumor (MMT) cell line to model breast cancer and includes activities designed to teach fundamental concepts while developing experimental design and analysis skills. Specifically, students learn to grow and characterize animal cells in culture and test the effects of traditional and non‐traditional chemotherapy agents on cell proliferation. Students determine the optimal cell concentration for plating and growing cells, learn how to prepare and dilute drug solutions, identify the best dosage and treatment time course of anti‐proliferative agents, and ascertain the rate of cell death in response to various treatments. The module employs both a standard cell counting technique using a hemocytometer and a novel cell counting method using microscopy software. The experimental procedure lends to open‐ended inquiry as students can modify critical steps of the protocol, including testing homeopathic agents and over‐the‐counter drugs. Here we report evaluation of the effectiveness of a pilot program that used this teaching module to train undergraduate researchers. Freshman and sophomore level research students performed the teaching module over a twenty week period during the 2017/2018 academic year. The students worked as a team and were mentored by an upper level undergraduate researcher in our lab. Students also met twice weekly with the lab PI. Assessment of student learning relied on evaluation of the initial skill and knowledge base of the students by the PI and student mentor, followed by weekly evaluation of achievement of learning objectives specific to the teaching module and research training. Assessment instruments included written, oral and practicum along with actual experimental results. Our initial findings strongly suggest that this educational module, which requires students to use the scientific process to apply foundational concepts while developing laboratory and experimental skills, is a highly effective means of training undergraduate researchers.Support or Funding InformationMarymount Manhattan College Faculty Research AwardSister Collette Mahoney Science Research GrantBadgeley FoundationJoseph A. Alexander FoundationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cerium oxide nanoparticles embedded thin-film nanocomposite nanofiltration membrane for water treatment

In this paper, a new approach to synthesize thin-film nanocomposite membranes using cerium oxide (CeO2) nanoparticles (NPs) by pre-seeding interfacial polymerization method was reported. Prepared membranes were examined using contact angle, molecular weight cut-off (MWCO), scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR) and scanning probe microscopy (SPM) to observe its hydrophilicity, pore size, morphology, surface chemistry, and roughness, respectively. Surface charges of the prepared membranes were also qualitatively calculated with the help of contact angle measurements by using the Grahame equation. MWCO studies revealed >90% polyethylene glycol (M.W. 1500 Da) rejection, which was fitted in the range of nanofiltration. By increasing the concentration of CeO2 NPs, flux (33.12 to 41.28 L/m2h), hydrophilicity (77.3 to 51.1°) and surface charges (−7.58 to −13.39 mC/m2) of the membranes was successfully improved, and also showed the high (>90%) salt rejections. The CeO2 embedded membrane was also found out in successful prevention from the attack of bacteria (Escherichia coli) compared to pure polyamide (PA) membrane and confirmed through SEM and viable cell count method. The membrane performances were also evaluated using seawater for fouling study and found that CeO2 embedded surface increased the rejection of hydrophobic contaminants, and notably reduced the fouling.

Quantification of Au Nanoparticle Biouptake and Distribution to Freshwater Algae Using Single Cell - ICP-MS.

Quantifying metal and nanoparticle (NP) biouptake and distribution on an individual cellular basis has previously been impossible, given available techniques which provide qualitative data that are laborious to acquire and prone to artifacts. Quantifying metal and metal NP uptake and loss processes in environmental organisms will lead to mechanistic understanding of biouptake and improved understanding of potential hazards and risks of metals and NPs. In this work, we present a new technique, single cell inductively coupled plasma mass spectrometry (SC-ICP-MS), which allows quantification of metal concentrations on an individual cell basis down to the attogram (ag) per cell level. We present data validating the novel method, along with the mass of metal per cell. Finally, we use SC-ICP-MS, with ancillary cell counting methods, to quantify the biouptake and strong sorption and distribution of both dissolved Au and Au NPs in a freshwater alga (Cyptomonas ovate). The data suggests differences between dissolved and NP uptake and loss. In the case of NPs, there was a dose and time dependent uptake, but individual cellular variations; at the highest realistic exposure conditions used in this study up to 40-50% of cells contained NPs, while 50-60% of cells did not.

Isolation and culture of primary adult skin fibroblasts from the Asian elephant (Elephas maximus).

BackgroundPrimary cultures from Asian elephants (Elephas maximus) allow scientists to obtain representative cells that have conserved most of their original characteristics, function, physiology and biochemistry. This technique has thus gained significant importance as a foundation for further cellular, cell biology and molecular research. Therefore, the aim of this study was to describe conditions for the successful establishment of primary adult fibroblasts from Asian elephant carcasses.MethodsEar tissue sample collection from Asian elephant carcasses and our recommendations are given. We describe here a simple modified protocol for successful isolation and maintenance of primary adult fibroblasts from elephant ear skin. Ear samples from each individual (five 3 × 3 cm2 pieces) were brought to the laboratory within 3 h after collection, kept in transportation medium at 0–4 °C. The ear tissues were prepared by a combination of 10% collagenase type II digestion procedure together with a simple explant procedure. Primary fibroblasts were cultured at 37 °C in Dulbecco’s modified Eagle’s medium (DMEM) with 20% fetal calf serum (FCS) in a humidified atmosphere containing 5% CO2. After the third passage, fibroblasts were routinely trypsinized with 0.25% trypsin/EDTA and cultured in DMEM with 10% FCS at 37 °C and 5% CO2. Traditional cell counting method was used to measure cell viability and growth curve. Long-term storage of cells used freezing medium consisting of 40% FCS (v/v).ResultsWe explored the most suitable conditions during sample collection (post-mortem storage time and sample storage temperature), which is the most important step in determining primary outgrowth. Our study successfully established and cultured primary adult skin fibroblasts obtained from post-mortem E. maximus ear skin tissues from six carcasses, with a success rate of around 83.3%. Outgrowth could be seen 4–12 days after explantation, and epithelial-like cells were found after 4–7 days of culture, while fibroblasts appeared at around day 7–10. The fibroblasts had viability and post-freezing recovery rates of around 97.3 ± 4.3% and 95.5 ± 7.3%, respectively, and doubling time was about 25 h (passage 6).DiscussionTo our knowledge, this report is the first to describe primary cell cultures derived from adult Asian elephant skin. Future studies should benefit from the information and useful suggestions herein, which may be used as a standard method for establishing primary skin fibroblast cultures in future experiments.

Hemolysis detection based on SVM of Adaboost classification algorithm

Aiming at the problem that clinical hemolysis is difficult to be observed and judged, a method of Adaboost learning classification based on SVM is proposed. The method firstly extracts the basic features of the target area of the blood sample, such as the average of the gray level, the standard deviation of the gray level and the appearance frequency of the particles, as the input eigenvectors of the learning, and carries out SVM weak learner learning. Subsequently, Adaboost algorithm is used to measure the weak learner Set linear weighting, so as to enhance the strong learning device; Finally, online testing, calculation of test sample hemolytic degree and classification. The Adaboost learning classification test based on SVM is compared with the macroscopic and red blood cell counting methods. The experimental results show that the learning-based classification testing method achieves higher detection accuracy without subjective factors and has the highest detection efficiency.

Development of antimicrobial chitosan based nanofiber dressings for wound healing applications

Objective(s): Chitosan based composite fine fibers were successfully produced via a centrifugal spinning technology. This study evaluates the ability of the composites to function as scaffolds for cell growth while maintaining an antibacterial activity. Materials and methods: Two sets of chitosan fiber composites were prepared, one filled with anti-microbial silver nanoparticles and another one with cinnamaldeyhde. Chitosan powder was dissolved in trifluoroacetic acid and dichloromethane followed by addition of the fillers. The fiber output was optimized by configuring the polymer weight concentration (7, 8, and 9 w/w% chitosan) and applied angular velocity (6000-9000 RPM) within the spinning process. Results: Scanning electron microscopy revealed fiber diameters ranging from 800-1500 nm. Cinnamaldehyde and silver nanoparticles helped to improve and control the anti-bacterial activity. Through a verified cell counting method and disk diffusion method, it was proven that the chitosan based composite fibers possess an enhanced anti-bacterial/microbial activity against gram-positive Staphylococcus aureus. Both composite systems showed anti-bacterial activity, inhibition zones fluctuating between 5 to 10 mm were observed depending on the size of the fiber matand no bacteria was found within the mats. The developed fiber scaffolds were found to be noncytotoxic serving as effective three-dimensional substrates for cell adhesion and viability. Conclusion: These results provide potential to use these scaffolds in wound healing and tissue regeneration applications.

Counting of viable C. burnetii cells by quantitative reverse transcription PCR using a recombinant plasmid (pCB-dotA) as a standard.

Coxiella burnetii is an intracellular pathogenic bacterium and etiological agent of Q fever in humans. Recently, the bacterium has been set free from the strictly intracellular condition by successful cultivation in acidified citrate cysteine medium. Here, we report a bacterial cell counting method that allows rapid quantification of the absolute or relative number of live cells of C. burnetii in a high throughput manner. The method utilizes TaqMan-based quantitative polymerase chain reaction (qPCR) targeting a single dotA gene for determination of genome equivalent (GE) presented either as DNA or complementary DNA (cDNA) synthesized via reverse transcription. The assay was shown to be specific, sensitive and efficiently reproducible. The quantification was linear over a range of 30 to 3x108 copies. Since there is only one copy of the dotA gene per Coxiella chromosome, the calculated dotA copy numbers can be compared to the number of bacterial cells. Finally, we demonstrated the potential of the method to assess effects of antibiotic on cell viability and to determine the antibiotic-tolerant fraction within a cell population. Keywords: Coxiella burnetii; Q fever; real-time polymerase chain reaction; copy number; antibiotic; axenic media; dotA gene.

Análisis de técnicas de recuento de Microorganismos.

El aislamiento y obtención de cultivos puros posiblemente hayan sido las bases más importantes en el desarrollo de la Microbiología. Los primeros trabajos en Microbiología se hicieron en unas condiciones experimentales que no permitían tener la certeza de que se hubieran obtenido cultivos puros. Más adelante, los microbiólogos observaron que, aparentemente, los microorganismos tenían una gran capacidad de variación en cuanto a su aspecto morfológico y características fisiológicas. Un cultivo puro es aquel que contiene una sola clase de microorganismo. En la práctica los cultivos puros son útiles por diferentes razones: mantienen los organismos viables, permiten hacer subcultivos para someterlos a diferentes análisis. Para obtener lo anterior es necesario recurrir a las técnicas de recuento. Existen métodos que permiten establecer el número de microorganismos en una muestra dada. Hay métodos que cuantifican el número de células, y existen otros que cuantifican la masa total de la población. El objetivo de este estudio es conocer las diversas técnicas utilizadas para el recuento de células de microorganismos, al igual que evaluar la efectividad de cada uno de estos métodos de conteo de células.

Adaptive and automatic red blood cell counting method based on microscopic hyperspectral imaging technology

Red blood cell counting, as a routine examination, plays an important role in medical diagnoses. Although automated hematology analyzers are widely used, manual microscopic examination by a hematologist or pathologist is still unavoidable, which is time-consuming and error-prone. This paper proposes a full-automatic red blood cell counting method which is based on microscopic hyperspectral imaging of blood smears and combines spatial and spectral information to achieve high precision. The acquired hyperspectral image data of the blood smear in the visible and near-infrared spectral range are firstly preprocessed, and then a quadratic blind linear unmixing algorithm is used to get endmember abundance images. Based on mathematical morphological operation and an adaptive Otsu’s method, a binaryzation process is performed on the abundance images. Finally, the connected component labeling algorithm with magnification-based parameter setting is applied to automatically select the binary images of red blood cell cytoplasm. Experimental results show that the proposed method can perform well and has potential for clinical applications.

Effect of light polarization on the efficiency of photodynamic therapy of basal cell carcinomas: an in vitro cellular study.

In an in vitro study, the effect of light polarization on the efficiency of 5-aminolaevulinic acid (ALA) photodynamic therapy (PDT) of basal cell carcinoma (BCC) was investigated. Three states of light polarization (non-polarized, linearly polarized, and circularly polarized) were considered. Cells were exposed to green (532 pm 20 nm) irradiation from light emitting diodes. Cell survival was measured by the colorimetric assay (WST-1) and Trypan blue staining. The colorimetric assay showed a pronounced decrease in the cell viability (up to 30%) using polarized light compared to the non-polarized one in the wavelength region used. Similar results were obtained by the cell counting method (20-30% increase in cell death). The observed effect was dependent on the concentration of photosensitizer. The effect is more expressed in the case of linearly polarized light compared to the circularly polarized one. Results show that the use of polarized light increases the efficiency of in vitro ALA-PDT of BCC. Utilizing polarized light, it is possible to obtain the same effect from PDT by lower concentrations of photosensitizer. Additionally, the concentration dependency of PDT response and photo-bleaching is also reduced.

MiR-30b regulates chondrogenic differentiation of mouse embryo-derived stem cells by targeting SOX9.

The present study aimed to investigate the mechanisms underlying microRNA (miRNA)-mediated regulation of chondrogenic differentiation. Mouse embryo-derived stem cells C3H10T1/2 were cultured and chondrogenic differentiation was induced using transforming growth factor-β3 (TGF-β3). In addition, miRNA expression profiles were detected via miRNA array analysis, and quantitative polymerase chain reaction was performed to verify the differentially expressed miRNAs. Furthermore, bioinformatics software was used to predict the putative targets and the prediction was validated by dual-luciferase reporter assays and western blot analysis. In addition, cell proliferation and glycosaminoglycans were measured by a direct cell count method and alcian blue staining, respectively. Compared with the control group, 86 miRNAs were identified as differentially expressed in TGF-β3-induced cells and the expression levels of 28 miRNAs were increased while the remaining 58 miRNAs exhibited a decline in expression. Amongst the differentially expressed miRNAs, miR-30b expression was observed to have significantly decreased during chondrogenic differentiation. SOX9 is a target gene of miR-30b, and miR-30b inhibits SOX9 expression during chondrogenic differentiation. Furthermore, the alcian blue staining results demonstrated that miR-30b inhibited early chondrogenic differentiation. However, the data of the present study indicated that miR-30b had no influence on C3H10T1/2 cell line proliferation. In conclusion, miR-30b is a key negative regulator of TGF-β3-induced C3H10T1/2 cell chondrogenic differentiation, which functions by directly targeting SOX9.

브로콜리 표면 미생물 계수와 미생물 군집 분석을 위한 시료 처리 방법

브로콜리 표면 미생물 계수와 미생물 군집 분석을 위한 시료 처리 방법

In Vitro Monitoring of a Cultured Human Retinal Pigment Epithelium Using 1375-nm Spectral-Domain Optical Coherence Tomography

Measurement of cell number is important in cell-based in vitro screenings for therapeutic drugs and tissue engineering such as cell culture maintenance, cell plating, and cell growth, as well as monitoring of cell viability, proliferation, and cytotoxicity. We performed cell counting using intensity-based in vitro spectral-domain optical coherence tomography (SD-OCT). Adult retinal pigment epithelium cells were cultured on a glass dish to prevent diffuse reflection from the surface, and the dish was tilted by 15° during OCT imaging to reduce coherence noise from specular reflection. Rasterized scanning was performed to generate a three-dimensional volume image with an en face image of the retinal pigment epithelium cell layers. Cell counting was achieved by measuring the density of bright spots after layer extraction and thresholding. Two other cell counting methods were also performed for the purpose of comparison, one using a hemocytometer and the other using water-soluble tetrazolium-1; cell counting by in vitro OCT yielded results better than those from the hemocytometer. Our results showed that in vitro OCT systems can be a powerful tool for estimating and analyzing cell density in a cultured sample without the need for dyeing the sample or causing cell death.

Low-Frequency Electrochemical Impedance Spectroscopy as a Monitoring Tool for Yeast Growth in Industrial Brewing Processes

Today’s yeast total biomass and viability measurements during the brewing process are dependent on offline methods such as methylene blue or florescence dye-based staining, and/or the usage of flow cytometric measurements. Additionally, microscopic cell counting methods decelerate an easy and quick prediction of yeast viability. These processes are time consuming and result in a time-delayed response signal, which not only reduces the knowledge of the performance of the yeast itself, but also impacts the quality of the final product. Novel approaches in process monitoring during the aerobic and anaerobic fermentation of Saccharomyces cerevisiae are not only limited to classical pH, dO2 and off-gas analysis, but they also use different in situ and online sensors based on different physical principles to determine the biomass, product quality and cell death. Within this contribution, electrochemical impedance spectroscopy (EIS) was used to monitor the biomass produced in aerobic and anaerobic batch cultivation approaches, simulating the propagation and fermentation unit operation of industrial brewing processes. Increases in the double-layer capacitance (CDL), determined at frequencies below 1 kHz, were proportional to the increase of biomass in the batch, which was monitored in the online and inline mode. A good correlation of CDL with the cell density was found. In order to prove the robustness and flexibility of this novel method, different state-of-the-art biomass measurements (dry cell weight—DCW and optical density—OD) were performed for comparison. Because measurements in this frequency range are largely determined by the double-layer region between the electrode and media, rather minor interferences with process parameters (aeration and stirring) were to be expected. It is shown that impedance spectroscopy at low frequencies is not only a powerful tool for the monitoring of viable yeast cell concentrations during operation, but it is also perfectly suited to determining physiological states of the cells, and may facilitate biomass monitoring in the brewing and yeast-propagating industry drastically.

Comparison of fibroblast cell regeneration in three different concentrations of Wharton’s Jelly mesenchymal stem cells conditioned medium (WJMSCs-CM)

Wharton’s Jelly-derived mesenchymal stem cells (WJMSCs) have gained interest as an alternative source of stem cells for regenerative medicine. Although many studies have characterized Wharton’s Jelly biologically, the effects of different concentrations in a cultured medium have not yet been compared. Damaged fibroblasts, the primary components of irreversible dental pulpitis, irreversibly impair the ability to regenerate and lead to the disruption of extracellular matrix. This study was performed to evaluate the potency of three WJMSCs-CM concentrations in improving serum-starved fibroblasts. Fibroblasts were cultivated in five passages, and divided into four groups. The first group (the control group) consisted of fibroblast cells that had been treated using starvation methods. The other groups (the treatment groups) were treated with various concentration of WJMSCs-CM (50%, 25% and 12.5%). Proliferative ability was evaluated using a cell count method and analyzed with a one-way ANOVA. Cultivation of serum-starved fibroblasts produced significantly higher cell counts in 12.5% WJMSCs-CM compared to the 50% group. It can be concluded that 12.5% WJMSCs-CM is the most efficient concentration for fibroblast proliferation.

AKT Is A Therapeutic Target in Myeloproliferative Neoplasms

To study the therapeutic effect of AKT inhibitor on the myeloproliferative neoplasms(MPN) and its significanse. The MPL W515L expression was induced by retroviral infection to construct the MPN cell model. The constitutive activation of the signal pathway was detected by Western blot assay. Cells were treated by AKT inhibitor and the proliferation of MPN cells were detected by cell counting method and clone formation unit test. Annexin V staining was used to detect cell apoptosis, and the cell cycle was detected by BrdU method. The animal survival experiment was performed to analyze the effect of AKT inhibitor on the survival rate of MPN mouse model. The overexpression of MPL W515L could significantly activate PI3K/AKT, JAK/STAT and other signaling pathways. Blocking these signal pathways could inhibit the proliferation and promote the apoptosis of MPN cell model. Among them, the PI3K/AKT inhibitor had the most significant effect, and the proportion of G1ME cells in the mitotic phase S decreased, while the proportion of G1/G0 phase increased, the cell cycle arrest function was shown. At the same time, in the blood samples of patients with MPN, blocking PI3K/AKT could also inhibit the colony forming units of bone marrow progenitor cells, and significantly increased the survival rate of animal model. AKT is a target for the treatment of bone marrow proliferative tumors.

Development and Efficacy Testing of a “Hollow Awl” That Leads to Patent Bone Marrow Channels and Greater Mesenchymal Stem Cell Mobilization During Bone Marrow Stimulation Cartilage Repair Surgery

To investigate whether microfracture with a cannulated hollow awl can yield more patent marrow channels and allow greater mobilization of the reparable cells to the defect compared to the conventional awl with blunt end in human knee joints. Patients who were planned for 1-stage bilateral total knee arthroplasty due to degenerative osteoarthritis with well-preserved lateral femoral condylar cartilage were retrospectively included. A 10-mm× 20-mm, rectangular, full-thickness chondral defect was made on the lateral femoral condyle on each knee joint. The 6-holed microfracture procedure, each at 9mm depth and 3mm separation of perforations, was followed using a hollow awl in one knee and using a conventional awl in the other knee, respectively. The bleeding through the microfracture holes was observed and collected using an absorbable gelatin sponge and was analyzed microscopically by colony forming unit-fibroblast assays and automated cell counting method. The representative 3 bony samples of the distal lateral femoral condyles obtained were also scanned with micro-computed tomography (micro-CT) for morphometric analysis (percent bone volume, trabecular separation, and total porosity) of subchondral bone microarchitecture of the microfracture holes. Twenty-two patients were enrolled, and the mean age was 70.8 ± 6.1 (58-83) years. Compared with the conventional awl group, the hollow awl group had a significantly greater amount of bleeding (1.8 ± 0.2g vs 1.1 ± 0.1g; P< .001) and a greater number of mesenchymal stem cells in the blood clot (21,474.0 ± 3,911.1 vs 13,329.7 ± 3,311.0; P= .004). The hollow awl group also showed overall more patent marrow channels around the adjacent subchondral bone of the microfracture hole, with greater trabecular separation on micro-CT analysis (P < .001). Compared to the conventional awl, microfracture with a cannulated hollow awl can yield more patent marrow channels at the adjacent subchondral bone of the microfracture hole and result in greater mobilization of the reparable cells to the defect in human knee joints. Level III, therapeutic case control.

Association of obesity with C reactive protein, total leukocyte count and erythrocyte sedimentation rate in Type 2 diabetes mellitus

Context: There is accumulating evidence implicating inflammation originating from adipose tissue as a potential pathway in the pathogenesis of type 2 diabetes mellitus (DM). This study is performed to ascertain the association of 3 markers of inflammation, leukocyte count (TLC), Erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) with obesity in diabetics and compare with non-diabetics. Aims: To determine the association of CRP, TLC and ESR levels with obesity in persons with type 2 DM and non-diabetics. Settings and Design: 50 diabetics and 50 non-diabetic subjects were enrolled prospectively and anthropometric and laboratory data were collected and analysed in a tertiary care hospital. Methods and Material: CRP by latex agglutination test, TLC by Cell counter method (BC-5380), ESR by Westerngrens method. Statistical analysis: Pearsons correlation, Chi square and Fishers exact test -Mean, Standard deviation, Frequency and Percentage. Results: Mean FBS, PPBS, ESR, CRP, TLC, Body mass index (BMI), Waist hip ratio (WHR) were 100.5mg/dl, 112.34mg/dl, 7.02mm/hr, 1.86mg/L,6984cells/mcL,24.77kg/m 2 ,0.88 respectively in non-diabetics and 190.88mg/dL, 275.58mg/dL, 21.56mm/hr, 3.91mg/L, 11760 cells/mcL, 25.86kg/m 2 , 0.98 respectively in diabetics. All these parameters except BMI are significantly different between diabetics and non-diabetics with p value <0.05. Pearson correlation showed statistically significant association of inflammatory markers with WHR but not with BMI. Conclusions: Inflammatory markers like TLC, ESR and CRP are significantly elevated in patients with diabetes, suggesting the important role of inflammation, prompting need for interventions reducing inflammation in effective diabetes management. WHR is significantly associated with elevated inflammatory markers suggesting the role of abdominal obesity in diabetes.

Abstract 2308: Sigma-2 receptor ligands induce apoptosis and inhibit proliferation in breast cancer cell line mda-mb-231

Abstract Introduction: Despite the reduction in mortality from breast cancer achieved in the last 30 years due to more effective targeted therapies, the survival rate for patients with triple negative breast cancer (TNBC) is poor and has virtually plateaued. Due to the absence of estrogen, progesterone, and human epidermal growth factor receptors 2, no targeted treatment options are currently available for TNBC. Moreover, the current standard systemic treatments are known to cause tremendous side effects. Thus, there is an urgent need to identify novel strategies for treating TNBC. Several reports have revealed that sigma-2 receptors are over-expressed in all solid tumors including TNBC, making it a plausible biomarker to explore for TNBC treatment. In this study, we investigated the effects of novel sigma-2 receptor ligands (XYZ-XI-14 and XYZ-VII-69) synthesized in our lab on the viability and survival of the TNBC, MDA-MB-231. Methods: MDA-MB-231 cells were treated for 48 h with XYZ-XI-14 and XYZ-VII-69, cell viability and proliferation were assessed using resazurin cell titre assay and cell count methods respectively. The effects of the ligands on spheroids formation, cell cycle, and mode of cell death were also investigated. Results: XYZ-XI-14 and XYZ-VII-69 decreased cell viability of MDA-MB-231 cells in a concentration-dependent manner. The EC50 for XYZ-XI-14 and XYZ-VII-69 were 12 and 13 µM respectively; and at 5 µM, the ligands inhibited cell proliferation, induced apoptosis, and arrested MDA-MB-231 cells at the G0/G1 phase of cell cycle. Additionally, concentrations of the ligand as low as 1 µM prevented the formation of spheroids as evidenced by the lack of compact spheroids, and caused the disintegration of preformed spheroids. Conclusion: This study indicates that targeting sigma-2 receptors with novel sigma-2 ligands (XYZ-XII-14 and XYZ-VII-69) effectively inhibits TNBC cancer cell growth by inducing apoptosis and cell cycle arrest, thus presenting a unique and effective pathway for treating TNBC. Additionally, the sigma-2 receptor ligands (XYZ-XI-14 and XYZ-VII-69) have the potential to halt tumor growth and prevent tumor relapse, as seen in our 3D culture assays. Thus, the sigma-2 receptor has the potential to be a valuable target for the development of novel agents for the treatment of TNBC. Citation Format: Gladys Asong, Felix Amissah, Olufisayo Salako, Rosemary Poku, Elizabeth Ntantie, Augustine Nkembo, Nazarius Lamango, Seth Ablordeppey. Sigma-2 receptor ligands induce apoptosis and inhibit proliferation in breast cancer cell line mda-mb-231 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2308. doi:10.1158/1538-7445.AM2017-2308