Standard Microscopy Techniques Research Articles

Assessment of urine metabolite biomarkers for the detection of S. haematobium infection in pre-school aged children in a rural community in Zimbabwe

BackgroundEarly diagnosis of urogenital schistosomiasis is key to its control and elimination. The current gold standard microscopic examination techniques lack sensitivity in detecting light Schistosomiasis infections in pre-school aged children thus it is urgent to develop diagnostic tools that may be integrated into control programs. In this study, we evaluated the diagnostic performance of urine metabolite biomarkers using a chemical reagent strip in the detection of S. haematobium infection in pre-school aged children. MethodsA case-control study was conducted involving 82 pre-school aged children that were age and sex matched. Urine samples were collected for 3 consecutive days and were evaluated using urine filtration gold techniques as the gold standard method. The samples were simultaneously measured for metabolite biomarkers specifically haematuria, proteins, ketones, nitrites, glucose, bilirubin and urobilinogen using chemical reagent strips. Pearson correlation test was used to measure the relationship between S. haematobium infection and the urine metabolite biomarkers. ResultsThe diagnostic performance of urine biomarkers were correlated with the microscopic examination urine filtration technique. Haematuria (r = 0.592, p = 0.0001) and proteinuria (r = 0.448, p = 0.0001) were correlated to S. haematobium infection. Negative correlations with p > 0.05 were recorded for ketones and urobilinogen. Highest sensitivity was 65.9 % (CI, 49.4 - 79.9) for haematuria whilst protein (albumin) biomarker had a lower specificity value of 43.9 % (28.5 – 60.3). Inversely, highest sensitivity was 87.8 % (73.8 – 95.9) for proteinuria whilst haematuria had a lower sensitivity value of 82.9 % (67.9 – 92.8). The positive predictive values ranged from 57.7 % (41.6 – 72.2) to 79.4 % (65.5 - 88.7) whereas negative predictive values ranged from 70.8 % (60.8 – 79.2) to 52.0 % (48.7 – 55.3). With respect to diagnostic efficiency, haematuria had a fair diagnostic performance with an area under the curve of 0.76 followed by proteinuria with proteinuria whilst the remaining metabolites fail discriminating ability with an area under the curve of <0.5. ConclusionAlthough haematuria and protein biomarkers in urine are moderately sensitive and specific, they are important morbidity indicators of urogenital schistosomiasis in pre-school aged that may be utilised during screening in schistosomiasis control programs. We recommend comprehensive analysis of biomarkers using metabolomics techniques to identify novel urine biomarkers.

Differential Fluorescent Chemosensing of Antibiotics Using a Luminescent Zn(II) Coordination Polymer Based on a 4-Amino-1,8-naphthalimide Tröger’s Base Fluorophore

The design and development of useful chemosensors for the ultra-trace detection of environmental pollutants and contaminants is a topical area of research. Herein, we report a new nanoscale emissive Zn(II) coordination polymer (TB-Zn-CP) for differential fluorescence sensing of various antibiotics in water. TB-Zn-CP was synthesized using a unique V-shaped green emitting 4-amino-1,8-naphthalimide Tröger’s base (TBNap) fluorophore. The structural and morphological features of TB-Zn-CP were characterized by various standard spectroscopic and microscopy techniques. The fluorescence titration studies in water demonstrated a remarkable sensitivity and differential fluorescence sensing properties of TB-Zn-CP for the fast detection of different antibiotics. Among different antibiotics, chloramphenicol (CRP), 1,2-dimethyl-5-nitroimidazole (DMZ), and sulfamethazine (SMZ) displayed the highest fluorescence-quenching efficiency and superior sensitivity in their detection. The differential sensing capability of TB-Zn-CP was also indicated by visualizable color changes. The Stern–Volmer quenching constant KSV was determined to be in the order of 103–104 M−1, and the sensitivity was shown to be at a nanomolar (10−9 M) level. All these results confirm that TB-Zn-CP can be a potential and practically useful polymeric sensor for differential fluorescence and visual detection of different antibiotics in water.

Trio fluorophore-based phenotypic assay for the detection of artemisinin-induced growth-arrested Plasmodium falciparum in human erythrocytes

Artemisinin combination therapy remains effective for the treatment of falciparum malaria. However, Plasmodium falciparum can escape the effects of artemisinin by arresting their growth. The growth-arrested parasites cannot be distinguished from nonviable parasites with standard microscopy techniques due to their morphological similarities. Here, we demonstrated the efficacy of a new laboratory assay that is compatible with the artemisinin susceptibility test. As a result of the differential cell permeabilities of two DNA-binding fluorophores, growth-arrested P. falciparum can be distinguished from parasites killed by artemisinin, since the latter lose cell membrane permeability. This fluorescence-based assay increased the sensitivity and specificity of the ring survival assay in the assessment of artemisinin susceptibility. When combined with a third fluorophore-conjugated anti-human leukocyte antibody, this trio fluorophore assay became more useful in identifying growth-arrested parasites in mock human blood samples. This novel assay is a simple and rapid technique for monitoring artemisinin resistance with greater sensitivity and accuracy compared with morphology-based observations under a light microscope.

Characterization of anatomical characters of Peperomia (Piperaceae) from Asia for taxonomy

Abstract The flowers of Peperomia (Piperaceae) are simple, minute, and unique structures within the angiosperms consisting of a rounded bract, two stamens and an ovary, but generally lack taxonomic value at species level. Because of this, morphological characters of vegetative parts have been mainly used for species level taxonomy. There is a need to supplement these vegetative characters with anatomical characters. We selected 17 native Thai species representatives of South East Asian Peperomia to investigate the utility of vegetative anatomical characters for species delimitation, taxon identification, and classification. Stem, petiole, and leaf anatomical characters were studied using standard microscopic techniques. The anatomical characters demonstrated high diversity and supported species identification. Unweighted Pair Group Method with Arithmetic Mean (UPGMA) culstering, Principal Coordinates analysis (PCoA), and Principal Component Anaysis (PCA) were used to investigate patterns in the multivariate anatomical data and identify groupings of value to species classification. Four broad groupings were retrieved and some subgroupings identified. The most important characters that differentiated the species in the multivariate analysis included stem shape (roundness and ridges), petiole indumentum and trichome type, vascular bundle pattern and number, stem pith cavity, mucilage canals, and stem cortex sclerenchyma. These data were further used to prepare anatomical descriptions at generic and species levels and to provide a key to species.

Functional leaf traits in congeneric species of tree communities in central Amazon

The Amazon is a Neotropical region that features various types of phytophysiognomies subject to multiple environmental filters. The Amazonian Savanna and Ombrophilous Forest areas support complex ecosystem functions and are sensitive to biotic and abiotic disturbances. The low availability of nutrients and water, along with the high solar exposure these vegetations experience, impose rigorous environmental filters that affect species distribution. Plant species with structural and functional adaptations can occur in specific locations due to environmental characteristics. We investigated potential variations in the functional leaf traits of congeneric species of Tachigali and Pouteria in different tree community vegetation types in savanna and forest areas in Central Amazon. For this purpose, using standard optical microscopy techniques and statistical models, we analyzed 9 congeneric species, with 3 individuals per species, of woody congeners from Ombrophilous Forest and Amazonian Savanna, both located in the state of Pará, Brazil. Fifteen anatomical traits, both quantitative and qualitative, possessing functional significance were evaluated by comparing tissues between forest and savanna plants, employing ANOVA and ordination analyses. The results indicate that variations in environmental conditions within different vegetation types might be linked to morphophysiological adjustments and strategies in leaves to resist environmental conditions. Species from the Amazonian Savanna present traits more related to conservative characteristics related to water retention and drought resistance, demonstrating a degree of leaf xeromorphism with greater thickness of leaves, palisade parenchyma and epidermal cells. On the other hand, forest species exhibited traits more related to acquisitive and diversified resource strategies, which may be characteristic of the greater availability of light, water and nutrients existing in this environment. The results show considerable variation in leaf traits among forested and savanna formations, and variations in leaf characteristics are expected to be essential for the survival and development of tree species within each type of vegetation.

Assessment of Invadopodium Formation and Gelatin Degradation in Vitro.

Some cancer cells form highly regulated structures, termed invadopodia, which mediate local, enzymatic degradation of extracellular matrix and facilitate cancer cell invasion and migration during metastatic progression. Understanding invadopodium formation and function in cancer cells is therefore an important strategy to find novel clinical approaches to interfere with metastasis. Invadopodia are F-actin-rich protrusions that form on the advancing edge of cells, supported by complex molecular interactions at the cell membrane. Invadopodia formation, structure, and function can be studied in vitro, using commonly cultured cancer cell lines and standard microscopic techniques. Here, these approaches are described in detail.

Correlative Imaging of Three-Dimensional Cell Culture on Opaque Bioscaffolds for Tissue Engineering Applications.

Three-dimensional (3D) tissue engineering (TE) is a prospective treatment that can be used to restore or replace damaged musculoskeletal tissues, such as articular cartilage. However, current challenges in TE include identifying materials that are biocompatible and have properties that closely match the mechanical properties and cellular microenvironment of the target tissue. Visualization and analysis of potential 3D porous scaffolds as well as the associated cell growth and proliferation characteristics present additional problems. This is particularly challenging for opaque scaffolds using standard optical imaging techniques. Here, we use graphene foam (GF) as a 3D porous biocompatible substrate, which is scalable, reproducible, and a suitable environment for ATDC5 cell growth and chondrogenic differentiation. ATDC5 cells are cultured, maintained, and stained with a combination of fluorophores and gold nanoparticles to enable correlative microscopic characterization techniques, which elucidate the effect of GF properties on cell behavior in a 3D environment. Most importantly, the staining protocol allows for direct imaging of cell growth and proliferation on opaque scaffolds using X-ray MicroCT, including imaging growth of cells within the hollow GF branches, which is not possible with standard fluorescence and electron microscopy techniques.

Beating heart cells: Using cultured cardiomyocytes to study cellular structure and contractility in laboratory exercises.

Heart muscle cells, or cardiomyocytes, exhibit intrinsic contractility in vitro. We found that commercially-available mammalian cardiomyocytes serve as an excellent model system for studying the cytoskeleton and cellular contractility, fundamental topics in undergraduate cell and molecular biology courses. Embryonic rat cardiomyocytes were plated on cell culture dishes or glass coverslips and visualized using an inverted phase-contrast microscope. The cardiomyocytes began contracting within 1-2 days after plating and continued to contract for many weeks, allowing their use in multiple laboratory sessions. Following background reading and instruction, students fixed and triple-stained the cardiomyocytes to examine the relative distributions of actin filaments and microtubules and the position of nuclei. Analysis and image capture with fluorescence microscopy provided striking examples of highly organized cytoskeletal elements. Students then designed experiments in which cardiomyocyte intrinsic contractility was explored. Changes in contraction rates were examined after treatment with signaling molecules, such as epinephrine. The addition of epinephrine to the culture medium, within a usable concentration window, increased the rate of contraction. These adaptable exercises provide undergraduate cell and molecular biology students with the exciting opportunity to study cardiomyocytes using standard cell culture and microscopy techniques.

Prevalence of malaria infection and Associated Risk Factors among students of Bauchi State University Gadau, Bauchi State, Nigeria

Malaria is one of the leading causes of mortality and morbidity in tropical and subtropical regions of the world. Most of the global malaria burden is in sub-Saharan African countries, including Nigeria. This study aimed to determine the prevalence and risk factors of malaria infection among students of Bauchi State University Gadau (BASUG), Bauchi State of Nigeria. A cross-sectional study involving 400 BASUG students was conducted between June and November 2020. A sample of 400 university students aged 15 to 30 years were tested for malaria infection. The thick and thin blood films were prepared and viewed using the standard parasitological microscopy technique to detect the Plasmodium parasite. Data on risk factors were obtained using a simple questionnaire. All collected data were analyzed with Statistical Package for Social Science (SPSS) version 24. A chi-square analysis was performed, and the strength of association was measured using Odds Ratio (OR) at 95 % CI. The overall prevalence of malaria among university students was 59.8%. The differences in the prevalence of malaria infection to sex and age were not statistically significant (p&gt;0.05). There is a statistically significant difference in the prevalence of malaria infection among students who live off-campus and their counterparts (χ2=23.4, df=1, p=0.90) and among students who do not use mosquito nets compared with other students who use it (χ2=23.4, df=1, p=0.90). The binary logistic regression analysis revealed that the students who stay off-campus are nearly two times more infected with malaria infection than those who lived on-campus [COR (95% CI): 1.56 (1.04-2.35)]. The students who do not sleep under the mosquito-treated net appeared to be at a high risk of malaria infection compared to those who use it [COR (95 % CI): 0.55 (0.36-0.83)]. Malaria is prevalent among the students of BASUG, Bauchi state, and this is glaring evidence indicating malaria is a public health challenge. The non-use of mosquito nets in the study area is one of the major risk factors influencing transmission. Health education on mosquito prevention should educate the students on the importance of using a mosquito bed net, fumigation, and indoor residual spray in their surroundings.

Polythiophene enveloped tungsten-oxide hybrid nanofillers dispersed oleo (Pithecellobium Dulce seed oil) polyurethane bi-functional nanocomposite coatings

Sustainable resource-based oleo-polyurethanes with low viscosity, superior fluidity, and good ability to spread on the surface of the substrate without involving any organic solvents, led to the formulation of volatile organic compounds (VOCs) free polymer coatings. Here, work reports the use of Pithecellobium Dulce seed oil (PDO), an eco-friendly, abundantly available, and sustainable resource for the synthesis of oleo-polyurethane (PDPU) and formulation of its nanocomposites coatings. To enhance their physicomechanical, anticorrosive, and antimicrobial properties, the tungsten oxide (WO), polythiophene (PTH), and polythiophene enveloped tungsten oxide (PTH/WO) hybrid nanofillers were dispersed in the proposed oleo-PDPU matrix. The physicochemical properties of PDPU and their nanocomposites along with the physicomechanical characterization of their coatings were conducted using standard laboratory/American Standard Testing Methods (ASTMs), spectroscopic methods (FTIR, XRD, NMR), microscopic techniques (SEM and TEM), and thermo-gravimetric analysis (TGA) techniques were used for structural, morphological, and thermal characterization. Their corrosion protective performances were analyzed using salt mist chamber, potentiodynamic polarization (PDP), and electrochemical impedance spectroscopy (EIS). Their microbial resistance performance against the S. aureus, E. coli, and P. aeruginosa bacteria was also investigated using the well diffusion method. These studies revealed that the hybrid nanofillers (PTH/WO) dispersed PDPU coatings possess highest antibacterial and anticorrosive activity as compared to plain PDO-based polyurethanes (PDPU) and other such systems reported in the literature.

Circadian Volume Changes in Hippocampal Glia Studied by Label-Free Interferometric Imaging.

Complex brain functions, including learning and memory, arise in part from the modulatory role of astrocytes on neuronal circuits. Functionally, the dentate gyrus (DG) exhibits differences in the acquisition of long-term potentiation (LTP) between day and night. We hypothesize that the dynamic nature of astrocyte morphology plays an important role in the functional circuitry of hippocampal learning and memory, specifically in the DG. Standard microscopy techniques, such as differential interference contrast (DIC), present insufficient contrast for detecting changes in astrocyte structure and function and are unable to inform on the intrinsic structure of the sample in a quantitative manner. Recently, gradient light interference microscopy (GLIM) has been developed to upgrade a DIC microscope with quantitative capabilities such as single-cell dry mass and volume characterization. Here, we present a methodology for combining GLIM and electrophysiology to quantify the astrocyte morphological behavior over the day-night cycle. Colocalized measurements of GLIM and fluorescence allowed us to quantify the dry masses and volumes of hundreds of astrocytes. Our results indicate that, on average, there is a 25% cell volume reduction during the nocturnal cycle. Remarkably, this cell volume change takes place at constant dry mass, which suggests that the volume regulation occurs primarily through aqueous medium exchange with the environment.

Impact of histopathological revision and molecular pathology in the diagnosis of sarcomas in a reference center in Brazil.

e23533 Background: The accurate classification of sarcomas remains a major challenge due to the diversity and rarity of this group of mesenchymal tumors. In addition to standard microscopy and immunohistochemical techniques, molecular analyses are often required to achieve an appropriate diagnosis. The limited availability of specialized pathology centers, multidisciplinary teams, and diagnostic tools may directly affect the initial evaluation and subsequent treatment plan. Therefore, we sought to characterize the impact of diagnostic review by reference pathologists and molecular assessments in a reference center in Brazil. Methods: We conducted a retrospective analysis of sarcoma samples initially analyzed at outside laboratories and subsequently revised in a reference center by two dedicated pathologists between 01/2014-12/2020. After extracting demographic and tumor characteristics, we assessed the presence of complete discordance (benign versus malignant, sarcoma versus other malignancy), partial concordance (similar diagnosis of connective tumor, but different grade/histological subtype/differentiation), and complete concordance. The concordance for histology or grade, as well as the role of molecular assessments supporting the diagnosis, were also independently determined. Statistical analyses were conducted through the kappa coefficient of agreement, adherence by χ2 test, χ2 test by Person, and Fisher's exact test. Results: A total of 197 cases were included, with a predominance of male patients (57.9%), localized tumors (86.8%), and the median age at diagnosis of 46.3 years. The most common primary sites were lower extremities (34.6%) and retroperitoneum (11.3%). Following the revision, the most frequent histologies were undifferentiated pleomorphic sarcoma (20.3%), liposarcomas (13.1%), and leiomyosarcoma (6.6%). Complete discordance was present in 13.2%, partial discordance in 45.2%, and complete concordance in 41.6% of reviews (p &lt; 0.001). We found a concordance for histology or grade of 53.5% (p &lt; 0.001) and 51.8% (p &lt; 0.001) respectively. Molecular assessments, comprising NGS panels (79.5%) and FISH (20.5%), were performed in 44 (22.3%) cases, with findings of diagnostic relevance in 31.8% of those. Conclusions: In almost 60% of the cases, the initial diagnosis was at least partially modified when revised in a reference center, with a relevant impact of molecular assessments. These findings highlight the importance of managing sarcoma patients in reference centers and justify the assembly of referral networks in countries with limited healthcare resources.

Microstructural Defects of Epoxy–Phenolic Polymers on Metal Substrates during Acidic Corrosion

This study uses confocal microscopy and image processing to investigate the microstructural changes of coating–metal systems immersed in a heated acidic bath. Unlike standard optical microscopy techniques, 3D confocal microscopy images can quantitatively reveal microscopic defects formed at early stages of cathodic delamination. The coatings are made of fluorescent epoxy–phenolic resins cured at high temperatures onto tinplate (T23) and tin-free steel (TFS) substrates. When the coated metal substrates are immersed in acetic acid, a series of microscopic corrosion events occur at the polymer–metal interface. These events are quantified by changes in the thickness distribution of the degraded samples relative to that of intact coatings. The degradation rate is highest for epoxy–phenolic polymers on TFS substrates, represented by multiple orders of magnitude increase in the number density of defect sites. Higher molecular weight coatings provide slightly better resistance against delamination. The coating thickness dictates the rate of oxygen diffusion and ion transport along the polymer–coating interface, where raised asperities serve as localized sites for metal oxidation and formation of alkaline species, leading to subsequent delamination of the cured polymers from the surfaces. The results show that the metal surface topology is as important as chemistry when designing the corrosion resistance of products containing acidic liquids, and that confocal microscopy is useful in quality control through early detection of mesoscale polymer failure.

Green-synthesized, pH-stable and biocompatible carbon nanosensor for Fe3+: An experimental and computational study

Brightly fluorescent Carbon Dots (CDs) were synthesized by green hydrothermal method using commonly available biomass (Aloe vera) as carbon precursor. Their physiochemical and optical characterization was done by standard microscopic and spectroscopic techniques. Photophysical features of their aqueous dispersion were investigated in detail. The influence of wide pH range (2–12), high ionic load (2M) and temperature on their photoluminescence behavior was investigated. Their in-vitro cytotoxicity examination was conducted on Human Cervical Cancer Cells (HeLa) using MTT assay. Testing of their ion-recognition property for common metal ions was done in aqueous medium. These CDs exhibited preferential interaction with Fe3+ over other tested metal ions, without any functionalization. Interaction between CDs and Fe3+ was analyzed in the light of Density Functional Theory (DFT). The work demonstrates that these CDs are acting as nanoprobe for Fe3+ and sensing it at ultra-trace level (5 nM).

Prevalence of Plasmodium falciparum Malaria among Children Residing in Urban and Peri-urban Settlements in Rivers State

Urban and peri-urban areas are considered to be at lower risk of malaria compared to rural areas because of improved housing, higher socioeconomic status, improved environmental sanitation, and a limited number of breeding sites for mosquitoes. Reports have shown that there is an increased prevalence of malaria in urban and peri-urban areas but it has not been established which of these areas is at risk. The aim of this study, therefore, is to determine the prevalence rate of Plasmodium falciparum malaria infection among children in urban and peri-urban areas of Rivers State. A total of 601 and 500 blood samples were collected from urban and peri-urban areas respectively and examined using standard microscopy technique for detection of malaria parasites in blood samples. From the urban area, 282 tested positive out of 601 blood samples that were examined, giving the prevalence rate of 47% while in the peri-urban settlement, a total of 210 out of 500 tested positive to Plasmodium falciparum giving the prevalence rate of 42%. When the age was considered, the highest prevalence was recorded in children between 1-3 years and 7-12 years in urban settlement while in peri-urban, the highest prevalence was recorded in children between the age of 4-6 years with a prevalence of 45% and 46% respectively. Although the prevalence rates are relatively high. There is no significant difference between the prevalence rate in urban areas and that of peri-urban areas. Conclusively, intensive efforts should be made in controlling malaria parasites not only in rural areas but also in urban and peri-urban areas in Rivers State.

Investigation of the Efficacy of Home-Based Oral Chloroquine Treatment among Under-Five Children with &amp;lt;i&amp;gt;Plasmodium falciparum&amp;lt;/i&amp;gt; Malaria in Some Parts of Jos, Plateau State, Nigeria

Background: Nigeria is currently a malaria endemic country with an estimated 76% of her population at risk of contracting malaria [1]. According to a study in Nigeria, the first line of action mothers took when their children under 5 years have malaria showed that over 50% of them used non-prescription drugs they have at home or bought from pharmacy stores. And 60% of the most commonly used drugs for malaria treatment were chloroquine [2]. Many recent studies have demonstrated re-emergence of chloroquine-sensitive P. falciparum, suggesting a possible role in future malaria control [3]. Objective: The aim of this study was to investigate the effect of home-based oral chloroquine treatment among children under 5 years with Plasmodium falciparum malaria attending Jos University Teaching Hospital and OLA Hospital in Jos Metropolis. Method: This is a cross-sectional study of 93 malaria and non-malaria children. Malaria diagnosis was carried out using microscopical examination of Leishman’s stained thick and thin blood films, P. falciparum parasitemia was assessed by standard microscopy techniques and complete blood count was done using Beckman Coulter Analyzer. Results: The body temperature on admission was significantly lower (p ˚C ± 0.07˚C) than in the three malaria groups. The mean body temperature of chloroquine treated children with malaria was significantly lower (p Plasmodium falciparum parasitemia and degree of anemia in children under 5 years with Plasmodium falciparum in Jos Metropolis.

COMPARISON BETWEEN FLUORESCENT MICROSCOPY AND DUPLEX PCR TO DETECT MYCOBACTERIUM BOVIS AND MYCOBACTERIUM TUBERCULOSIS IN TUBERCULOSIS SUSPECTED PATIENTS

Objective: The aim of this study was to compare the gold-standard fluorescent microscopy as a diagnostic tech­nique with the PCR test, an advance molecular technique. Methodology: A total of 200 suspected pulmonary and extra-pulmonary samples were taken and stored for analysis in Quetta city. Samples were tested for Mycobacterium tuberculosis and Mycobacterium bovis by both the gold standard fluorescent microscopy (auramine-O FM) and molecular technique duplex PCR. By duplex PCR, Mycobacterium tuberculosis’s 245bp sequence and Mycobacterium bovis’s 500bp sequence was detected by using specific primers. Results: Among 200 pulmonary and extra-pulmonary samples, fluorescent microscopy detected 31 positive cas­es, while PCR detected 60 and 2 positive cases for Mycobacterium tuberculosis and Mycobacterium bovis respec­tively. The PCR analysis showed 28% of male patients and 32% female patients as positive for M. tuberculosis. Moreover, about52/164 pulmonary samples and 8/36 Extra-pulmonary samples were detected to be positive by PCR analysis. Conclusion: The PCR results were more accurate, rapid, sensitive and specie specific for detection of tuberculosis showing 60 positive cases for Mycobacterium tuberculosis and 2 positives for Mycobacterium bovis with a sig­nificant p-value. On the other hand, FM detected Mycobacterium tuberculosis with comparatively lower sensitivity with only 31 positive cases and had failed to distinguish between species.

Evaluating Mouse Fibroblast Interaction with Implant Surfaces in a 3D Microenvironment

Previous studies assessing fibroblast interactions with implants have mainly relied on measurements such as cell migration, gene expression, and cell adhesion. For these studies, testing cellular behavior at the implant surface was done by imaging the cell-implant interface using standard microscopy techniques in 2D tissue culture dishes. The true behavior of cells relative to the implant can best be assessed in a more physiologic 3D microenvironment. The embedding of the implant disks in 3D collagen gels was standardized with labeled fibroblasts to allow the imaging of fibroblast morphology and behavior when proximal to or binding to the implant disks. This allowed comparison of the behavior of laser-microgrooved and machined implant disk surfaces quantitatively in an in vitro 3D microenvironment. This in vitro imaging assay revealed for the first time in a 3D microenvironment setting the statistically significant impact laser-microgrooved disk surfaces have on both cell adherence and recruitment of cells in proximity to the disk. It also allowed visualization of membrane protrusivity and cytoskeletal organization in cells adherent to the implant disk. This assay provides a simple and effective way of observing cell behavior on and around the implant disk surface in a more physiologic 3D setting. Within the limits of this study, it revealed that the laser-microgrooved implant surface demonstrates significant superiority in fibroblast recruitment and binding in a 3D microenvironment.

Stable magneto-fluorescent gadolinium-doped AgInS2 core quantum dots (QDs) with enhanced photoluminescence properties

Gadolinium paramagnetic ion-doped quantum dots (QDs) have been recognized as attractive materials for fluorescence and magnetic field-driven water-based applications. However, the current Gd-based magneto-fluorescent materials exhibit low magnetic loading (low Gd molar ratio) and poor fluorescence characteristics (loss of photoluminescence at higher Gd loading), thus limiting their efficiency and applications. This paper demonstrates the facile synthesis of the citrate-capped gadolinium-doped AgInS2 QDs with stable magnetic properties and enhanced photoluminescence characteristics over a long period of time. To the best of our knowledge, the excellent fluorescent magnetic AgInS2 QDs with Gd’s molar ratio of up to 20 is being realized for the first time. The materials were characterized using standard optical and microscopic techniques. The fluorescence properties of the nanomaterials were enhanced over six months of storage without loss of the magnetic properties. The high Gd-loading makes the as-synthesized materials attractive for sensing and removal of pollutants from the water environment.

Lipid and neutrophil accumulation and active inflammasome in high BMI tumor regions of human pancreatic ductal adenocarcinoma.

e16271 Background: Obesity is an established risk factor for cancer and cancer-related mortality. Adipocytes may act as a “damage” signal resulting in the accumulation of innate immune cells, fueling an inflammatory micro-environment. Among the innate immune cells, tumor-associated neutrophils (TANs) are confirmed to accumulate inside the tumor. As obesity is a significant risk factor for a poorer prognosis in pancreatic ductal adenocarcinoma (PDAC), we hypothesize that obesity-driven adipose fuels the progression of PDAC in a TAN and inflammasome-facilitated manner. This may happen through several mechanisms, including genomic instability of aggressive PDAC clonal populations. Methods: Tissue from resected tumors of PDAC patients (n = 44) with a body-mass index (BMI) &gt; 27 (obese [n = 21]) and BMI &lt; 22 (normal [n = 23]) were incubated with anti-human CD66b antibodies and detected using standard immunofluorescent microscopy techniques. Images were acquired in a Zeiss Axioplan microscope workstation and analyzed by ZEN and Metamorph software. Results: We identified the presence of adipocytes in all tissue sections and CD66b+ neutrophils in all tissue sections of normal BMI patients and in 81% of high BMI patients. There is a significantly greater density of adipocytes in the tumor environment when compared to adjacent normal areas, suggesting that these cells are acting as a damage signal. We have also shown that there are fewer total cells per area in the tumor region, suggesting a disorganization that may allow more adipose tissue to form in these areas of high BMI patients. Our novel data also shows that high BMI results in a significantly higher neutrophil count in tumor specific tissue when compared to patients with a low BMI. These data suggest an association between BMI and inflammasome accumulation in the tumor environment. Conclusions: Our data uniquely shows that CD66b+ neutrophils tend to accumulate inside PDAC in high BMI patients and this is associated with intra-tumoral active inflammasomes. The higher concentrations of these inflammasomes in higher BMI patients may be of mechanistic relevance in understanding how inflammation induces progression of PDAC. A larger cohort of tissues from PDAC obtained from low and high BMI individuals will be needed to test our hypothesis and to begin to decipher the role of obesity on inflammation and PDAC progression.