Cytological Properties Research Articles

Adjusting physicochemical and cytological properties of biphasic calcium phosphate by magnesium substitution: An in vitro study

Biphasic calcium phosphate (BCP) is a highly study bone defect repair material with adjustable degradation, perfect osteoconduction and good osteoinduction. As one of the essential trace elements, magnesium (Mg) possesses the abilities of pro-osteogenesis and pro-angiogenesis. Therefore, Mg doping may further expand the application of BCP in bone defect repair, but few studies focus on promoting the osteogenesis and angiogenesis of BCP simultaneously by Mg doping, and the optimal doping amount of Mg remains to be explored. In this study, the physicochemical and biological properties of BCP scaffold affected by Mg doping were systematically study. Results showed that Mg doping enhanced the sintering of BCP scaffold, resulting in the decrease of degradation rate at the initial soaking period. However, the introduction of Mg damaged the lattice stability of BCP, leading to the increase of BCP degradation rate at the later soaking period. BCP scaffolds with Mg doping content ≥3 mol.% could achieve a long-term sustained release of Mg. The ion microenvironment created by Mg-doped scaffolds was simultaneously conducive to the osteogenic differentiation of stem cells and the enhanced angiogenic activity of endothelial cells. The scaffold doped with 5 mol.% of Mg (Mg5–S) showed the highest efficiency in promoting osteogenic differentiation. Mg-doped BCP scaffolds with a doping content ≥3 mol.%, especially Mg5–S, significantly improved the proliferation and angiogenic differentiation of endothelial cells. Based on these, we believe that the optimal doping content of Mg in BCP is 5 mol.%, and Mg5–S has great application potential in bone defect repair.

Physicochemical and cytological properties of poorly crystalline calcium-deficient hydroxyapatite with different Ca/P ratios

Compared to highly crystalline hydroxyapatite (HA), poorly crystalline hydroxyapatite has been proved to have better bioactivity and degradability, owing to its similar crystallographic structure to natural bone. However, there are few systematic comparative studies on poorly crystalline HA with different Ca/P ratios. In this work, poorly crystalline HA with different Ca/P molar ratios (1.50, 1.55, 1.60 and 1.67) was prepared by chemical precipitation method. The effects of Ca/P ratio on its structure, composition, morphology, surface properties, protein adsorption behaviors, ion adsorption and release abilities, cytological properties were systematically investigated. Results showed that the prepared HA was poorly crystalline, nano-sized and with the gradient change of Ca/P ratios. Protein adsorption capacity and P release of calcium deficient HA (CDHA) were effectively improved by reducing the Ca/P ratio, but CDHA with lower Ca/P ratio would also adsorb more Ca ions in the culture medium, which jointly affected the cytological properties of CDHA. In vitro cell experiments indicated that when mouse bone mesenchymal stem cells were co-cultured with CDHA with a Ca/P ratio of 1.55, their proliferation, ALP activity and osteogenesis-related genes expression were the strongest. This study provides a theoretical support and potential for further improving the biological performance of poorly crystalline CDHA materials.

Facile Fabrication of Hollow Hydrogel Microfiber via 3D Printing-Assisted Microfluidics and Its Application as a Biomimetic Blood Capillary

Simulating the structure and function of blood capillaries is very important for an in-depth insight into their role in the human body and treatment of capillary-related diseases. Due to the similar composition and structure, hollow hydrogel microfibers are well-recognized as potential biomimetic blood capillaries. In this paper, we report a novel, facile, and reproducible method to fabricate coaxial microfluidic chips via 3D printing-assisted soft lithography and then hollow hydrogel microfibers using the as-prepared coaxial microfluidic chips. Instead of traditional photoresist-based lithography, 3D printing of gelatin hydrogel under various extrusion pressures is used to construct sacrificial templates of coaxial microfluidic chips. Various solid and hollow hydrogel microfibers with complicated and hierarchical structures can be obtained via multitype coaxial microfluidic chips or a combination of coaxial microfluidic fabrication and post-treatment. The as-formed hollow hydrogel microfibers are evaluated in detail as biomimetic blood capillaries, including physicochemical and cytological properties. Our results prove that the hollow hydrogel microfibers exhibit excellent mass transport capacity, hemocompatibility, semipermeability, and mechanical strength, and their barrier function can be further enhanced in the presence of endothelial cells. Overall, our 3D printing-assisted fabrication strategy provides a new technique to construct microfluidic chips with complicated 3D microchannels, and the resulting hollow hydrogel microfibers are promising candidates for blood capillaries.

Impact of preterm birth on the cellular characteristics of neonatal buccal cells.

To demonstrate the impact of preterm birth on the cytological, cytomorphometrical, and nuclear parameters of neonatal buccal smears. This study consisted of Early Preterm Neonates (EPN; ≤34th gestational week [gw]; n=36), Late Preterm Neonates (LPN; 34th to <37th gw; n=46), and Term Neonates (control; ≥37th gw; n=56). Cytological evaluation and buccal cytome assay were performed using Papanicolaou and Feulgen methods, respectively. Cytological evaluation demonstrated that smear background was cleaner (P<.05) and there were less macrophages in the control group (P<.001). Cyto-morphometric analysis showed that the measurements of nuclear diameter, nuclear area, and nucleus-to-cytoplasm ratio were higher in the preterm (EPN and LPN) versus the control groups (P=.016, P<.001, and P<.001, respectively). We also demonstrated that staining intensity of the nucleus and cytoplasm were less intense in the EPN and LPN groups (P<.001). There was no statistically significant difference between the EPN and LPN groups for any parameters (P>.05). Buccal cytome assay showed that nuclear buds were more prevalent in term newborns compared to preterm neonates (P<.001). Morphological and cytological properties of neonatal buccal cells are influenced by preterm birth status, and buccal smears may be used as a tool to detect biological markers of neonatal health problems.

Complete sequence of an isolate of snake melon asteroid mosaic virus confirms that it is a member of a distinct sobemovirus species.

A virus tentatively named "snake melon asteroid mosaic virus" (SMAMV) was found in Sudan in cucurbit crops (10% of 600 samples) between 1992 and 2003. Biological and cytological properties as well as sequence data on a 345-nt fragment suggested that SMAMV was a member of the genus Sobemovirus. However, no complete sequence had been obtained, and the relationship between SMAMV and the acknowledged sobemoviruses had not been ascertained. In this work, we obtained the full-length sequence of an SMAMV isolate. The sequence was 4225 nt long, with a typical sobemovirus genetic organization. Sequence identity to other sobemoviruses was below 50%, both for the full-length genome and for individual proteins. These data confirm that SMAMV belongs to a novel sobemovirus species.

Improved response of triploid citrus varieties to water deficit is related to anatomical and cytological properties

Polyploidy plays a major role in citrus plant breeding to improve the adaptation of polyploid rootstocks as well as scions to adverse conditions and to enhance agronomic characteristics. In Citrus breeding programs, triploidy could be a useful tool to react to environmental issues and consumer demands because the produced fruits are seedless. In this study, we compared the physiological, biochemical, morphological, and ultrastructural responses to water deficit of triploid and diploid citrus varieties obtained from ‘Fortune’ mandarin and ‘Ellendale’ tangor hybridization. One diploid clementine tree was included and used as a reference. All studied scions were grafted on C-35 citrange rootstock. Triploidy decreased stomatal density and increased stomata size. The number of chloroplasts increased in 3x varieties. These cytological properties may explain the greater photosynthetic capacity (Pnet, gs, Fv/Fm) and enhanced water-holding capacity (RWC, proline). In addition, reduced degradation of ultrastructural organelles (chloroplasts and mitochondria) and thylakoids accompanied by less photosynthetic activity and low oxidative damages were found in 3x varieties. Triploid varieties, especially T40-3x, had a better ability to limit water loss and dissipate excess energy (NPQ) to protect photosystems. Higher starch reserves in 3x varieties suggest a better carbon and energy supply and increases in plastoglobuli size suggest less oxidative damage (H2O2, MDA), especially in T40-3x, and preservation of photosynthetic apparatus. Taken together, our results suggest that desirable cytological and ultrastructural traits induced by triploidy improve water stress response and could be a useful stress marker during environmental constraints.

Purification of cryopreserved camel spermatozoa following protease-based semen liquefaction by lectin-functionalized DNA-defrag magnetic nanoparticles.

Although incorporating proteases into sperm medium is considered the most effective procedure to eliminate camel semen viscosity, it drastically affects viability, morpho-functional properties and, hence, fertilization potential of spermatozoa. The present work aimed at evaluating adequacy of employing magnetic nanoparticles-based sperm purification technique for eluting impaired and apoptotic camel spermatozoa from cryopreserved semen doses following protease-based semen liquefaction. Thirty cryopreserved semen doses (50 x 106 sperm/straw) representing the following liquefaction treatments: control (untreated), 0.1mg/ml papain or 5 U/ml bromelain were used (n=10 straws per treatment). Immediately after thawing (38°C for 40s), sperm concentration of each straw within treatment was readjusted to 15 x 106 sperm/mL by dilution in PBS (37°C). Sperm physical and cytological properties were then assessed (non-purified semen). Thereafter, each specimen was subjected to lectin-functionalized DNA-defrag magnetic nanoparticles sperm purification, and the same sperm traits were re-evaluated after undergoing purification (purified semen). Sperm DNA fragmentation level within each group, prior to and after magnetic nano-purification, was also determined by fluorescent imaging. The results showed a dramatic improvement (p<.05) in post-thaw motility (%), viability (%), normal sperm (%), intact acrosome (%) and HOST-reacted (%) spermatozoa in protease-liquefied semen following sperm magnetic nano-purification. Additionally, the highest (p<.05) DNA fragmentation level was recorded in all cryopreserved semen groups prior to purification, whereas the lowest (p<.05) was observed in the protease-treated specimens after magnetic nano-purification. These results indicate that protease-based semen liquefaction prior to cryopreservation in conjunction with magnetic nano-purification post-thawing holds potential for reducing the proportion of damaged and dead spermatozoa, hence improving camel sperm fertilization competence.

Cytological approach for pleural fluid analysis – One year study

Background: Collection of an abnormal quality and quantity of fluid in the pleural cavity is called pleural effusion. Cytological analysis of pleural effusions plays an important role in the diagnosis of various lesions. The objective of the study is to evaluate the utility of pleural effusion cytology as an attempt to diagnose the underlying etiology. Materials and Methods: A prospective study for one year duration from September 2018 - August 2019 was conducted in the Department of Pathology- S N M C and H.S.K hospital and Research Centre, Bagalkot. Pleural fluids received for cytological analysis from all the departments were included in the study. After a thorough physical examination, microscopic evaluation was done. The smears were stained with Giemsa, Papanicolaou stain and Hematoxylin and Eosin Stains. ZN stain was done in suspected cases of tuberculosis. Results: 65 cases of pleural effusion were evaluated out of which67% of patients were males and 33% were females. Out of 65 cases 53.8% showed right sided pleural effusion, 24.6% showed left sided pleural effusion and 21.5% showed bilateral pleural effusion. All the smears were screened for malignancy and resulted in 14% of positive cases. The 86% non-malignant cases were further classified based on the cytology findings; 46.1% lymphocytic predominant, 32.3% neutrophilic predominant, 4.6% reactive mesothelial cell predominant and 1.5% eosinophilic predominant. Conclusion: This study showed that meticulous evaluation of the pleural fluids for their cytological properties will help the clinicians in pointing towards the underlying etiological factor and to choose an appropriate management. Keywords: Pleural effusion, Non-Malignant Effusion, Malignant Effusion, Metastatic Adenocarcinoma, Lymphoma.

Macro- and Micro-morphological, Anatomical, Cytological and Phytochemical Properties of &lt;i&gt;Tridax procumbens&lt;/i&gt; Linn. (Asteraceae)

This study examined the macro- and micro-morphological, Anatomical, Cytological and Phytochemical Properties of Tridax procumbens L. in the family Asteraceae. Observations of plant parts aided by measurements were done and these were sectioned following Wahua’s method; root tips squashed with FLP Orcein and qualitative phytochemical analyses were carried out. The slides were viewed using the light compound microscope and photomicrographs were taken after careful examination from good preparations. Macromorphological studies showed the plant grows up to 40cm or more in height. Foliar features revealed opposite, pinnate, oblong to ovate with coarsely serrated margin and acute apex, 4±1.5cm long and 2±1cm wide with petiole up to 1.5±0.5cm in length. The floral structure show cased diameter of each flower head as 1.0±0.4cm while the peduncle is elongated and up to 10±5cm in length; the petal is 0.7±0.2cm in length alongside tubular sepals up to 0.9±0.3cm in length. The stamen is 0.9±0.2cm in length while the carpel is 1.0±0.1cm. Presence of anomocytic stomata which is amphistomatic. Anatomical sections on the mid-ribs, petiole, internodes, nodes and roots revealed peculiar internal features. Mitotic chromosome is 2n=36. Phytochemical studies revealed the presence of saponins, alkaloids, flavonoids, tannins, steroids, cardiac glycosides, phenols. The information generated from this study would further aid in the delimitation of the species.&#x0D; Keywords: Tridax procumbens, Morphology, anatomy, cytology, palynology, phytochemistry

Enhancing liquid-chilled storage and cryopreservation capacities of ram spermatozoa by supplementing the diluent with different additives.

ObjectiveIn the present study, we determined efficiency of incorporating caffeine, melatonin or omega-3 polyunsaturated fatty acid in the diluent on mitigating consequences of (a) liquid chilled- and (b) cryo-storage of ram spermatozoa.MethodsIn the first experiment, ejaculates (n = 30) were collected from 5 adult rams and were pooled, diluted (1:10) with Tris-citric acid (base diluent) and were split into 4 aliquots assigned for: control (untreated), caffeine (0.1 mM), melatonin (0.3 mM) or omega-3 fatty acids (0.3 mM) (T0). The diluted specimens were stored at 4°C for 48 h, during which sperm physical and cytological properties were evaluated along with oxidative stress indices (T24, T48). In the second experiment, 15 ejaculates (3 per male) were pooled, diluted with glycerolized base diluent (4% glycerol, v/v) and were split corresponding to the same previous treatment groups before being processed for cryopreservation. Post-thaw physical and kinematic sperm properties were assessed by a computer-assisted sperm analysis system.ResultsThe results clarified superiority of both melatonin and omega-3 supplementation on maintaining (p<0.05) sperm properties, while reducing (p<0.05) lipid peroxidase reaction and enzymatic activities of alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase in preservation medium, compared to caffeine either during liquid-chilled storage or cryopreservation of spermatozoa.ConclusionMelatonin and omega-3 are regarded efficient alternatives to caffeine when processing ram spermatozoa for application of artificial insemination or in vitro fertilization.

α-Lys424 Participates in Insertion of FeMoco to MoFe Protein and Maintains Nitrogenase Activity in Klebsiella oxytoca M5al.

Our previous investigation of substrates reduction catalyzed by nitrogenase suggested that α-Ile423 of MoFe protein possibly functions as an electron transfer gate to Mo site of active center-“FeMoco”. Amino acid residue α-Lys424 connects directly to α-Ile423, and they are located in the same α-helix (α423-431). In the present study, function of α-Lys424 was investigated by replacing it with Arg (alkaline, like Lys), Gln (neutral), Glu (acidic), and Ala (neutral) through site-directed mutagenesis and homologous recombination. The mutants were, respectively, termed 424R, 424Q, 424E, and 424A. Studies of diazotrophic cell growth, cytological, and enzymatic properties indicated that none of the substitutions altered the secondary structure of MoFe protein, or normal expression of nifA, nifL, and nifD. Substitution of alkaline amino acid (i.e., 424R) maintained acetylene (C2H2) and proton (H+) reduction activities at normal levels similar to that of wild-type (WT), because its FeMoco content did not reduce. In contrast, substitution of acidic or neutral amino acid (i.e., 424Q, 424E, 424A) impaired the catalytic activity of nitrogenase to varying degrees. Combination of MoFe protein structural simulation and the results of a series of experiments, the function of α-Lys424 in ensuring insertion of FeMoco to MoFe protein was further confirmed, and the contribution of α-Lys424 in maintaining low potential of the microenvironment causing efficient catalytic activity of nitrogenase was demonstrated.

A fractal based approach to evaluate the progression of esophageal squamous cell dysplasia

Abstract Esophageal squamous cell carcinoma (ESCC) is the most prevalent malignancy of the esophagus with a very poor prognosis. Nevertheless, squamous cell dysplasia (ESD) has been identified as the only histological precursors of ESCC. Since, tissue alterations are slight in the early stage of ESD, human diagnosis is subjective. Hence, this work presents a first computer-aided system to differentiate low-grade dysplasia (LGD) from normal esophageal mucosa according to Vienna grading system, which is the most widespread method for histological grading of esophagus tissues. We captured microscopic images of a well-oriented region of Normal and LGD biopsies to characterize the architectural and cytological properties of specimens based on the computational analysis. We produced two sets of enhanced images. Then, by considering the fractal concept, we defined a new scale-dependent function in the generalized fractal dimension formulation to include the special information of both preprocessed images together. Then, for each image, a pattern was computed from variations of tissue fractal geometry across the pathway of dysplasia development. We proposed features extracted from these patterns to classify deviations of tissue characteristics from the normal stage. This method successfully differentiated the two diagnosis classes with statistical significance and high performance (accuracy = 97.78% ± 0.05, p

Effects of phospholipid and polyethylene glycol monostearate (100) on the in vitro and in vivo physico-chemical characterization of poly(n-butyl cyanoacrylate) nanoparticles.

In this study, poly(n-butyl cyanoacrylate) nanoparticles (PBCA-NPs) modified with various amounts of soybean phospholipid (PC) and polyethylene glycol monostearate (S100) were prepared in order to investigate the effects of PC and S100 on the nanoparticles' physico-chemical properties, cytological properties and in vivo gastrointestinal absorption. Coumarin-6 (C6) was used as a fluorescent probe; C6-loaded PBCA-NPs modified with both PC and S100 (C6-PS-PBCA-NPs) were prepared using miniemulsion polymerization, and C6-loaded PBCA-NPs modified with either S100 (C6-S-PBCA-NPs) or PC (C6-P-PBCA-NPs) were used as references. All of the different NPs were shown to be stably dispersed and to have a small particle size. A cytotoxicity study indicated that all of the blank PBCA-NPs were safe and nontoxic. The uptake of NPs by Caco-2 cells was shown to be increased when the amount of PC was increased from 0% to 1.25% and the amount of S100 was increased from 0% to 0.725%. The use of a ligated intestinal loop model demonstrated that C6-PS-PBCA-NPs could rapidly penetrate a highly viscoelastic mucous layer, leading to an improvement in the absorption efficiency. During a pharmacokinetic study, C6-PS-PBCA-NPs improved the absorption of C6, as indicated by their higher Cmax and AUC0-t values compared with those of C6-S-PBCA-NPs or C6-P-PBCA-NPs. Overall, the results of this study indicate that PBCA-NPs modified with PC and S100 can enhance the absorption of orally administered drugs.

Effects of gold core size on regulating the performance of doxorubicin-conjugated gold nanoparticles

Studies on the influence of one critical parameter (e.g., size), targeting a specific disease, while keeping other factors unchanged, are important for improving understanding and application of the molecular design of biomedical nanomaterials. In this study, we used doxorubicin (Dox)-conjugated gold nanoparticles (GNPs) to investigate the effects of the size of the gold core (10, 20, or 60 nm) on the performance of their conjugates. We found that all three conjugates differed slightly in their physicochemical properties, facilitating a direct and accurate assessment of the size effects of GNP-Dox conjugates on their in vitro and in vivo performance. The cytological properties (the cell penetration rate and efficiency, as well as the cytotoxicity) and antitumor performance (the intratumoral penetration, treatment efficacy, and biodistribution) were highly correlated to the size of the inorganic core. Among all test groups, although the conjugate with a 60-nm gold core had the highest drug loading and release efficiency, the conjugate with a 10-nm gold core displayed the best antitumor efficacy toward the liver cancer models. This was because it showed the deepest tumor permeability and the highest tumor cell-killing ability of Dox transported by the relatively small GNPs. This study provides important evidence for better understanding the effect of size on in vitro and in vivo properties of potential therapeutic nanosystems and their structure design.

Prognostic Significance of Blood, Serum, and Ascites Parameters in Patients with Malignant Peritoneal Mesothelioma or Peritoneal Carcinomatosis.

To determine effects of the biochemical and cytological properties of blood, serum, and ascites on survival of patients with malignant peritoneal effusion (MPeE), including malignant peritoneal mesothelioma (MPeM) and peritoneal carcinomatosis (PC), we conducted a retrospective study of patients with MPeE and healthy controls. Potential prognostic factors were identified as follows: age, sex, blood neutrophil-to-lymphocyte ratio (NLR), serum parameters, ascites parameters, serum-ascites albumin gradient, and the ascites-serum LDH ratio. Compared to those of the control group, serum albumin levels were significantly lower, and the NLR and serum LDH levels were significantly higher in the MPeE group. Overall survival (OS) was longer in patients with MPeM compared to that in patients with PC. Compared with patients in the MPeM, patients with PC had higher NLRs, ascites glucose levels, serum-ascites albumin gradients, and serum LDH levels. In contrast, their ascites albumin levels and ascites-serum LDH ratios were lower. Univariate analyses indicated that the NLR, serum LDH levels, ascites LDH levels, ascites coenocyte levels, and the ascites coenocyte-to-monocyte ratios affected the OS. Multivariate analyses identified only serum and ascites LDH levels as independent prognostic factors.

Cytological Study of Pleural Effusions and its Utility in Clinical Approach

Background: Cytological analyses of pleural effusions play an important role in the diagnosis of various lesions. Most importantly it gives a significant contribution in diagnosis and management. Methods: A retrospective study for one year duration from January 2015 to December 2015 was conducted in the Cytology section, Department of Pathology, Govt. Medical College & Sir- T hospital Bhavnagar. Pleural fluids which were come for cytological analysis from all the departments are included in the study, then physical examination and microscopy done by staining with Gimsa, Papanicolaou stain and Hematoxylin and Eosin Stains for evaluation. Result: 295 cases of pleural effusion evaluated in which 29% are female and 71% are male and 12% of effusions are Bilateral, 32% are left sided, 57% right sided pleural effusion, 95% non Malignant lesions and only in 5% of cases were reported as malignant and further non-malignant lesions comprises of pleural effusions with reactive mesothelial cells 11% while remaining fluids 40% are of lymphocytic predominant effusions and 13% are of acute inflammatory conditions with abundant polymorphs, remaining 36% of pleural effusions were scant-cellular and have no suggestive role by cytological evaluation. Conclusion: This study showed that meticulous evaluation of the pleural fluids for their cytological properties will help the clinicians in clinching the diagnosis as well as early approach in management of these cases. DOI: 10.21276/APALM.1277

Diagnostic peritoneal fluid aspiration: a simple maneuver to diagnose a major question: a prospective study surgical point of view

Background: A variety of fluids can accumulate in the peritoneal and pelvic cavities including ascites, blood, pus, bile, urine, lymph, cerebral spinal fluid, and mucin. The appearance of some of these fluids by imaging may suggest a specific etiology, but a diagnostic aspiration must be performed in the majority of cases. This review aims to assess critically the value of peritoneal fluid analysis in the diagnosis.Methods: There was prospectively evaluated 80 patients presented with intraperitoneal fluid collection at Sohag University Hospital from July 2016 till July 2017. Physical, chemical, bacteriological and cytological properties of the aspirated peritoneal fluid was studied.Results: The study included 80 patients with intra-peritoneal collection of surgical importance (due to trauma, inflammation, malignancy). Diagnostic aspiration from intra-abdominal collection was routinely done in all cases giving the following results: 20 cases (25%) showed blood in the aspirate, 18 cases (22.5%) showed pus, 11 cases (13.75%) showed bile stained fluid, 9 cases (11.25%) showed gastric contents, 8 cases (10%) showed serosangenous aspirate, 7 cases (8.75%) showed clear fluid aspirate, 4 cases (5%) showed turbid fluid and 3 cases (3.75%) showed small intestinal contents.Conclusions: Diagnostic aspiration from intraperitoneal fluid collection is considered a simple available test that helps in diagnosis of major problematic cases.

Establishment and evaluation of immortalized human epidermal keratinocytes for an alternative skin irritation test

Human skin is located at the outermost part of the body, and various cosmetics and chemicals that may come in contact with human skin need to be evaluated for their potential to cause irritation. Until recently, the Draize test was considered the standard method for skin irritation; however, this technique has disadvantages such as the need to sacrifice many rabbits and subjective scoring. Thus, to contribute to the development of an animal-free alternative skin irritation test, we investigated the cytotoxicity and inflammatory response to standard skin irritants in SV40 large T antigen-transformed human epidermal keratinocyte 2 cells (SV-HEK2 cells). In this study, we established an SV-HEK2 cell line immortalized by SV40 large T antigen (SV40 T) and characterized the inherent morphological and cytological properties. We next used 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) or neutral red uptake (NRU) assays of cell viability to investigate the optimal experimental conditions for determining SV-HEK2 cell viability after exposure to sodium dodecyl sulfate at 6.25×10−3% to 1×10−1% as a standard skin irritant. We then examined the viability of SV-HEK2 cells in response to five skin irritants (benzalkonium chloride, isopropanol, sodium dodecyl sulfate, Triton X-100 and Tween20) at 5×10−3% to 1×10−1% by MTT or NRU assay. Finally, we estimated the level of cytokines secretion in response to stimulation by skin irritants in SV-HEK2 cells. The results revealed that SV-HEK2 cells responded well to skin irritants in a concentration-dependent manner and that there was good correlation between irritant concentration and cytotoxicity (or cytokine secretion) when cells were exposed to skin irritants for 10min at room temperature (RT) using an MTT assay. Overall, these findings suggest that SV-HEK2 cells could be a good alternative in vitro model for skin irritation tests.

Analysis of Meiotic Sister Chromatid Cohesion in Caenorhabditis elegans.

In sexually reproducing organisms, the formation of healthy gametes (sperm and eggs) requires the proper establishment and release of meiotic sister chromatid cohesion (SCC). SCC tethers replicated sisters from their formation in premeiotic S phase until the stepwise removal of cohesion in anaphase of meiosis I and II allows the separation of homologs and then sisters. Defects in the establishment or release of meiotic cohesion cause chromosome segregation errors that lead to the formation of aneuploid gametes and inviable embryos. The nematode Caenorhabditis elegans is an attractive model for studies of meiotic sister chromatid cohesion due to its genetic tractability and the excellent cytological properties of the hermaphrodite gonad. Moreover, mutants defective in the establishment or maintenance of meiotic SCC nevertheless produce abundant gametes, allowing analysis of the pattern of chromosome segregation. Here I describe two approaches for analysis of meiotic cohesion in C. elegans. The first approach relies on cytology to detect and quantify defects in SCC. The second approach relies on PCR and restriction digests to identify embryos that inherited an incorrect complement of chromosomes due to aberrant meiotic chromosome segregation. Both approaches are sensitive enough to identify rare errors and precise enough to reveal distinctive phenotypes resulting from mutations that perturb meiotic SCC in different ways. The robust, quantitative nature of these assays should strengthen phenotypic comparisons of different meiotic mutants and enhance the reproducibility of data generated by different investigators.

In situ detection of histone variants and modifications in mouse brain using imaging mass spectrometry.

Histone posttranslational modifications and histone variants control the epigenetic regulation of gene expression and affect a wide variety of biological processes. A complex pattern of such modifications and variants defines the identity of cells within complex organ systems and can therefore be used to characterize cells at a molecular level. However, their detection and identification in situ has been limited so far due to lack of specificity, selectivity, and availability of antihistone antibodies. Here, we describe a novel MALDI imaging MS based workflow, which enables us to detect and characterize histones by their intact mass and their correlation with cytological properties of the tissue using novel statistical and image analysis tools. The workflow allows us to characterize the in situ distribution of the major histone variants and their modification in the mouse brain. This new analysis tool is particularly useful for the investigation of expression patterns of the linker histone H1 variants for which suitable antibodies are so far not available.