Total Number Of Nuclei Research Articles

Nonlinear dynamics of a bubble cluster in liquid cavity wrapped by elastic medium

Abstract According to the modified nucleation theory, gas nuclei can generate and grow into cavitation bubbles when’s the liquid pressure exceeds a threshold in a cavity. Based on the modified nucleation theory, the growth process of the gas nuclei in a liquid cavity can be simplified to two “events”, and the full confinement effects of the surrounding elastic medium of the cavity was considered by including the bulk modulus in the equation of state. The dynamical equations were derived to model the nonlinear oscillation of the multi-bubble system in the cavity. The effects of acoustic parameters, the initial radius and the total number of bubbles on bubble dynamics were investigated numerically. It is found that bubble behaviors are sensitive to the acoustic field. In the low frequency region (f <1MHz), bubble oscillation exhibits a modulated waveform, and fundamental, subharmonic and harmonic modes can be found in the acoustic response curve. The oscillations of confined bubbles diminish with increasing frequency. In the high frequency region (f >1MHz), the confinement state affects the acoustic response of the gas nuclei, and the growth rate differ by a factor of 5 difference in the presence of shelled or unbounded elastic confinement of the cavity. Therefore, the suppression or enhancement of cavitation growth and collapse in confined cavity can be modulated by a variety of factors, such as the total number of nuclei, cavity size, acoustic frequency and amplitude, negative pressure, and elastic medium, which have a synergistic influence on the confined cavitation process.

Comprehensive analytics for virus-cell and cell-cell multinucleation system

Cell-fusion mediated generation of multinucleated syncytia represent critical feature during viral infection and in development. Efficiency of syncytia formation is usually illustrated as fusion efficiency under given condition by quantifying total number of nuclei in syncytia normalized to total number of nuclei (both within syncytia and unfused cell nuclei) in unit field of view. However heterogeneity in multinucleated syncytia sizes poses challenge in quantification of cell-fusion multinucleation under diverse conditions. Taking in-vitro SARS-CoV-2 spike-protein variants mediated virus-cell fusion model and placenta trophoblast syncytialization as cell-cell fusion model; herein we emphasize wide application of simple unbiased detailed measure of virus-cell and cell-cell multinucleation using experiential cumulative distribution function (CDF) and fusion number events (FNE) approaches illustrating comprehensive metrics for syncytia interpretation.

Megasporogenesis and megagametogenesis in Hydrocleys nymphoides, Alisma plantago-aquatica, and Sagittaria montevidensis (Alismataceae).

Ovule morphology, megasporogenesis, and megagametogenesis processes were examined in Hydrocleys nymphoides, Alisma plantago-aquatica, and Sagittaria montevidensis. Each of these species belongs to a different clade within the Alismataceae family. It is worth mentioning that the genus Hydrocleys previously belonged to the Limnocharitaceae family but is now classified within the Alismataceae. Flowers in different developmental stages were processed following classical histological methods for their observation with bright-field microscope. The three species present an anatropous and bitegmic mature ovule. This is tenuinucellate in A. plantago-aquatica and S. montevidensis and pseudo-crassinucellate in H. nymphoides. Although all three species have the same type of megasporogenesis, they differ in the megagametogenesis and in the total number of nuclei and cells that form the mature gametophyte. H. nymphoides has a female gametophyte composed of four cells and four nuclei, while A. plantago-aquatica and S. montevidensis have a female gametophyte of five cells and six nuclei. The results are discussed according to the phylogenetic position of each of the species. Moreover, new types of megagametophyte development are described: Hydrocleys and Sagittaria types. The reduction of the female gametophyte with respect to the Polygonum type is found in families belonging to the ANA grade and in other aquatic families within the order Alismatales. We infer that the reduction in the number of cells and nuclei in the female gametophyte is characteristic of species that inhabit aquatic environments. Future studies in aquatic species belonging to other families would be necessary to confirm this hypothesis.

Alcalase-Assisted Mytilus edulis Hydrolysate: A Nutritional Approach for Recovery from Muscle Atrophy.

Muscle atrophy is a complex physiological condition caused by a variety of reasons, including muscle disuse, aging, malnutrition, chronic diseases, immobilization, and hormonal imbalance. Beyond its effect on physical appearance, this condition significantly reduces the quality of human life, thus warranting the development of preventive strategies. Although exercising is effective in managing this condition, it is applicable only for individuals who can engage in physical activities and are not bedridden. A combination of exercise and nutritional supplementation has emerged as a more advantageous approach. Here, we evaluated the effects of enzyme-assisted hydrolysates of Mytilus edulis prepared using Protamex (PMH), Alcalase (AMH), or Flavourzyme (FMH) in protecting against muscle atrophy in a dexamethasone (Dex)-induced muscular atrophy model in vitro and in vitro. Alcalase-assisted M. edulis hydrolysate (AMH) was the most efficient among the tested treatments and resulted in higher protein recovery (57.06 ± 0.42%) and abundant amino acid composition (43,158 mg/100 g; 43.16%). AMH treatment also escalated the proliferation of C2C12 cells while increasing the total number of nuclei, myotube coverage, and myotube diameter. These results were corroborated by a successful reduction in the levels of proteins responsible for muscle atrophy, including E3 ubiquitin ligases, and an increase in the expression of proteins associated with muscle hypertrophy, including myogenin and MyHC. These results were further solidified by the successful enhancement of locomotor ability and body weight in zebrafish following AMH treatment. Thus, these findings highlight the potential of AMH in recovery from muscle atrophy.

Effect of Serum and Oxygen on the In Vitro Culture of Hanwoo Korean Native Cattle-Derived Skeletal Myogenic Cells Used in Cellular Agriculture

Skeletal muscle-derived myogenic cells (SKMCs) are novel protein sources capable of replacing animal meat. However, SKMCs have not been commercialized owing to poor productivity and the high cost of in vitro cell culture. Therefore, we cultured SKMCs in varying serum (5-20%) and oxygen concentrations (5-20%) to investigate the parameters that most impact cell productivity (serum, hypoxia, and culture medium) and examined cell proliferation ability and genes involved in myogenesis/proliferation/apoptosis/reactive oxygen species (ROS). In fetal bovine serum (FBS) groups, hypoxia induction doubled cell number, and the 20% FBS/normoxia group exhibited similar cell numbers as 5% FBS/5% hypoxia, confirming that 5% hypoxia reduced serum requirement by four-fold. The use of 20% FBS downregulated MTF5/MYOD1/MYOG/MYH1, whereas hypoxia induction with ≤10% FBS upregulated them. Although 20% FBS lowered TERT expression through rapid cell proliferation, NOX1, a major factor of ROS, was suppressed. DMEM/F12 demonstrated better differentiation potential than F10 by upregulating MYF3/MYOD1/MYOG/MYH1 and downregulating MSTN, particularly DMEM/F12 with 2% FBS/5% hypoxia. The myogenic fusion index was higher in DMEM/F12 without FBS than in DMEM/F12 with FBS (0.5-5%); however, the total nuclei number was reduced owing to apoptosis. Therefore, high serum levels are essential in influencing SKMC growth, followed by hypoxia as a synergistic component.

Crystallization of Aluminum droplet at low undercooling: homogeneous nucleation approach

Crystallization in an undercooled Aluminum (Al) droplet occurs via the formation of crystalline nuclei by homogeneous or heterogeneous nucleation and successive growth of nuclei. Clusters of a new phase appear in the system due to fluctuations and after reaching a critical size grow up to microscopic size. Crystallization event at low undercooling 6.4°C of 9.91 mg in compact Al sample was detected using isothermal differential scanning calorimetry (DSC) after several tenths of minutes. In this case, standard analysis based on Johnson-Mehl-Avrami-Kolmogorov (JMAK) model is not appropriate as Avrami parameter n obtained by fitting of DSC data is too low (n ≤ 1 and thus the dimensionality of the system d = n – 1 ≤ 0). Al growth rate is extremely high and that is why we presume that the time delay of crystallization, detected by DSC, is slightly higher than nucleation time delay. A homogeneous nucleation model is applied to determine the basic characteristics of nucleation: the size distribution of nuclei, nucleation rate, total number of nuclei and crystallization fraction at the nucleation process. The number of atoms in a liquid Aluminum droplet decreases with time as the formation of a new crystalline phase occurs. As a consequence, a decrease in stationary nucleation occurs. It is shown that decrease in the number of atoms in a liquid Al droplet is predominantly caused by formation of subcritical clusters.

Cyclooxygenase-2 is a critical determinant of angiotensin II-induced vascular remodeling and stiffness in resistance arteries of ouabain-treated rats.

To investigate the role of angiotensin II/AT 1 receptor signaling and/or cyclooxygenase-2 (COX-2) activation on vascular remodeling and stiffening of the mesenteric resistance arteries (MRA) of ouabain-treated rats. Ouabain-treated (OUA, 30 μg kg/day for 5 weeks) and vehicle (VEH)-treated Wistar rats were co-treated with losartan (LOS, AT 1 R antagonist), nimesulide (NIM, COX-2 inhibitor) or hydralazine hydrochloride plus hydrochlorothiazide. MRA structure and mechanics were assessed with pressure myography and histology. Picrosirius red staining was used to determine the total collagen content. Western blotting was used to detect the expression of collagen I/III, MMP-2, Src, NFκB, Bax, Bcl-2 and COX-2. Reactive oxygen species (ROS) and plasma angiotensin II levels were measured by fluorescence and ELISA, respectively. Blockade of AT 1 R or inhibition of COX-2 prevented ouabain-induced blood pressure elevation. Plasma angiotensin II level was higher in OUA than in VEH. LOS, but not hydralazine hydrochloride with hydrochlorothiazide, prevented inward hypotrophic remodeling, increased collagen deposition and stiffness, and oxidative stress in OUA MRA. LOS prevented the reduction in the total number of nuclei in the media layer and the Bcl-2 expression induced by OUA in MRA. The higher pSrc/Src ratio, NFκB/IκB ratio, and COX-2 expression in OUA MRA were also prevented by LOS. Likewise, COX-2 inhibition prevented vascular remodeling, mechanical changes, oxidative stress and inflammation in OUA MRA. The results suggest that, regardless of hemodynamic adjustments, the angiotensin II/AT 1 R/pSrc/ROS/NFκB/COX-2 pathway is involved in the development of MRA inward hypotrophic remodeling and stiffness in ouabain-treated rats.

Syncytia formation by SARS-CoV-2-infected cells.

Severe cases of COVID‐19 are associated with extensive lung damage and the presence of infected multinucleated syncytial pneumocytes. The viral and cellular mechanisms regulating the formation of these syncytia are not well understood. Here, we show that SARS‐CoV‐2‐infected cells express the Spike protein (S) at their surface and fuse with ACE2‐positive neighboring cells. Expression of S without any other viral proteins triggers syncytia formation. Interferon‐induced transmembrane proteins (IFITMs), a family of restriction factors that block the entry of many viruses, inhibit S‐mediated fusion, with IFITM1 being more active than IFITM2 and IFITM3. On the contrary, the TMPRSS2 serine protease, which is known to enhance infectivity of cell‐free virions, processes both S and ACE2 and increases syncytia formation by accelerating the fusion process. TMPRSS2 thwarts the antiviral effect of IFITMs. Our results show that SARS‐CoV‐2 pathological effects are modulated by cellular proteins that either inhibit or facilitate syncytia formation.

Inhibition of leucine aminopeptidase affects myocyte proliferation and differentiation

BackgroundLoss of skeletal muscle mass in the elderly, namely sarcopenia, is a rapidly growing global health problem. Understanding the mechanism of muscle mass regulation is essential to confront this problem. The balance of muscle protein synthesis and degradation is thought to be one of the key issues. Previous reports showed that muscle‐specific deletion of a crucial proteasomal gene, Rpt3 (also known as Psmc4), resulted in severe skeletal muscle atrophy (Kitajima Y et al., 2014). Although proteasomal proteolysis plays an essential role in the maintenance of muscle mass, the underlying mechanism remains unknown. Proteasome generates oligopeptides in the cytoplasm, which are further degraded quickly to amino acids depending on several aminopeptidases (Hershko A et al., 1992, Saric T et al., 2004). Although aminopeptidases may have a significant role in the maintenance of muscle mass, not much is known. Microarray analysis of transcripts in Rpt3 knockdown myocytes revealed an upregulated leucine aminopeptidase related genes. In this study, therefore, we examined the contribution of leucine aminopeptidase in the proliferation and differentiation of cultured myocytes.MethodsWe examined the consequence of leucine aminopeptidase inhibition on C2C12 myocytes under different culture conditions. Leucine aminopeptidase was inhibited by bestatin methyl ester (Bes‐ME; 100 μM), an aminopeptidase inhibitor. Proliferation was evaluated by counting the number of Ki67 (proliferation marker) positive cells. Differentiation was evaluated by myosin heavy chain (MyHC) staining and fusion index. Fusion index was calculated as the ratio of the nuclei number in myocytes with two or more nuclei versus the total number of nuclei. Additionally, phosphorylation of mammalian (or mechanistic) target of rapamycin (mTOR) and eukaryotic translation initiation factor 4E‐binding protein (4EBP1), were evaluated by Western Blotting because they are key factors for muscle protein synthesis.ResultsLeucine aminopeptidase inhibition by Bes‐ME significantly suppressed the activity of leucine aminopeptidase. The proliferation of C2C12 myoblasts was significantly reduced and the differentiation of C2C12 myoblasts was also markedly restricted after Bes‐ME administration. The protein expression levels of MyHC, Myogenin, and MyoD responsible for myodifferentiation were also significantly reduced. Furthermore, we also discovered that the phosphorylation of mTOR was significantly suppressed after leucine aminopeptidase inhibition.ConclusionsLeucine aminopeptidase is essential for myoblast proliferation and differentiation; however, further studies are required to clarify the role of aminopeptidase in skeletal muscle growth.Support or Funding InformationAcknowledgementsThis work was supported by JSPS KAKENHI Grant Number 18H04080.

Histone hyperacetylation may improve the preimplantation development and epigenetic status of cloned embryos

The current study investigated the mechanism of mini pig fetal fibroblasts in improving the epigenetic modification and preimplantation development of cloned embryos. The results showed that the increased AcH3K14 level was dose- and time-dependent. Histone hyperacetylation had no significant effect on cell morphology, cell viability, cell cycle, and relative gene (HDAC1, HAT1, DNMT3A, and BAX) expression. The treated cloned embryos had significantly higher development rates and the total nuclei number than the control (27.62 ± 6.94 % vs. 16.14 ± 10.55 %; 43.90 ± 18.39 vs. 33.06 ± 15.87; P < 0.05). The AcH3K14 level in the treated cloned blastocysts was close to that of IVF blastocysts (5.17 ± 0.93 vs. 5.45 ± 1.91, P > 0.05). The gene transcription (CDX2 and OCT4) of the treated cloned blastocysts was significantly up-regulated than the control (3.32 ± 0.51 vs. 2.05 ± 0.30; 1.21 ± 0.18 vs. 0.81 ± 0.09; P < 0.05). The improvement in the cloned embryo development and the partial correction of abnormal acetylation modification were not necessarily related to the cellular characteristics. This could be caused by histone hyperacetylation of mini pig fetal fibroblasts.

Inhibition of RNA synthesis during Scriptaid exposure enhances gene reprogramming in SCNT embryos.

Insufficient epigenetic reprogramming is incompatible with normal development of embryos produced by somatic cell nuclear transfer (SCNT), but treatment with histone deacetylases inhibitors (HDACi) enhances development of SCNT embryos. However, the mechanisms underpinning HDACi benefits in SCNT embryos remain largely uncharacterized. We hypothesized that, in addition to enhancing reprogramming, HDACi treatment may promote expression of genes not required for early development of SCNT embryos. To test this hypothesis, RNA synthesis was inhibited by treating bovine SCNT embryos with 5,6-dichlorobenzimidazole 1-β-D-ribofuranoside (DBR), which were concomitantly treated or not with Scriptaid (Scrip; an HDACi). Development to the blastocyst stage was significantly increased by treatment with Scrip alone (26.6%) or associated with DRB (28.6%) compared to Control (17.9%). The total number of nuclei was significantly improved only in embryos that were treated with both Scrip + DRB. Nuclear decondensation after SCNT was significantly increased by DRB treatment either alone or associated with Scrip. The relative mRNA expression, evaluated during the embryo genome activation (EGA) transition, revealed that some KDMs (KDM1A, KDM3A, KDM4C and KDM6A) and DNMT1 where prematurely expressed in Scrip-treated embryos. However, treatment with Scrip + DRB inhibited early mRNA expression of those genes, as well as several other KDMs (KDM4A, KDM4B, KDM5A, KDM5B, KDM5C and KDM7A) compared to embryos treated with Scrip alone. These findings revealed that HDACi improved development in SCNT embryos compared to Control, but altered the expression of genes involved in epigenetic regulation and did not improve embryo quality. Inhibition of RNA synthesis during HDACi treatment enhanced nuclear chromatin decondensation, modulated gene expression and improved SCNT embryo quality.

71 Removal of hypotaurine from porcine embryo culture medium decreases message for pro-apoptotic genes but does not affect development at low oxygen tension

Hypotaurine (HT) is a routine additive to embryo culture medium, acting primarily as an oxygen radical scavenger. However, the practice of culturing embryos at ~5% O2 has been widely adopted because this is more physiologically relevant to the oxygen tensions detected in the oviduct and uterus. Thus, the utility of HT may be diminished as fewer oxygen radicals are produced during culture at 5% O2. The objective here was to determine the effects of removing HT from our porcine embryo culture medium (MU2) on the development of embryos incubated at a lower oxygen tension (5% O2) compared with atmospheric oxygen (~20% O2). Porcine cumulus-oocyte complexes were aspirated, matured, and fertilized with standard procedures from our laboratory. Presumptive zygotes were cultured in 1 of 4 conditions: MU2 with 5mM HT at 5% O2 (low O2 +HT; control), MU2 without HT at 5% O2 (low O2 −HT), MU2 with 5mM HT at 20% O2 (high O2 +HT), or MU2 without HT at 20% O2 (high O2 −HT). The percentage of presumptive zygotes that developed to the blastocyst stage on Day 6 and total number of nuclei in the blastocysts were determined. The RNA was extracted from pools of 30 blastocysts, and cDNA was synthesised for quantitative PCR for genes associated with HT synthesis, oxidative stress, and apoptosis. Damage to DNA was assessed by TUNEL staining of Day 6 blastocysts. Data were analysed for normality by using a Shapiro-Wilk test, and differences between means were detected by using 2-way ANOVA followed by Tukey’s honest significant difference test with P&amp;lt;0.05 considered significant. Embryos cultured with high O2 −HT had significantly decreased blastocyst development compared with all other groups (26.2±2.5% v. 36.9-41.7±3.3-4.3%). Embryos cultured with low O2 −HT had significantly more nuclei than those cultured with high O2 +HT (50.5±1.5v. 45.5±1.2). Notably, differences in blastocyst development (41.7±3.3% v. 36.9±3.3%) or total number of nuclei (50.0±1.8v. 50.5±1.5) were not detected between embryos cultured with low O2 +HT or low O2 −HT. The abundance of messages for genes involved in HT synthesis (cysteine sulfinic acid decarboxylase [CSAD]) and oxidative stress (superoxide dismutase 1 [SOD1] and glutathione peroxidase 6 [GPX6]) did not differ between groups. Contrarily, messages for 2 pro-apoptotic markers (BCL2 associated agonist of cell death [BAD] and caspase 3 [CASP3]) were significantly increased in embryos cultured with +HT regardless of oxygen tension; however, percentages of DNA-damaged nuclei in blastocysts after TUNEL staining were not different between groups (5.4-6.5±0.5-1.0%). These results indicate that HT is not necessary for porcine pre-implantation development at 5% O2 but is beneficial at atmospheric oxygen tension. Further investigation is required to confirm if HT promotes apoptosis in pre-implantation embryos. This research was supported by Food for the 21st Century at the University of Missouri and R01 HD080636.

Varied approach of using MSCs for bovine embryo in vitro culture

The objective of the present study was to evaluate the effect of porcine Mesenchymal Stem Cells (MSCs) secreted factors on bovine in vitro embryo development by using MSCs in different culture systems: SOF medium, SOF medium conditioned by MSCs in monolayer and in reverse drop and by embryo culture in co-culture with MSCs. Statistically highly significant differences were noted between the number of blastocysts derived cultures in all tested culture systems. The in vitro culture in SOF turned out to be the most optimal. Statistically highly significant differences were observed in the number of blastocyst obtained between SOF and SOF in co-culture with MSCs (p < 0.0001), and between SOF and SOF conditioned (monolayer and drop) (p < 0.00001). The trials to produce blastocysts in SOF conditioned by MSCs in reverse drops and monolayer failed. The blastocysts were obtained and analysed by TUNEL only in two out of four experimental groups: SOF and SOF in co-culture with MSCs. There were no significant differences between any of analysed blastocysts’ groups neither in the total number of nuclei nor in the apoptotic features. Neither medium conditioning by MSCs in monolayer and in reverse drop nor embryo culture in co-culture with MSC turned out to be effective.

Abstract 3645: Quantifying cell populations in CTC enriched samples with the open-source imaging program ACCEPT

Abstract Introduction. Composition of cell suspensions enriched for Circulating Tumor Cells (CTC) by the CellSearch system has not yet been explored as the system only presents DAPI+/Cytokeratin+ objects to the operator to identify CTC. We used ACCEPT (http://github.com/LeonieZ/ACCEPT) to identify and characterize all DAPI+ nuclei in EpCAM enriched samples and explored avenues to increase the number of nuclei that can be assigned to a cell lineage. Addition of CD16, expressed on granulocytes and NK-cells, the detection of a cellular plasma membrane and the use of LED light, as opposed to the mercury arc lamp, to improve the excitation of CD45-APC were tested. Methods. In 127 controls and 300 samples from 192 metastatic lung cancer patients, CD16-PerCP was added to the CellSearch CTC identification antibody cocktail. A CellTracks Analyzer equipped with six filter cubes was used to acquire the images. For exploration, after the CellTracks image acquisition 5 cartridges were scanned again on a microscope that contains a LED light source to determine if this would improve the detection of CD45-APC. Also, wheat germ agglutinin (wga) conjugated to AlexaFluor488 stains the cellular plasma membrane and was added to the CellSearch antibody cocktail to discriminate between bare nuclei and unstained cells in 4 samples. The stack of fluorescence images was analyzed with the open source imaging program ACCEPT to define and identify various cell populations. Results. In 427 samples 26,285 ±26,590 [603-156,519] (mean, SD, [range]) nuclei were identified with ACCEPT in the CellSearch images of which only 14% ±15 were identified as leukocytes or CTC, remaining 86% ±15 cells unidentified. Adding CD16 increased the leukocyte population by 47% ±17, thereby reducing the unidentified population to 39% ±19 of the total number of nuclei. CD16-staining was found on 13% of the CTC. Using a LED-light source improved the identification of APC+ cells, and reduced the amount of unidentified nuclei in these samples from 29% (±5) to 14% (±10). Exploration with wga indicated that 43% (±11) (mean 987 cells, range 331-2,638 cells) of the unstained cells contained a cellular membrane. Conclusion. In 427 blood samples run on CellSearch, only 14% of the nuclei obtained after EpCAM enrichment could be assigned as a leukocyte or a CTC. By adding CD16 to the antibody cocktail, the unidentified population is reduced from 86% to 39%. Also, 13% of nuclei scored as CTC express CD16, suggesting these are granulocytes that are incorrectly designated as tumor cells. Using a LED light source improves the identification of dimly stained CD45+ cells. Exploration with wga indicates that 57% of the unidentified nuclei are simply bare nuclei and the origin of 43% still remains unknown. Further studies are needed to determine whether EpCAM+/Cytokeratin- CTC are present among the nucleated cells of which the origin is not yet known. Citation Format: Sanne De Wit, Leonie L. Zeune, T. Jeroen N. Hiltermann, Harry J. Groen, Leon W.M. Terstappen. Quantifying cell populations in CTC enriched samples with the open-source imaging program ACCEPT [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3645.

Evaluation of Endothelial and Vascular-Derived Progenitor Cell Populations in the Proximal and Distal UCL of the Elbow: A Comparative Study.

Background:Vascular-derived progenitor and endothelial cell populations (CD31, CD34, CD146) are capable of multipotent differentiation at the site of injured ligamentous tissue to aid in the intrinsic healing response. Proximal ulnar collateral ligament (UCL) tears have been reported to have better healing capability when compared with distal UCL tears.Purpose:To compare the vascular composition of the proximal and distal insertions of the anterior bundle of the UCL of the elbow via known markers of endothelial and vascular-derived progenitor cells (CD31, CD34, CD146).Study Design:Descriptive laboratory study.Methods:UCLs were harvested from 10 nonpaired fresh-frozen human cadaveric elbows and transected into proximal and distal portions. Endothelial and vascular-derived progenitor cell densities were assessed with 4 staining groups: CD31 (immunohistochemistry) and CD31/α-smooth muscle actin (α-SMA), CD34/α-SMA, and CD146/α-SMA (immunofluorescence). CD31 immunohistochemistry identified endothelial progenitor cells in the UCL. Later staining of the same slides with α-SMA demonstrated the relationship of progenitor cells to the surrounding vasculature. Fluorescent staining was quantified by calculating the proportion of positively stained nuclei versus the total number of nuclei in the proximal and distal UCL.Results:CD31+ cells were present in the proximal and distal sections of all 10 UCLs. Fluorescent staining revealed no significant differences in the ratio of CD31 to total nuclei between the distal (median, 36% [range, 23%-53%]) and proximal UCL (39% [22%-56%]) (P = .432, Wilcoxon signed-rank test). Similarly, no differences were seen between CD34 distal (39% [24%-64%]) and proximal regions (46% [28%-63%]) (P = .846, Wilcoxon signed-rank test) or CD146 distal (40% [12%-65%]) and proximal regions (40% [22%-51%]) (P ≥ .999, Wilcoxon signed-rank test).Conclusion:Analysis of UCL tissues demonstrated equal distributions of vascular endothelial and vascular-derived progenitor cell markers throughout the proximal and distal UCL. Unlike that of the medial collateral ligament of the knee, the microvascular composition of the proximal and distal UCL insertions was not different, suggesting a well-vascularized ligament throughout its course.Clinical Relevance:These findings investigate one of the possible contributors to UCL healing after injury, which may provide insight into operative and nonoperative management of UCL injuries in the future. This study also indicates that reasons other than differences in progenitor cell density alone may explain the clinical healing differences seen between proximal and distal UCL tears. A better understanding of the microvascular environment and associated blood supply is warranted to understand the healing capability of the UCL.

Glutamine supplementation enhances development of in vitro-produced porcine embryos and increases leucine consumption from the medium.

Improper composition of culture medium contributes to reduced viability of in vitro-produced embryos. Glutamine (Gln) is a crucial amino acid for preimplantation embryos as it supports proliferation and is involved in many different biosynthetic pathways. Previous transcriptional profiling revealed several upregulated genes related to Gln transport and metabolism in in vitro-produced porcine blastocysts compared to in vivo-produced counterparts, indicating a potential deficiency in the culture medium. Therefore, the objective of this study was to determine the effects of Gln supplementation on in vitro-produced porcine embryo development, gene expression, and metabolism. Cleaved embryos were selected and cultured in MU2 medium supplemented with 1 mM Gln (control), 3.75 mM Gln (+Gln), 3.75 mM GlutaMAX (+Max), or 3.75 mM alanine (+Ala) until day 6. Embryos cultured with+Gln or+Max had increased development to the blastocyst stage and total number of nuclei compared to the control (P<0.05). Moreover, expression of misregulated transcripts involved in glutamine and glutamate transport and metabolism was corrected when embryos were cultured with+Gln or+Max. Metabolomics analysis revealed increased production of glutamine and glutamate into the medium by embryos cultured with+Max and increased consumption of leucine by embryos cultured with+Gln or+Max. As an indicator of cellular health, mitochondrial membrane potential was increased when embryos were cultured with+Max which was coincident with decreased apoptosis in these blastocysts. Lastly, two embryo transfers by using embryos cultured with+Max resulted in viable piglets, confirming that this treatment is consistent with in vivo developmental competence.

Nuclear IHC enumeration: A digital phantom to evaluate the performance of automated algorithms in digital pathology.

Automatic and accurate detection of positive and negative nuclei from images of immunostained tissue biopsies is critical to the success of digital pathology. The evaluation of most nuclei detection algorithms relies on manually generated ground truth prepared by pathologists, which is unfortunately time-consuming and suffers from inter-pathologist variability. In this work, we developed a digital immunohistochemistry (IHC) phantom that can be used for evaluating computer algorithms for enumeration of IHC positive cells. Our phantom development consists of two main steps, 1) extraction of the individual as well as nuclei clumps of both positive and negative nuclei from real WSI images, and 2) systematic placement of the extracted nuclei clumps on an image canvas. The resulting images are visually similar to the original tissue images. We created a set of 42 images with different concentrations of positive and negative nuclei. These images were evaluated by four board certified pathologists in the task of estimating the ratio of positive to total number of nuclei. The resulting concordance correlation coefficients (CCC) between the pathologist and the true ratio range from 0.86 to 0.95 (point estimates). The same ratio was also computed by an automated computer algorithm, which yielded a CCC value of 0.99. Reading the phantom data with known ground truth, the human readers show substantial variability and lower average performance than the computer algorithm in terms of CCC. This shows the limitation of using a human reader panel to establish a reference standard for the evaluation of computer algorithms, thereby highlighting the usefulness of the phantom developed in this work. Using our phantom images, we further developed a function that can approximate the true ratio from the area of the positive and negative nuclei, hence avoiding the need to detect individual nuclei. The predicted ratios of 10 held-out images using the function (trained on 32 images) are within ±2.68% of the true ratio. Moreover, we also report the evaluation of a computerized image analysis method on the synthetic tissue dataset.

Varied Approach of Using MSCs for Bovine Embryo In Vitro Culture

ABSTRACTThe objective of the present study was to evaluate the effect of porcine mesenchymal stem cells (MSCs) secreted factors on bovine in vitro embryo development using MSCs in different culture systems: SOF medium, SOF medium conditioned by MSCs in monolayer, and in reverse drop and by embryo culture in coculture with MSCs. Statistically highly significant differences were noted between the number of blastocysts derived cultures in all tested culture systems. The in vitro culture in SOF turned out to be the most optimal. Statistically highly significant differences were observed in the number of blastocyst obtained between SOF and SOF in coculture with MSCs (P < 0.0001), and between SOF and SOF conditioned (monolayer and drop) (P < 0.00001). The trials to produce blastocysts in SOF conditioned by MSCs in reverse drops and monolayer failed. The blastocysts were obtained and analyzed by TUNEL only in two out of four experimental groups: SOF and SOF in coculture with MSCs. There were no significant differences between any of analyzed blastocysts’ groups neither in the total number of nuclei nor in the apoptotic features. Neither medium conditioning by MSCs in monolayer and in reverse drop nor embryo culture in coculture with MSC turned out to be effective.

Correlation of proliferative index with various clinicopathologic prognostic parameters in primary breast carcinoma: A study from North India.

Routine assessment of cell proliferation is recommended in the pathologic evaluation for all breast cancers. Considering the poor reproducibility and interobserver variability in mitotic counts, Ki-67 is an easily available and reliable substitute for mitotic counts and has been shown to have a significant relationship with the histologic grade of malignancy and the mitotic activity of tumors. Our study aimed at exploring Ki-67 expression and studying its correlation with other established prognostic parameters. Seventy-five cases of primary breast cancer undergoing radical or modified radical mastectomy constituted the study group. Estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2)/neu, and Ki-67 was assessed in each case. Ki-67 labeling index (Ki-67-LI) was estimated as the number of positive nuclei divided by total number of nuclei scanned counting a minimum of 1000 cells in 10 selected high power fields that displayed the highest immunoreactivity and was expressed as percentage. Ki-67 expression was correlated with various clinicopathologic prognostic parameters including age, tumor size, tumor type, axillary lymph node status, and histologic tumor grade. A statistically significant direct association was observed between Ki-67-LI and tumor size, histologic grade and Nottingham prognostic index. A statistically significant inverse association was observed between Ki-67-LI and ER and PR expression. However, no association was observed between Ki-67-LI and menopausal status, lymph node involvement and HER2/neu expression. Based on our results, we concluded that modified Bloom-Richardson (MBR) grading has been recognized as a treatment related indicator. The accuracy and reliability in grading have always been a matter of concern, hence, the reproducibility of grading should be enhanced. Ki-67-a proliferation marker is easily identified and provides comparable accurate information. In contrast to poor reproducibility of mitotic counts, Ki-67 can achieve high agreement between pathologists; is more reproducible; adds complementary value to the MBR grading system and correlates well with other clinicopathologic parameters. It may act as a significant prognostic indicator for routine clinical use and be helpful for selection of adjuvant treatment. It can also add value in categorizing Grade II tumors into two prognostic subgroups with prognosis equivalent to Grades I and III, respectively.

Apoptosis in trophoblast in pre-eclampsia and intrauterine growth retardation: A light microscopy study

Background: Pre-eclampsia and Intrauterine growth retardation (IUGR) affects 3% and 10% of all pregnancies. Apoptosis, a programmed cell death is known to occur as a part of both physiological and pathological process. This study was done to calculate apoptotic index in placental trophoblasts of pre-eclampsia, IUGR and compare it with normal term placenta. The aim of this study is to calculate apoptotic index in cytotrophoblast and syncytiotrophoblast in preeclampsia and IUGR and compare it with placenta from normal pregnancyMethods: A total of fifty four placentas were included in the study. 18/54 cases were from pre-eclampsia patients, 18/54 cases were from IUGR patients, 18/54 were age matched controls. Hematoxylin and eosin stained sections from representative were screened under oil immersion using Olympus microscope (100X) for apoptotic bodies and apoptotic index was calculated by counting the total number of apoptotic bodies and dividing it by total number of nuclei counted in the trophoblasts multipled by 100. Data was entered into Microsoft excel data sheet and was analyzed using SPSS 22 version software. ANOVA was the test of significance used. P value<0.05 was considered significant. Result: Apoptotic bodies and index were maximum in cytotrophoblast and syncytiocytotrophoblast of preeclampsia as compared with IUGR and normal placentas. Statistically significant difference was observed in apoptotic index in cytotrophoblast and apoptotic index in Syncytiotrophoblast between three groups.Conclusion: Increased number of apoptotic bodies and index were seen in trophoblast of pre-eclampsia cases as compared with IUGR and normal placentas which may be related to onset of maternal inflammatory syndrome.DOI:10.21276/APALM.1693