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The phytoplankton composition of Sawa Lake was studied monthly for 18 months, from October 1999 to March 2001. A phytoplankton net (20 $) was used to collect phytoplankton samples. A total of 51 algal taxa was identified belong to diatoms (Bacillariophyceae), blue greens (Cyanophyceae), green algae (Chlorophyceae) and euglenoids (Euglenophyceae). Diatoms were dominated in species number (64.7%) and total cell number (68.6%) followed by blue greens (23.5%) and (15.5%), and the green (7.8%) and (14.5%) respectively. Bimodal seasonal peaks of chlorophyll-a and phytoplankton population were observed in the lake.

Microorganisms can be defined as a small living forms of life, even can't be viewable with the naked eyes. The three types of microorganisms named Bacteria, Yeasts and Molds in which almost always there may be confusion with their functions, but they are just as real and alive as we are. They eat, grow, reproduce and die also. The tumour cells has also ability to reproduce themselves, to grow in numbers and die to reduce their density in some specific region. In this work we try show the growth for both tumour cells and microorganism with the help of mathematical model and also try to compare the numerical results with the help of Python. This model consists of a system of partial differential equations incorporated with different factors like migrating cells, proliferating cells and nutrient which effects the growth most. At last the results shows the variation of different classes of cell densities with respect to growth function and as well as with the total cell density.

The present study was designed to explore the relationship between radioactivity at Al-Rohban soil in Al-Najaf Governorate, located 30 km away from Najaf city center, and its microbial content. The radiological survey was conducted by γ–ray spectrometry, using purity Germanium (HPGe) detector. A selected surface soil layer (10 cm depth, 50 and 100 m expansion) was tested. The physical analyses were conducted in the Ministry of Environment, Center for Prevention of Radiation. The results showed that the estimated concentrations of Bi-214, Ra-226, Ac-228, Th-232, K-40 and Cs-137 were 47.93, 81.87, 5.03, 1.63, 126.3 and 3.5 Bq/Kg, respectively. Isotopes average concentrations were equivalent to the lowest specified International Atomic Energy standards. As related to the analysis of bacterial content in the soil sample, the total cell count (in cells per gram of soil) in the different areas studied (R1, R2, R3 and R4) had values of 70000, 200, 60000 and 300 cell/gm., respectively. The statistical analysis of these results revealed no relationship between radioactivity and microorganisms existence.

Search to a new alternative to control bovine ticks (Rhipicephalus microplus), the present study aimed to evaluate the acaricidal activity of organo-modified siloxane alone and in association with different commercial products or with piperonyl butoxide (BPO). Engorged females were subjected to an in vitro immersion test and 10 groups were used: control, 0.5% siloxane, 1% siloxane, 2% siloxane, 0.5% siloxane + 5% BPO, 1% siloxane + 5% BPO, 2% siloxane + 5% BPO, commercial product, 0.5% siloxane + commercial product, and 1% siloxane + commercial product. After immersion, engorged females were incubated for 14 days for oviposition and hatchability tests. Another immersion test was performed with 5% siloxane and 2.5% siloxane + 10% BPO to evaluate the histopathological changes. Then, engorged females were incubated for 0, 2, 4, 8, 12, 24, 36, 48, 60, 72, 84, 96, 120, 144, and 168 h and immersed in 10% formaldehyde for later analyses. The unassociated siloxane showed an acaricide efficacy of 93.88% at 2% concentration, and when associated with 5% BPO, it reached 100% efficacy at all tested concentrations. The tested commercial products showed enhanced efficacy when associated with siloxane. Histopathological analysis showed cell changes in both treatments and total cell disintegration after 120 h in the 5% siloxane group and after 96 h in the 2.5% siloxane + 10% BPO group. Therefore, siloxane alone or in combination is an alternative against R. microplus, and siloxane enhances the efficacy of available commercial products.

This study aims to determine the effect of type and concentration of additives in production of local microorganisms (MOL) on overall qualities (physical, chemical, sensory, and microbiological). The method used in this study was a factorial completely randomized design (CRD) consisting of two factors. The first factor was the type of additive (J) which consisted of 4 levels, namely rice (J1), soil (J2), leaf bokashi (J3), and leaf compost (J4). The second factor was the concentration of additives (K) which consisted of 2 levels, namely 7.5% (K1) and 10% (K2). The product was analysed total cell counts (TCC), pH, temperature, and organoleptic tests (hedonic test) on color and organoleptic (descriptive test) on aroma. The best MOL was J4K2 (combination between leaf compost and 10%). The J4K2 MOL had the following characteristics: TCC of 9.6x107 CFU / ml, pH value of 3.7, color hedonic test of 4.3 (preferred by panelists) and aroma descriptive test of 4.3 (tape aroma / medium acid).

Purpose The aim of the present study was to investigate the effect of glutathione ethyl ester (GSH‐OEt) in the recovery medium on the developmental competence of mouse vitrified‐warmed MII oocytes. Methods Vitrified‐warmed oocytes were incubated for 1 h in recovery medium in the presence or absence of 0.5 mM GSH‐OEt. The authors examined the effects of GSH‐OEt, first on the levels of glutathione (GSH) and reactive oxygen species (ROS) in vitrified‐warmed oocytes, and second, on in vitro blastocyst development, division speed to blastocysts, and total cell numbers of blastocysts from vitrified‐warmed oocytes fertilized by Intracytoplasmic sperm injection (ICSI). Results Adding GSH‐OEt to the recovery medium significantly (p

The efficiency with which polycationic peptides penetrate the cytosol depends on the number and overall patterning of arginine residues. While general trends and unusually penetrant patterns of arginine residues have been discovered, prior work has not effectively leveraged high-throughput screens to measure cytosolic penetration, rather than total cell uptake. In this work, we adapted the chloroalkane penetration assay, which exclusively measures cytosolic penetration, to screen peptide libraries in a high-throughput, quantitative, and automation-ready manner. We demonstrate the usefulness of the screening platform by efficiently exploring how the number, patterning, and stereochemistry of arginine residues affect the cytosolic penetration of a model 10-residue peptide.

This study aimed to investigate the physicochemical and textural characteristics of bread produced from doughs composed of high-pressure homogenization (HPH) treated flour slurries. For this purpose, bread samples were produced from doughs composed of HPH treated flour slurries at 0 (control), 20, 30, 50, and 70 MPa. HPH treatment resulted in decreased particle size with more uniformity in flour slurries. A monomodal particle size distribution was determined in slurries. HPH obtained higher firmness, consistency, and cohesivity in slurries. In bread samples, HPH increased yellowness and lightness values. The bread samples prepared from HPH treatment had 1.09–1.25 times higher specific volumes than the control sample. HPH treatment provided decreased hardness and increased cohesiveness and springiness values. Image analysis results showed HPH treatment decreased total cell count, whereas increased mean cell area of bread samples. The findings revealed that HPH treatment results in higher quality bread compared to control.

The changes of the total cell number in doenjang (Korean traditional fermented soybean paste) by conventional conduction heating, high pressure non-thermal treatment, and ohmic heating were compared. A total of (101–102) CFU/g cells were decreased by heating at (100–105) °C for 10 min. The inactivation rate was improved when heated to a temperature higher than 110 °C, but the taste, color of doenjang were severely changed. Inactivation by high pressure at (200–800) MPa was not achieved, because the total cell did not reach a reduction of 101 CFU/g. The total bacterial counts of 103 CFU/g were decreased during ohmic heating at 15 V and 60 Hz for 10 min, and showed the most effective inactivation. Therefore, application of the ohmic heating in doenjang with high viscosity can kill target microorganisms related to quality deterioration, and rapid and uniform ohmic heating leads to reduction in sensory quality damage.

Objectives The aim of the study is to synthesize the evidence and evaluate the total cell-free deoxyribonucleic (cfDNA) associated with the prediction of preeclampsia (PE). Total cfDNA is constituted by both cell-free fetal DNA (cffDNA) originated mainly from the placenta, and maternal cfDNA derived from maternal leukocytes. Methods A systematic review was conducted by searching PubMed and Medline. Literature reporting levels of total cfDNA in the development of PE was included. Studies that only reported cffDNA, but no cfDNA concentrations were not included in this review. Results Eight studies were included. Seven reported values of cfDNA in PE patients, regardless of early or late onset PE, six of which demonstrated a significant increase of cfDNA in patients who subsequently developed PE. Seven studies evaluated cfDNA levels in the first trimester, six of which showed significant increase of cfDNA concentrations in women who later developed PE. Five studies investigated cfDNA levels in the second trimester, all presenting increased total cfDNA levels in the PE group compared with normal controls. Conclusion Total cfDNA may play a role as a biochemical marker of PE, compared with fetal cfDNA. Large prospective studies with homogeneous populations and standardized methodology are needed to further confirm its predictive value.

Embryonic morphofunctional competence features regulating post-cryopreservation resumption of development are still poorly understood. In this study, we investigated the correlation between embryonic viability and the speed and ability to resume post-cryopreservation development. Thus, in vitro produced blastocysts were vitrified by the Cryotop method using standard protocols. Subsequently, the embryos were warmed, re-cultured, and classified into groups according to their speed and ability to resume post-cryopreservation development: embryos not re-expanded at 12h (NE12); embryos re-expanded at 12h and hatched at 24h (E12H24); embryos re-expanded at 12h and hatched at 48h (E12H48); embryos re-expanded at 12h and not hatched at 48h (E12NH48). Subsequently, the embryos were subjected to monitoring of total cell number and apoptosis. We identified that the blastocoel's ability to re-expand was negatively affected by the significant higher percentage of apoptotic cells observed in the NE12 group than in the other groups. A greater (P 0.05) number of total cells, found in groups E12H24 and E12H48, seems to have a positive influence on the hatching capacity of blastocysts after cryopreservation. In conclusion, the total number of cells and apoptotic index correlated with the speed and ability to resume post-cryopreservation development. Apoptosis was a determinant for embryonic re-expansion, and the total cell number was crucial for blastocyst hatching.

Our aim is to clarify the association of the bioelectrical impedance-derived 50-kHz phase angle (φ50 ) with electrical parameters regarding the Cole-Cole model and clinical parameters. A total of 440 sets of bioelectrical impedance data from pre- and post-hemodialysis from 157 patients were used. Resistance at infinite frequency (Rinf ), resistance at 0 frequency (R0 ), capacitance of the cell membrane (Cm), and a parameter for the distribution of the time constant (α) were examined as electrical parameters. Normally hydrated lean tissue mass as a percentage of the dry weight (PNHLT ), excess fluid mass as a percentage of the dry weight (PExF ), body mass index (BMI), age, and sex (Nsex , 0 for male, 1 for female) were examined as clinical parameters. φ50 increased with the decrease in Rinf /R0 and α and also with the increase in Cm (multiple regression coefficients [β]: pre/post, -0.886/-0.936, -0.175/-0.212, and 0.167/0.141), which determined the ratio of intra- to extracellular fluid volume (ICV/ECV), tissue homogeneity, and total cell mass. φ50 increased with an increase in PNHLT and BMI and decrease in PExF , Nsex , and age (β: pre/post, 0.654/0.581, 0.466/0.412, -0.483/-0.473, -0.216/-0.154, and -0.145/-0.127). The concordance correlation coefficient between φ50 for pre- and post-hemodialysis (ρ = 0.772) may be improved (ρ = 0.950) by adding a multiplication of 0.2 and PExF to φ50 for correction. φ50 may be used to estimate body composition through the association with ICV/ECV, tissue homogeneity, and total cell mass. The correction for excess fluid is essential in order to use φ50 as a marker of body composition related to nutrition.

Elevation of total cell-free DNA (cfDNA) in patients with preeclampsia is well-known; however, whether this change precedes the onset of symptoms remains inconclusive. Here, we conducted a nested case–control study to determine the elevation of cfDNA levels in women who subsequently developed preeclampsia. Methylated HYP2 (m-HYP2) levels were determined in 68 blood samples collected from women with hypertensive disorders of pregnancy, along with 136 control samples, using real-time quantitative PCR. The measured m-HYP2 levels were converted to multiples of the median (MoM) values for correction of maternal characteristics. The m-HYP2 levels and MoM values in patients with preeclampsia were significantly higher than in controls during the third trimester (P P P

This study aimed at investigating for the first time whether thermosonication (TS), a non-thermal processing technique, could induce the beer-spoilage bacterium, Lactobacillus acetotolerans CGMCC 7.150, into the viable but putatively non-culturable (VPNC) state. After determining culturable, viable and total cell counts, we found that L. acetotolerans in beer could enter the VPNC state by TS at a constant frequency of 24 kHz at 50 °C at three relatively low volumetric powers (0.6, 1.2 and 1.8 W/mL, but not 2.4 W/mL). Furthermore, with the increase of volumetric power, the time required for L. acetotolerans cells to enter the VPNC state shortened. The addition of Tween 80 at a concentration of 0.1% v/v into de Man Rogosa Sharpe agar enabled the VPNC cells to regain culturability with a recovery time of approximately 3 days. However, their recovery capability decreased with the increasing volumetric power. Scanning electron microscopy analysis showed that cells decreased in size and gradually changed morphology from short rods to coccoids upon entering the VPNC state. An insignificant difference in beer-spoilage ability was found between the exponential-phase and VPNC cells. The resistance of these VPNC cells to 0.025 M NaOH and 0.025 M KOH was enhanced compared with the exponential-phase cells.

The present study aimed to examine the effects of adding different concentrations of resveratrol during in vitro culture (IVC) alone and during both in vitro maturation (IVM) and IVC on ovine blastocyst yield and quality. Therefore, this study was conducted in two separate experiments. The first experiment was carried out to test the effect of different concentrations of resveratrol (0, 0.1, 0.25, 0.5, 2.0, and 5.0 μM) in the IVC medium on cleavage, morula, developmental potential of blastocyst, and total cell number (TCN) of the embryos. Addition of 0.25 and 0.5 μM of resveratrol during IVC significantly enhanced morula and blastocyst rates as compared with other groups (P0.05). Also, supplementation of the IVC medium with 0.5 μM of resveratrol had beneficial effects on trophectoderm cells (TE), inner cell mass (ICM), and TCN of blastocysts. In the second experiment, the same concentrations of resveratrol (0, 0.1, 0.25, 0.5, 2.0, and 5.0 μM) were applied during IVM and IVC. Therefore, oocytes were matured in vitro in the presence of different concentrations of resveratrol for 22-24 h. After in vitro fertilization, presumptive zygotes were cultured in media containing 0, 0.1, 0.25, 0.5, 2.0, and 5.0 μM of resveratrol for 8 d. No significant difference was found in the percentage of oocytes developed to MII (0, 0.1, 0.25, 0.5, and 2.0 μM of resveratrol), but the percentage of oocytes developed to MII were significantly lower in 5.0 μM of resveratrol in comparison with other groups. Addition of 0.5 μM of resveratrol to the maturation and culture media significantly increased morula and blastocyst rates compared with other groups (P0.05). However, a too high concentration of resveratrol (5.0 μM) during IVM and IVC decreased cleavage, morula, and blastocyst rates compared with low concentrations (P0.05). Treatment with 0.5/0.5 μM of resveratrol during IVM/IVC significantly improved the TE, ICM, and TCN of blastocysts. In conclusion, sequential treatment with 0.5 μM of resveratrol during IVM and IVC and during IVC alone improved the developmental competence of oocytes, which was reflected in higher blastocyst rates and TCN of blastocysts.

Selecting patients for early clinical trials is a challenging process and clinicians lack sufficient tools to predict overall survival (OS). Circulating cell-free DNA (cfDNA) has recently been shown to be a promising prognostic biomarker. The aim of this study was to investigate whether baseline cfDNA measurement could improve the prognostic information of the Royal Marsden Hospital (RMH) score. Solid tumour patients referred for phase I trials were included in the Copenhagen Personalized Oncology (CoPPO) programme. Baseline characteristics were collected prospectively, including the RMH prognostic score, Eastern Cooperative Oncology Group (ECOG) performance status and concentration of cfDNA per millilitre plasma. Cox proportional hazards model was used to assess the prognostic value of baseline variables. Plasma cfDNA concentration was quantifiable in 302 patients out of a total of 419 included in the study period of 2 years and 5 months. The RMH score was confirmed to be associated with OS. Cell-free DNA was shown to be an independent prognostic marker of OS and improved the risk model, including RMH, performance status and age. Furthermore, both plasma cfDNA concentration and RMH score were associated with treatment allocation (p

Blood counting is one of the most commonly ordered clinical assays, and is often part of the basis for initial diagnosis and screening for disease. While substantial prior research has shown the ability of portable instruments to accurately produce blood counts through image- or flow-based cytometry, these methods require complex sample preparation using either costly commercial imaging chambers or complicated reagents. To address these issues, in this paper we developed a method to prepare trace volumes of whole blood aimed at portable blood counting. The strategy is based on pre-storing dry-form reagents and fabricating a specifically designed cell counter. In order to obtain total cell counts for red blood cells, platelets, and 3-part differentials of white blood cells, two parallel counting chambers with different depths are made from cost- and environmentally friendly materials using soft lithography. As little as 1 μl of whole blood is prepared with pre-stored reagents in centrifuge vials, whereas red blood cells are sphered and white blood cells are stained at the same time. Driven by the capillary force, prepared blood samples enter the hydrophilic chambers automatically. Monolayers of cells are formed when the blood dilution factors and the chamber depths are co-optimized. Combined with our previous custom-built instrument and automated analysis algorithm, the sample preparation strategy allows producing counting results with excellent agreement to a gold-standard clinical hematology instrument. The success of this preparation method may further advance applications of our technology for global use in low-resource settings where central hematology laboratories are not accessible. Graphical abstract Graphical Abstract.

The evaluation of surgical damage is challenging because of the lack of specific biomarkers. Total cell-free DNA (cfDNA) levels have been reported to increase with external trauma and may be a biomarker for tissue damage. To investigate the utility of perioperative total cfDNA levels in evaluating surgical damage in urological surgeries. This multicenter, prospective, observational study included 196 patients scheduled for urological surgeries between September 2020 and July 2021. The primary outcome was the change in total cfDNA levels before and after urological surgery. The secondary outcome was the effect of surgical type on total cfDNA ratio before and after urological surgery. The postoperative median total cfDNA level of the 196 patients was significantly increased 2.5-fold compared to the preoperative level (185.2 ng/mL vs. 406.7 ng/mL, P

Background Placental derived cell‐free DNA (cfDNA), widely utilized for prenatal screening, may serve as a biomarker for preeclampsia. To determine whether cfDNA parameters are altered in preeclampsia, we conducted a case‐control study using prospectively collected maternal plasma (n=20 preeclampsia, n=22 normal) using our in‐house validated prenatal screening assay. Methods and Results Isolated cfDNA was quantified, sequenced using Illumina NextSeq 500, and the placental‐derived fraction was determined. Clinical and test characteristics were compared between preeclampsia and controls, followed by comparisons within the preeclampsia cohort dichotomized by cfDNA concentration. Lastly, cfDNA parameters in preeclampsia were correlated with markers of disease severity. Maternal age, body mass index, gestational age at delivery, cesarean rate, and neonatal birthweight were expectedly different between groups ( P ≤0.05). The placental‐derived cfDNA fraction did not differ between groups (21.4% versus 16.9%, P =0.06); however, total cfDNA was more than 10 times higher in preeclampsia (1235 versus 106.5 pg/µL, P P =0.01). The dichotomized preeclampsia group with the highest cfDNA concentration delivered earlier (33.2 versus 36.6 weeks, P =0.02) and had lower placental‐derived fractions (9.1% versus 21.4%, P =0.04). Among preeclampsia cases, higher total cfDNA correlated with earlier gestational age at delivery ( P =0.01) and higher maximum systolic blood pressure ( P =0.04). Conclusions At diagnosis, total cfDNA is notably higher in preeclampsia, whereas the placental derived fraction remains similar to healthy pregnancies. In preeclampsia, higher total cfDNA correlates with earlier gestational age at delivery and higher systolic blood pressure. These findings may indicate increased release of cfDNA from maternal tissue injury.

Cell quantification in 3D atherosclerotic plaque immunofluorescent images is critical in improving understanding of plaque composition and possibly preventing rupture. Manual annotation of these cells is required to build predictive models that classify cell types within the lesion. Due to the difficulty and the amount of time required to label a substantial number of cells, researchers must often aggregate multiple annotation strategies with various limitations. We present several methods to create segmentation masks to include polygon outlines, basic shapes, and small dots. The combination of these methods leverages the nuclei polygon annotations that must be linked to the other cellular markers. A U-Net segmentation model was trained with these annotations and used for evaluation. The models achieved a test set total cell count percent error of -2.7 when assessing total cell count alone; and 5.3, and 1.4 when counting ACTA2+ or LGALS3+ cells respectively. Further analysis of these cell counts with patient phenotype data may lead to findings that ultimately reduce heart attack and stroke rates.

In this study, the minimal residual disease (MRD) levels in patients with multiple myeloma (MM) were assessed by comparing the new 8-color single-tube multiparameter flow cytometry method (DuraClone), which reduces the cost of antibodies and labor burden of laboratories, with the EuroFlow next-generation flow (NGF) method. A total of 96 samples derived from 69 patients with MM were assessed to determine the total cell acquisition number (tCAN), percentages of total and normal plasma cells (PCs), and MRD levels using two methods. We found that the tCAN was significantly higher with EuroFlow-NGF than with DuraClone (median 8.6 × 106 vs. 5.7 × 106; p p

Conventional lithography techniques are unable to achieve the resolution required by advance technology nodes. Multiple patterning lithography (MPL) has been introduced as a viable solution. Besides, new standard cell structure with multiple middle-of-line (MOL) layers is adopted to improve intra-cell routability. A mixed-cell-height standard cell library, consisting of cells of single-row and multiple-row heights, is also used in designs for power, performance and area concerns. As a result, it becomes increasingly difficult to get a feasible placement for a mixed-cell-height design where multiple cell layers require MPL. In this paper, we present a methodology to refine a given mixed-cell-height standard cell placement for satisfying MPL requirements on multiple cell layers as much as possible, while minimizing the total cell displacement. We introduce the concept of uncolored cell group (UCG) to facilitate the effective removal of coloring conflicts. By eliminating UCGs without generating any new coloring conflict around them, the number of UCGs is effectively reduced in the local and global refinement stages of our methodology. We report promising experimental results to demonstrate the efficacy of our methodology.

Objective: In biomanufacturing there is a need for quantitative methods to map cell viability and density inside 3D bioreactors to assess health and proliferation over time. Recently, noninvasive MRI readouts of cell density have been achieved. However, the ratio of live to dead cells was not varied. Herein we present an approach for measuring the viability of cells embedded in a hydrogel independently from cell density to map cell number and health. Methods: Independent quantification of cell viability and density was achieved by calibrating the $\boldsymbol{^1}$ H magnetization transfer- (MT) and diffusion-weighted NMR signals to samples of known cell density and viability using a multivariate approach. Maps of cell viability and density were generated by weighting NMR images by these parameters post-calibration. Results: Using this method, the limits of detection (LODs) of total cell density and viable cell density were found to be $\boldsymbol{3.88 \times 10^{8}}$ cells $\boldsymbol{\cdot }$ mL $\boldsymbol{^{-1}\cdot }$ Hz $\boldsymbol{^{-1/2}}$ and $\boldsymbol{2.36 \times 10^{9}}$ viable cells $\boldsymbol{\cdot }$ mL $\boldsymbol{^{-1}\cdot }$ Hz $\boldsymbol{^{-1/2}}$ respectively. Conclusion: This mapping technique provides a noninvasive means of visualizing cell viability and number density within optically opaque bioreactors. Significance: We anticipate that such nondestructive readouts will provide valuable feedback for monitoring and controlling cell populations in bioreactors.

Cell counting has become an essential method for monitoring the viability and proliferation of cells. A hemacytometer is the standard device used to measure cell numbers in most laboratories which are typically automated to increase throughput. The principle of both manual and automated hemacytometers is to calculate cell numbers with a fixed volume within a set measurement range (105 ~ 106 cells/ml). If the cell concentration of the unknown sample is outside the range of the hemacytometer, the sample must be prepared again by increasing or decreasing the cell concentration. We have developed a new hemacytometer that has a multi-volume chamber with 4 different depths containing different volumes (0.1, 0.2, 0.4, 0.8 µl respectively). A multi-volume hemacytometer can measure cell concentration with a maximum of 106 cells/ml to a minimum of 5 × 103 cells/ml. Compared to a typical hemacytometer with a fixed volume of 0.1 µl, the minimum measurable cell concentration of 5 × 103 cells/ml on the multi-volume hemacytometer is twenty times lower. Additionally, the Multi-Volume Cell Counting model (cell concentration calculation with the slope value of cell number in multi-chambers) showed a wide measurement range (5 × 103 ~ 1 × 106 cells/ml) while reducing total cell counting numbers by 62.5% compared to a large volume (0.8 µl-chamber) hemacytometer.

Summary This protocol describes an easy and reliable in-gel proteasome assay to quantify the activity and composition of different proteasome complexes in cells and tissues. The assay works well with limited amounts of total cell protein lysates. Although this assay is optimized specifically for the proteasome chymotrypsin-like activity, it can be expanded to other proteasome activities as well. Using antibodies that detect distinct proteasome subunits or regulators, we can determine the composition and relative quantity of active proteasome complexes. For complete details on the use and execution of this protocol, please refer to Meul et al. (2020)., Graphical abstract, Highlights • Maintain the activity of proteasome complexes by nondenaturing conditions • Quantify the specific activities of each proteasome complex • Determine the subunits and the bound regulators of proteasome complexes • Analyze the relative distribution of proteasome complexes upon treatments, This protocol describes an easy and reliable in-gel proteasome assay to quantify the activity and composition of different proteasome complexes in cells and tissues. The assay works well with limited amounts of total cell protein lysates. Although this assay is optimized specifically for the proteasome chymotrypsin-like activity, it can be expanded to other proteasome activities as well. Using antibodies that detect distinct proteasome subunits or regulators, we can determine the composition and relative quantity of active proteasome complexes.

A claim for cellular inflammation in tissues needs the full picture, with total cell count and full differential counts, and not just data on one specific cell type, in addition to details of what normal looks likehttps://bit.ly/2G0Tpob

Diurnal vertical migration (DVM) is a widespread phenomenon in the upper ocean, but it remains unclear to what degree it also involves passively transported micro- and meso-zooplankton. These organisms are difficult to monitor by in situ sensing and observations from discrete samples are often inconclusive. Prime examples of such ambiguity are planktonic foraminifera, where contradictory evidence for DVM continues to cast doubt on the stability of species vertical habitats, which introduces uncertainties in geochemical proxy interpretation. To provide a robust answer, we carried out highly replicated randomized sampling with 41 vertically resolved plankton net hauls taken within 26 hours in a confined area of 400 km2 in the tropical North Atlantic, where DVM in larger plankton occurs. Manual enumeration of planktonic foraminifera cell density consistently reveals the highest total cell concentrations in the surface mixed layer (top 50 m) and analysis of cell density in seven individual species representing different shell sizes, life strategies and presumed depth habitats reveals consistent vertical habitats not changing over the 26 hours sampling period. These observations robustly reject the existence of DVM in planktonic foraminifera in a setting where DVM occurs in other organisms.

Background Total cell counts (TC-BF) and percent polymorphonuclear cells (%PMN) of synovial fluid (SF) aspirates provide important cues for the timely diagnosis and management of septic arthritis. To facilitate faster turnaround time, we compared automated to manual TC-BF and differential counts in order to identify reporting cut-offs for automated TC-BF and %PMN that would allow release of automated results concordant with manual counts and differentials. Methods Automated TC-BF and %PMN counts of a non-validated analyzer (Analyzer-B in STAT laboratory) were compared to a validated analyzer (Analyzer-A) and manual TC-BF counts and cytospin differentials. Concordance and %differences of Analyzer-B versus Analyzer-A and manual counts were assessed by linear regression analysis and Bland-Altman comparison. Results Overall, automated and manual counts displayed good correlation. A majority of samples demonstrated unacceptable (>20%) differences between automated and manual counts at lower TC-BF ( Conclusions Based on good overall correlation and fewer samples with unacceptable (>20%) differences between automated and manual counts, we adopted TC-BF > 10,000 cells/μl and %PMN > 60% as cutoffs for reporting automated counts. These cutoffs minimize differences between automated and manual cell counts and differentials and would allow rapid automated reporting in the vast majority of septic arthritis cases.

An upcoming surgical treatment method for segmental vitiligo and piebaldism is non-cultured autologous cell suspension transplantation (CST). Currently the criteria for successful transplantation using this methodology is not fully understood. Repigmentation might be determined by the number and/or viability of melanocytes or total cell numbers transplanted per square millimeter (mm2 ).

We investigated shifts in growth rates and cell size reduction-restoration processes in two species of diatoms, Skeletonema japonicum and Skeletonema dohrnii. Growth rates greatly fluctuated from 1.0 to 2.1 per day, even under the same conditions. Rates increased from 1.1 to 2.1 per day with decreasing valve diameter; however, rates quickly decreased to 1.0 per day when cell size reached the threshold for initiating auxosporulation. We also conducted co-culture experiments using different size combinations of the two species. The experiment was started using the same cell densities (75 cells · mL-1 ) of both species, and when batch cultures reached late exponential phase, the cultures were reinoculated twice into new medium. When large (valve diameter of 17 μm) S. japonicum cells and small (6 μm) S. dohrnii cells were co-cultured, the S. dohrnii contributed 99% of the total cell density on day 16 (S. dohrnii: 263,900 cells · mL-1 ; S. japonicum: 2,000 cells · mL-1 ). In contrast, when small (9 μm) S. japonicum cells and large (15 μm) S. dohrnii cells were co-cultured, small S. japonicum cells accounted for 97% of the total cell density after only 13 d (S. dohrnii: 1,900 cells · mL-1 ; S. japonicum: 62,500 cells · mL-1 ). This study demonstrated that diatom growth rates covary with cell size, and this phenomenon potentially determines the outcome of competition between species.

Background We havebeen attempting to use cardiac spheroids to construct three-dimensional contractilestructures for failed hearts. Recent studies have reported that neuralprogenitors (NPs) play significant roles in heart regeneration. However, theeffect of NPs on the cardiac spheroid has not yet been elucidated. Objective This studyaims to demonstrate the influence of NPs on the function of cardiac spheroids. Methods Thespheroids were constructed on a low-attachment-well plate by mixing humaninduced pluripotent stem (hiPS) cell-derived cardiomyocytes and hiPScell-derived NPs (hiPS-NPs). The ratio of hiPS-NPs was set at 0%, 10%, 20%,30%, and 40% of the total cell number of spheroids, which was 2500. The motionwas recorded, and the fractional shortening and the contraction velocity weremeasured. Results Spheroidswere formed within 48 h after mixing the cells, except for the spheroidscontaining 0% hiPS-NPs. Observation at day 7 revealed significant differencesin the fractional shortening (analysis of variance; p = 0.01). The bestfractional shortening was observed with the spheroids containing 30% hiPS-NPs.Neuronal cells were detected morphologically within the spheroids under aconfocal microscope. Conclusion Theaddition of hiPS-NPs influenced the contractile function of the cardiacspheroids. Further studies are warranted to elucidate the underlying mechanism.

The monitoring and control of bioprocesses is of the utmost importance in order to provide a consistent, safe, and high-quality product for consumers. Current monitoring and control schemes rely on infrequent and time consuming offline sampling methods, which inherently leads to some variability in the process which may impact the product quality profile. As part of Lonza's dedication to process analytical technology (PAT) initiatives this study evaluated the ability to generate generic calibration models, which are independent of the cell line, using Raman probes to monitor changes in glucose, lactate, glutamate, ammonium, viable cell concentration (VCC), total cell concentration (TCC) and product concentration. Calibration models were developed from cell culture using two different CHOK1SV GS-KOTM cell lines producing different monoclonal antibodies (mAbs). Developed predictive models, measured changes in glucose, lactate, ammonium, VCC, and TCC with average prediction errors of 0.44, 0.23, 0.03 g L-1 , 1.90 × 106 cells mL-1 , and 1.85 × 106 cells mL-1 , respectively over the course of cell culture with minimal cell line dependence. The development of these generic models allows the application of spectroscopic PAT techniques in clinical and commercial manufacturing environments, where processes are typically run once or twice in GMP manufacturing based on a common platform process. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:730-737, 2018.

Slight postharvest bruises of loquats remarkably affect the quality and shelf life of the fruits, but they are difficult to identify using visual inspection. Sub-surface structural changes in cells caused by mechanical injury or impact can be detected using spectroscopy-based methods from different angles. Optical coherence tomography (OCT), a non-destructive technology, can acquire cross-sectional images to analyze sub-surface structures of loquats, thus offering the potential to identify fruit bruises. This study proposes an automated OCT image processing method for extracting large cells from loquat images, which involves a series of steps including image denoising, boundary detection, filtering, binarization, segmentation, and region selection. Parenchyma cells in loquat tissue were visualized and characterized, and the five-cell morphological parameters, including total cell surface area, average cell surface area, average cell Feret diameter, equivalent diameter, and cell amount were measured. The bruised and non-bruised loquat groups showed significant differences in the total cell surface area and cell amount, suggesting that these two parameters might be used as indictors for bruise identification. No significant differences in other parameters were observed between the two groups. The microcosm approach proposed in this study sheds some light on ways to improve fruit quality evaluation. Overall, combined with appropriate image processing, OCT is an efficient and non-destructive tool for loquat bruise detection. The proposed strategy might also be expanded to other agricultural applications.

Ki67 scoring is required for the grading of pancreatic neuroendocrine tumors. Ongoing debate exists about the best scoring method in terms of accuracy and practicality. Manual counting of cells in camera-captured/printed images is a widely used and accepted method and considered the most reliable one among the manual methods. It requires counting 500 to 2000 cells to determine the Ki67 score accurately and it is time and energy consuming. We investigated the possibility of achieving the same results by counting only a particular fraction of tumor cells in a printed image in a series of 45 (24 grade 1 and 21 grade 2) pancreatic neuroendocrine tumors. After counting Ki67-positive tumor cells in the whole image, the tumor cells were counted within one-tenth of the same image with the aid of a previously prepared grid on an acetate sheet. The cell number obtained was multiplied by 10 to estimate the total cell count and the Ki67 score was calculated. The agreement between the results of the acetate grid and conventional whole-image counting method was assessed. Near-perfect agreement was achieved regarding the total cell count and Ki67 score. The agreement on tumor grade between the two methods was perfect. The time spent on the process was significantly less than that spent on the conventional method. Although it needs to be validated in a larger series, the acetate grid method might be considered an alternative method for Ki67 scoring in neuroendocrine tumors.

We investigated the aspects of S. Typhimurium biofilms and their resistance (thermal and chemical) depending on the different morphotypes at room temperature (rdar; strain 43971 and bdar; strain 19585) and maturation temperature. Strain 19585 biofilm had a relatively low cell mass and polysaccharide content compared to strain 43971 in all cases, and when matured for 9 days at 15 °C, both properties were significantly higher than at 25 and 37 °C maturation. The difference in secretion ability influenced the pattern in which the biofilm increased resistance. The thermal and chemical resistance levels of strain 19585 biofilms increased at 15 °C, and those of strain 43971 biofilms increased at 15 and 25 °C. When the two strains were mixed, the aspect of biofilm formation was similar to that of strain 43971 except that the heat and chemical resistance were higher at 37 °C maturation. Following 9 days of biofilm maturation, we concluded that 15 °C compared with 25 and 37 °C was the optimal condition for rdar and bdar strains in terms of quantity of total cell mass, EPS secretions and resistance against heat and chemical treatments.

Contents The purpose of this study was to investigate the effect of seminal plasma (SP) from bulls of known fertility on bovine endometrial epithelial cells (bEEC) in culture. The bEEC from passage 5, approximately 5.0–13 × 105 cells per flask, were challenged with SP from bulls of high or low fertility (n = 3 and 2, respectively) or PBS (control), at 1% (75 μl) or 4% (300 μl) and were incubated for 72 hr (n = 13 per challenge). Total cell number and viability of bEEC after challenge with 1% SP from either high- or low-fertility bulls (75H or 75L, respectively) did not differ from controls. In contrast, challenge with 4% of SP from high- or low-fertility bulls (300H or 300L) negatively affected bEEC cell number and viability. Challenge with 300 L had a greater adverse effect than 300H. These results suggest that the negative effect of bovine SP on bEEC is both dose-dependent and fertility-dependent.

Wheat bran and rice bran were concentrated in terms of dietary fiber and were dephytinized by two different methods (fermentation and hydrothermal). Untreated, concentrated, concentrated-dephytinized by fermentation method, and concentrated-dephytinized by hydrothermal method bran samples were each incorporated into flour at levels of 0, 10, 15, and 20%, and their effects on bread properties were investigated. Unprocessed wheat bran and rice bran addition decreased the volume yield, and the specific volume of the bread depends on the incorporation level. A further decrease was observed with the addition of concentrated bran. However, the dephytinization treatments slightly improved these values. The same pattern was observed for the total number of cells and the total cell area of bread crumbs. Both wheat bran and rice bran were observed to cause a darker crumb color, and the effects of bran samples on crumb color were more pronounced after the dephytinization treatment. The addition of concentrated wheat bran and rice bran significantly increased hardness, as well as decreasing springiness, cohesiveness, and resilience of the bread, depending on the bran levels. Although dephytinization treatments enhanced the textural properties of bread, these results were still inferior to those obtained using bread produced with untreated bran. Regarding the dephytinization treatments, the influence of fermentation treatment on bread properties was slightly beneficial compared to hydrothermal treatment.Consumption of dietary fiber offers a range of health benefits. Cereal bran has great potential as a dietary fiber source. However, this bran's high phytic acid content and adverse effects on bread quality limit that potential. It is possible to obtain high dietary fiber and low phytic acid bran samples through bran concentration and dephytinization treatments. The dephytinization treatment is an effective method for degradation of phytic acid. The negative effects of wheat bran and rice bran on bread quality decreased significantly following the dephytinization treatments. This study demonstrated that fiber-enriched bread with low phytic acid content and acceptable texture can be produced using concentrated and dephytinized bran.

Many adult organs grow or shrink to accommodate different physiological demands. Often, as total cell number changes, stem cell number changes proportionally in a phenomenon called ‘stem cell scaling’. The cellular behaviors that give rise to scaling are unknown. Here we study two complementary theoretical models of the adult Drosophila midgut, a stem cell-based organ with known resizing dynamics. First, we derive a differential equations model of midgut resizing and show that the in vivo kinetics of growth can be recapitulated if the rate of fate commitment depends on the tissue’s stem cell proportion. Second, we develop a twodimensional simulation of the midgut and find that proportion-dependent commitment rate and stem cell scaling can arise phenomenologically from the stem cells’ exploration of physical tissue space during its lifetime. Together, these models provide a biophysical understanding of how stem cell scaling is maintained during organ growth and shrinkage.

Introduction Dialyzer reprocessing machines have replaced human labor in preparing re-usable dialyzers. It also made the process of total cell volume (TCV) measurement become faster. Nevertheless, there has been a lack of data on efficacy of weight evaluation on TCV by machine compared to volume evaluation by the conventional method. The aim of this study was to evaluate the efficacy of TCV measurement performed by Kidney-Kleen® reprocessing machine, produced by MEDITOP Company in Thailand, compared to that of the conventional method. Methods This prospective cohort study was performed during September 2014 to December 2015.The low-flux (N = 101) and high-flux dialyzers (N = 100) were included for TCV evaluation. Reused times were up to 5 in the low-flux and 20 in the high-flux dialyzers. The Bland Altman analysis was used to evaluate value measured by different methods. Results The values measured by weight evaluation (by machine) were higher than those obtained by volumetric evaluation of the conventional method in the low-flux (0.81 ± 0.20%) and high-flux (1.32 ± 0.39%) dialyzers. The correlation of TCV values of the two methods were r = 0.98, p, Highlights • Over the past decade, dialyzer reprocessing machines have replaced human labor and time spent in preparing re-usable dialyzers. • It also made the process of total cell volume (TCV) measurement become faster. • Volumetric evaluation was considered as the standard to compare with the TCV values from the reprocessing machine. • Nevertheless, there has been a lack of data on efficacy of weight evaluation on TCV by machine compared to volume evaluation by the conventional method. • The aim of study was to evaluate the efficacy of TCV measurement performed by the reprocessing machine compared to that of the conventional method.

Biofilm heterogeneity has been characterized on various scales for both natural and engineered ecosystems. This heterogeneity has been attributed to spatial differences in environmental factors. Understanding their impact on localized biofilm heterogeneity in building plumbing systems is important for both management and representative sampling strategies. We assessed heterogeneity within the confined engineered ecosystem of a shower hose by high-resolution sampling (200 individual biofilm sections per hose) on varying scales (μm to m). We postulated that a biofilm grown on a single material under uniform conditions should be homogeneous in its structure, bacterial numbers, and community composition. A biofilm grown for 12 months under controlled laboratory conditions, showed homogeneity on large-scale. However, some small-scale heterogeneity was clearly observed. For example, biofilm thickness of cm-sections varied up to 4-fold, total cell concentrations (TCC) 3-fold, and relative abundance of dominant taxa up to 5-fold. A biofilm grown under real (i.e., uncontrolled) use conditions developed considerably more heterogeneity in all variables which was attributed to more discontinuity in environmental conditions. Interestingly, biofilm communities from both hoses showed comparably low diversity, with, Frontiers in Microbiology, 10, ISSN:1664-302X

The relative abundance and diversity of lactobacilli present in feces of infants fed with breastmilk and fructooligosaccharide-galactooligosaccharide (FOS-GOS)-, and inulin-galactooligosaccharide (inulin-GOS)-supplemented infant formulae and combination of both were compared. Fecal lactobacilli rapidly colonized and reached maximum total cell counts, which were significantly higher in the infants fed by combining breastmilk with a formula containing either FOS-GOS (C1-A infant) or inulin-GOS (C2-C infant) and the exclusively formula fed ones (F1-F and F2-H infants) than those detected in the exclusively breast-fed (B1-D and B2-E infants) (P

Complex human biomolecular processes are made possible by the diversity of human proteoforms. Constructing proteoform families, groups of proteoforms derived from the same gene, is one way to represent this diversity. Comprehensive, high-confidence identification of human proteoforms remains a central challenge in mass spectrometry-based proteomics. We have previously reported a strategy for proteoform identification using intact-mass measurements, and we have since improved that strategy by mass calibration based on search results, the use of a global post-translational modification discovery database, and the integration of top-down proteomics results with intact-mass analysis. In the present study, we combine these strategies for enhanced proteoform identification in total cell lysate from the Jurkat human T lymphocyte cell line. We collected, processed, and integrated three types of proteomics data (NeuCode-labeled intact-mass, label-free top-down, and multi-protease bottom-up) to maximize the number of confident proteoform identifications. The integrated analysis revealed 5,950 unique experimentally observed proteoforms, which were assembled into 848 proteoform families. Twenty percent of the observed proteoforms were confidently identified at a 3.9% false discovery rate, representing 1,207 unique proteoforms derived from 484 genes.

Protein sialylation is ubiquitous and essential in a wide range of biological processes. Herein, a mass defect-based chemical-directed proteomics method (MdCDPM) was presented for targeted analysis of intact sialylglycopeptides (SGPs). The process starts by specific oxidation of dihydroxy in sialic acid to aldehyde, which was then chemically labeled by two arginine isotopologues (Arg-15N4 and Arg-D4, differs by 36 mDa). The equally mixed precursor partners, spacing tens of mDa apart, enable the direct recognition of SGPs in MS1 level and benefit the subsequent targeted MS2 characterization. The mass envelope of two labeled forms falling into a narrow m/z window strengthens recognition uniqueness greatly, and the proposed 1:1 intensity ratio of doublets will not be readily distorted. More important, such subtle mass differences permit multiple sialic acids labeling without additional complexity of precursor patterns. Also, the partner m/z shifts detail the number of sialic acids contained in the precursor species. By applying MdCDPM, femtomole quantities of SGPs could be detected from total cell lysates, even at a signal-to-noise ratio of as low as 3:1. In addition, assays were performed to estimate the false positive rate and demonstrated high confidence of MdCDPM. Furthermore, it was designed and successfully exploited to analyze SGPs in human serum, which highlighted the feasibility of this strategy for biological applications.

Flexible hydrogen-bonded organic frameworks (FHOFs) are quite rare but promising for applications in separation, sensing and host-guest chemistry. They are difficult to stabilize, making their constructions a major challenge. Here, a flexible HOF (named 8PN) with permanent porosity has been successfully constructed. Nine single crystals of 8PN with different pore structures are obtained, achieving a large-scale void regulation from 4.4% to 33.2% of total cell volume. In response to external stimuli, multimode reversible structural transformations of 8PN accompanied by changes in luminescence properties have been realized. Furthermore, a series of high-quality co-crystals containing guests of varying shapes, sizes, aggregation states and even amounts are obtained, showing that 8PN can adapt to different guests by regulating the molecular conformations and assembling forms of its building blocks. The unexpected flexibility of 8PN makes it a promising material for enriching the applications of existing porous materials., Flexible hydrogen-bonded organic frameworks (FHOFs) are challenging in fabrication but promising materials for applications in separation and sensing. Here, the authors report a stimuli responsive and flexible HOF which can adapt to different guests by regulating the molecular conformation.

The Acridine orange direct count (AODC) enables the determination of total cell count (TCC) in building materials. The AODC technique is considered as an analytic standard in many different areas but is not common in the indoor environment. In this paper AODC was tested with various building materials to investigate TCC. Colony forming units (CFU) only detect viable cells in contrast to the TCC assay which estimates total microbial contamination. The results show that the total cell count is a reliable and sensitive method to investigate the indoor environment.

Flow cytometry (FCM) is routinely used in fundamental and applied research, clinical practice, and clinical trials. In the last three decades, this technique has also become a routine tool used in immunological studies of molluscs to analyse physical and chemical characteristics of haemocytes. Here, we briefly review the current implementation of FCM in the field of molluscan immunology. These applications cover a diverse range of practices from straightforward total cell counts and cell viability to characterize cell subpopulations, and further extend to analyses of DNA content, phagocytosis, oxidative stress and apoptosis. The challenges and prospects of FCM applications in immunological studies of molluscs are also discussed.

BackgroundThe Isotropic Fractionator (IF) is a method used to determine the cellular composition of nervous tissue. It has been mostly applied to assess variation across species, where differences are expected to be large enough not to be masked by methodological error. However, understanding the sources of variation in the method is important if the goal is to detect smaller differences, for example, in same-species comparisons. Comparisons between different mice strains suggest that the IF is consistent enough to detected these differences. Nevertheless, the internal validity of the method has not yet been examined directly.MethodIn this study, we evaluate the reliability of the IF method for the determination of cellular and neuronal numbers. We performed repeated cell counts of the same material by different experimenters to quantify different sources of variation.ResultsIn total cell counts, we observed that for the cerebral cortex most of the variance was at the counter level. For the cerebellum, most of the variance is attributed to the sample itself. As for neurons, random error along with the immunological staining correspond to most of the variation, both in the cerebral cortex and in the cerebellum. Test-retest reliability coefficients were relatively high, especially for cell counts.ConclusionsAlthough biases between counters and random variation in staining could be problematic when aggregating data from different sources, we offer practical suggestions to improve the reliability of the method. While small, this study is a most needed step towards more precise measurement of the brain’s cellular composition.HighlightsMost variance in cell counts was between counters (η = 0.58) for cerebral cortices.For cerebella, most of the variance was attributed to the samples (η = 0.49).Variance in immunocytochemical counts was mostly residual/random (η > 0.8).Test-retest reliability was high (same counter, same sample).Practical suggestions are offered to improve the reliability of the method.

One of the dysbioses often observed in Crohn's disease (CD) patients is an increased abundance of Escherichia coli (10–100 fold compared to healthy individuals) (Gevers et al., 2014). The data reported is a large-scale proteome profile for E. coli isolates collected from CD patients and healthy individuals. 43 isolates were achieved from 30 CD patients (17 male, 12 female, median age 30) and 19 isolates from 7 healthy individuals (7 male, median age 19). Isolates were cultivated on LB medium at aerobic conditions up to medium log phase. Protein extraction was performed with sodium deoxycholate (DCNa) and urea, alcylation with tris(2-carboxyethyl)phosphine and iodacetamide. Protein trypsinolysis was performed as described in (Matyushkina et al., 2016). Total cell proteomes were analysed by shotgun proteomics with HPLC-MS/MS on a maXis qTOF mass-spectrometer. The data including HPLC-MS/MS raw files and exported Mascot search results was deposited to the PRIDE repository project accession: PXD010920, project https://doi.org/10.6019/PXD010920. Keywords: E. coli, Proteome, Crohn's disease, HPLC-MS/MS