Search

Emerging evidence indicates that aberrant changes of lncRNAs expression induced by hypoxia participate in the development of HCC. The present study aimed to identify novel hypoxia-responsive lncRNAs and reveal its role and mechanism in HCC. Hypoxia exposure in HCC tissues was comprehensively estimated based on public data using multiple hypoxia gene signatures. Huh7 cells were treated with hypoxia and RNA-seq was performed. Then we analyzed the changes of lncRNAs in HCC tissues and cells exposed to hypoxia. We found that lncRNA BSG-AS1 was highly expressed in tissues with high hypoxia score. Then we verified the response of lncRNA BSG-AS1 to hypoxia in the cell hypoxia model in vitro. Through functional phenotypic analysis, we found that lncRNA BSG-AS1 can mediate the promoting effect of hypoxia on the proliferation and migration in HCC cells. RNA-seq was used to find the downstream target genes of lncRNA BSG-AS1. Sequencing data and wet experiments showed that mRNA of BSG decreased after knockout of lncRNA BSG-AS1, and mediated the promotive effect of lncRNA BSG-AS1 on proliferation and migration in HCC cells. The mechanism is that lncRNA BSG-AS1 can enhance the stability of BSG mRNA as antisense lncRNA. Finally, the data based on the public cohort and the cohort we collected suggested that the overexpression of lncRNA BSG-AS1 and BSG are related to the poor prognosis. In conclusion, lncRNA BSG-AS1 is a novel hypoxia-responsive lncRNA. LncRNA BSG-AS1 can positively regulate BSG, by maintaining the mRNA stability of BSG, thus promoting the proliferation and migration of HCC. High expression of lncRNA BSG-AS1 and BSG are risk factors for prognosis.

Selective IgM deficiency (SIgMD) is a rare immunodeficiency characterized by serum IgM below two standard of mean, and normal IgG and IgA levels. Both in human and mice with selective IgM deficiency, germinal centers cells are decreased. The development of germinal center and humoral immunity are regulated in part by follicular helper T (TFH) and follicular regulatory T (TFR) cells. However, the analysis of circulating TFH (cTFH) and TFR (cTFR) cells in the pathogenesis of SIgMD has not been explored. We observed lower percentage of cTFR cells in SIgMD patients than in control group. However, we did not observe any significant difference in the percentage of cTFH cells and their subsets between both experimental groups. When data were analyzed according to specific antibody response to pneumococcal polysaccharide, we observed a higher percentage of cTFH cells in SIgMD patients with specific antibody deficiency than in SIgMD patients with normal specific antibody response. Our results suggest that cTFH cells and their subsets are preserved in SIgMD patients. However, the role of lower percentage of cTFR cells in the pathogenesis of this immunodeficiency is not clear.

Adoptive cell therapy (ACT) of tumor-infiltrating lymphocytes (TILs) has demonstrated clinical benefits in metastatic melanoma treatment. However, the clinical application of TILs produced by a widely used standard protocol from non-small cell lung cancer (NSCLC) can be quite challenging because of the limited clinical benefits. A comprehensive phenotypic knowledge of TILs obtained from NSCLC is important for the development and improvement of personalized TIL therapy for NSCLC patients.In this study, we successfully expanded TILs from 141 NSCLC tissues which can be used in clinical ACT after expansion by a rapid expansion protocol (REP).Our study indicates that the clinicopathological characteristics of patients have considerable impacts on the phenotype of

International audience; Schistosomiasis is a Neglected Tropical Disease caused by trematode species of the genus Schistosoma. Both, autochthonous and imported cases of urogenital schistosomiasis have been described in Europe. The present study focuses on eggs, considered pure S. haematobium by genetic characterisation (intergenic ITS region of the rDNA and cox1 mtDNA). A phenotypic characterisation of S. haematobium eggs was made by morphometric comparison with experimental populations of S. bovis and S. mansoni, to help in the diagnosis of S. haematobium populations infecting sub-Saharan migrants in Spain. Analyses were made by Computer Image Analysis System (CIAS) applied on the basis of new standardised measurements and geometric morphometric tools. The principal component analysis (PCA), including seventeen non-redundant measurements, showed three phenotypic patterns in eggs of S. haematobium, S. bovis and S. mansoni. PCA showed that the S. bovis population presented a large egg size range with a pronouncedly larger maximum size. Similarly, S. bovis shows bigger spine values than S. haematobium. Mahalanobis distances between each pair of groups were calculated for each discriminant analysis performed. In general, S. mansoni and S. bovis present larger distances between them than with S. haematobium, i.e. they present the greatest differences. Regarding the spine, S. haematobium and S. mansoni are the most distant species. Results show the usefulness of this methodology for the phenotypic differentiation between eggs from these Schistosoma species, capable of discerning morphologically close eggs, as is the case of the haematobium group. Schistosoma egg phenotyping approaches may be applied to assess not only hybrid forms but also potential influences of a variety of other factors

It is a challenge to establish and use model systems for the study of complex neuropsychiatric diseases such as schizophrenia (SZ) and autism spectrum disorder (ASD). The development of induced pluripotent stem cell (iPSC) technologies now offers a promising approach which faithfully represents the varying genetic background of individual patients. Phenotypic analysis of iPSC-derived neurons reveals differences at the level of transcriptomes and calcium signalling allowing for a specific discrimination between SZ and ASD phenotypes.

Phenotypic analysis of Caenorhabditis elegans has greatly advanced our understanding of the molecular mechanisms implicated in the aging process as well as in age-related pathologies. However, conventional high-resolution imaging methods and survival assays are labor-intensive and subject to operator-based variations and decreased reproducibility. Recent advances in microfluidics and automated flatbed scanner technologies have significantly improved experimentation by eliminating handling errors and increasing the sensitivity in measurements. Here, we introduce a medium-throughput microfluidic platform, which efficiently positions and immobilizes single worms through pressurization for high resolution imaging. Worms are sorted based on select imaging criteria, and subsequently transferred into multi-well plates for automated lifespan assessment. To illustrate the applicability of this method, we imaged α-synuclein deposits in a C. elegans model of Parkinson's Disease (PD). We found that age synchronized individuals expressing human α-synuclein vary greatly in the quantity and size of intracellular α-synuclein foci at early stages in life. Subsequent lifespan analysis of the individuals, however, did not reveal any correlation between the number or extent of α-synuclein deposits and subsequent lifespan. These studies suggest that the observed natural variations in α-synuclein deposits found in C. elegans models of PD do not originate from inherent differences in the fitness of the organism or contribute to alterations in lifespan.

Background Atopic dermatitis (AD) is the most common skin disorder in infancy. However, the diagnosis and definite significance of infantile AD remains a debated issue. Objective To analyse the phenotypes of AD in infancy, to establish diagnostic criteria and to estimate the prevalence of this condition in China. Methods This is a multicentric study, in which 12 locations were chosen from different metropolitan areas of China. Following careful and complete history-taking and skin examination, the definite diagnosis of AD was made and the severity based on the SCORAD index was determined by local experienced dermatologists. Based on the detailed phenotyping, the major and representative clinical features of infantile AD were selected to establish the diagnostic criteria and evaluate their diagnostic efficacy. Results A total of 5967 infants were included in this study. The overall point prevalence of AD was 30.48%. The infantile AD developed as early as at the second month of life, and its incidence peaked in the third month of life at 40.81%. The proportion of mild, moderate and severe AD was 67.40%, 30.57% and 2.03%, respectively. The most commonly seen manifestations in the infantile AD were facial dermatitis (72.07%), xerosis (42.72%) and scalp dermatitis (27.93%). We established the novel diagnostic criteria of infants, which included: (i) onset after 2 weeks of birth; (ii) pruritus and/or irritability and sleeplessness comparable with lesions; and (iii) all two items above with one of the following items can reach a diagnosis of AD: (i) eczematous lesions distributed on cheeks and/or scalp and/or extensor limbs, and (ii) eczematous lesions on any other parts of body accompanied by xerosis. Conclusions In China, the prevalence of AD in infancy is 30.48% according to clinical diagnosis of dermatologists. The novel Chinese diagnostic criteria for AD in infants show a higher sensitivity and comparable specificity.

To improve understanding of treatment-resistant depression (TRD) in a large population of individuals with depression, a self-reported antidepressant efficacy survey was designed and administered to 23andMe research participants. Participants with a current depressive episode or with a depressive episode within the last 5 years were queried for the effect of pharmacotherapy during the episode. TRD was defined as non-response to at least two antidepressants taken for at least 5-6 weeks. Non-TRD (NTRD) was defined as responsive to either the first or second medication taken for at least 3-4 weeks. Participants who could not be classified as TRD or NTRD were excluded from the analysis. Approximately 56,000 participants completed the survey, among which approximately 33,000 took medication for a depressive episode. The 3409 participants with self-reported TRD tended to have younger age of onset, and a more persistent course prior to initiation of treatment (e.g., a longer prior average episode duration and residual symptoms between episodes) than the 18,511 participants classified as NTRD. This survey identified depression characteristics, comorbidities, trigger events, and early childhood trauma that distinguish TRD from NTRD.

International audience; OBJECTIVE: We aimed to report a community outbreak of an uncommon methicillin-resistant Staphylococcus aureus (MRSA) originating in a maternity ward. PATIENTS AND METHODS: Cases were defined by epidemiological, clinical, and microbiological investigations. Microbiological investigations included phenotypic analysis, molecular typing, and whole genome sequencing. To control the outbreak, we applied both national recommendations to prevent in-hospital transmission and the French High Council for Public Health guidelines on the management of community-acquired MRSA infections. RESULTS: Between March and July 2016, seven patients with MRSA infections were identified: six skin and soft tissue infections and one pulmonary infection, including six microbiologically confirmed infections. Infections occurred in community settings, but a link with the same maternity ward was found for all patients. All MRSA strains had a t690 spa type, were tetracycline-resistant, and produced Panton-Valentine leukocidin. All isolates belonged to the sequence type 88 (ST88). CONCLUSION: This outbreak highlights the largely underestimated risk of healthcare-associated infections in maternity wards. Healthcare workers should be aware of the importance of standard hygiene precautions and use of alcohol-based hand sanitizers for neonates and mothers.

Background: The Frieswal cattle a crossbred cattle breed having 62.50 per cent of exotic (inheritance) having total lactation milk yield of 4000 Kg with average lactation length 300 days (PDC Annual Report, 2015-16). Presently, the Frieswal Animal is mainly maintained at 36 Military Farms located in various agro-climatic regions in the country. Methods: The present study was undertaken on first Part lactation production, reproduction and part lactation records of 1470 Frieswal cows maintained over a period of 10 years (2003-2012) two military farms viz Meerut and Ambala at CIRC, Meerut. Result: The estimate the heritability of production and reproduction traits were 0.29 ±0 .098,0.142±.081, 0.130±0.079, 0.087 ±.074, 0.631 ± 0.255, 0.345 ± 0.247 and 0.100 ± 0.153 for standard 305 days milk yield, age of first calving (AFC), First lactation total milk yield (FLMY), First lactation 305-day or less milk yield (FL305DMY), First lactation length (FLL), Inter calving interval (ICI), First dry period (FDP), First Service Period (FSP), respectively. All the genetic and phenotypic correlation among different production and reproduction traits were high and positive. The estimates of heritability of first lactation individual part monthly yields in Frieswal cattle were found to be 0.100 ± 0.069, 0.105 ± .070, 0.100 ± 0.069, 0.112 ± 0.071, 0.204 ± 0.086, 0.160±0.079, 0.176 ± 0.081, 0.221 ± 0.088, 0.213 ± 0.087 and 0.192 ± 0.09, respectively.

Background Knowledge on the main clinical and prognostic characteristics of older multimorbid subjects with liver cirrhosis (LC) admitted to acute medical wards is scarce. Objectives To estimate the prevalence of LC among older patients admitted to acute medical wards and to assess the main clinical characteristics of LC along with its association with major clinical outcomes and to explore the possibility that well-distinguished phenotypic profiles of LC have classificatory and prognostic properties. Methods A cohort of 6,193 older subjects hospitalised between 2010 and 2018 and included in the REPOSI registry was analysed. Results LC was diagnosed in 315 patients (5%). LC was associated with rehospitalisation (age–sex adjusted hazard ratio, [aHR] 1.44; 95% CI, 1.10–1.88) and with mortality after discharge, independently of all confounders (multiple aHR, 2.1; 95% CI, 1.37–3.22), but not with in-hospital mortality and incident disability. Three main clinical phenotypes of LC patients were recognised: relatively fit subjects (FIT, N = 150), subjects characterised by poor social support (PSS, N = 89) and, finally, subjects with disability and multimorbidity (D&M, N = 76). PSS subjects had an increased incident disability (35% vs 13%, P Conclusions LC has a relatively low prevalence in older hospitalised subjects but, when present, accounts for worse post-discharge outcomes. Phenotypic analysis unravelled the heterogeneity of LC older population and the association of selected phenotypes with different clinical and prognostic features.

Background: Chloroquine, 4-N-(7-chloroquinolin-4-yl)-1-N,-N-diethylpentane-1,4-diamine has promising activity against corona virus disease 2019 (COVID-19) and as such, it is imperative to thoroughly understand and determine the rate at which individual body systems metabolizes the drug. Chloroquine a known antimalarial drug belongs to the chemical class of 4-aminoquinolines. Objectives: The study aimed to analyze Chloroquine and its metabolite in biological fluids of healthy subjects by simple thin layer chromatography (TLC), which is an efficient, and inexpensive method for quantifying Chloroquine and its metabolites. Methods: A total of 30 healthy volunteers participated in the study by ingesting 500 mg of chloroquine, and the results were compared with side effects experienced by these subjects. Two brands of Chloroquine phosphate were used for the analysis and the urine were collected pre and post-drug administration and the intensities of the spots observed were compared with the reference standard stock solution. The same or greater intensity of sample spot indicates poor metabolizer, less intensity when compared to the stock spot indicates intermediate metabolizer while a much lesser intensity indicates an extensive metabolizer. Results: There was a statistically significant difference between the brands of chloroquine used at P

Inherited epidermolysis bullosa defines a heterogeneous group of genodermatoses characterized by skin and/or mucosa fragility resulting in blistering. The junctional variant (JEB) is associated with mutations affecting the genes expressing the components of the dermo-epidermal junction (DEJ) [1-2]. We report 34 JEB patients with COL17A1 genetic mutations diagnosed in our Center between 1993 and 2019. Medical and biological records were collected with a standardized questionnaire.

One of the major goals of vaccination is to prepare the body to rapidly secrete specific Abs during an infection. Assessment of the vaccine quality is often difficult to perform, as simple measurements like Ab titer only partly correlate with protection. Similarly, these simple measurements are not always sensitive to changes in the preceding immunization scheme. Therefore, we introduce in this paper a new, to our knowledge, method to assay the quality of immunization schemes for mice: shortly after a recall with pure Ag, we analyze the frequencies of IgG-secreting cells (IgG-SCs) in the spleen, as well as for each cells, the Ag affinity of the secreted Abs. We observed that after recall, appearance of the IgG-SCs within the spleen of immunized mice was fast (

This study aimed to characterize milk coagulation properties (rennet coagulation time, curd-firming time and curd firmness 30 min after rennet addition to milk) and major mineral contents (Ca, Mg, P, K and Na) in Pinzgauer dual-purpose cattle breed. The edited dataset consisted of 7763 milk observations from 851 cows reared in 60 herds in the Alpine area of Bolzano province (Italy). Data were analysed through a linear mixed model which included stage of lactation, parity and their interaction as fixed effects, and cow and herd test date as random effects. Rennet coagulation time, curd-firming time and curd firmness 30 min after rennet addition to milk averaged 22.66 min, 5.53 min and 16.79 mm, respectively. The most abundant minerals were P (1495 mg kg−1) and Ca (1344 mg kg−1), and the least abundant Mg (141 mg kg−1). Compared to their older contemporaries, early-lactating younger animals yielded milk that was more favourable for cheese production (i.e. with shorter coagulation time and stronger curd firmness). Mineral contents were lower in milk of primiparous than multiparous cows, except for Na. Moreover, Ca, Mg, P and Na contents decreased from parturition to peak of lactation and increased thereafter, except for K, which exhibited an opposite trend. Our results showed that Pinzgauer breed produced milk with better coagulation properties and mineral content, from a technological point of view, in first than later parities and in early than late lactation. The characterization of milk coagulation properties and mineral content in autochthonous breeds is important to increase their value and marketability of their products.

Advances in flow cytometry (FCM) increasingly demand adoption of computational analysis tools to tackle the ever-growing data dimensionality. In this study, we tested different data input modes to evaluate how cytometry acquisition configuration and data compensation procedures affect the performance of unsupervised phenotyping tools. An analysis workflow was set up and tested for the detection of changes in reference bead subsets and in a rare subpopulation of murine lymph node CD103+ dendritic cells acquired by conventional or spectral cytometry. Raw spectral data or pseudospectral data acquired with the full set of available detectors by conventional cytometry consistently outperformed datasets acquired and compensated according to FCM standards. Our results thus challenge the paradigm of one-fluorochrome/one-parameter acquisition in FCM for unsupervised cluster-based analysis. Instead, we propose to configure instrument acquisition to use all available fluorescence detectors and to avoid integration and compensation procedures, thereby using raw spectral or pseudospectral data for improved automated phenotypic analysis. We thank Irene Palacios, Elena Prieto, Mariano Vito´n, and Raquel Nieto for excellent technical assistance and Dr. Salvador Iborra for helpful discussion of dendritic cell studies. Editorial assistance was provided by Simon Bartlett. Sí

Purpose The present study aimed to investigate chromosomal microarray (CMA) and clinical data in patients with unexplained developmental delay/intellectual disability (DD/ID) accompanying dysmorphism, congenital anomalies, or epilepsy. We also aimed to evaluate phenotypic clues in patients with pathogenic copy number variants (CNVs). Materials and methods We collected clinical and CMA data from patients at Konyang University Hospital between September 2013 and October 2014. We included patients who had taken the CMA test to evaluate the etiology of unexplained DD/ID. Results All of the 50 patients identified had DD/ID. Thirty-nine patients had dysmorphism, 19 patients suffered from epilepsy, and 12 patients had congenital anomalies. Twenty-nine of the 50 patients (58%) showed abnormal results. Eighteen (36%) were considered to have pathogenic CNVs. Dysmorphism (p=0.028) was significantly higher in patients with pathogenic CNVs than in those with normal CMA. Two or more clinical features were presented by 61.9% (13/21) of the patients with normal CMA and by 83.3% (15/18) of the patients with pathogenic CMA. Conclusion Dysmorphism can be a phenotypic clue to pathogenic CNVs. Furthermore, pathogenic CNV might be more frequently found if patients have two or more clinical features in addition to DD/ID.

In the developing central nervous system, many neurons migrate tangentially, parallel to the pial surface and independently of radial glia processes. Tangential migration has been first described in the embryonic hindbrain where neurons of the precerebellar nuclei (PCN) and of the facial branchiomotor nucleus (FBM) follow complex migratory routes along the anterior–posterior and dorsoventral axis. We are reviewing here the cellular and molecular mechanisms which control the tangential migration of PCN and FBM neurons. To a large extent these models are based on the phenotypic analysis of multiple mouse and fish mutants. Although mounting evidence suggests that various diffusible molecules, such as slits and netrin1, influence the ventral migration of PCN neurons and their behavior at the midline, the mechanisms controlling their anterior–posterior distribution are still largely unknown. By contrast, the caudal migration of FBM neurons has been extensively studied and shown to be controlled by wnt, growth factors, and components of the PCP pathways, even though chemotropic attractive and repulsive molecules might also be involved. Interestingly, various secreted molecules such as CXCL12 and retinoic acid seem responsible of the preferential subpial position of both PCN and FBM migrating neurons. For the two types of neurons, the final switch from a tangential mode of migration to a radial one also seems to obey to some common rules.

Background Recently, it was found that 1% Phytagel plates used to conduct Arabidopsis thaliana seedling phenotypic analysis no longer reproduced previously published results. This Phytagel, which is produced in China (Phytagel C), has replace American-made Phytagel (Phytagel), which is no longer commercially available. In this study, we present the impact of Phytagel produced in the United States vs. China on seedling phenotypic analysis. As a part of this study, an alternative gelling agent has been identified that is capable of reproducing previously published seedling morphometrics. Results Phytagel and Phytagel C were investigated based on their ability to reproduce the subtle phenotype of the sob3-4 esc-8 double mutant. Fluence-rate-response analysis of seedlings grown on 1% Phytagel C plates failed to replicate the sob3-4 esc-8 subtle phenotype seen on 1% Phytagel. Furthermore, root penetrance analysis showed a significant difference between sob3-4 esc-8 seedlings grown on 1% Phytagel and 1% Phytagel C. It was also found that 1% Phytagel C was significantly harder than 1% Phytagel. As a replacement for Phytagel C, Gellan was tested. 1% Gellan was able to reproduce the subtle phenotype of sob3-4 esc-8. Furthermore, there was no significant difference in root penetration of the wild type or sob3-4 esc-8 seedlings between 1% Phytagel and 1% Gellan. This may be due to the significant reduction in hardness in 1% Gellan plates compared to 1% Phytagel plates. Finally, we tested additional concentrations of Gellan and found that seedlings on 0.6% Gellan looked more uniform while also being able to reproduce previously published results. Conclusions Phytagel has been the standard gelling agent for several studies involving the characterization of subtle seedling phenotypes. After production was moved to China, Phytagel C was no longer capable of reproducing these previously published results. An alternative gelling agent, Gellan, was able to reproduce previously published seedling phenotypes at both 1% and 0.6% concentrations. The information provided in this manuscript is beneficial to the scientific community as whole, specifically phenomics labs, as it details key problematic differences between gelling agents that should be performing identically (Phytagel and Phytagel C).

Microfluidic platforms integrating phenotyping and genotyping approaches have the potential to advance the understanding of single cell genotype-to-phenotype correlations. These correlations can play a key role in tackling antibiotic heteroresistance, cancer cell heterogeneity, and other related fundamental problems. Herein, we report a novel platform that enables both high-throughput digital molecular detection and single-cell phenotypic analysis, utilizing nanoliter-sized biocompatible polyethylene glycol hydrogel beads produced by a convenient and disposable centrifugal droplet generation device. The hydrogel beads have been demonstrated enhanced thermal stability, and achieved uncompromised efficiencies in digital polymerase chain reaction, digital loop-mediated isothermal amplification, and single cell phenotyping. The crosslinked hydrogel network highlights the prospective linkage of various subsequent molecular analyses to address the genotypic differences between cellular subpopulations exhibiting distinct phenotypes. Our platform shows great potential for applications in clinical practice and medical research, and promises new perspectives in mechanism elucidation of environment-evolution interaction and other basic research areas.

Flow cytometers have been utilized for the analysis of submicron-sized particles since the late 1970s. Initially, virus analyses preceded extracellular vesicle (EV), which began in the 1990s. Despite decades of documented use, the lack of standardization in data reporting has resulted in a growing body of literature that cannot be easily interpreted, validated, or reproduced. This has made it difficult for objective assessments of both assays and instruments, in-turn leading to significant hindrances in scientific progress, specifically in the study of EVs, where the phenotypic analysis of these submicron-sized vesicles is becoming common-place in every biomedical field. Methods for fluorescence and light scatter standardization are well established and the reagents to perform these analyses are commercially available. However, fluorescence and light scatter calibration are not widely adopted by the small particle community as methods to standardize flow cytometry data. In this proof-of-concept study carried out as a resource for use at the CYTO2019 workshop, we demonstrate for the first-time simultaneous fluorescence and light scatter calibration of small particle data to show the ease and feasibility of this method for standardized flow cytometry data reporting. This data was acquired using standard configuration commercial flow cytometers, with commercially available materials, published methods, and freely available software tools. We show that application of light scatter, fluorescence, and concentration calibration can result in highly concordant data between flow cytometry platforms independent of instrument collection angle, gain/voltage settings, and flow rate; thus, providing a means of cross-comparison in standard units.

SUMMARYThe relative contributions of genetic and environmental factors to variation in immune responses are still poorly understood. Here, we performed a deep phenotypic analysis of immunological parameters of laboratory mice released into an outdoor enclosure, carrying susceptibility genes (Nod2 and Atg16l1) implicated in the development of inflammatory bowel diseases. Variations of immune cell populations were largely driven by environment, whereas cytokine production in response to stimulation was affected more by genetic mutations. Multi-omic models identified transcriptional signatures associated with differences in T cell populations. Subnetworks associated with responses against Clostridium perfringens, Candida albicans and Bacteroides vulgatus were also coupled with rewilding. Hence, exposing laboratory mice carrying different genetic mutations to a natural environment uncovered important contributors to immune variation.One sentence summaryNatural environment exposure in laboratory mice primarily promotes variation in population frequencies of immune cells, whereas cytokine responses to stimulation are affected more by genetic susceptibility to inflammatory bowel disease.

The diagnosis of hemophagocytic lymphohistiocytosis (HLH) can be a difficult one, and the distinction between primary versus secondary HLH can be particularly challenging during the early stages of diagnosis. This distinction is important to make as primary HLH requires allogeneic hematopoietic cell transplantation for a definitive cure. Flow cytometric screening tests for many of the genetic forms of HLH are available. However, not all patients with primary HLH are captured by these screening tests, due to the fact that no screening test is 100% sensitive, and additionally, some patients with "primary" forms of HLH may have mutations in genes which are yet to be discovered. In this issue of the European Journal of Immunology, Ammann et al. [Eur. J. Immunol. 2017. 47: 364-373] compare T-cell phenotype patterns among patients with primary and secondary HLH, and find that assessment of T-cell activation and differentiation may assist with the diagnosis of HLH. Furthermore, this phenotypic analysis has the potential to help make the important distinction between primary and secondary HLH.

The objective was to test the hypothesis that more frequent but less accurately analyzed milk components may give a more representative measure of a cow's total lactation production. Daily records for milk production and fat and protein concentration collected by the AfiLab recording system (Afimilk, Kibbutz Afikim, Israel) from January 2014 to January 2016 from 47 large kibbutz (communal) herds distributed throughout Israel with a total of 37,486 Israeli Holstein cows were compared with the same statistics derived from monthly test day records derived by Bentley and Foss milk analyzers at the central laboratory of the Israel Cattle Breeders Association. The lactation means for all traits were quite similar for the 2 methods in both parities, except for fat production, which was lower for the daily records. This finding corresponded to fat lactation curves, which showed that daily results were lower with low days in milk (DIM) but almost equal to the monthly results after 125 DIM. Relative to monthly records, daily records overestimated protein percentage before 150 DIM and underestimated protein percentage in the second half of the lactation. The standard deviation for first- and second-parity daily records scored by the monthly and daily system were least similar for fat percentage, but even for this trait the difference was no more than 0.1 percentage points. The standard deviations for complete lactation production were slightly lower for the daily results for all traits but protein production. First-parity heritabilities were higher for lactations computed from daily records for all traits except for protein percentage, but differences were not significant. For daily records, coefficients of determination to predict future milk, fat, and protein lactation production from truncated lactations were greatest and root mean squared errors were least if the mean production from the last 2 weeks before the truncation date was used to estimate future production. Daily first-parity partial lactations for milk, fat, and protein production with

Central nervous system malignancies (CNSMs) are categorized among the most aggressive and deadly types of cancer. The low median survival in patients with CNSMs is partly explained by the objective difficulties of brain surgeries as well as by the acquired chemoresistance of CNSM cells. Flow Cytometry is an analytical technique with the ability to quantify cell phenotype and to categorize cell populations on the basis of their characteristics. In the current review, we summarize the Flow Cytometry methodologies that have been used to study different phenotypic aspects of CNSMs. These include DNA content analysis for the determination of malignancy status and phenotypic characterization, as well as the methodologies used during the development of novel therapeutic agents. We conclude with the historical and current utility of Flow Cytometry in the field, and we propose how we can exploit current and possible future methodologies in the battle against this dreadful type of malignancy.

Introduction. Von Willebrand factor (vWF) is an important component of hemostatic system and deviations from the norm of its content or composition are the cause of various types of von Willebrand disease development. Determination of ristoce- tin-cofactor activity of vWF (vWF:RCo) is particularly important for identifying 2A, 2B and 2M types of von Willebrand disease, in which the content of vWF antigen is within the normal range, and vWF:RCo significantly reduced. Aim: to obtain a reagent for the measurement of vWF:RCo from platelet concentrates with a long shelf life, providing correct diagnostics. Materials and methods. Fully functional platelets were obtained from donor platelet concentrates with a shelf life of 7–14 days. Results. We discovered the method for selection of fully functional donor platelets to produce formaldehyde-fixed cells. On the basis of these platelets we developed a reagent that allows to measure vWF:RCo using both aggregation and agglutina- tion methods. The appropriateness was found between the results of vWF determination by both methods with a correlation coefficient 0.96. The aggregation method of measuring vWF:RCo was evaluated by a result of participation in the internation- al program of external quality control UK NEQAS for Blood Coagulation (United Kingdom). Conclusion. It is shown that the use of a reagent obtained from fixed platelets allows for the correct measurement of vWF:RCo. REFERENCES 1. Zubairov D.М. Von Willebrand factor. In: Molecular basis of blood coagulation and thrombus formation: monograph. [Fak- tor fon Villebranda. V kn.: Molekulyarnye osnovy svertyvaniya krovi i tromboobrazovaniya: monografiya]. Kazan’: FEN. 2000: 111–21 (in Russ.). 2. Mannucci P.M., Coppola R. Von Willebrand factor. In: Laboratory techniques in thrombosis — a manual [Eds. J. Jespersen, R.M. Ber- tina, F. Haverkate]. 2nd revised ed. Dordrecht/Boston/London: Kluwer Academic Publishers. 2000: 115–9. 3. Meyer D., Girma J.P. Von Willebrand factor: structure and function. Thromb Haemost. 1993; 70 (1): 99–104. 4. Barkagan Z.S., Momot A.P. Determination of von Willebrand factor on formalized platelets. In: The fundamentals of the diagnosis of hemostasis disorders. [Opredelenie faktora Villebranda na formalinizirovannyh trombocitah. V kn.: Osnovy diagnostiki narushenij gemostaza]. M.: N’yudiamed-AO. 1999: 58–62 (in Russ.). 5. Kitchen S., McCraw A., Echenagucia M. Ristocetin cofactor activity/von Willebrand factor activity (vWF:RCo or vWF:Act). In: Diagnosis of hemophilia and other bleeding disorders: a laboratory manual. 2nd ed. World Federation of Hemophilia. 2010: 83–6. 6. Favaloro E.J. Laboratory evaluation of von Willebrand desease: phenotypic analysis. In: Kitchen S., Olson J.D., Preston F.E. Quality in laboratory hemostasis and thrombosis. Leiden: Wiley-Black- well. 2009: 125–36. 7. Evans R.J., Austen D.E. Assay of ristocetin cofactor using fixed platelets and platelet counting technique. Brit J Hematol. 1997; 37: 289–94. 8. Howard M.A., Firkin B.G. A new tool in the investigation of platelet aggregation. Thromb Diath Haemorrh. 1971; 26: 362–5. 9. Malakhov V.N., Karinova I.N., Zaikin E.V., Dobrovolsky A.B. Interlaboratory variation of the results of coagulation analyzes according to the data of the FC VOC. [Mezhlaboratornaya variaciya rezul’tatov koagulologicheskih analizov po dannym FS VOK]. URL: www.fs-vok.ru/Publications.htm (in Russ.). [Accessed: 28.06.2018].

Optically decodable beads link the identity of a sample to a measurement through an optical barcode, enabling libraries of biomolecules to be captured on beads in solution and decoded by fluorescence. This approach has been foundational to microarray, sequencing, and flow-based expression profiling technologies. We combine microfluidics with optically decodable beads and show that phenotypic analysis of living cells can be linked to single-cell sequencing. As a proof-of-concept, we demonstrate the accuracy and scalability of our tool called Single Cell Optical Phenotyping and Expression sequencing (SCOPE-Seq) to combine live cell imaging with single-cell RNA sequencing. Electronic supplementary material The online version of this article (10.1186/s13059-018-1607-x) contains supplementary material, which is available to authorized users.

Salmonella typhiinfection occur in most endemic areas. Patients suspected of typhoid fever and diarrhea attending health facilities in Bauchi metropolis were the population studied. 384 stool and blood specimens were collected. The aim of the study was to investigateSalmonellatyphiusing phenotypic analysis to determine whether the organism pose significance attributes among age group or gender and it is specific with fever and diarrhea in Bauchi metropolis, Bauchi, Nigeria. Blood and stool specimens were first enriched in tetrathionate and selenite F broth respectively before sub-cultured on selective medium while identification was conducted with some biochemical analysis. Of the 384 blood and stool specimens screened, 178(46.4 %) yielded significant bacterial growth, while 206(53.6 %) showed no evidence of bacterial growths.Salmonellatyphiaccounted for 6.2 % of the total bacteria isolated while other Enterobacteriaceae accounted for 93.8 %. Distribution ofSalmonellatyphiwere insignificant in the selected health facility using Cochran-Mantel’s analysis with male and female at (P=0.827), and (P=0.866) in blood and stool specimens respectively. Age groups also shows insignificant attributes toSalmonellatyphiinvestigation at (P=0.44). It continues to maintain a mainstream focus of difficulty to isolate the organism via culture despite the selective medium used for the study. Therefore, screening and identification forSalmonellatyphiat phenotypic level still pose a problem in many Health facility. The research has considerably shown the adverse variability ofSalmonellatyphifrom both samples collected among research inclusions.

This study was planned to gain more about E. coli causing diarrhea in newly weaned rabbits in Ismailia governorate. A total of (192) rectal swab was collected from newly weaned dirrhoiec rabbits at different seasonal period extended from (April 2014 –March 2015) to examine the presence of E. coli. These isolates were further characterized by biochemical tests and polymerase chain reaction. E. coli was isolated in 84 of the 192 samples (43.75%). Seasonal prevalence of Escherichia coli infection revealed that the highest incidence occurred in summer season 29/84 (65.9%). Followed by spring 21/84(40.4%), then in winter 15/84 (32.6%), and finally in autumn 19/84 (38%). The isolated E. coli strain were found to belong to O stereotypes in order of frequency O158, O128, O125, O27, O18, O20, O148 and Untypable (20%, 13.33%, 13.33 13.33%, 6.67%, 6.67%, 6.67%, and 20% isolates respectively). Based on PCR, All examined E. coli were positive100 %( 7/7) for eae virulence gene, while 87.8 %( 6/7) of the tested E. coli isolates were positive to tsh gene. It was concluded that Combination of genotypic with phenotypic analysis of E. coli isolates is more valuable as an epidemiological tool for differentiation and identification of the isolates than one test alone.

Some patients with chronic hepatitis B (CHB) receiving entecavir (ETV) exhibit partial virological response (PVR) to ETV and the mechanism is not clear. In this study, we aim to investigate the in vitro susceptibility of residual clinical strains isolated from the sera of nucleos(t)ide-naive hepatitis B virus e antigen (HBeAg)-positive patients with CHB and PVR to ETV, and to evaluate the clinical and virological responses to prolonged ETV monotherapy in these patients. We followed 69 nucleos(t)ide-naive HBeAg-positive CHB patients receiving ETV treatment, with 13 partial responders to ETV. And we found that no genotypic resistance mutants were detected among the 13 PVR patients. Phenotypic analysis revealed that the residual HBV strains had normal replication capacity, and were as susceptible to ETV as wild-type HBV. All PVR patients continued to receive ETV monotherapy, and serum HBV DNA of the majority became undetectable after prolonged treatment. However, none of these patients achieved HBeAg loss. In contrast, 25.6% and 23.2% of the patients with virological response achieved HBeAg loss (P < 0.001) and HBeAg seroconversion (P < 0.001) at week 144, respectively. Thus, we conclude suboptimal response to ETV might not be due to reduced HBV susceptibility to ETV, and prolonging ETV monotherapy in patients with PVR is recommended.

Recent advances in cellular therapies for patients with cancer, including checkpoint blockade and ex vivo-expanded, tumor-specific T cells, have demonstrated that targeting the immune system is a powerful approach to the elimination of tumor cells. Clinical efforts have also demonstrated limitations, however, including the potential for tumor cell antigenic drift and neoantigen formation, which promote tumor escape and recurrence, as well as rapid onset of T cell exhaustion in vivo. These findings suggest that antigen unrestricted cells, such as natural killer (NK) cells, may be beneficial for use as an alternative to or in combination with T cell based approaches. Although highly effective in lysing transformed cells, to date, few clinical trials have demonstrated antitumor function or persistence of transferred NK cells. Several recent studies describe methods to expand NK cells for adoptive transfer, although the effects of ex vivo expansion are not fully understood. We therefore explored the impact of a clinically validated 12-day expansion protocol using a K562 cell line expressing membrane-bound IL-15 and 4-1BB ligand with high-dose soluble IL-2 on the phenotype and functions of NK cells from healthy donors. Following expansions using this protocol, we found expression of surface proteins that implicate preferential expansion of NK cells that are not fully mature, as is typically associated with highly cytotoxic NK cell subsets. Despite increased expression of markers associated with functional exhaustion in T cells, we found that ex vivo-expanded NK cells retained cytokine production capacity and had enhanced tumor cell cytotoxicity. The preferential expansion of an NK cell subset that is phenotypically immature and functionally pleiotropic suggests that adoptively transferred cells may persist better in vivo when compared with previous methods using this approach. Ex vivo expansion does not quell killer immunoglobulin-like receptor diversity, allowing responsiveness to various factors in vivo that may influence activation and inhibition. Collectively, our data suggest that in addition to robust NK cell expansion that has been described using this method, expanded NK cells may represent an ideal cell therapy that is longer lived, highly potent, and responsive to an array of activating and inhibitory signals.

In vivo muscle function testing has become of great interest as primary phenotypic analysis of muscle performance. This protocol provides detailed procedures to perform the treadmill exhaustion test and the whole-limb grip strength assay, two methods commonly used in the neuromuscular research field.

The Centers for Disease Control and Prevention, Arbovirus Reference Collection (ARC) contains viral isolates from both environmental and human sources that are maintained in the laboratory through passage in suckling mouse brain and/or vertebrate and invertebrate cell culture. There has been increased concern regarding the effect of mycoplasma contamination on virus growth and its impact on research and phenotypic analysis. Therefore, quality control testing of virus preparations has become a routine part of the ARC quality assurance program. We compared the performance of three kits - the PCR Mycoplasma Detection Kit (ABM), the VenorGem Mycoplasma Detection Kit (Sigma), and the MycoAlert Mycoplasma Detection Kit (Lonza) - against a reference mycoplasma detection assay from the American Tissue Culture Collection (ATCC) using 744 virus preparations in the ARC, representing 721 unique viruses comprising twelve families and unclassified viruses. We found the ABM kit had the highest sensitivity and specificity, followed by the Sigma kit and Lonza kit, when compared to the ATCC kit. An increase in false positives was observed for the Lonza kit for preparations recently passaged in suckling mouse. Our data supports previously reported observations; that once introduced a specific species of mycoplasma is maintained within a lab.

Background Inflammatory Bowel Disease (IBD) includes Ulcerative Colitis (UC) and Crohn Disease (CD). In the last decade, there have been introduced therapeutic changes that have revolutionised the pharmacologic and surgical management of patients with IBD. The incidence of UC and CD has been increasing in Latin America but the exact prevalence is unknown. Our objective is to describe the demographic characteristics, clinical and therapeutic aspects of the IBD in patients that have presented in the University Hospital Fundación Santa Fe de Bogotá (UH-FSFB), Colombia. Methods Retrospective Descriptive Cohort Study. Clinical histories, pathology reports, and endoscopic results from data base HI-ISIS of the UH-FSFB and medical data between January 1996 and February 2019 were recollected, stored in Excel and analysed using IBM SPSS Statistics Visor. Patients with diagnosis of IBD were included. Patients with incomplete clinical histories were excluded. Results From 398 patients included in this study, 72.1% had UC, 25.6% CD and 2.3% Indeterminate Colitis. The average age of diagnosis was 43.54 years (range: 12–91). In both patients with UC and CD there were smaller proportions of men than women (0.9:1 for UC and 0.7:1 for CD). Of the patients with UC, 46.3% had been hospitalised. 37.2% presented with proctitis, 23.8% left colitis and 39% with pancolitis. 13.5% had an asymptomatic clinical disease, 22.4% mild, 15.3% moderate, and 48.8% severe. 12.9% received biological therapy (BT). 24.3% of patients received a second line BT. Fifteen per cent required surgical interventions (SI), of which there were no mortalities. 27% who were receiving BT required SI. Of the patients with CD, 82.4% required hospitalisation. 43.1% had an ileal, 9.8% colonic, 39.2% ileal- colonic, 0% isolated upper digestive and 21.6% perianal compromise. 34.3% had non-stenosing behaviour, 49% stenosing and 16.7% penetrating. 44.1% of patients with CD received BT of which 40% required a second line BT. 55.9% required SI, of which 1 mortality was reported. 71.1% who were receiving BT required a SI. Conclusion Our study contributes to the epidemiology and integral management required by patients with IBD in our environment. More studies are recommended that replicate our methodology in the population with IBD in both Colombia and Latin America.

The present work evaluated the species distribution associated with the antimicrobial susceptibility pattern by phenotypic analysis and mec and bla gene detection in 100 Staphylococcus strains from 219 clinical samples from cats and dogs in the state of Rio de Janeiro, Brazil. Oxacillin susceptibility profiles were detected by the Clinical and Laboratory Standards Institute (CLSI) recommended tests. The amplification of the mecA gene was positive in 25% of the Staphylococcus spp. The whole mec complex (mecA-mecI-mecR1) was detected in four phenotypically oxacillin-resistant isolates. The CLSI recommended nitrocefin-based test detected 38% (38/100) β-lactamase producers Staphylococcus strains. Also, 32% (32/100) Staphylococcus spp. strains tested positive for bla genes. The whole bla gene complex, blaZ-blaI-blaR1, was detected in 7.8% (3/38) of the nitrocefinase-positive isolates. β-Lactamases was well spread among the samples and it seems to be a prevalent mechanism in resistant staphylococci strains from pet animals. The mec and bla gene regulatory systems can interfere in expression of resistance mediated by PBPs and β-lactamases, conferring the heterogeneous oxacillin-resistance in Staphylococcus spp. detected by phenotypic tests. Key words: Staphylococcus spp., oxacillin, β-lactamase, mecA, pet animals.

SUMMARYA community outbreak of legionellosis occurred in Barrow-in-Furness, Cumbria, during July and August 2002. A descriptive study and active case-finding were instigated and all known wet cooling systems and other potential sources were investigated. Genotypic and phenotypic analysis, and amplified fragment length polymorphism of clinical human and environmental isolates confirmed the air-conditioning unit of a council-owned arts and leisure centre to be the source of infection. Subsequent sequence-based typing confirmed this link. One hundred and seventy-nine cases, including seven deaths [case fatality rate (CFR) 3·9%] were attributed to the outbreak. Timely recognition and management of the incident very likely led to the low CFR compared to other outbreaks. The outbreak highlights the responsibility associated with managing an aerosol-producing system, with the potential to expose and infect a large proportion of the local population and the consequent legal ramifications and human cost.

To clarify comprehensive immunological signature patterns of tumour tissue-infiltrating lymphocytes in patients with renal cell carcinoma and show its clinical significance. We investigated the surface marker expressions of tumour tissue-infiltrating lymphocytes quantitatively and classified them based on their functional populations. We extracted 109 sets of tumour tissue-infiltrating lymphocytes from 80 patients who underwent surgical resection of renal cell carcinoma, of which 44 tumour tissue-infiltrating lymphocytes were multiply extracted from 15 patients. Each tumour tissue-infiltrating lymphocyte was characterised on the basis of functional T-cell populations using ten surface marker expressions measured by flow cytometry. All sets of the tumour tissue-infiltrating lymphocytes were classified into three groups, which correlated significantly with Fuhrman grade (OR 0.253, 95% CI 0.094–0.678, P = 0.006). Importantly, both overall metastasis-free survival (HR 0.449, 95% CI 0.243–0.832, P = 0.011) and recurrence-free survival (HR 0.475, 95% CI 0.238–0.948, P = 0.035) of the patients with the higher marker expressions were significantly inferior to those of the patients with the lower marker expressions by multivariate analysis. Six specific genes for this classification identified by microarray analysis verified our results using the TCGA KIRC data set. In addition, we discovered the presence of intra-tumoural diversity in the classification of 3 (20%) of the 15 patients. This study showed that the presence of classable diversity in the immunological signature of tumour tissue-infiltrating lymphocytes correlated with prognosis and tumour aggressiveness that was observed even within individual tumours in some patients with renal cell carcinoma.

The Western world is in the midst of an epidemic of obesity, which is the cause of severe clinical complications such as type 2 diabetes, hypertension, and cardiovascular disease. Obesity develops when energy intake chronically exceeds energy expenditure; thus, either reducing the energy intake and/or increasing the energy expenditure has been used in the treatment and prevention of obesity. On a cellular level, energy storage is mediated by white adipocyte tissue (WAT). In contrast, brown adipose tissue (BAT) contributes to body temperature and metabolic homeostasis by metabolizing lipids and glucose. Adipose tissue is a notoriously difficult tissue to work with, due to the high content of triglycerides and the fragility of the cells. In this unit, several approaches to analysis of BAT and WAT are described that overcome these limitations. Curr. Protoc. Mouse Biol. 3:205-216 © 2013 by John Wiley & Sons, Inc. Keywords: white adipose tissue; brown adipose tissue; adipogenesis; adipocytes; FACS; cryosectioning

Whether or not T- and B-cell memory is antigen driven remains unresolved. Recent studies indicate that, in humans, naive and memory T cells can be distinguished by their expression of different CD45 isoforms. Extensive phenotypic analysis of naive and memory T cells shows that the latter express greater amounts of several adhesion molecules as well as low levels of several antigens indicative of activation. These features suggest to Peter Beverley that memory T cells may be more readily activated and that memory may be maintained by crossreactive restimulation. © 1990.

The association of hepatitis B virus (HBV) preS1 and preS2 deletions with progressive liver diseases are not fully understood.The study aimed to investigate characteristics of HBV preS deletion in HBV-infected patients with different illness categories.Total of 539 HBV-infected patients were enrolled in the study, including 146 with chronic hepatitis B (CHB), 111 with HBV-related liver cirrhosis (LC), 146 with HBV-related acute-on-chronic liver failure (ACLF), and 136 with HBV-related hepatocellular carcinoma (HCC). PreS deletion was determined by sequencing. Replicons containing representative preS1 and preS2 deletion mutants and wild-type were respectively constructed and transfected into HepG2 cells for phenotypic analysis.The detection rates of overall preS deletion were 15.8%, 26.1%, 24.0%, and 34.6% in CHB, LC, ACLF, and HCC patients, respectively. PreS1 deletion was most frequently detected in LC patients while preS2 deletion was most frequently detected in HCC patients, both frequencies were significantly higher than that in CHB patients (17.1% vs. 4.8%, P0.01; 19.1% vs. 4.8%, P0.01). The deletion patterns across preS gene were different among the 4 illness categories. Compared with wild-type strain, the preS1 deletion mutant had defected preS1 expression, significantly decreased viral mRNA level and SP II promoter activity; while preS2 deletion mutant had defected preS2 expression, and significantly decreased viral mRNA level.HBV preS deletion was associated with advancement of liver diseases not only presented in preS deletion incidence, but also in the deletion pattern. Patients with preS2 deletion might have a higher risk to develop HCC.