Multivariate calibration approach for quantitative determination of cell-line cross contamination by intact cell mass spectrometry and artificial neural networks

Cross-contamination of eukaryotic cell lines used in biomedical research represents a highly relevant problem. Analysis of repetitive DNA sequences, such as Short Tandem Repeats (STR), or Simple Sequence Repeats (SSR), is a widely accepted, simple, and commercially available technique to authenticate cell lines. However, it provides only qualitative information that depends on the extent of reference databases for interpretation. In this work, we developed and validated a rapid and routinely applicable method for evaluation of cell culture cross-contamination levels based on mass spectrometric fingerprints of intact mammalian cells coupled with artificial neural networks (ANNs). We used human embryonic stem cells (hESCs) contaminated by either mouse embryonic stem cells (mESCs) or mouse embryonic fibroblasts (MEFs) as a model. We determined the contamination level using a mass spectra database of known calibration mixtures that served as training input for an ANN. The ANN was then capable of correct quantification of the level of contamination of hESCs by mESCs or MEFs. We demonstrate that MS analysis, when linked to proper mathematical instruments, is a tangible tool for unraveling and quantifying heterogeneity in cell cultures. The analysis is applicable in routine scenarios for cell authentication and/or cell phenotyping in general

Neurological signs and MRI findings in 12 dogs with multiple myeloma

Vertebral lesions and associated neurological signs occur in dogs with multiple myeloma, however, veterinary literature describing MRI findings is currently lacking. The objective of this multicenter, retrospective, case series study was to describe neurological signs and MRI findings in a group of dogs that presented for spinal pain or other neurological deficits and had multiple myeloma. Electronic records of four veterinary referral hospitals were reviewed. Dogs were included if they had a pathologically confirmed diagnosis of multiple myeloma, had presented for spinal pain or other neurological signs, and had undergone MRI of the vertebral column. The MRI studies were evaluated and the anatomical location of lesion(s), signal intensity, presence of extra‐dural material, degree of spinal cord compression, extent of vertebral lesions, and contrast enhancement were recorded. Twelve dogs met inclusion criteria. Most dogs (n = 8) had a chronic progressive history, with varying degrees of proprioceptive ataxia and paresis (n = 11), and spinal pain was a feature in all dogs. The MRI findings were variable but more consistent features included the presence of multiple expansile vertebral lesions without extension beyond the outer cortical limits of affected vertebrae, and associated extradural material causing spinal cord compression. The majority of lesions were hyper‐ to isointense on T2 (n = 12) and T1‐weighted (n = 8) sequences, with variable but homogeneous contrast‐enhancement (n = 12). These described MRI characteristics of multiple myeloma may be used to aid early identification and guide subsequent confirmatory diagnostic steps, to ultimately improve therapeutic approach and long‐term outcome

Determinants of inappropriate acute pain management in old people unable to communicate verbally in the emergency department

Objectives: Poor pain management is relevant among individuals unable to communicate verbally (UCV). Analgesia may be due to three determinants: patients' status, physician's characteristics and pain etiology. Our aim is to investigate the association between prescription of ED pain treatment and these determinants. Materials and Methods: An observational prospective study including UCV patients was conducted. Severity of pain was evaluated by ALGOPLUS Scale and a score P â¥Â 2 out of 5 on the pain scale was retained as the threshold for the presence of acute pain in elderly UCV patients. Results: Our data showed that only 31,9% of UCV patients received a pharmacological treatment. The presence of the caregiver would influence the rate of therapy administration [OR 6,19 (95% CI 2,6â14,75)]. The presence of leg pain [OR 0,32 (95% CI 0,12â0,86)] and head pain [OR 0,29 (95% CI 0,10â0,84)] were less likely associated to receive analgesia. Pain related to trauma [OR 4.82 (95% CI 1.17 to 19.78)] and youngest physicians [OR 1.08 (95% CI 1.001 to 1.18)] were variables associated with the administration of drugs opiates. Discussion: Older UCV patients presenting to the ED with pain are at high risk of inadequate analgesia. Providers should always suspect presence of pain and an increasing need for behavioural pain evaluation is necessary for a complete assessment. Conclusions: Presence of a caregiver influences a more appropriate pain management in these patients. Staff training on pain management could result in better assessment, treatment, and interaction with caregivers. Keywords: Emergency department, Pain, Oligoanalgesi

Multiplex staining depicts the immune infiltrate in colitis-induced colon cancer model

Assessment of the host immune response pattern is of increasing importance as highly prognostic and diagnostic, in immune-related diseases and in some types of cancer. Chronic inflammation is a major hallmark in colon cancer formation, but, despite the extent of local inflammatory infiltrate has been demonstrated to be extremely informative, its evaluation is not routinely assessed due to the complexity and limitations of classical immunohistochemistry (IHC). In the last years, technological advance helped in bypassing technical limits, setting up multiplex IHC (mIHC) based on tyramide signal amplification (TSA) method and designing software suited to aid pathologists in cell scoring analysis. Several studies verified the efficacy of this method, but they were restricted to the analysis of human samples. In the era of translational medicine the use of animal models to depict human pathologies, in a more complete and complex approach, is really crucial. Nevertheless, the optimization and validation of this method to species other than human is still poor. We took advantage of Multispectral Imaging System to identify the immunoprofile of Dextran Sulphate Sodium (DSS)-treated mouse colon. We optimized a protocol to sequentially stain formalin fixed paraffin embedded murine colon samples for CD3, CD8a, CD4, and CD4R5B0 antigens. With this approach we obtained a detailed lymphocyte profile, while preserving the morphological tissue context, generally lost with techniques like gene expression profiling or flow cytometry. This study, comparing the results obtained by mIHC with immunophenotyping performed with cytofluorimetric and standard IHC methods validates the potentiality and the applicability of this innovative approach

Comportamento de suínos suplementados com ractopamina.

Projeto: 02.06.01.001

Dinâmica do herbicida METRIBUZIN aplicado sobre palha de cana-de açúcar (Saccarum officinarum).

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