Evaluation of Host Protein Biomarkers by ELISA From Whole Lysed Peripheral Blood for Development of Diagnostic Tests for Active Tuberculosis

Tuberculosis (TB) remains a significant global health crisis and the number one cause of death for an infectious disease. The health consequences in high-burden countries are significant. Barriers to TB control and eradication are in part caused by difficulties in diagnosis. Improvements in diagnosis are required for organisations like the World Health Organisation (WHO) to meet their ambitious target of reducing the incidence of TB by 50% by the year 2025, which has become hard to reach due to the COVID-19 pandemic. Development of new tests for TB are key priorities of the WHO, as defined in their 2014 report for target product profiles (TPPs). Rapid triage and biomarker-based confirmatory tests would greatly enhance the diagnostic capability for identifying and diagnosing TB-infected individuals. Protein-based test methods e.g. lateral flow devices (LFDs) have a significant advantage over other technologies with regard to assay turnaround time (minutes as opposed to hours) field-ability, ease of use by relatively untrained staff and without the need for supporting laboratory infrastructure. Here we evaluate the diagnostic performance of nine biomarkers from our previously published biomarker qPCR validation study; CALCOCO2, CD274, CD52, GBP1, IFIT3, IFITM3, SAMD9L, SNX10 and TMEM49, as protein targets assayed by ELISA. This preliminary evaluation study was conducted to quantify the level of biomarker protein expression across latent, extra-pulmonary or pulmonary TB groups and negative controls, collected across the UK and India, in whole lysed blood samples (WLB). We also investigated associative correlations between the biomarkers and assessed their suitability for ongoing diagnostic test development, using receiver operating characteristic/area under the curve (ROC) analyses, singly and in panel combinations. The top performing single biomarkers for pulmonary TB versus controls were CALCOCO2, SAMD9L, GBP1, IFITM3, IFIT3 and SNX10. TMEM49 was also significantly differentially expressed but downregulated in TB groups. CD52 expression was not highly differentially expressed across most of the groups but may provide additional patient stratification information and some limited use for incipient latent TB infection. These show therefore great potential for diagnostic test development either in minimal configuration panels for rapid triage or more complex formulations to capture the diversity of disease presentations

Emodin Suppresses Migration and Invasion through the Modulation of CXCR4 Expression in an Orthotopic Model of Human Hepatocellular Carcinoma

10.1371/journal.pone.0057015PLoS ONE83

Postoperative catecholamine resistance following fetal methamphetamine exposure

Methamphetamine and its related compounds are among the most widely abused recreational drugs worldwide. While a myriad of clinical complications of methamphetamine use have been described, there is a paucity of literature regarding the effects of maternal abuse during pregnancy on neonatal hearts. In this report, we describe a neonate who underwent Norwood-type palliation and subsequently developed catecholamine-resistant cardiogenic shock, likely related to methamphetamine exposure, which recovered after a period of venoarterial extracorporeal membrane oxygenation support

Antioxidant response elements: Discovery, classes, regulation and potential applications

© 2018 The Authors Exposure to antioxidants and xenobiotics triggers the expression of a myriad of genes encoding antioxidant proteins, detoxifying enzymes, and xenobiotic transporters to offer protection against oxidative stress. This articulated universal mechanism is regulated through the cis-acting elements in an array of Nrf2 target genes called antioxidant response elements (AREs), which play a critical role in redox homeostasis. Though the Keap1/Nrf2/ARE system involves many players, AREs hold the key in transcriptional regulation of cytoprotective genes. ARE-mediated reporter constructs have been widely used, including xenobiotics profiling and Nrf2 activator screening. The complexity of AREs is brought by the presence of other regulatory elements within the AREs. The diversity in the ARE sequences not only bring regulatory selectivity of diverse transcription factors, but also confer functional complexity in the Keap1/Nrf2/ARE pathway. The different transcription factors either homodimerize or heterodimerize to bind the AREs. Depending on the nature of partners, they may activate or suppress the transcription. Attention is required for deeper mechanistic understanding of ARE-mediated gene regulation. The computational methods of identification and analysis of AREs are still in their infancy. Investigations are required to know whether epigenetics mechanism plays a role in the regulation of genes mediated through AREs. The polymorphisms in the AREs leading to oxidative stress related diseases are warranted. A thorough understanding of AREs will pave the way for the development of therapeutic agents against cancer, neurodegenerative, cardiovascular, metabolic and other diseases with oxidative stress

Feeding Neonates and Infants Prior to Surgery for Congenital Heart Defects: Systematic Review and Meta-Analysis

Background: Necrotising enterocolitis (NEC) is a significant cause of mortality and morbidity in neonates requiring cardiac surgery. Feeding practices vary significantly across institutions and remain controversial. We conducted a systematic review of the literature and a meta-analysis to identify associations between feeding practices and necrotising enterocolitis. Methods: This study was carried out in accordance with the PRISMA guidelines. A literature search was performed in November 2022 using the Cochrane Central Register, Embase, and Pubmed. Two investigators then independently retrieved eligible manuscripts considered suitable for inclusion. Data extracted included gestational age, birth weight, sex, nature of congenital heart lesion, type of operation performed, time on ventilator, ICU stay, hospital stay, post-operative feeding strategy, and complications. The methodological quality was assessed using the Downs and Black score for all randomised control trials and observational studies. Results: The initial search yielded 92 studies. After removing duplicates, there were 85 abstracts remaining. After excluding ineligible studies, 8 studies were included for the meta-analysis. There was no significant risk of NEC associated with pre-operative feeding [OR = 1.22 (95% CI 0.77,1.92)] or umbilical artery catheter placement [OR = 0.91 (95% CI 0.44, 1.89)] and neither outcome exhibited heterogeneity [I2 = 8% and 0%, respectively]. There was a significant association between HLHS and NEC [OR = 2.56 (95% CI 1.56, 4.19)] as well as prematurity and NEC [OR 3.34 (95% CI 1.94, 5.75)] and neither outcome exhibited heterogeneity [I2 = 0% and 0%, respectively]. Conclusions: There was no association between NEC and pre-operative feeding status in neonates awaiting cardiac surgery. Pre-operative feeding status was not associated with prolonged hospital stay or need for tube assisted feeding at discharge. HLHS and prematurity were associated with increased incidence of NEC

Universal implantation of temporary epicardial pacing wires after surgery for congenital heart disease: necessity or luxury?

OBJECTIVES: Routine implantation of temporary epicardial pacing wires after surgery for congenital heart disease (CHD) has recently been questioned. We evaluated the incidence of arrhythmias, arrhythmias causing haemodynamic compromise and the safety of a strategy of selective implantation of pacing wires in our unit.METHODS: All patients who underwent surgery for CHD using cardiopulmonary bypass between September 2015 and December 2016 were retrospectively enrolled in the study (n = 313). Patients were stratified into group A (universal implantation) and group B (selective implantation). Group B received pacing wires only when postoperative rhythm disturbances were anticipated based on the operating surgeon's judgement. The primary outcome was arrhythmia causing haemodynamic compromise. Outcomes were compared between unmatched and propensity matched groups.RESULTS: Forty-eight patients experienced an arrhythmia causing haemodynamic compromise (15.3%). Twenty-three patients (7.3%) experienced an arrhythmia causing haemodynamic compromise that required the use of pacing wires for therapeutic purposes (group A n = 13, group B n = 10, P = 0.34). There were no pacing wire related complications in either group. All patients in group A and 90% in group B had pacing wires when needed (P = 0.435). In group A, 89% of patients had pacing wires which were not used compared with 13% in group B (P < 0.001). Results were unchanged when repeated using propensity matching (81 pairs).CONCLUSIONS: The probability of developing a postoperative arrhythmia requiring therapeutic pacing can be predicted using the risk factors identified in our study. The routine implantation of pacing wires after surgery for CHD is not necessary. A measured reduction from universal implantation is safe

Emodin suppresses lung metastasis and CXCR4 expression in orthotopic mice model.

<p>Spontaneous metastatic model of human liver cancer cells orthotopically implanted in nude mice. Cubes of HCCLM3_Luc tumor were implanted orthotopically into the liver of female Balb/c nude mice. Mice were euthanized when the detectable liver tumor signal was >1×10¹¹ p/s. A, At the time of necropsy, bioluminescent imaging was employed to monitor signals from lung metastasis. The lung biopsy was imaged to detect metastasis in the lungs. The color bar depicts the photon flux (p/s) emitted. # = mouse ID; D = day at necropsy. Lung and liver signals are in p/s unit. B, Comparison of the lung metastasis signals detected at time of necropsy for the untreated and emodin-treated groups. C, Tumor tissue extracts were prepared as described in Methods. 30 µg of protein was taken and analyzed by Western blot analysis with antibodies against CXCR4. The same blots were stripped and reprobed with β-actin antibody to show equal protein loading. D, Immunohistochemical analysis of representative section of tumor tissues stained with anti-CXCR4 antibody demonstrate strong cytoplasmic staining for CXCR4 in the control section and weak cytoplasmic staining in the emodin treated section E, Emodin decreases CXCR4 levels in tumor tissues. Tumor tissue homogenate was prepared as described in Methods. 150 µg of the tissue supernatant was taken for ELISA assay. Assay was performed according to manufacturer’s instructions using ELISA kit to determine the levels of CXCR4 in the control and treated group. Data is represented as Mean ± SE. * indicates p value <0.05.</p

Emodin suppresses migration and invasion of HepG2 cells.

<p>A, Wound-healing assay was performed for evaluating the inhibitory effect of emodin on HepG2 cell migration. Confluent monolayers of HepG2 cells were scarred, and repair was monitored microscopically after 12 h of pre-treatment with emodin (50 µM) before being exposed to 100ng/mL CXCL12 for 24 h. Width of wound was measured at time zero and 24 h of incubation with and without emodin in the absence or presence of CXCL12. The representative photographs showed the same area at time zero and after 24 h of incubation. B, HepG2 (2×10⁵ cells) were seeded in the top-chamber of the Matrigel. After pre-incubation with or without emodin (50 µM) for 12 h, transwell chambers were then placed into the wells of a 24-well plate, in which we had added either the basal medium only or basal medium containing 100 ng/mL CXCL12 for 24 h. After incubation, they were assessed for cell invasion as described in Materials and Methods. Columns represent percentage of invaded cells; bars, S.E. *indicates p value <0.05. Representative results of two independent experiments are shown.</p

Emodin suppresses CXCR4 mRNA level in HCC cells.

<p>A and B, Emodin suppresses CXCR4, neither by proteasomal nor by lysosomal degradation. HepG2 cells (1×10⁶) were treated with indicated concentrations of ALLN or chloroquine for 1 h at 37°C, followed by treatment of 50 µM emodin for 6 h. Whole-cell extracts were prepared and analyzed by Western blot analysis using antibodies against CXCR4. The same blots were stripped and reprobed with β-actin antibody to show equal protein loading. Representative results of two independent experiments are shown. C, Emodin suppresses the expression of CXCR4 mRNA in HepG2 cells. HepG2 cells were treated with 50µM emodin for the indicated time intervals, after which cells were harvested after treatment and total RNA samples were extracted. 1µg portions of the respective RNA extracts then proceed for Reverse Transcription to generate corresponding cDNA. Real time PCR was performed to measure the relative quantities of CXCR4 mRNA using targeted TaqMan probes, with GAPDH as endogenous control for measurement of equal loading of RNA samples. Results were analyzed using Sequence Detection Software version 1.3 provided by Applied Biosystems. Relative gene expression was obtained after normalization with endogenous GAPDH and determination of the difference in threshold cycle (Ct) between treated and untreated cells using 2-ΔΔCt method.</p

Emodin suppresses migration and invasion of Hep3B cells.

<p>A, Wound-healing assay for evaluating the inhibitory effect of emodin on cell migration. Confluent monolayers of Hep3B cells were scarred, and repair was monitored microscopically after 12 h of pre-treatment with emodin (50 µM) before being exposed to 100 ng/mL CXCL12 for 24 h. Width of wound was measured at time zero and 24 h of incubation with and without emodin in the absence or presence of CXCL12. The representative photographs of two independent experiments showed the same area at time zero and after 24 h of incubation. B, Hep3B (2×10⁵ cells) were seeded in the top-chamber of the Matrigel. After pre-incubation with or without emodin (50 µM) for 12 h, transwell chambers were then placed into the wells of a 24-well plate, in which we had added either the basal medium only or basal medium containing 100 ng/mL CXCL12 for 24 h. After incubation, the chambers were assessed for cell invasion as described in Materials and Methods. Columns represent percentage of invaded cells; bars, SE. * indicates p value <0.05. Representative results of two independent experiments are shown.</p