An ELISA-based procedure for assaying proteins in digests of human leukocytes and cell lines, using specifically selected peptides and appropriate antibodies

BACKGROUND: We describe the application of an ELISA-based assay (the Peptidomatrix) that can be used to simultaneously identify and quantitate a number of proteins in biological samples. The biological sample (blood component, biopsy, culture or other) is first lysed to release all the proteins, without any additional separation. The denatured proteins in the sample are then digested in bulk with the desired proteolytic enzyme(s). The peptides in the digest are then assayed by appropriate antibodies, using a competition ELISA protocol. RESULTS: As an example of its use, the present paper applies the Peptidomatrix to the assay of four membrane proteins MDR1 (P-glycoprotein or ABCB1), MRP1 (ABCC1), BCRP/MXR (ABCG2) and the alpha subunit of the Na, K_ATPase (ATP1A1), present in a number of cell lines and in human lymphocytes. We show that we can detect and quantitate these proteins, using a series of peptide-antibody pairs, and that we can differentiate between cell lines or cell preparations that express the target proteins and those that do not. CONCLUSION: We have devised a simple, ELISA-based proteomics assay that enables the quantitation of designated proteins in a cell or tissue sample, and that can be used in any laboratory, with minimal specialized equipment

Antipsychotics and Lorazepam During Delirium: Are We Harming Older Patients? A Real-Life Data Study

Background: Delirium affects approximately one out of three older hospitalized patients and is associated with poor clinical outcomes. Approaches used to manage delirium consist of non-pharmacological and pharmacological interventions. Antipsychotics and lorazepam are commonly used to treat symptoms of delirium, but conflicting data exist on the effect of these drugs on the outcomes of delirium. Objective: The aim of this study was to investigate whether the use of antipsychotics, with or without lorazepam, increases the risk of prolonged hospital stay, post-discharge institutionalization, and in-hospital mortality in older patients with delirium. Methods: In this retrospective chart review study, we included acutely ill patients aged ≥ 65 years who were admitted to a geriatric ward and diagnosed with delirium. Patients were stratified into three groups based on whether or not they received antipsychotics and lorazepam to manage delirium: (0) no antipsychotics; (1) antipsychotics only; and (2) antipsychotics plus lorazepam. Length of hospital stay (LOS) and frequencies of post-discharge institutionalization and in-hospital mortality were compared. Results: In total, 212 patients with delirium were included (mean age 81.9 ± 5.6 years); 40 did not receive antipsychotics (18.9%), 123 received antipsychotics only (58.0%) and 49 received antipsychotics and lorazepam (23.1%). There was a trend to a longer LOS in patients who received both antipsychotics and lorazepam (median LOS group 0 = 8.0 days, group 1 = 10.0 days, and group 2 = 12.0 days). Furthermore, trends to a higher incidence of post-discharge institutionalization and in-hospital mortality were observed in patients who received both treatments (institutionalization group 0 = 45.0%, group 1 = 59.3%, group 2 = 81.6%; and in-hospital mortality group 0 = 7.5%, group 1 = 10.6%, group 2 = 16.3%). Conclusion: The use of antipsychotics, with or without lorazepam, during delirium is associated with increased risks of poor outcomes. These findings suggest that clinicians should be cautious about routine prescribing of these drugs to older patients with delirium. Further investigation is needed to clarify this association

Anticholinergic Drug Burden and Delirium: A Systematic Review

Objectives: To investigate the association between anticholinergic drug burden (ADB), measured with anticholinergic drug scales, and delirium and delirium severity. Design: Systematic review. Setting and Participants: All available studies. Methods: A systematic literature search was performed in Medline, Embase, PsycINFO, Web of Science, CINAHL, Cochrane library, and Google Scholar. Studies evaluating the association between ADB (measured as a total score) and delirium or delirium severity, published in English, were eligible for inclusion. Results: Sixteen studies, including 148,756 persons, were included. Fifteen studies investigated delirium. ADB was measured with the Anticholinergic Risk Scale (ARS, n = 5), the Anticholinergic Cognitive Burden Scale (ACB, n = 6), the list of Chew (n = 1), the Anticholinergic Drug Scale (ADS, n = 5), a modified version of the ARS (n = 1), and a modified version of the ACB (n = 1). A high ADB, measured with the ARS, was associated with delirium (5/5). Also with the modified version of the ARS and ACB, an association was found between a high ADB and delirium during 3-month (1/1) and 1-year follow-up (1/1), respectively. When ADB was assessed with other scales, the results were inconclusive, with only 1 positive association for the ACB (1/6) and ADS (1/5) each. The possible association between ADB and delirium severity has also been investigated (ADS n = 2, Summers Drug Risk Number n = 1). One study found an association between a high ADB, measured with the ADS, and an increase in severity of delirium. Conclusions and Implications: ADB assessed with the ARS is consistently associated with delirium. The association found between the modified versions of the ARS and ACB and delirium needs confirmation. When ADB was assessed with other scales, the findings were inconclusive. The current findings suggest that the ARS might be a useful tool to identify patients at increased risk for delirium

Structure of the Full-Length Major Pilin from Streptococcus pneumoniae: Implications for Isopeptide Bond Formation in Gram-Positive Bacterial Pili

The surface of the pneumococcal cell is adorned with virulence factors including pili. The major pilin RrgB, which forms the pilus shaft on pathogenic Streptococcus pneumoniae, comprises four immunoglobulin (Ig)-like domains, each with a common CnaB topology. The three C-terminal domains are each stabilized by internal Lys-Asn isopeptide bonds, formed autocatalytically with the aid of an essential Glu residue. The structure and orientation of the crucial N-terminal domain, which provides the covalent linkage to the next pilin subunit in the shaft, however, remain incompletely characterised. We report the crystal structure of full length RrgB, solved by X-ray crystallography at 2.8 Å resolution. The N-terminal (D1) domain makes few contacts with the rest of the RrgB structure, and has higher B-factors. This may explain why D1 is readily lost by proteolysis, as are the N-terminal domains of many major pilins. D1 is also found to have a triad of Lys, Asn and Glu residues in the same topological positions as in the other domains, yet mass spectrometry and the crystal structure show that no internal isopeptide bond is formed. We show that this is because β-strand G of D1, which carries the Asn residue, diverges from β-strand A, carrying the Lys residue, such that these residues are too far apart for bond formation. Strand G also carries the YPKN motif that provides the essential Lys residue for the sortase-mediated intermolecular linkages along the pilus shaft. Interaction with the sortase and formation of the intermolecular linkage could result in a change in the orientation of this strand, explaining why isopeptide bond formation in the N-terminal domains of some major pilins appears to take place only upon assembly of the pili

Dynamic Computational Model Suggests That Cellular Citizenship Is Fundamental for Selective Tumor Apoptosis

Computational models in the field of cancer research have focused primarily on estimates of biological events based on laboratory generated data. We introduce a novel in-silico technology that takes us to the next level of prediction models and facilitates innovative solutions through the mathematical system. The model's building blocks are cells defined phenotypically as normal or tumor, with biological processes translated into equations describing the life protocols of the cells in a quantitative and stochastic manner. The essentials of communication in a society composed of normal and tumor cells are explored to reveal “protocols” for selective tumor eradication. Results consistently identify “citizenship properties” among cells that are essential for the induction of healing processes in a healthy system invaded by cancer. These properties act via inter-cellular communication protocols that can be optimized to induce tumor eradication along with system recovery. Within the computational systems, the protocols universally succeed in removing a wide variety of tumors defined by proliferation rates, initial volumes, and apoptosis resistant phenotypes; they show high adaptability for biological details and allow incorporation of population heterogeneity. These protocols work as long as at least 32% of cells obey extra-cellular commands and at least 28% of cancer cells report their deaths. This low percentage implies that the protocols are resilient to the suboptimal situations often seen in biological systems. We conclude that our in-silico model is a powerful tool to investigate, to propose, and to exercise logical anti-cancer solutions. Functional results should be confirmed in a biological system and molecular findings should be loaded into the computational model for the next level of directed experiments

Association of CD40 Gene Polymorphisms with Sporadic Breast Cancer in Chinese Han Women of Northeast China

BACKGROUND: Breast cancer is a polygenetic disorder with a complex inheritance pattern. Single nucleotide polymorphisms (SNPs), the most common genetic variations, influence not only phenotypic traits, but also interindividual predisposition to disease, treatment outcomes with drugs and disease prognosis. The co-stimulatory molecule CD40 plays a prominent role in immune regulation and homeostasis. Accumulating evidence suggests that CD40 contributes to the pathogenesis of cancer. Here, we set out to test the association between polymorphisms in the CD40 gene and breast carcinogenesis and tumor pathology. METHODOLOGY AND PRINCIPAL FINDINGS: Four SNPs (rs1800686, rs1883832, rs4810485 and rs3765459) were genotyped by the polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) method in a case-control study including 591 breast cancer patients and 600 age-matched healthy controls. Differences in the genotypic distribution between breast cancer patients and healthy controls were analyzed by the Chi-square test for trends. Our preliminary data showed a statistically significant association between the four CD40 gene SNPs and sporadic breast cancer risk (additive P = 0.0223, 0.0012, 0.0013 and 0.0279, respectively). A strong association was also found using the dominant, recessive and homozygote comparison genetic models. In the clinical features analysis, significant associations were observed between CD40 SNPs and lymph node metastasis, human epidermal growth factor receptor 2 (C-erbB2), estrogen receptor (ER), progesterone receptor (PR) and tumor protein 53 (P53) statuses. In addition, our haplotype analysis indicated that the haplotype C(rs1883832)G(rs4810485), which was located within the only linkage disequilibrium (LD) block identified, was a protective haplotype for breast cancer, whereas T(rs1883832)T(rs4810485) increased the risk in the studied population, even after correcting the P value for multiple testing (P = 0.0337 and 0.0430, respectively). CONCLUSIONS AND SIGNIFICANCE: Our findings primarily show that CD40 gene polymorphisms contribute to sporadic breast cancer risk and have a significant association with clinicopathological features among Chinese Han women from the Heilongjiang Province

Prospective Association of Daily Steps with Cardiovascular Disease: A Harmonized Meta-Analysis

Background: Taking fewer than the widely promoted “10 000 steps per day” has recently been associated with lower risk of all-cause mortality. The relationship of steps and cardiovascular disease (CVD) risk remains poorly described. A meta-analysis examining the dose–response relationship between steps per day and CVD can help inform clinical and public health guidelines. Methods: Eight prospective studies (20 152 adults [ie, ≥18 years of age]) were included with device-measured steps and participants followed for CVD events. Studies quantified steps per day and CVD events were defined as fatal and nonfatal coronary heart disease, stroke, and heart failure. Cox proportional hazards regression analyses were completed using study-specific quartiles and hazard ratios (HR) and 95% CI were meta-analyzed with inverse-variance–weighted random effects models. Results: The mean age of participants was 63.2±12.4 years and 52% were women. The mean follow-up was 6.2 years (123 209 person-years), with a total of 1523 CVD events (12.4 per 1000 participant-years) reported. There was a significant difference in the association of steps per day and CVD between older (ie, ≥60 years of age) and younger adults (ie, <60 years of age). For older adults, the HR for quartile 2 was 0.80 (95% CI, 0.69 to 0.93), 0.62 for quartile 3 (95% CI, 0.52 to 0.74), and 0.51 for quartile 4 (95% CI, 0.41 to 0.63) compared with the lowest quartile. For younger adults, the HR for quartile 2 was 0.79 (95% CI, 0.46 to 1.35), 0.90 for quartile 3 (95% CI, 0.64 to 1.25), and 0.95 for quartile 4 (95% CI, 0.61 to 1.48) compared with the lowest quartile. Restricted cubic splines demonstrated a nonlinear association whereby more steps were associated with decreased risk of CVD among older adults. Conclusions: For older adults, taking more daily steps was associated with a progressively decreased risk of CVD. Monitoring and promoting steps per day is a simple metric for clinician–patient communication and population health to reduce the risk of CVD

Matrix Metalloproteinase-2 and -9 Secreted by Leukemic Cells Increase the Permeability of Blood-Brain Barrier by Disrupting Tight Junction Proteins

Central nervous system (CNS) involvement remains an important cause of morbidity and mortality in acute leukemia, the mechanisms of leukemic cell infiltration into the CNS have not yet been elucidated. The blood-brain barrier (BBB) makes CNS become a refugee to leukemic cells and serves as a resource of cells that seed extraneural sites. How can the leukemic cells disrupt this barrier and invasive the CNS, even if many of the currently available chemotherapies can not cross the BBB? Tight junction in endothelial cells occupies a central role in the function of the BBB. Except the well known role of degrading extracellular matrix in metastasis of cancer cells, here we show matrix metalloproteinase (MMP)-2 and -9, secreted by leukemic cells, mediate the BBB opening by disrupting tight junction proteins in the CNS leukemia. We demonstrated that leukemic cells impaired tight junction proteins ZO-1, claudin-5 and occludin resulting in increased permeability of the BBB. However, these alterations reduced when MMP-2 and -9 activities were inhibited by RNA interference strategy or by MMP inhibitor GM6001 in an in vitro BBB model. We also found that the disruption of the BBB in company with the down-regulation of ZO-1, claudin-5 and occludin and the up-regulation of MMP-2 and -9 in mouse brain tissues with leukemic cell infiltration by confocal imaging and the assay of in situ gelatin zymography. Besides, GM6001 protected all mice against CNS leukemia. Our findings suggest that the degradation of tight junction proteins ZO-1, claudin-5 and occludin by MMP-2 and -9 secreted by leukemic cells constitutes an important mechanism in the BBB breakdown which contributes to the invasion of leukemic cells to the CNS in acute leukemia

Functional Characterization of a Newly Identified Group B Streptococcus Pullulanase Eliciting Antibodies Able to Prevent Alpha-Glucans Degradation

Streptococcal pullulanases have been recently proposed as key components of the metabolic machinery involved in bacterial adaptation to host niches. By sequence analysis of the Group B Streptococcus (GBS) genome we found a novel putative surface exposed protein with pullulanase activity. We named such a protein SAP. The sap gene is highly conserved among GBS strains and homologous genes, such as PulA and SpuA, have been described in other pathogenic streptococci. The SAP protein contains two N-terminal carbohydrate-binding motifs, followed by a catalytic domain and a C-terminal LPXTG cell wall-anchoring domain. In vitro analysis revealed that the recombinant form of SAP is able to degrade α-glucan polysaccharides, such as pullulan, glycogen and starch. Moreover, NMR analysis showed that SAP acts as a type I pullulanase. Studies performed on whole bacteria indicated that the presence of α-glucan polysaccharides in culture medium up-regulated the expression of SAP on bacterial surface as confirmed by FACS analysis and confocal imaging. Deletion of the sap gene resulted in a reduced capacity of bacteria to grow in medium containing pullulan or glycogen, but not glucose or maltose, confirming the pivotal role of SAP in GBS metabolism of α-glucans. As reported for other streptococcal pullulanases, we found specific anti-SAP antibodies in human sera from healthy volunteers. Investigation of the functional role of anti-SAP antibodies revealed that incubation of GBS in the presence of sera from animals immunized with SAP reduced the capacity of the bacterium to degrade pullulan. Of interest, anti-SAP sera, although to a lower extent, also inhibited Group A Streptococcus pullulanase activity. These data open new perspectives on the possibility to use SAP as a potential vaccine component inducing functional cross-reacting antibodies interfering with streptococcal infections