Site-specific labeling of annexin V with F-18 for apoptosis imaging

Annexin V is useful in detecting apoptotic cells by binding to phosphatidylserine (PS) that is exposed on the outer surface of the cell membrane during apoptosis. In this study, we examined the labeling of annexin V-128, a mutated form of annexin V that has a single cysteine residue at the NH 2 terminus, with the thiol-selective reagent (18)F-labeling agent N-[4-[(4-[(18)F]fluorobenzylidene)aminooxy]butyl]maleimide ([(18)F]FBABM). We also examined the cell binding affinity of the (18)F-labeled annexin V-128 ([(18)F]FAN-128). [(18)F]FBABM was synthesized in two-step, one-pot method modified from literature procedure. (Toyokuni et al., Bioconjugate Chem. 2003, 14, 1253-1259). The average yield of [(18)F]FBABM was 23 +/- 4% (n = 4, decay-corrected) and the specific activity was approximately 6000 Ci/mmol. The total synthesis time was approximately 92 min. The critical improvement of this study was identifying and then developing a purification method to remove an impurity N-[4-[(4-dimethylaminobenzylidene)aminooxy]butyl]maleimide 4, whose presence dramatically decreased the yield of protein labeling. Conjugation of [(18)F]FBABM with the thiol-containing annexin V-128 gave [(18)F]FAN-128 in 37 +/- 9% yield (n = 4, decay corrected). Erythrocyte binding assay of [(18)F]FAN-128 showed that this modification of annexin V-128 did not compromise its membrane binding affinity. Thus, an in vivo investigation of [ (18)F]FAN-128 as an apoptosis imaging agent is warranted.     

C-reactive protein as an early predictor of COVID-19 severity

BackgroundData for predicting severity of patients with COVID-19 infection are sparse and still under investigation. We retrospectively studied whether the admission serum C-reactive protein level (CRP) can serve as nearly predictor of disease severity during COVID-19 infection in comparison with other hematologic and inflammatory markers.MethodsWe included all consecutive patients who were admitted in Cheikh Khalifa International University Hospital, Casablanca, Morocco, between February to April 2020, with a confirmed diagnosis of COVID-19 infection using SARS-CoV-2 viral nucleic acid via RT-PCR. The complete blood count and serum CRP level were routinely measured on admission. All clinical and laboratory data of patients were collected and analyzed. The classification of the disease severity was in accordance with the clinical classification of the WHO interim guidance, and the management of patients were adapted to the national management guideline. We estimated receiver operating characteristic (ROC) curves of blood routine parameters as well as their association with COVID-19 disease severity.Results145 COVID-19 patients were included in the study. The median age (range) was 50 (32-63) years, and 75 (51.7%) were men. 101 patients were classified in the non-severe group and 44 patients in the severe group. Based on disease severity, significant differences were observed in the age, gender, comorbidities, and respiratory symptom. Similarly, the biological analysis found significant differences for the neutrophil count, lymphocyte count, eosinophil count, and CRP level. However, according to ROC curves of these laboratory biomarkers, the AUC of CRP at 0.872 was significantly higher than all other parameters. Further, CRP was independently associated with severity of COVID-19 disease (OR = 1.11, 95% IC (1.01-1.22) and or = 1.13, 95% IC (1.04-1.23)).ConclusionsThis study found that the CRP level at admission represent a simple and independent factor that can be useful for early detection of severity during COVID-19 and the easy guidance of primary care.     

Computational analysis of mRNA expression profiles identifies microRNA-29a/c as predictor of colorectal cancer early recurrence

Colorectal cancer (CRC) is one of the leading malignant cancers with a rapid increase in incidence and mortality. The recurrences of CRC after curative resection are sometimes unavoidable and often take place within the first year after surgery. MicroRNAs may serve as biomarkers to predict early recurrence of CRC, but identifying them from over 1,400 known human microRNAs is challenging and costly. An alternative approach is to analyze existing expression data of messenger RNAs (mRNAs) because generally speaking the expression levels of microRNAs and their target mRNAs are inversely correlated. In this study, we extracted six mRNA expression data of CRC in four studies (GSE12032, GSE17538, GSE4526 and GSE17181) from the gene expression omnibus (GEO). We inferred microRNA expression profiles and performed computational analysis to identify microRNAs associated with CRC recurrence using the IMRE method based on the MicroCosm database that includes 568,071 microRNA-target connections between 711 microRNAs and 20,884 gene targets. Two microRNAs, miR-29a and miR-29c, were disclosed and further meta-analysis of the six mRNA expression datasets showed that these two microRNAs were highly significant based on the Fisher p-value combination (p = 9.14 × 10(-9) for miR-29a and p = 1.14 × 10(-6) for miR-29c). Furthermore, these two microRNAs were experimentally tested in 78 human CRC samples to validate their effect on early recurrence. Our empirical results showed that the two microRNAs were significantly down-regulated (p = 0.007 for miR-29a and p = 0.007 for miR-29c) in the early-recurrence patients. This study shows the feasibility of using mRNA profiles to indicate microRNAs. We also shows miR-29a/c could be potential biomarkers for CRC early recurrence.     

Pemphigus vulgaris identifies plakoglobin as key suppressor of c-Myc in the skin

The autoimmune disease pemphigus vulgaris (PV) manifests as loss of keratinocyte cohesion triggered by autoantibody binding to desmoglein (Dsg)3, an intercellular adhesion molecule of mucous membranes, epidermis, and epidermal stem cells. Here we describe a so far unknown signaling cascade activated by PV antibodies. It extends from a transient enhanced turn over of cell surface-exposed, nonkeratin-anchored Dsg3 and associated plakoglobin (PG), through to depletion of nuclear PG, and as one of the consequences, abrogation of PG-mediated c-Myc suppression. In PV patients (6/6), this results in pathogenic c-Myc overexpression in all targeted tissues, including the stem cell compartments. In summary, these results show that PV antibodies act via PG to abolish the c-Myc suppression required for both maintenance of epidermal stem cells in their niche and controlled differentiation along the epidermal lineage. Besides a completely novel insight into PV pathogenesis, these data identify PG as a potent modulator of epithelial homeostasis via its role as a key suppressor of c-Myc.     

A simplified method for purification of annexin V from human placenta

A simplified procedure for purification of annexin V from human placenta was developed. At first, the protein was separated from other proteins in membrane bound form in the presence of Ca2+, then was extracted with EDTA and purified by affinity chromatography on PAAG-immobilized phosphatidylserine. The purified protein gave a single band with a molecular weight of 35,000 in SDS-PAGE.     

Palatal length in cleft palate as a predictor of speech outcome

In an attempt to predict which patients might benefit from primary posterior pharyngeal flaps done at the time of palatal repair, palatal length was assessed before palatal repair and the patient was placed in one of four categories. Patients with longer palates preoperatively had statistically better speech outcomes than patients with shorter palates. Statistical significance was found for most speech parameters. Information on presurgical palatal length can be useful in predicting which patients might profit from primary "pharyngoplasties."     

Infrared body temperature measurement of mice as an early predictor of death in experimental fungal infections

Temperatures of mice were measured using an infrared high performance non-contact thermometer, after the device had been calibrated using implantable microchips containing temperature transponders. Mice were infected with three species of Candida (isolates) and the resultant disseminated infections monitored. Mouse temperatures could be reliably measured using the infrared device and this measurement caused little distress to the mice. We were further able to demonstrate that mice rarely recovered if their body temperature dropped below 33.3 degrees C (sensitivity 68%, specificity 97%). Adoption of a 33.3 degrees C endpoint in fungal sepsis experiments measured by infrared non-contact thermometer would significantly reduce the suffering in the terminal stages of this type of infection model.     

A systems approach identifies HIPK2 as a key regulator of kidney fibrosis

Kidney fibrosis is a common process that leads to the progression of various types of kidney disease. We used an integrated computational and experimental systems biology approach to identify protein kinases that regulate gene expression changes in the kidneys of human immunodeficiency virus (HIV) transgenic mice (Tg26 mice), which have both tubulointerstitial fibrosis and glomerulosclerosis. We identified homeo-domain interacting protein kinase 2 (HIPK2) as a key regulator of kidney fibrosis. HIPK2 was upregulated in the kidneys of Tg26 mice and in those of patients with various kidney diseases. HIV infection increased the protein concentrations of HIPK2 by promoting oxidative stress, which inhibited the seven in absentia homolog 1 (SIAH1)-mediated proteasomal degradation of HIPK2. HIPK2 induced apoptosis and the expression of epithelial-to-mesenchymal transition markers in kidney epithelial cells by activating the p53, transforming growth factor β (TGF-β)-SMAD family member 3 (Smad3) and Wnt-Notch pathways. Knockout of HIPK2 improved renal function and attenuated proteinuria and kidney fibrosis in Tg26 mice, as well as in other murine models of kidney fibrosis. We therefore conclude that HIPK2 is a potential target for anti-fibrosis therapy.     

Quantitative image analysis identifies pVHL as a key regulator of microtubule dynamic instability

Von Hippel-Lindau (VHL) tumor suppressor gene mutations predispose carriers to kidney cancer. The protein pVHL has been shown to interact with microtubules (MTs), which is critical to cilia maintenance and mitotic spindle orientation. However, the function for pVHL in the regulation of MT dynamics is unknown. We tracked MT growth via the plus end marker EB3 (end-binding protein 3)-GFP and inferred additional parameters of MT dynamics indirectly by spatiotemporal grouping of growth tracks from live cell imaging. Our data establish pVHL as a near-optimal MT-stabilizing protein: it attenuates tubulin turnover, both during MT growth and shrinkage, inhibits catastrophe, and enhances rescue frequencies. These functions are mediated, in part, by inhibition of tubulin guanosine triphosphatase activity in vitro and at MT plus ends and along the MT lattice in vivo. Mutants connected to the VHL cancer syndrome are differentially compromised in these activities. Thus, single cell–level analysis of pVHL MT regulatory function allows new predictions for genotype to phenotype associations that deviate from the coarser clinically defined mutant classifications.     

Systematic profiling identifies PDLIM2 as a novel prognostic predictor for oesophageal squamous cell carcinoma (ESCC)

Till now, no appropriate biomarkers for high‐risk population screening and prognosis prediction have been identified for patients with oesophageal squamous cell carcinoma (ESCC). In this study, by the combined use of data from the Gene Expression Omnibus (GEO) datasets and The Cancer Genome Atlas (TCGA)‐oesophageal carcinoma (ESCA), we aimed to screen dysregulated genes with prognostic value in ESCC and the genetic and epigenetic alterations underlying the dysregulation. About 222 genes that had at least fourfold change in ESCC compared with adjacent normal tissues were identified using the microarray data in GDS3838. Among these genes, only PDLIM2 was associated with nodal invasion and overall survival (OS) at the same time. The high PDLIM2 expression group had significantly longer OS and its expression was independently associated with better OS (HR: 0.64, 95% CI: 0.43‐0.95, P = 0.03), after adjustment for gender and pathologic stages. The expression of its exon 7/8/9/10 had the highest AUC value (0.724) and better prognostic value (HR: 0.43, 95% CI: 0.22‐0.83, P = 0.01) than total PDLIM2 expression. PDLIM2 DNA copy deletion was common in ESCC and was associated with decreased gene expression. The methylation status of two CpG sites (cg23696886 and cg20449614) in the proximal promoter region of PDLIM2 showed a moderate negative correlation with the gene expression in PDLIM2 copy neutral/amplification group. In conclusion, we infer that PDLIM2 expression might be a novel prognostic indicator for ESCC patients. Its exon 7/8/9/10 expression had the best prognostic value. Its down‐regulation might be associated with gene‐level copy deletion and promoter hypermethylation.     

Fundal height as a predictor of early preterm triplet delivery.

The purpose of this study was to create graphs of fundal height parameters in triplet pregnancies compared with those in twin pregnancies, and to investigate whether larger fundal heights in triplet pregnancies would predispose them to earlier delivery (before 34 weeks). The subjects were 727 twin pregnant women and 133 triplet pregnant women, who delivered after 1984. Triplet pregnancies showed significantly higher fundal heights compared with twin pregnancies at each gestational age (weeks). In triplet pregnancies, a single fundal height measurement above the 90th percentile before 34 weeks yielded a sensitivity of 31.3% and specificity of 82.4% for delivery before 34 weeks, with a positive predictive value of 50.0% and a negative predictive value of 68.0%. After adjusting for each associated factor using logistic regression, the risk of preterm labour was not significantly associated with a single fundal height measurement above the 90th percentile recorded before 34 weeks.     

V-ATPase as an effective therapeutic target for sarcomas

Malignant tumors show intense glycolysis and, as a consequence, high lactate production and proton efflux activity. We investigated proton dynamics in osteosarcoma, rhabdomyosarcoma, and chondrosarcoma, and evaluated the effects of esomeprazole as a therapeutic agent interfering with tumor acidic microenvironment. All sarcomas were able to survive in an acidic microenvironment (up to 5.9–6.0 pH) and abundant acidic lysosomes were found in all sarcoma subtypes. V-ATPase, a proton pump that acidifies intracellular compartments and transports protons across the plasma membrane, was detected in all cell types with a histotype-specific expression pattern.     

Cellular imaging assay for early evaluation of an apoptosis inducer

The objective of this study was to propose a feasibility of a cellular imaging assay as an alternative to the conventional cytotoxicity assay, such as MTS assay, for apoptosis monitoring. As an apoptosis monitoring parameter, affinity interaction between phosphatidylserine (PS) and annexin V was chosen. First, the specific binding affinity between annexin V and PS in phospholipid bilayers consisting of various molar (0–15%) composition of PS was measured using a surface plasmon resonance biosensor. As PS composition increased, the binding level of annexin V increased proportionally. Second, various concentrations (0.1–10 μM) of staurosporine were used as to induce apoptosis and introduced to MCF-7 breast carcinoma cells. The cellular fluorescence images from annexin V-FITC conjugate were obtained by confocal microscopy, and their fluorescence intensities were quantified by image scanning. Dose–apoptosis (or cell death) relationships were very similar to those from MTS and FACS assays. In summary, our cellular imaging method could serve as a quicker and simpler alternative to MTS (end point assay) and FACS (flow cytometry) to screen potential apoptosis inducers.     

Functional identification of alpha 1-giardin as an annexin of Giardia lamblia

A protein with a relative molecular mass of 31 kDa was specifically extracted by EGTA from a detergent-insoluble fraction of Giardia lamblia. N-terminal sequencing showed this protein to be identical to alpha 1-giardin, a component of the ventral disc which, based on its predicted amino acid sequence, has been classified as annexin XIX. Purified alpha 1-giardin associated with multilamellar phosphatidyl serine-containing vesicles in a Ca(2+)-dependent manner, confirming that it is a functional annexin. Molecular modelling of the amino acid sequence of the giardial annexin into the X-ray structure of annexin V suggests that the Ca(2+)-binding sites, which, as in other annexins, are all located on the convex surface of the molecule, are of the low-affinity type III.     

Competition of annexin V and anticardiolipin antibodies for binding to phosphatidylserine containing membranes

Annexin V, an intracellular protein with a calcium-dependent high affinity for anionic phospholipid membranes, acts as an inhibitor of lipid-dependent reactions of the blood coagulation. Antiphospholipid antibodies found in the plasma of patients with antiphospholipid syndrome generally do not interact with phospholipid membranes directly, but recognize (plasma) proteins associated with lipid membranes, mostly prothrombin or beta(2)-glycoprotein I (beta(2)GPI). Previously, it has been proposed that antiphospholipid antibodies may cause thrombosis by displacing annexin V from procoagulant cell surfaces. We used ellipsometry to study the binding of annexin V and of complexes of beta(2)GPI with patient-derived IgG antibodies to beta(2)GPI, commonly referred to as anticardiolipin antibodies (ACA), to phospholipid bilayers composed of phosphatidylcholine (PC) and 20% phosphatidylserine (PS). More specifically, we investigated the competition of these proteins for the binding sites at these bilayers. We show that ACA-beta(2)GPI complexes, adsorbed to PSPC bilayers, are displaced for more than 70% by annexin V and that annexin V binding is unaffected by the presence of ACA-beta(2)GPI complexes. Conversely, annexin V preadsorbed to these bilayers completely prevents adsorption of ACA-beta(2)GPI complexes, and none of the preadsorbed annexin V is displaced by ACA-beta(2)GPI complexes. Using ellipsometry, we also studied the effect of ACA-beta(2)GPI complexes on the interaction of annexin V with the membranes of ionophore-activated blood platelets as a more physiological relevant model of cell membranes. The experiments with blood platelets confirm the high-affinity binding of annexin V to these membranes and unequivocally show that annexin V binding is unaffected by the presence of ACA-beta(2)GPI. In conclusion, our data unambiguously show that ACA-beta(2)GPI complexes are unable to displace annexin V from procoagulant membranes to any significant extent, whereas annexin V does displace the majority of preadsorbed ACA-beta(2)GPI complexes from these membranes.