Screening of anti‐HPA‐1a antibodies in HPA‐1a‐negative mothers

Results from a study of more than 100 000 pregnant women show that the pathophysiology of neonatal alloimmune thrombocytopenia (NAIT) and haemolytic disease of the newborn (HDN) has many equalities [ 1 ]. It has been reported that NAIT is more than three times more frequent compared with HDN. This makes it tempting to consider a design for screening for HPA 1a negativity and follow up in/after the pregnancy based on the same principles as for RhD‐negative pregnant women. Even without available vaccination, it is strongly indicated that screening and Caesarean section 2–4 weeks prior to term in mothers with the HPA‐1a‐negative platelet type, reduce neonatal morbidity and mortality due to NAIT.     

Synthesis of a Polyphenol with a Mesoionic 6‐Oxo‐1,6‐dihydropyrimidin‐3‐ium‐4‐olate as Pendant Group and Its Photochemical Behavior

This study deals with the synthesis of a polyphenol bearing photosensitive groups, and its photochemical behavior upon UV irradiation. Mesoionic 5‐(4‐hydroxybenzyl)‐6‐oxo‐1,2,3‐triphenyl‐1,6‐dihydropyrimidin‐3‐ium‐4‐olate was prepared by condensation of 4‐benzyloxybenzylmalonic acid with N,N′‐diphenylbenzamidine using dicyclohexylcarbodiimide as condensing agent, followed by deprotection of the benzyl group. The monomer was polymerized by an iron‐salen complex to give a polymer with a mesoionic 6‐oxo‐1,6‐dihydropyrimidin‐3‐ium‐4‐olate moiety as the pendant group. Spin coated polymer films were prepared and characterized. It was shown by IR spectroscopy that irradiation with UV light converted the mesoionic structure to a bis(β‐lactam) structure. Waveguide spectroscopy showed a large decrease in the film thickness without refractive index changes during irradiation.

Time development of the refractive index and the film thickness measured with p‐polarized light.     

Development and application of a one‐step real‐time RT‐PCR using a minor‐groove‐binding probe for the detection of a novel bunyavirus in clinical specimens

A highly sensitive one‐step real‐time RT‐PCR method using a minor‐groove‐binding (MGB) probe was developed for detection and quantitation of severe febrile with thrombocytopenia syndrome virus (SFTSV). The assay could discriminate SFTSV infection from other related viral diseases in human with a minimum detection limit of 10 viral RNA copies/µl and was 1,000 times more sensitive than the conventional PCR. Strong linear correlations (r² > 0.99) between the C_t values and viral RNA standards over a linear range were obtained. The coefficients of variation of intra‐ and inter‐assay reproducibility were both less than 2%. The RT‐PCR was also shown to be highly specific, as no positive signals were detected for other related viruses. Evaluation of this assay with serum samples from laboratory confirmed cases and healthy donors showed 100% clinical diagnostic sensitivity and over 99% specificity. Clinical application with samples from 287 patients admitted to the hospital with suspected SFTSV infection showed that 15% were infected by SFTSV. This assay was rapid, requiring just over 2 hr, including the nucleic acid extraction step. J. Med. Virol. 85:370–377, 2013. © 2012 Wiley Periodicals, Inc.     

Down‐regulation of miR‐301a suppresses pro‐inflammatory cytokines in Toll‐like receptor‐triggered macrophages

In many types of tumours, especially pancreatic adenocarcinoma, miR‐301a is over‐expressed. This over‐expression results in negative regulation of the target gene of miR‐301a, the nuclear factor‐κB (NF‐κB) repressing factor (NKRF), increasing the activation of NF‐κB and production of NF‐κB‐responsive pro‐inflammatory cytokines such as interleukin‐8, interferon‐β, nitric oxide synthase 2A and cytochrome oxidase subunit 2 (COX‐2). However, in immune cells, mechanisms that regulate miR‐301a have not been reported. Similar to tumour cells, Toll‐like receptor (TLR) ‐activated macrophages produce NF‐κB‐responsive pro‐inflammatory cytokines. Therefore, it is of considerable interest to determine whether miR‐301a regulates the secretion of cytokines by immune cells. In the present study, we demonstrate that the expression of miR‐301a was decreased in TLR‐triggered macrophages. Through targeting NKRF, miR‐301a affected the activity of NF‐κB and the expression of pro‐inflammatory genes downstream of NF‐κB such as COX‐2, prostaglandin E₂ and interleukin‐6. In addition, when lipopolysaccharide‐treated macrophages were simultaneously stimulated with trichostatin A, an inhibitor of histone deacetylases, the expression of miR‐301a increased, whereas NKRF and pro‐inflammatory cytokine expression decreased. However, further investigation revealed that there was no correlation between the induction of miR‐301a and the inhibitory effect of trichostatin A on lipopolysaccharide‐induced gene expression in macrophages. In summary, our study indicates a new mechanism by which miR‐301a regulates inflammatory cytokine expression in macrophages, which may clarify the regulatory role of microRNAs in immune‐mediated inflammatory responses.     

Velo‐cardio‐facial syndrome: Clinical report of a 70‐year‐old woman

A clinical report is presented of a 70‐year‐old female in whom, after more than 40 years residential psychiatric care, the diagnosis of velo‐cardio‐facial syndrome (VCFS) was ultimately established; the patient has a 46, XX.ish del (22)(q11.2q11.2)(D₂₂S₇₅‐) karyotype. It is advocated that a rather specific psychopathological profile is present in patients with VCFS, for which the term psychopathological phenotype is introduced, that should include data from genetics, neuropathology, development, psychology, and psychiatry. © 2002 Wiley‐Liss, Inc.     

Effectiveness of mite‐impermeable covers: a hypothesis‐generating meta‐analysis

Asthma is a heterogeneous disease. The subject of mite allergen control has evolved into a debate dominated by a Cochrane review by Gøtzsche and Johansen (Cochrane Database of Systematic Reviews, 2008, Art. No: CD001187). A not well‐discussed aspect of that study is the selection by those authors of a univariate meta‐analysis including various interventions. This study extends the meta‐analysis by Gøtzsche and Johansen and aims to generate hypotheses on the effectiveness of various bedding interventions, including the coverage of all bedding elements. Trials were selected based on environmental criteria. The interventions were classified according to the number of barriers used. Standardized mean differences yielded the mite load, three physiological outcomes and asthma symptom scores. The influence of covariates was examined with a mixed‐effect model using the metafor package for meta‐analysis in R. Twelve trials included 1187 observations. The interventions included one barrier or product (six trials), two barriers or partial control (four trials) and three barriers or integral control (two trials). The exposure data showed considerable heterogeneity (I² = 93%). The risk of bias significantly (P = 0.04) influenced the final load, the square root of the interaction between the baseline load and the type of intervention as well (95% CI: ?0.66 to ?0.07 μg/g; P = 0.02). Changes in load showed similar tendencies. Health outcomes showed moderate to considerable heterogeneity (physiological outcomes I² = 44–94%; symptom score I² = 93%). A meta‐regression of bedding interventions indicates that integral control most significantly reduced mite load when the load was high at baseline. The number of trials was too small to allow an appropriate examination of health outcomes. Future studies are suggested to test the hypothesis that allergic patients benefit from integral control when the baseline mite load is high.     

Synthesis and Characterization of a Phthalimide‐Containing Redox‐Active Polymer for High‐Voltage Polymer‐Based Redox‐Flow Batteries

A phthalimide‐containing redox‐active polymer is designed and synthesized in particular for the application in all‐organic redox‐flow batteries. 4‐Vinylbenzyl phthalimide is prepared in a one‐step reaction and copolymerized with a solubility promoting comonomer. The novel polymer is electrochemically characterized in various electrolytes via cyclic voltammetry and features a quasireversible reduction reaction at E⁰_½ = ?1.89 V versus AgNO₃/Ag. Afterward, the polymer is applied as anode‐active material in a first preliminary battery test in combination with a 2,2,6,6‐tetramethylpiperdinyl‐N‐oxyl‐containing polymer as cathode‐active material and a porous membrane as separator, yielding a polymer‐based all‐organic battery system with a high cell voltage of 2.0 V.     

Assessment of sleep‐disordered breathing using a non‐contact bio‐motion sensor

Obstructive sleep apnoea is a highly prevalent but under‐diagnosed disorder. The gold standard for diagnosis of obstructive sleep apnoea is inpatient polysomnography. This is resource intensive and inconvenient for the patient, and the development of ambulatory diagnostic modalities has been identified as a key research priority. SleepMinder (BiancaMed, NovaUCD, Ireland) is a novel, non‐contact, bedside sensor, which uses radio‐waves to measure respiration and movement. Previous studies have shown it to be effective in measuring sleep and respiration. We sought to assess its utility in the diagnosis of obstructive sleep apnoea. SleepMinder and polysomnographic assessment of sleep‐disordered breathing were performed simultaneously on consecutive subjects recruited prospectively from our sleep clinic. We assessed the diagnostic accuracy of SleepMinder in identifying obstructive sleep apnoea, and how SleepMinder assessment of the apnoea–hypopnoea index correlated with polysomnography. Seventy‐four subjects were recruited. The apnoea–hypopnoea index as measured by SleepMinder correlated strongly with polysomnographic measurement (r = 0.90; P ≤ 0.0001). When a diagnostic threshold of moderate–severe (apnoea–hypopnoea index ≥15 events h^?1) obstructive sleep apnoea was used, SleepMinder displayed a sensitivity of 90%, a specificity of 92% and an accuracy of 91% in the diagnosis of sleep‐disordered breathing. The area under the curve for the receiver operator characteristic was 0.97. SleepMinder correctly classified obstructive sleep apnoea severity in the majority of cases, with only one case different from equivalent polysomnography by more than one diagnostic class. We conclude that in an unselected clinical population undergoing investigation for suspected obstructive sleep apnoea, SleepMinder measurement of sleep‐disordered breathing correlates significantly with polysomnography.     

Social Support as a Moderator of the Big‐Fish‐in‐a‐Little‐Pond Effect in Online Self‐Help Support Groups1

Downward social comparisons to others in a relatively unsuccessful group can bolster mood, a phenomenon known as the big‐fish‐in‐a‐little‐pond effect (BFLPE). The current study examined social support as a moderator of the BFLPE in online weight‐management support groups (SGs). Participants (N= 149) were recruited from weight‐management message boards. In an Internet survey, participants made weight‐related social comparisons to the average person and the average SG member. Big fish indicated that they would feel more self‐pride after reading a downward social comparison message than did other participants, but the BFLPE occurred only for participants with lower weight‐related social support. Social support could foster collective identity in online self‐help support groups, reducing the BFLPE.     

Aromatic Polyethers with a 4‐Chloro‐3‐trifluoromethylphenyl Group

Summary: A novel bisphenol, (4‐chloro‐3‐trifluoromethyl)phenylhydroquinone (3FC‐PH), was synthesized via a three‐step synthetic procedure. Four aromatic polyethers based on 3FC‐PH were prepared via a nucleophilic aromatic substitution polycondensation. These polymers had a high thermal stability, and the temperatures at a 5% weight loss were above 516 °C in air. The solubility of the polyethers was improved by the introduction of bulky pendant groups. The average refractive indices of the polymer films at 1 320 nm were in the range 1.5381–1.6145. The dielectric constants of the polyether films estimated from the refractive indices were 2.69–2.98. Highly fluorinated 3FC‐PAE exhibited lower light absorption in the near‐IR region.

Part of the ¹H NMR spectra of 3FC‐PAE.     

Synthesis of a Multiblock Copolymer of cis‐1,4‐Polybutadiene and Poly(3‐buten‐1‐ol)

Synthesis of a multiblock copolymer composed of cis‐1,4‐polybutadiene (PBd) segments and poly(3‐buten‐1‐ol) segments is performed via successive hydroboration and oxidation of cis‐1,4/syn‐1,2‐multiblock PBd. The ratio of functionalization can be controlled by changing the amount of the borane reagent. The obtained polymer shows two distinctive glass‐transition temperatures, which correspond to the cis‐1,4‐PBd block and the poly(3‐buten‐1‐ol) block. These thermal properties clearly show that the functionalization of the PBd proceeds keeping the elastic property derived from cis‐1,4 segment.

     

The DC‐HIL ligand syndecan‐4 is a negative regulator of T‐cell allo‐reactivity responsible for graft‐versus‐host disease

Acute graft-versus-host disease (GVHD) is the most important cause of mortality after allogeneic haematopoietic stem cell transplantation. Allo-reactive T cells are the major mediators of GVHD and the process is regulated by positive and negative regulators on antigen-presenting cells (APC). Because the significance of negative regulators in GVHD pathogenesis is not fully understood, and having discovered that syndecan-4 (SD-4) on effector T cells mediates the inhibitory function of DC-HIL on APC, we proposed that SD-4 negatively regulates the T-cell response to allo-stimulation in acute GVHD, using SD-4 knockout mice. Although not different from their wild-type counterparts in responsiveness to anti-CD3 stimulation, SD-4^−/− T cells lost the capacity to mediate the inhibitory function of DC-HIL and were hyper-reactive to allogeneic APC. Moreover, infusion of SD-4^−/− T cells into sub-lethally γ-irradiated allogeneic mice worsened mortality, with hyper-proliferation of infused T cells in recipients. Although there my be little or no involvement of regulatory T cells in this model because SD-4 deletion had no deleterious effect on T-cell-suppressive activity compared with SD-4^+/+ regulatory T cells. We conclude that SD-4, as the T-cell ligand of DC-HIL, is a potent inhibitor of allo-reactive T cells responsible for GVHD and a potentially useful target for treating this disease.     

Single‐shot single‐voxel lactate measurements using FOCI‐LASER and a multiple‐quantum filter

Measurement of tissue lactate using ¹H MRS is often confounded by overlap with intense lipid signals at 1.3 ppm. Single‐voxel localization using PRESS is also compromised by the large chemical shift displacement between voxels for the 4.1 ppm (–CH) resonance and the 1.3 ppm –CH₃ resonance, leading to subvoxels with signals of opposite phase and hence partial signal cancellation. To reduce the chemical shift displacement to negligible proportions, a modified semi‐LASER sequence was written (“FOCI‐LASER”, abbreviated as fLASER) using FOCI pulses to permit high RF bandwidth even with the limited RF amplitude characteristic of clinical MRI scanners. A further modification, MQF‐fLASER, includes a selective multiple‐quantum filter to detect lactate and reject lipid signals. The sequences were implemented on a Philips 3 T Achieva TX system. In a solution of brain metabolites fLASER lactate signals were 2.7 times those of PRESS. MQF‐fLASER lactate was 47% of fLASER (the theoretical maximum is 50%) but still larger than PRESS lactate. In oil, the main 1.3 ppm lipid peak was suppressed to less than 1%. Enhanced suppression was possible using increased gradient durations. The minimum detectable lactate concentration was approximately 0.5 mM. Coherence selection gradients needed to be at the magic angle to avoid large water signals derived from intermolecular multiple‐quantum coherences. In pilot patient measurements, lactate peaks were often observed in brain tumours, but not in cervix tumours; lipids were effectively suppressed. In summary, compared with PRESS, the fLASER sequence yields greatly superior sensitivity for direct detection of lactate (and equivalent sensitivity for other metabolites), while the single‐voxel single‐shot MQF‐fLASER sequence surpasses PRESS for lactate detection while eliminating substantial signals from lipids. This sequence will increase the potential for in vivo lactate measurement as a biomarker in targeted anti‐cancer treatments as well as in measurements of tissue hypoxia. Copyright © 2015 John Wiley & Sons, Ltd.     

Single‐molecule detection of cancer mutations using a novel PCR‐LDR‐qPCR assay

Detection of low‐abundance mutations in cell‐free DNA is being used to identify early cancer and early cancer recurrence. Here, we report a new PCR‐LDR‐qPCR assay capable of detecting point mutations at a single‐molecule resolution in the presence of an excess of wild‐type DNA. Major features of the assay include selective amplification and detection of mutant DNA employing multiple nested primer‐binding regions as well as wild‐type sequence blocking oligonucleotides, prevention of carryover contamination, spatial sample dilution, and detection of multiple mutations in the same position. Our method was tested to interrogate the following common cancer somatic mutations: BRAF:c.1799T>A (p.Val600Glu), TP53:c.743G>A (p.Arg248Gln), KRAS:c.35G>C (p.Gly12Ala), KRAS:c.35G>T (p.Gly12Val), KRAS:c.35G>A (p.Gly12Asp), KRAS:c.34G>T (p.Gly12Cys), and KRAS:c.34G>A (p.Gly12Ser). The single‐well version of the assay detected 2–5 copies of these mutations, when diluted with 10,000 genome equivalents (GE) of wild‐type human genomic DNA (hgDNA) from buffy coat. A 12‐well (pixel) version of the assay was capable of single‐molecule detection of the aforementioned mutations at TP53, BRAF, and KRAS (specifically p.Gly12Val and p.Gly12Cys), mixed with 1,000–2,250 GE of wild‐type hgDNA from plasma or buffy coat. The assay described herein is highly sensitive, specific, and robust, and potentially useful in liquid biopsies.     

An evaluation of Com‐B27, a fluorescein‐conjugated mouse anti‐HLA‐B27 reagent

We evaluated the HLA‐B27 typing reagent Com‐B27, which is claimed to show minimal cross‐reactivity with HLA‐B7, for use in flow cytometry‐based typing. This combination reagent consists of the fluorescein‐conjugated HLA‐B27 mouse monoclonal antibody ABC‐m3 and a non‐conjugated HLA‐B7 monoclonal antibody that is claimed to block the reactivity of ABC‐m3 to B7 without affecting its reactivity to B27. It reacted well with B27 (B*2702, B*2705) and B2708 [mean median channel fluorescence intensity (MCFI) 8.40] and weakly with B7 (including cells from homozygous B*07 donors), B42/B73 and B22/B37/B44 reference cells (mean MCFI 2.05, 3.09 and 1.10, respectively). It showed a uniform discrimination between B7 and B27/B2708 with no ‘overlap’ in MCFI values, which was seen with the standard ABC‐m3 antibody. There was complete agreement when our standard three‐antibody‐based B27/B2708 flow cytometry assay and the Com‐B27 reagent alone were used to independently assign B27/B2708 status to 651 random patients. Thus, the Com‐B27 reagent provided improved discrimination between B7 and B27/B2708 over the ABC‐m3 antibody, and its B27/B2708 assignments were comparable with our standard flow cytometry assay. However, for consistently reliable B27/B2708 typing we continue to recommend the use of a minimum of two B27 reagents in a protocol that includes DNA‐based testing of ‘equivocal’ B27/B2708 assignments.     

Evaluation of in vivo genotoxicity induced by N‐ethyl‐N‐nitrosourea,benzo[a]pyrene,and 4‐nitroquinoline‐1‐oxide in the Pig‐a and gpt assays

The recently developed Pig‐a mutation assay is based on flow cytometric enumeration of glycosylphosphatidylinositol (GPI) anchor‐deficient red blood cells caused by a forward mutation in the Pig‐a gene. Because the assay can be conducted in nontransgenic animals and the mutations accumulate with repeat dosing, we believe that the Pig‐a assay could be integrated into repeat‐dose toxicology studies and provides an alternative to transgenic rodent (TGR) mutation assays. The capacity and characteristics of the Pig‐a assay relative to TGR mutation assays, however, are unclear. Here, using transgenic gpt delta mice, we compared the in vivo genotoxicity of single oral doses of N‐ethyl‐N‐nitrosourea (ENU, 40 mg/kg), benzo[a]pyrene (BP, 100 and 200 mg/kg), and 4‐nitroquinoline‐1‐oxide (4NQO, 50 mg/kg) in the Pig‐a (peripheral blood) and gpt (bone marrow and liver) gene mutation assays. Pig‐a assays were conducted at 2, 4, and 7 weeks after the treatment, while gpt assays were conducted on tissues collected at the 7‐week terminal sacrifice. ENU increased both Pig‐a and gpt mutant frequencies (MFs) at all sampling times, and BP increased MFs in both assays but the Pig‐a MFs peaked at 2 weeks and then decreased. Although 4NQO increased gpt MFs in the liver, only weak, nonsignificant increases (two‐ or threefold above control) were detected in the bone marrow in both the Pig‐a and the gpt assay. These findings suggest that further studies are needed to elucidate the kinetics of the Pig‐a mutation assay in order to use it as an alternative to the TGR mutation assay. Environ. Mol. Mutagen. 54:747–754, 2013. © 2013 Wiley Periodicals, Inc.