The consequences of tetraploidy on Caenorhabditis elegans physiology and sensitivity to chemotherapeutics.

Polyploid cells contain more than two copies of each chromosome. Polyploidy has important roles in development, evolution, and tissue regeneration/repair, and can arise as a programmed polyploidization event or be triggered by stress. Cancer cells are often polyploid. C. elegans nematodes are typically diploid, but stressors such as heat shock and starvation can trigger the production of tetraploid offspring. In this study, we utilized a recently published protocol to generate stable tetraploid strains of C. elegans and compared their physiological traits and sensitivity to two DNA-damaging chemotherapeutic drugs, cisplatin and doxorubicin. As prior studies have shown, tetraploid worms are approximately 30% longer, shorter-lived, and have a smaller brood size than diploids. We investigated the reproductive defect further, determining that tetraploid worms have a shorter overall germline length, a higher rate of germ cell apoptosis, more aneuploidy in oocytes and offspring, and larger oocytes and embryos. We also found that tetraploid worms are modestly protected from growth delay from the chemotherapeutics but are similarly or more sensitive to reproductive toxicity. Transcriptomic analysis revealed differentially expressed pathways that may contribute to sensitivity to stress. This study reveals phenotypic consequences of whole-animal tetraploidy that make C. elegans an excellent model for ploidy differences.

The consequences of tetraploidy on Caenorhabditis elegans physiology and sensitivity to chemotherapeutics.

Polyploid cells contain more than two copies of each chromosome. Polyploidy has important roles in development, evolution, and tissue regeneration/repair, and can arise as a programmed polyploidization event or be triggered by stress. Cancer cells are often polyploid. C. elegans nematodes are typically diploid, but stressors such as heat shock and starvation can trigger the production of tetraploid offspring. In this study, we utilized a recently published protocol to generate stable tetraploid strains of C. elegans and compared their physiological traits and sensitivity to two DNA-damaging chemotherapeutic drugs, cisplatin and doxorubicin. As prior studies have shown, tetraploid worms are approximately 30% longer, shorter-lived, and have a smaller brood size than diploids. We investigated the reproductive defect further, determining that tetraploid worms have a shorter overall germline length, a higher rate of germ cell apoptosis, more aneuploidy in oocytes and offspring, and larger oocytes and embryos. We also found that tetraploid worms are modestly protected from growth delay from the chemotherapeutics but are similarly or more sensitive to reproductive toxicity. Transcriptomic analysis revealed differentially expressed pathways that may contribute to sensitivity to stress. Overall, this study reveals the phenotypic consequences of whole-animal tetraploidy in C. elegans.

Diverse and novel RHD variants in Australian blood donors with a weak D phenotype: implication for transfusion management.

BACKGROUND AND OBJECTIVES: Variant RHD genes associated with the weak D phenotype can result in complete or partial D-epitope expression on the red cell. This study examines the genetic classification in Australian blood donors with a weak D phenotype and correlates RHD variants associated with the weak D phenotype against D-epitope profile. MATERIALS AND METHODS: Following automated and manual serology, blood samples from donors reported as 'weak D' (n = 100) were RHD genotyped by a commercial SNP-typing platform and Sanger sequencing. Two commercial anti-D antibody kits were used for extended serological testing for D-epitope profiles. RESULTS: Three samples had wild-type RHD exonic sequences, and 97 samples had RHD variants. RHD*weak D type 1, RHD*weak D type 2 or RHD*weak D type 3 was detected in 75 donors. The remaining 22 samples exhibited 17 different RHD variants. One donor exhibited a novel RHD*c.939+3A>C lacking one D-epitope. Weak D types 1·1, 5, 15, 17 and 90 showed a partial D-epitope profile. CONCLUSION: The array of RHD variants detected in this study indicated diversity in the Australian donor population that needs to be accommodated for in future genotyping strategies.

Optical measurement of the temporal delay between two ultra-short and focussed laser pluses.

Temporal overlapping of ultra-short and focussed laser pulses is a particularly challenging task, as this timescale lies orders of magnitude below the typical range of fast electronic devices. Here we present an optical technique that allows for the measurement of the temporal delay between two focussed and ultra-short laser pulses. This method is virtually applicable to any focussing geometry and relative intensity of the two lasers. Experimental implementation of this technique provides excellent quantitative agreement with theoretical expectations. The proposed technique will prove highly beneficial for high-power multiple-beam laser experiments.

International society of blood transfusion working party on red cell immunogenetics and terminology: report of the Seoul and London meetings.

The Working Party has met twice since the last report: in Seoul, South Korea 2014, and in London, UK 2015, both in association with the International Society of Blood Transfusion (ISBT) Congress. As in previous meetings, matters pertaining to blood group antigen nomenclature were discussed. Eleven new blood group antigens were added to seven blood group systems. This brings the current total of blood group antigens recognized by the ISBT to 346, of which 308 are clustered within 36 blood groups systems. The remaining 38 antigens are currently unassigned to a known blood group system.

Duffy blood group phenotype-genotype correlations using high-resolution melting analysis PCR and microarray reveal complex cases including a new null FY*A allele: the role for sequencing in genotyping algorithms.

BACKGROUND AND OBJECTIVES: Duffy blood group phenotypes can be predicted by genotyping for single nucleotide polymorphisms (SNPs) responsible for the Fy(a) /Fy(b) polymorphism, for weak Fy(b) antigen, and for the red cell null Fy(a-b-) phenotype. This study correlates Duffy phenotype predictions with serotyping to assess the most reliable procedure for typing. MATERIALS AND METHODS: Samples, n = 155 (135 donors and 20 patients), were genotyped by high-resolution melt PCR and by microarray. Samples were in three serology groups: 1) Duffy patterns expected n = 79, 2) weak and equivocal Fy(b) patterns n = 29 and 3) Fy(a-b-) n = 47 (one with anti-Fy3 antibody). RESULTS: Discrepancies were observed for five samples. For two, SNP genotyping predicted weak Fy(b) expression discrepant with Fy(b-) (Group 1 and 3). For three, SNP genotyping predicted Fy(a) , discrepant with Fy(a-b-) (Group 3). DNA sequencing identified silencing mutations in these FY*A alleles. One was a novel FY*A 719delG. One, the sample with the anti-Fy3, was homozygous for a 14-bp deletion (FY*01N.02); a true null. CONCLUSION: Both the high-resolution melting analysis and SNP microarray assays were concordant and showed genotyping, as well as phenotyping, is essential to ensure 100% accuracy for Duffy blood group assignments. Sequencing is important to resolve phenotype/genotype conflicts which here identified alleles, one novel, that carry silencing mutations. The risk of alloimmunisation may be dependent on this zygosity status.

A novel FY*A allele with the 265T and 298A SNPs formerly associated exclusively with the FY*B allele and weak Fy(b) antigen expression: implication for genotyping interpretative algorithms.

BACKGROUND AND OBJECTIVES: An Australian Caucasian blood donor consistently presented a serology profile for the Duffy blood group as Fy(a+b+) with Fy(a) antigen expression weaker than other examples of Fy(a+b+) red cells. Molecular typing studies were performed to investigate the reason for the observed serology profile. MATERIAL AND METHODS: Blood group genotyping was performed using a commercial SNP microarray platform. Sanger sequencing was performed using primer sets to amplify across exons 1 and 2 of the FY gene and using allele-specific primers. RESULTS: The propositus was genotyped as FY*A/B, FY*X heterozygote that predicted the Fy(a+b+(w) ) phenotype. Sequencing identified the 265T and 298A variants on the FY*A allele. This link between FY*A allele and 265T was confirmed by allele-specific PCR. CONCLUSION: The reduced Fy(a) antigen reactivity is attributed to a FY*A allele-carrying 265T and 298A variants previously defined in combination only with the FY*B allele and associated with weak Fy(b) antigen expression. This novel allele should be considered in genotyping interpretative algorithms for generating a predicted phenotype.

International Society of Blood Transfusion Working Party on red cell immunogenetics and blood group terminology: Cancun report (2012).

The International Society of Blood Transfusion Working Party on red cell immunogenetics and blood group terminology convened during the International congress in Cancun, July 2012. This report details the newly identified antigens in existing blood group systems and presents three new blood group systems.

Heterogeneous drying stresses in stratum corneum.

We study the drying of stratum corneum, the skin's outermost layer and an essential barrier to mechanical and chemical stresses from the environment. Even though stratum corneum exhibits structural features across multiple length-scales, contemporary understanding of the mechanical properties of stratum corneum is based on the assumption that its thickness and composition are homogeneous. We quantify spatially resolved in-plane traction stress and deformation at the interface between a macroscopic sample of porcine stratum corneum and an adherent deformable elastomer substrate. At length-scales greater than a millimeter, the skin behaves as a homogeneous elastic material. At this scale, a linear elastic model captures the spatial distribution of traction stresses and the dependence of drying behavior on the elastic modulus of the substrate. At smaller scales, the traction stresses are strikingly heterogeneous and dominated by the heterogeneous structure of the stratum corneum.

Seroprevalence of 2009 pandemic influenza A(H1N1) virus in Australian blood donors, October - December 2009.

Assessment of the severity of disease due to the 2009 pandemic influenza A(H1N1) in Australian states and territories has been hampered by the absence of denominator data on population exposure. We compared antibody reactivity to the pandemic virus using haemagglutination inhibition assays performed on plasma specimens taken from healthy adult blood donors (older than 16 years) before and after the influenza pandemic that occurred during the southern hemisphere winter. Pre-influenza season samples (April ­ May 2009, n=496) were taken from donation collection centres in North Queensland (in Cairns and Townsville); post-outbreak specimens (October ­ November 2009, n=779) were from donors at seven centres in five states. Using a threshold antibody titre of 40 as a marker of recent infection, we observed an increase in the influenza-seropositive proportion of donors from 12% to 22%, not dissimilar to recent reports of influenza A(H1N1)-specific immunity in adults from the United Kingdom. No significant differences in seroprevalence were observed between Australian states, although the ability to detect minor variations was limited by the sample size. On the basis of these figures and national reporting data, we estimate that approximately 0.23% of all individuals in Australia exposed to the pandemic virus required hospitalisation and 0.01% died. The low seroprevalence reported here suggests that some degree of prior immunity to the virus, perhaps mediated by broadly reactive T-cell responses to conserved influenza viral antigens, limited transmission among adults and thus constrained the pandemic in Australia.

Two distinct types of murine blast colony-forming cells are multipotential hematopoietic precursors.

Two types of blast colonies can be stimulated to develop in semisolid agar cultures of murine bone marrow cells. Typically, these are either multicentric colonies stimulated by stem cell factor (SCF) plus interleukin-6 (IL-6) or dispersed colonies stimulated by Flt3 ligand (FL) plus IL-6. Both types of blast colony-forming cells (BL-CFCs) can generate large numbers of lineage-committed granulocyte-macrophage progenitor cells and exhibit some capacity for self-generation and the formation of eosinophil and megakaryocyte progenitor cells. However, the two populations of BL-CFCs are largely distinct and partially separable by fluorescence-activated cell sorting and are distinguished by differing capacity to form granulocyte-committed progeny. Both types of BL-CFCs can generate dendritic cells and small numbers of lymphocytes but the FL-responsive BL-CFCs have a greater capacity to form both B and T lymphocytes. Both types of blast colonies offer remarkable opportunities to analyze multilineage commitment at a clonal level in vitro.

Follow-up of six blood donors highlights the complementary role and limitations of hepatitis C virus antibody and nucleic acid amplification tests.

BACKGROUND AND OBJECTIVES: The purpose of this study was to analyse the follow-up results for six blood donors who screened positive for hepatitis C virus (HCV) by nucleic acid amplification technology (NAT) but were non-reactive in the primary antibody immunoassay (HCV NAT yield). MATERIALS AND METHODS: Volunteer blood donations were screened, in parallel, for antibodies to hepatitis C virus (anti-HCV) and for human immunodeficiency virus (HIV)/HCV RNA using the Abbott PRISM HCV Chemiluminescent immunoassay (ChLIA) and the Chiron Procleix HIV-1/HCV RNA assays, respectively. NAT yield donor samples were further tested using supplemental assays, including an alternate HCV antibody enzyme immunoassay (EIA) (Abbott Murex anti-HCV Version 4), an immunoblot (Ortho RIBA-3 or Genelabs Diagnostics HCV Blot 3.0) and two alternative HCV NAT assays [Roche HCV Amplicor and an assembled HCV polymerase chain reaction (PCR)]. Five of the six donors were available for follow-up testing. RESULTS: The six NAT yield donations were identified as constituents of 24-member minipools among 2,212,695 donations screened over the 28-month study period. All samples were positive when tested, undiluted, using the Roche Amplicor and assembled reverse transcription-polymerase chain reaction (RT-PCR) alternate NAT assays. One of the donors, subsequent to seroconversion, showed RNA levels that fluctuated above and below the limit of detection of the NAT screening assay. Three of the six were reactive on the secondary EIA and showed reactivity to the core c22(p) antigen by immunoblot at the index donation. Two others subsequently became reactive in the ChLIA prior to the EIA, showing reactivity against c100 and/or c33c antigens by immunoblot. The remaining donor became reactive in the ChLIA and EIA at the same time, showing RIBA reactivity against all of the following three peptides: c100; c33c; and c22(p). CONCLUSIONS: This study demonstrated that at least five of six HCV NAT yield donors were in the pre- or early antibody seroconversion phase of infection. The observation that one yield donor demonstrated HCV RNA that fluctuated above and below the limit of detection of the primary NAT-screening assay supports the maintenance of both NAT and antibody screening for HCV. Follow-up testing of suspected yield donors revealed that the primary and alternate anti-HCV immunoassays had different performance characteristics, depending on the specificity of the donor's early anti-HCV response.

Nucleic acid technology screening of Australian blood donors for hepatitis C and human immunodeficiency virus-1 RNA: comparison of two high-throughput testing strategies.

BACKGROUND AND OBJECTIVES: The aim of this study was to compare the performance of two high-throughput strategies for hepatitis C virus (HCV) and human immunodeficiency virus-1 (HIV-1) RNA nucleic acid technology (NAT) screening in a volunteer blood-donor population. MATERIALS AND METHODS: The semiautomated Chiron Procleix HIV-1/HCV transcription mediated amplification (TMA) assay was used to screen 1 439 765 donations in two different testing configurations. Three sites (termed PDT sites) performed a mixture of individual donation (ID) and minipool (MP) testing, where 1 113 288 donations were screened as pools of 24 and an additional 32 003 donations were screened in ID format. A further two sites (termed SDT sites) screened 294 474 donations exclusively in ID format. RESULTS: A significantly higher proportion of initial NAT reactives that failed to react on follow-up testing [termed non-repeatably reactive (NRR)] was observed for ID testing at SDT sites than at PDT sites (0.082 vs. 0.047%: P < 0.01). Within the PDT sites, however, there was no significant difference between the NRR rate for MP or ID samples (0.037 vs. 0.047%; not significant). There was a significant difference in failed run rates between PDT and SDT sites (P < 0.01), with PDT sites having a higher run failure rate owing to non-amplification of the internal control. The PDT sites also had a significantly higher overall invalid sample rate. However, the invalid sample rate, specifically caused by known equipment failure, was significantly higher in the SDT sites, possibly attributable to greater usage (P < 0.0001). Four HCV NAT-positive/antibody-negative samples were identified in the course of the study. CONCLUSIONS: The comparison of the performance of PDT with SDT sites identified only minor differences that did not adversely impact on the timely release of blood products. Although both ID and MP strategies showed excellent specificity, irrespective of site configuration, the significantly increased NRR rate, observed exclusively for ID testing performed at SDT sites, indicates a potential for contamination that may limit the number of samples that can optimally be processed using ID testing. The performance data for ID testing in particular should serve as a useful benchmark for evaluating candidate NAT systems that are fully automated.

Better care and better teaching. New model of postpartum care for early discharge programs.

PROBLEM BEING ADDRESSED: Rapid postpartum discharge has reduced opportunities to detect early newborn or parenting problems and to teach neonatal assessment and maternal postpartum care to medical trainees. OBJECTIVE OF PROGRAM: Development of a program to not only ensure adequate care of mothers and newborns after early hospital discharge, but also to teach outpatient assessment skills to family medicine residents. MAIN COMPONENTS OF PROGRAM: In an urban, secondary care, university-affiliated teaching hospital predominantly training family medicine residents, an interdisciplinary committee created and supervised a neonatal and maternal postpartum assessment program. Newborn infants and their mothers are seen by a family physician, a family medicine resident, and a nurse within 48 hours of discharge, after which care is assumed in the community by the child's primary care physician. An assessment protocol developed by the interdisciplinary group promotes standardized mother and child care and a structured learning experience for trainees. CONCLUSION: Rapid follow up of early discharged infants and their mothers can be facilitated by a program of standardized assessment by a roster of pooled, interacting family physicians and nurses. When this assessment occurs in a teaching milieu, a comprehensive learning experience can be combined with defined objectives that emphasize and encourage newborn and maternal assessment for ambulatory patients.

Membrane interaction of Escherichia coli hemolysin: flotation and insertion-dependent labeling by phospholipid vesicles.

The 1,024-amino-acid acylated hemolysin of Escherichia coli subverts host cell functions and causes cell lysis. Both activities require insertion of the toxin into target mammalian cell membranes. To identify directly the principal toxin sequences dictating membrane binding and insertion, we assayed the lipid bilayer interaction of native protoxin, stably active toxin, and recombinant peptides. Binding was assessed by flotation of protein-liposome mixtures through density gradients, and insertion was assessed by labeling with a photoactivatable probe incorporated into the target lipid bilayer. Both the active acylated hemolysin and the inactive unacylated protoxin were able to bind and also insert. Ca(2+) binding, which is required for toxin activity, did not influence the in vitro interaction with liposomes. Three overlapping large peptides were expressed separately. A C-terminal peptide including residues 601 to 1024 did not interact in either assay. An internal peptide spanning residues 496 to 831, including the two acylation sites, bound to phospholipid vesicles and showed a low level of insertion-dependent labeling. In vitro acylation had no effect on the bilayer interaction of either this peptide or the full-length protoxin. An N-terminal peptide comprising residues 1 to 520 also bound to phospholipid vesicles and showed strong insertion-dependent labeling, ca. 5- to 25-fold that of the internal peptide. Generation of five smaller peptides from the N-terminal region identified the principal determinant of lipid insertion as the hydrophobic sequence encompassing residues 177 to 411, which is conserved among hemolysin-related toxins.