Liquid flow and control without solid walls.

When miniaturizing fluidic circuitry, the solid walls of the fluid channels become increasingly important1 because they limit the flow rates achievable for a given pressure drop, and they are prone to fouling2. Approaches for reducing the wall interactions include hydrophobic coatings3, liquid-infused porous surfaces4-6, nanoparticle surfactant jamming7, changes to surface electronic structure8, electrowetting9,10, surface tension pinning11,12 and use of atomically flat channels13. A better solution may be to avoid the solid walls altogether. Droplet microfluidics and sheath flow achieve this but require continuous flow of the central liquid and the surrounding liquid1,14. Here we demonstrate an approach in which aqueous liquid channels are surrounded by an immiscible magnetic liquid, both of which are stabilized by a quadrupolar magnetic field. This creates self-healing, non-clogging, anti-fouling and near-frictionless liquid-in-liquid fluidic channels. Manipulation of the field provides flow control, such as valving, splitting, merging and pumping. The latter is achieved by moving permanent magnets that have no physical contact with the liquid channel. We show that this magnetostaltic pumping method can be used to transport whole human blood with very little damage due to shear forces. Haemolysis (rupture of blood cells) is reduced by an order of magnitude compared with traditional peristaltic pumping, in which blood is mechanically squeezed through a plastic tube. Our liquid-in-liquid approach provides new ways to transport delicate liquids, particularly when scaling channels down to the micrometre scale, with no need for high pressures, and could also be used for microfluidic circuitry.

Fluid Drag Reduction by Magnetic Confinement.

The frictional forces of a viscous liquid flow are a major energy loss issue and severely limit microfluidics practical use. Reducing this drag by more than a few tens of percent remain elusive. Here, we show how cylindrical liquid-in-liquid flow leads to drag reduction of 60-99% for sub-mm and mm-sized channels, regardless of whether the viscosity of the transported liquid is larger or smaller than that of the confining one. In contrast to lubrication or sheath flow, we do not require a continuous flow of the confining lubricant, here made of a ferrofluid held in place by magnetic forces. In a laminar flow model with appropriate boundary conditions, we introduce a modified Reynolds number with a scaling that depends on geometrical factors and viscosity ratio of the two liquids. It explains our whole range of data and reveals the key design parameters for optimizing the drag reduction values. Our approach promises a new route for microfluidics designs with pressure gradient reduced by orders of magnitude.

Behaviour of flexible superhydrophobic striped surfaces during (electro-)wetting of a sessile drop.

We study here the microscopic deformations of elastic lamellae constituting a superhydrophobic substrate under different wetting conditions of a sessile droplet using electrowetting. The deformation profiles of the lamellae are experimentally evaluated using confocal microscopy. These experimental results are then explained using a variational principle formalism within the framework of linear elasticity. We show that the local deformation profile of a lamella is mainly controlled by the net horizontal component of the capillary forces acting on its top due to the pinned droplet contact line. We also discuss the indirect role of electrowetting in dictating the deformation characteristics of the elastic lamellae. One important conclusion is that the small deflection assumption, which is frequently used in the literature, fails to provide a quantitative description of the experimental results; a full solution of the non-linear governing equation is necessary to describe the experimentally obtained deflection profiles.

Spry1 as a novel regulator of erythropoiesis, EPO/EPOR target, and suppressor of JAK2.

Sprouty proteins are established modifiers of receptor tyrosine kinase (RTK) signaling and play important roles in vasculogenesis, bone morphogenesis, and renal uteric branching. Little is understood, however, concerning possible roles for these molecular adaptors during hematopoiesis. Within erythroid lineage, Spry1 was observed to be selectively and highly expressed at CFU-e to erythroblast stages. In analyses of possible functional roles, an Mx1-Cre approach was applied to conditionally delete Spry1. At steady state, Spry1 deletion selectively perturbed erythroid development and led to reticulocytosis plus heightened splenic erythropoiesis. When challenged by hemolysis, Spry1-null mice exhibited worsened anemia and delayed recovery. During short-term marrow transplantation, Spry1-null donor marrow also failed to efficiently rescue the erythron. In each anemia model, however, hyperexpansion of erythroid progenitors was observed. Spry function depends on phosphorylation of a conserved N-terminal PY motif. Through an LC-MS/MS approach, Spry1 was discovered to be regulated via the erythropoietin receptor (EPOR), with marked EPO-induced Spry1-PY53 phosphorylation observed. When EPOR signaling pathways were analyzed within Spry1-deficient erythroid progenitors, hyperactivation of not only Erk1,2 but also Jak2 was observed. Studies implicate Spry1 as a novel regulator of erythropoiesis during anemia, transducer of EPOR signals, and candidate suppressor of Jak2 activity.

The clinical year dilemma: Examining the stressors and alternatives of pre-radiology training.

Since its reinstatement in 1997, the effectiveness of the clinical year prior to radiology residency has been a contentious topic concerning its role in cultivating skilled radiologists. This review evaluates the limitations of the one-year internship and explores alternative approaches. Utilizing databases such as PubMed, Google Scholar, and Scopus, this study identified pertinent articles that aligned with the inclusion criteria for post-graduate year 1 (PGY-1) training before radiology residency. Through a qualitative analysis of the literature, the review identifies prevalent themes concerning the drawbacks of the preliminary clinical year and potential alternative strategies. Many current trainees express skepticism about the value of the clinical year, noting a disconnect between its generalist nature and the specialized demands of subsequent radiology training. Interns felt uncertain about radiology exam indications and found radiology departments to be unapproachable, reflecting the need for alternative educational strategies to improve the preparedness and confidence of radiology interns as they transition from academic environments to clinical practice. The preparatory clinical year prior to entering radiology residency presents a mix of utility, along with alternative approaches to structuring this year. These alternatives include incorporating it into the undergraduate medical curriculum, restructuring or designing radiology-focused clinical years, and reevaluating the overall effectiveness of the clinical year in training.

During EPO or anemia challenge, erythroid progenitor cells transit through a selectively expandable proerythroblast pool.

Investigations of bone marrow (BM) erythroblast development are important for clinical concerns but are hindered by progenitor cell and tissue availability. We therefore sought to more specifically define dynamics, and key regulators, of the formation of developing BM erythroid cell cohorts. A unique Kit(-)CD71(high)Ter119(-) "stage E2" proerythroblast pool first is described, which (unlike its Kit(+) "stage E1" progenitors, or maturing Ter119(+) "stage E3" progeny) proved to selectively expand ∼ 7-fold on erythropoietin challenge. During short-term BM transplantation, stage E2 proerythroblasts additionally proved to be a predominantly expanded progenitor pool within spleen. This E1→E2→E3 erythroid series reproducibly formed ex vivo, enabling further characterizations. Expansion, in part, involved E1 cell hyperproliferation together with rapid E2 conversion plus E2 stage restricted BCL2 expression. Possible erythropoietin/erythropoietin receptor proerythroblast stage specific events were further investigated in mice expressing minimal erythropoietin receptor alleles. For a hypomorphic erythropoietin receptor-HM allele, major defects in erythroblast development occurred selectively at stage E2. In addition, stage E2 cells proved to interact productively with primary BM stromal cells in ways that enhanced both survival and late-stage development. Overall, findings reveal a novel transitional proerythroblast compartment that deploys unique expansion devices.

In vitro-differentiated embryonic stem cells give rise to male gametes that can generate offspring mice.

Male gametes originate from a small population of spermatogonial stem cells (SSCs). These cells are believed to divide infinitely and to support spermatogenesis throughout life in the male. Here, we developed a strategy for the establishment of SSC lines from embryonic stem (ES) cells. These cells are able to undergo meiosis, are able to generate haploid male gametes in vitro, and are functional, as shown by fertilization after intracytoplasmic injection into mouse oocytes. Resulting two-cell embryos were transferred into oviducts, and live mice were born. Six of seven animals developed to adult mice. This is a clear indication that male gametes derived in vitro from ES cells by this strategy are able to induce normal fertilization and development. Our approach provides an accessible in vitro model system for studies of mammalian gametogenesis, as well as for the development of new strategies for the generation of transgenic mice and treatment of infertility.

Erythropoietin receptor response circuits.

PURPOSE OF REVIEW: In 1985-1989, erythropoietin (EPO), its receptor (EPOR), and janus kinase 2 were cloned; established to be essential for definitive erythropoiesis; and initially intensely studied. Recently, new impetus, tools, and model systems have emerged to re-examine EPO/EPOR actions, and are addressed in this review. Impetus includes indications that EPO affects significantly more than standard erythroblast survival pathways, the development of novel erythropoiesis-stimulating agents, increasing evidence for EPO/EPOR cytoprotection of ischemically injured tissues, and potential EPO-mediated worsening of tumorigenesis. RECENT FINDINGS: New findings are reviewed in four functional contexts: (pro)erythroblast survival mechanisms, new candidate EPO/EPOR effects on erythroid cell development and new EPOR responses, EPOR downmodulation and trafficking, and novel erythropoiesis-stimulating agents. SUMMARY: As Current Opinion, this monograph seeks to summarize, and provoke, new EPO/EPOR action concepts. Specific problems addressed include: beyond (and before) BCL-XL, what key survival factors are deployed in early-stage proerythroblasts? Are distinct EPO/EPOR signals transduced in stage-selective fashions? Is erythroblast proliferation also modulated by EPO/EPOR signals? What functions are subserved by new noncanonical EPO/EPOR response factors (e.g. podocalyxin like-1, tribbles 3, reactive oxygen species, and nuclear factor kappa B)? What key regulators mediate EPOR inhibition and trafficking? And for emerging erythropoiesis-stimulating agents, to what extent do activities parallel EPOs (or differ in advantageous, potentially complicating ways, or both)?

EPO receptor circuits for primary erythroblast survival.

EPO functions primarily as an erythroblast survival factor, and its antiapoptotic actions have been proposed to involve predominantly PI3-kinase and BCL-X pathways. Presently, the nature of EPO-regulated survival genes has been investigated through transcriptome analyses of highly responsive, primary bone marrow erythroblasts. Two proapoptotic factors, Bim and FoxO3a, were rapidly repressed not only via the wild-type EPOR, but also by PY-deficient knocked-in EPOR alleles. In parallel, Pim1 and Pim3 kinases and Irs2 were induced. For this survival gene set, induction failed via a PY-null EPOR-HM allele, but was restored upon reconstitution of a PY343 STAT5-binding site within a related EPOR-H allele. Notably, EPOR-HM supports erythropoiesis at steady state but not during anemia, while EPOR-H exhibits near wild-type EPOR activities. EPOR-H and the wild-type EPOR (but not EPOR-HM) also markedly stimulated the expression of Trb3 pseudokinase, and intracellular serpin, Serpina-3G. For SERPINA-3G and TRB3, ectopic expression in EPO-dependent progenitors furthermore significantly inhibited apoptosis due to cytokine withdrawal. BCL-XL and BCL2 also were studied, but in highly responsive Kit(pos)CD71(high)Ter119(neg) erythroblasts, neither was EPO modulated. EPOR survival circuits therefore include the repression of Bim plus FoxO3a, and EPOR/PY343/STAT5-dependent stimulation of Pim1, Pim3, Irs2 plus Serpina-3G, and Trb3 as new antiapoptotic effectors.

A newborn with hereditary haemorrhagic telangiectasia and an unusually severe phenotype.

UNLABELLED: Hereditary haemorrhagic telangiectasia (HHT), associated with arteriovenous malformations, is a genetic disease of the vascular system with a frequency of approx. 1:10,000. Genetic diagnosis serves to identify individuals at risk of developing the disease and is a useful tool for genetic counselling purposes. QUESTIONS UNDER STUDY: Here we report on a child presenting severe arteriovenous malformations leading to heart failure. Her mother and grandmother present fewer symptoms of hereditary haemorrhagic telangiectasia. In this study we identify the cause of HHT in the family. METHODS: Clinical examination, PCR, DNA sequencing, quantitative PCR, Southern blot, xray, ultrasound, cardiac catheterisation and angiocardiography. RESULTS: Initially the sequence variant in c.392C>T in the endoglin gene was detected in the grandmother, but not in other affected family members. Further analyses revealed a deletion of exon 1 of endoglin, segregating with the phenotype. CONCLUSIONS: This report points out the need for careful evaluation of molecular genetic findings, particularly in diseases with highly variable phenotype.

Governing roles for Trib3 pseudokinase during stress erythropoiesis.

In response to anemia, the heightened production of erythropoietin (EPO) can sharply promote erythroid progenitor cell (EPC) formation. Specific mediators of such EPO- accelerated erythropoiesis, however, are not well understood. Presently, we first report that the expression of Trib3 in adult bone marrow EPCs in vivo is nominal at steady state, but strongly activated on EPO challenge. In a knockout mouse model, Trib3 disruption modestly increased steady-state erythrocyte numbers and decreased mean corpuscular volume. Following 5-fluorouracil myeloablation, however, rebound red blood cell production and hemoglobin levels were substantially (and selectively) compromised in Trib3-/- mice versus Trib3+/+ congenic controls. Erythrocytes from 5-fluorouracil-treated Trib3-/- mice additionally were more prone to lysis and exhibited elevated peroxide-induced reactive oxygen species. Ex vivo, the development of CD71posTer119pos erythroblasts from Trib3-/- bone marrow progenitors was attenuated, and this was associated with heightened EPO-dependent Erk1/2 activation and moderately increased Akt activation. For developmentally staged EPCs, gene profiling provided further initial insight into candidate mediators of EPO-induced Trib3 gene expression, including Cebp-beta, Atf4, Egr-1, and Nab1. Overall, Trib3 is indicated to act as a novel EPC-intrinsic governor of stress erythropoiesis.

Erythropoietin-directed erythropoiesis depends on serpin inhibition of erythroblast lysosomal cathepsins.

Erythropoietin (EPO) and its cell surface receptor (EPOR) are essential for red blood cell production and exert important cytoprotective effects on select vascular, immune, and cancer cells. To discover novel EPO action modes, we profiled the transcriptome of primary erythroid progenitors. We report Serpina3g/Spi2A as a major new EPO/EPOR target for the survival of erythroid progenitors. In knockout mice, loss of Spi2A worsened anemia caused by hemolysis, radiation, or transplantation. EPO-induced erythropoiesis also was compromised. In particular, maturing erythroblasts required Spi2A for cytoprotection, with iron and reactive oxygen species as cytotoxic agents. Spi2A defects were ameliorated by cathepsin-B/L inhibition, and by genetic co-deletion of lysosomal cathepsin B. Pharmacological inhibition of cathepsin B/L enhanced EPO-induced red cell formation in normal mice. Overall, we define an unexpected EPO action mode via an EPOR-Spi2A serpin-cathepsin axis in maturing erythroblasts, with lysosomal cathepsins as novel therapeutic targets.

Defining an EPOR- regulated transcriptome for primary progenitors, including Tnfr-sf13c as a novel mediator of EPO- dependent erythroblast formation.

Certain concepts concerning EPO/EPOR action modes have been challenged by in vivo studies: Bcl-x levels are elevated in maturing erythroblasts, but not in their progenitors; truncated EPOR alleles that lack a major p85/PI3K recruitment site nonetheless promote polycythemia; and Erk1 disruption unexpectedly bolsters erythropoiesis. To discover novel EPO/EPOR action routes, global transcriptome analyses presently are applied to interrogate EPO/EPOR effects on primary bone marrow-derived CFUe-like progenitors. Overall, 160 EPO/EPOR target transcripts were significantly modulated 2-to 21.8-fold. A unique set of EPO-regulated survival factors included Lyl1, Gas5, Pim3, Pim1, Bim, Trib3 and Serpina 3g. EPO/EPOR-modulated cell cycle mediators included Cdc25a, Btg3, Cyclin-d2, p27-kip1, Cyclin-g2 and CyclinB1-IP-1. EPO regulation of signal transduction factors was also interestingly complex. For example, not only Socs3 plus Socs2 but also Spred2, Spred1 and Eaf1 were EPO-induced as negative-feedback components. Socs2, plus five additional targets, further proved to comprise new EPOR/Jak2/Stat5 response genes (which are important for erythropoiesis during anemia). Among receptors, an atypical TNF-receptor Tnfr-sf13c was up-modulated >5-fold by EPO. Functionally, Tnfr-sf13c ligation proved to both promote proerythroblast survival, and substantially enhance erythroblast formation. The EPOR therefore engages a sophisticated set of transcriptome response circuits, with Tnfr-sf13c deployed as one novel positive regulator of proerythroblast formation.

DYRK3 dual-specificity kinase attenuates erythropoiesis during anemia.

During anemia erythropoiesis is bolstered by several factors including KIT ligand, oncostatin-M, glucocorticoids, and erythropoietin. Less is understood concerning factors that limit this process. Experiments performed using dual-specificity tyrosine-regulated kinase-3 (DYRK3) knock-out and transgenic mice reveal that erythropoiesis is attenuated selectively during anemia. DYRK3 is restricted to erythroid progenitor cells and testes. DYRK3-/- mice exhibited essentially normal hematological profiles at steady state and reproduced normally. In response to hemolytic anemia, however, reticulocyte production increased severalfold due to DYRK3 deficiency. During 5-fluorouracil-induced anemia, both reticulocyte and red cell formation in DYRK3-/- mice were elevated. In short term transplant experiments, DYRK3-/- progenitors also supported enhanced erythroblast formation, and erythropoietic advantages due to DYRK3-deficiency also were observed in 5-fluorouracil-treated mice expressing a compromised erythropoietin receptor EPOR-HM allele. As analyzed ex vivo, DYRK3-/- erythroblasts exhibited enhanced CD71posTer119pos cell formation and 3HdT incorporation. Transgenic pA2gata1-DYRK3 mice, in contrast, produced fewer reticulocytes during hemolytic anemia, and pA2gata1-DYRK3 progenitors were compromised in late pro-erythroblast formation ex vivo. Finally, as studied in erythroid K562 cells, DYRK3 proved to effectively inhibit NFAT (nuclear factor of activated T cells) transcriptional response pathways and to co-immunoprecipitate with NFATc3. Findings indicate that DYRK3 attenuates (and possibly apportions) red cell production selectively during anemia.

Mice deficient for RNA-binding protein brunol1 show reduction of spermatogenesis but are fertile.

RNA-binding proteins are involved in post-transcriptional processes like mRNA stabilization, alternative splicing, and transport. Brunol1 is a novel mouse gene related to elav/Bruno family of genes encoding for RNA-binding proteins. We report here the expression and functional analysis of murine Brunol1. Expression analysis of Brunol1 during embryogenesis by RT-PCR showed that Brunol1 expression starts at 9.5 dpc and continues to the later stages of embryonic development. In adult mice, the Brunol1 expression is restricted to brain and testis. We also analyzed the Brunol1 expression in testes of different mutants with spermatogenesis defects: W/W(V), Tfm/y, Leyl(-/-), olt/olt, and qk/qk. Brunol1 transcript was detectable in Leyl(-/-), olt/olt, and qk/qk mutant but not in W/W(V) and Tfm/y mutants. We also showed by transfection of a fusion protein of green fluorescent protein and Brunol1 protein into NIH3T3 cells, that Brunol1 is localized in cytoplasm and nucleus. In order to elucidate the function of the Brunol1 protein in spermatogenesis, we disrupted the Brunol1 locus in mouse by homologous recombination, which resulted in a complete loss of the Brunol1 transcript. Male and female Brunol1(+/-) and Brunol1(-/-) mice from genetic backgrounds C57BL/6J x 129/Sv hybrid and 129X1/SvJ when inbred exhibited normal phenotype and are fertile, although the number and motility of sperms are significantly reduced. An intensive phenotypic analysis showed no gross abnormalities in testis morphology. Collectively our results demonstrate that Brunol1 might be nonessential protein for mouse embryonic development and spermatogenesis.

Expression, localization and tau exon 10 splicing activity of the brain RNA-binding protein TNRC4.

Elucidating the mechanisms of alternative splicing in the brain is a prerequisite to the understanding of the pathogenesis of major neurological diseases linked to impairment of pre-mRNA alternative splicing. The gene trinucleotide repeat-containing 4 (TNRC4) is predicted to encode a member of the CELF (CUG-BP- and ETR-3-like factors) family of RNA-binding proteins containing a 15-18-residue polyglutamine sequence. The TNRC4 transcript is selectively expressed in the brain. Using an anti-peptide antibody against the predicted sequence, we establish the presence of TNRC4 as a approximately 50 kDa protein in the brain. Full-length TNRC4 displays nuclear and cytoplasmic localizations in transfected cells, whereas a C-terminally truncated mutant is essentially confined to the cytoplasm. TNRC4 is not recruited into inclusions formed by polyglutamine-expanded ataxin-1 or huntingtin. TNRC4 activates tau exon 10 (E10) inclusion at high efficiency in transfected cells. TNRC4 contains two consecutive N-terminal RNA recognition motifs (RRMs) separated from the C-terminal RRM. Deletion and point mutant analysis show that the activity of TNRC4 on tau E10 splicing is mainly mediated by the RNA-binding activity of the second RRM and involves an intronic element of the tau pre-mRNA. The polyglutamine sequence has no effect on the activity of TNRC4 on tau E10 splicing. This study represents the first characterization of TNRC4 and provides further insight into the mechanisms of brain-specific alternative splicing and their possible pathological implications.

Premature translation of transition protein 2 mRNA causes sperm abnormalities and male infertility.

During mammalian spermiogenesis somatic histones are replaced at first by transition proteins, which are in turn replaced by the protamines, forming the sperm nucleoprotamines. It is believed that transition protein 2 (Tnp2) is necessary for maintaining the normal processing of protamines and, consequently, the completion of chromatin condensation. The transition protein mRNAs are stored in translationally inert messenger ribonucleoprotein particles for up to 7 days until translational activation in elongated spermatids. Substantial evidence suggests an involvement of 3'untranslated region (UTR) in the translational regulation of the Tnp2 mRNAs. In order to determine the role of Tnp2 3'UTR in translational regulation and to study whether the translational repression of Tnp2 mRNA is necessary for normal spermatid differentiation in mice, we generated transgenic mice that carry a Tnp2-hGH transgene. In this transgene, 3'UTR of Tnp2 gene was replaced by 3' 3'UTR of human growth hormone gene. In these transgenic animals, transcription and translation of Tnp2 occur simultaneously in round spermatids which is an evidence for involvement of Tnp2 3'UTR in its translation repression. Premature translation of Tnp2 mRNA caused abnormal head morphogenesis, reduced sperm motility and male infertility. These results show clearly that a strict temporal and stage-specific Tnp2 translation is necessary for the correct differentiation of round spermatids into mature spermatozoa and for male fertility.

Putative human male germ cells from bone marrow stem cells.

Germ cells must develop along distinct male or female paths to produce the spermatozoa or oocyte required for sexual reproduction. Male germline stem cells maintain spermatogenesis in the postnatal human testis. Here we show that a small population of bone marrow cells is able to transdifferentiate to male germ cell-like cells. We show expression of early germ cell markers (Oct4, Fragilis, Stella and Vasa) and male germ cell specific markers (Dazl, TSPY, Piwil2 and Stra8) in these cells. Our preliminary findings provide direct evidence that human bone marrow cells can differentiate to putative male germ cells and identify bone marrow as a potential source of male germ cells that could sustain sperm production.