Loss of B Cells in Patients with Heterozygous Mutations in IKAROS.

BACKGROUND: Common variable immunodeficiency (CVID) is characterized by late-onset hypogammaglobulinemia in the absence of predisposing factors. The genetic cause is unknown in the majority of cases, and less than 10% of patients have a family history of the disease. Most patients have normal numbers of B cells but lack plasma cells. METHODS: We used whole-exome sequencing and array-based comparative genomic hybridization to evaluate a subset of patients with CVID and low B-cell numbers. Mutant proteins were analyzed for DNA binding with the use of an electrophoretic mobility-shift assay (EMSA) and confocal microscopy. Flow cytometry was used to analyze peripheral-blood lymphocytes and bone marrow aspirates. RESULTS: Six different heterozygous mutations in IKZF1, the gene encoding the transcription factor IKAROS, were identified in 29 persons from six families. In two families, the mutation was a de novo event in the proband. All the mutations, four amino acid substitutions, an intragenic deletion, and a 4.7-Mb multigene deletion involved the DNA-binding domain of IKAROS. The proteins bearing missense mutations failed to bind target DNA sequences on EMSA and confocal microscopy; however, they did not inhibit the binding of wild-type IKAROS. Studies in family members showed progressive loss of B cells and serum immunoglobulins. Bone marrow aspirates in two patients had markedly decreased early B-cell precursors, but plasma cells were present. Acute lymphoblastic leukemia developed in 2 of the 29 patients. CONCLUSIONS: Heterozygous mutations in the transcription factor IKAROS caused an autosomal dominant form of CVID that is associated with a striking decrease in B-cell numbers. (Funded by the National Institutes of Health and others.).

Stability Limit of Electrified Droplets.

In many physical processes, including cloud electrification, electrospray, and demulsification, droplets and bubbles are exposed to electric fields and may either remain whole or burst in response to electrical stresses. Determining the stability limit of a droplet exposed to an external electric field has been a long-standing mathematical challenge, and the only analytical treatment to date is an approximate calculation for the particular case of a free-floating droplet. Here we demonstrate, experimentally and theoretically, that the stability limit of a conducting droplet or bubble exposed to an external electric field is described by a power law with broad generality that, in practice, applies to the cases in which the droplet or bubble is pinned or sliding on a conducting surface or free floating. This power law can facilitate the design of devices for liquid manipulation via a simple formula that captures the parameter range of bubbles and droplets that can be supported on electrified surfaces.

Bouncing droplet dynamics above the Faraday threshold.

We present the results of an experimental investigation of the dynamics of droplets bouncing on a vibrating fluid bath for forcing accelerations above the Faraday threshold. Two distinct fluid viscosity and vibrational frequency combinations (20 cS-80 Hz and 50 cS-50 Hz) are considered, and the dependence of the system behavior on drop size and vibrational acceleration is characterized. A number of new dynamical regimes are reported, including meandering, zig-zagging, erratic bouncing, coalescing, and trapped regimes. Particular attention is given to the regime in which droplets change direction erratically and exhibit a dynamics akin to Brownian motion. We demonstrate that the effective diffusivity increases with vibrational acceleration and decreases with drop size, as suggested by simple scaling arguments.

A review of the theoretical modeling of walking droplets: Toward a generalized pilot-wave framework.

The walking droplet system has extended the range of classical systems to include several features previously thought to be exclusive to quantum systems. We review the hierarchy of analytic models that have been developed, on the basis of various simplifying assumptions, to describe droplets walking on a vibrating fluid bath. Particular attention is given to detailing their successes and failures in various settings. Finally, we present a theoretical model that may be adopted to explore a more generalized pilot-wave framework capable of further extending the phenomenological range of classical pilot-wave systems beyond that achievable in the laboratory.

Ratcheting droplet pairs.

Millimetric droplets may be levitated on the surface of a vibrating fluid bath. Eddi et al. [Europhys. Lett. 82, 44001 (2008)] demonstrated that when a pair of levitating drops of unequal size are placed nearby, they interact through their common wavefield in such a way as to self-propel through a ratcheting mechanism. We present the results of an integrated experimental and theoretical investigation of such ratcheting pairs. Particular attention is given to characterizing the dependence of the ratcheting behavior on the droplet sizes and vibrational acceleration. Our experiments demonstrate that the quantized inter-drop distances of a ratcheting pair depend on the vibrational acceleration, and that as this acceleration is increased progressively, the direction of the ratcheting motion may reverse up to four times. Our simulations highlight the critical role of both the vertical bouncing dynamics of the individual drops and the traveling wave fronts generated during impact on the ratcheting motion, allowing us to rationalize the majority of our experimental findings.

Polygonal instabilities on interfacial vorticities.

We report the results of a theoretical investigation of the stability of a toroidal vortex bound by an interface. Two distinct instability mechanisms are identified that rely on, respectively, surface tension and fluid inertia, either of which may prompt the transformation from a circular to a polygonal torus. Our results are discussed in the context of three experiments, a toroidal vortex ring, the hydraulic jump, and the hydraulic bump.

Periderm Fate during Palatogenesis: TGF-ß and Periderm Dedifferentiation.

Failure of palatogenesis results in cleft palate, one of the most common congenital disabilities in humans. During the final phases of palatogenesis, the protective function of the peridermal cell layer must be eliminated for the medial edge epithelia to adhere properly, which is a prerequisite for the successful fusion of the secondary palate. However, a deeper understanding of the role and fate of the periderm in palatal adherence and fusion has been hampered due to a lack of appropriate periderm-specific genetic tools to examine this cell type in vivo. Here we used the cytokeratin-6A (Krt-6a) locus to develop both constitutive (Krt6ai-Cre) and inducible (Krt6ai-CreERT2) periderm-specific Cre driver mouse lines. These novel lines allowed us to achieve both the spatial and temporal control needed to dissect the periderm fate on a cellular resolution during palatogenesis. Our studies suggest that, already before the opposing palatal shelves contact each other, at least some palatal periderm cells start to gradually lose their squamous periderm-like phenotype and dedifferentiate into cuboidal cells, reminiscent of the basal epithelial cells seen in the palatal midline seam. Moreover, we show that transforming growth factor-ß (TGF-ß) signaling plays a critical periderm-specific role in palatogenesis. Thirty-three percent of embryos lacking a gene encoding the TGF-ß type I receptor (Tgfbr1) in the periderm display a complete cleft of the secondary palate. Our subsequent experiments demonstrated that Tgfbr1-deficient periderm fails to undergo appropriate dedifferentiation. These studies define the periderm cell fate during palatogenesis and reveal a novel, critical role for TGF-ß signaling in periderm dedifferentiation, which is a prerequisite for appropriate palatal epithelial adhesion and fusion.

Randomized phase II clinical trial of avotermin versus placebo for scar improvement.

BACKGROUND: Scarring is a major problem following skin injury. In early clinical trials, transforming growth factor ß3 (avotermin) improved scar appearance. The aim of this study was to determine whether an injection of avotermin at the time of wound closure is effective in improving scar appearance. METHODS: Study RN1001-0042, a double-blind, randomized, within-patient, placebo-controlled trial, investigated the efficacy and safety of four doses of avotermin given once. Patients undergoing bilateral surgery to remove varicose leg veins by saphenofemoral ligation and long saphenous vein stripping were enrolled at 20 European centres. A total of 156 patients were randomized to receive one of four doses of avotermin (5, 50, 200 or 500 ng per 100 µl, at 100 µl per linear cm of wound margin), administered by intradermal injection to the groin and distal wound margins of one leg; placebo was administered to the other leg. Scar appearance was evaluated by an independent panel of lay people (lay panel), investigators and patients. The primary efficacy variable was lay panel Total Scar Score (ToScar), derived from visual analogue scale scores for groin scars between 6 weeks and 7 months. RESULTS: Avotermin 500 ng significantly improved groin scar appearance compared with placebo (mean lay panel ToScar difference 16·49 mm; P = 0·036). CONCLUSION: Avotermin 500 ng per 100 µl per linear cm of wound margin given once is well tolerated and significantly improves scar appearance.

Testicular germ cell tumours in dogs are predominantly of spermatocytic seminoma type and are frequently associated with somatic cell tumours.

Unlike seminomas in humans, seminomas in animals are not typically sub-classified as classical or spermatocytic types. To compare testicular germ cell tumours (TGCT) in dogs with those of men, archived tissues from 347 cases of canine testicular tumours were morphologically evaluated and characterized using human classification criteria. Histopathological and immunohistological analysis of PLAP, KIT, DAZ and DMRT1 expression revealed that canine seminomas closely resemble human spermatocytic seminomas. In addition, a relatively frequent concomitant presence of somatic cell tumours was noted in canine TGCT. None of the canine TGCT evaluated demonstrated the presence of carcinoma in situ cells, a standard feature of human classical seminomas, suggesting that classical seminomas either do not occur in dogs or are rare in occurrence. Canine spermatocytic seminomas may provide a useful model for this rare human neoplasm.

Rolling ribbons.

We present the results of a combined experimental and theoretical investigation of rolling elastic ribbons. Particular attention is given to characterizing the steady shapes that arise in static and dynamic rolling configurations. In both cases, above a critical value of the forcing (either gravitational or centrifugal), the ribbon assumes a two-lobed, peanut shape similar to that assumed by rolling droplets. Our theoretical model allows us to rationalize the observed shapes through consideration of the ribbon's bending and stretching in response to the applied forcing.

YAP/TAZ Regulate Elevation and Bone Formation of the Mouse Secondary Palate.

Clefting of the secondary palate is one of the most common congenital anomalies, and the multiple corrective surgeries that individuals with isolated cleft palate undergo are associated with major costs and morbidities. Secondary palate development is a complex, multistep process that includes the elevation of the palatal shelves from a vertical to horizontal position, a process that is not well understood. The Hippo signaling cascade is a mechanosensory pathway that regulates morphogenesis, homeostasis, and regeneration by controlling cell proliferation, apoptosis, and differentiation, primarily via negative regulation of the downstream effectors, Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ). We deleted Yap/Taz throughout the palatal shelf mesenchyme as well as specifically in the posterior palatal shelf mesenchyme, using the Osr2Cre and Col2Cre drivers, respectively, which resulted in palatal shelf elevation delay and clefting of the secondary palate. In addition, the deletion resulted in undersized bones of the secondary palate. We next determined downstream targets of YAP/TAZ in the posterior palatal shelves, which included Ibsp and Phex, genes involved in mineralization, and Loxl4, which encodes a lysyl oxidase that catalyzes collagen crosslinking. Ibsp, Phex, and Loxl4 were expressed at decreased levels in the ossification region in the posterior palatal shelf mesenchyme upon deletion of Yap/Taz. Furthermore, collagen levels were decreased specifically in the same region prior to elevation. Thus, our data suggest that YAP/TAZ may regulate collagen crosslinking in the palatal shelf mesenchyme, thus controlling palatal shelf elevation, as well as mineralization of the bones of the secondary palate.

Inactivation of two Dictyostelium discoideum genes, DdPIK1 and DdPIK2, encoding proteins related to mammalian phosphatidylinositide 3-kinases, results in defects in endocytosis, lysosome to postlysosome transport, and actin cytoskeleton organization.

Phosphatidylinositide 3-kinases (PI3-kinases) have been implicated in controlling cell proliferation, actin cytoskeleton organization, and the regulation of vesicle trafficking between intracellular organelles. There are at least three genes in Dictyostelium discoideum. DdPIK1, DdPIK2, and DdPIK3, encoding proteins most closely related to the mammalian 110-kD PI-3 kinase in amino acid sequence within the kinase domain. A mutant disrupted in DdPIK1 and DdPIK2 (delta ddpik1/ddpik2) grows slowly in liquid medium. Using FITC-dextran (FD) as a fluid phase marker, we determined that the mutant strain was impaired in pinocytosis but normal in phagocytosis of beads or bacteria. Microscopic and biochemical approaches indicated that the transport rate of fluid-phase from acidic lysosomes to non-acidic postlysosomal vacuoles was reduced in mutant cells resulting in a reduction in efflux of fluid phase. Mutant cells were also almost completely devoid of large postlysosomal vacuoles as determined by transmission EM. However, delta ddpik1/ddpik2 cells functioned normally in the regulation of other membrane traffic. For instance, radiolabel pulse-chase experiments indicated that the transport rates along the secretory pathway and the sorting efficiency of the lysosomal enzyme alpha-mannosidase were normal in the mutant strain. Furthermore, the contractile vacuole network of membranes (probably connected to the endosomal pathway by membrane traffic) was functionally and morphologically normal in mutant cells. Light microscopy revealed that delta ddpik1/ddpik2 cells appeared smaller and more irregularly shaped than wild-type cells; 1-3% of the mutant cells were also connected by a thin cytoplasmic bridge. Scanning EM indicated that the mutant cells contained numerous filopodia projecting laterally and vertically from the cell surface, and fluorescent microscopy indicated that these filopodia were enriched in F-actin which accumulated in a cortical pattern in control cells. Finally, delta ddpik1/ddpik2 cells responded and moved more rapidly towards cAMP. Together, these results suggest that Dictyostelium DdPIK1 and DdPIK2 gene products regulate multiple steps in the endosomal pathway, and function in the regulation of cell shape and movement perhaps through changes in actin organization.

Assessment of the allelopathic potential of Juniperus ashei on germination and growth of Bouteloua curtipendula.

Potential allelopathic compounds of Juniperus ashei Buchh. (Ashe juniper) and their effect on a native grass were determined in laboratory and field studies. Solid-phase microextraction and gas chromatography/mass spectrometry were used to determine if monoterpenes found in the essential oils of J. ashei are released in leaf and litter leachate, as well as volatilized from leaf tissue. Camphor, bornyl acetate, and limonene were found in leaf and fresh litter leachates; however, degraded litter did not contain any of these compounds. Camphor was the most common potentially allelopathic compound found in J. ashei leaf and litter leachate and in volatiles from leaf tissue. The effects of leaf and litter tissue on the germination of Bouteloua curtipendula (Michx.) Torr. (side-oats grama) was tested by using the "sandwich agar method". The highest germination of B. curtipendula (29.6%) occurred in the control, which was significantly higher than fresh litter (13.2%) and degraded litter (16.2%). The lowest germination (6.2%) occurred with J. ashei leaves. In the field experiment, aboveground dry mass of B. curtipendula was evaluated in relation to position within the canopy and intercanopy of J. ashei adult trees when light and water were held constant across locations. Aboveground dry mass of B. curtipendula was significantly greater in the intercanopies of J. ashei (163.7 g m(2)) compared to the dry mass in the understory (44.8 g m(2)) and dripline (44.5 g m(2)), suggesting some negative influence by J. ashei. Chemical analyses indicate that monoterpenes are released through leaching and volatilization from J. ashei, and germination and field studies suggest that these compounds inhibit B. curtipendula.

Views on chemical safety information and influences on chemical disposal behaviour in the UK.

This study examined how groups representing four tiers in the chemical supply chain (manufacturers, vendors, workers and consumers) understood safety information, and the factors that influenced disposal behaviour. Data from seven, semi-structured, focus groups was analysed both qualitatively (textual analysis) and quantitatively (network analysis). Such combined analytical methods enabled us to achieve both detailed insights into perceptions and behaviour and an objective understanding of the prevailing opinions that occurred within and between the focus group discussions. We found issues around awareness, trust, access and disposal behaviours differed between groups within the supply chain. Participants from the lower tiers perceived chemical safety information to be largely inaccessible. Labels were the main source of information on chemical risks for the middle and bottom tiers of the supply chain. Almost all of the participants were aware of the St Andrew's Cross and skull and crossbones symbols but few were familiar with the Volatile Organic Compound logo or the fish and tree symbol. Both the network and thematic analysis demonstrated that whilst frequent references to health risks associated with chemicals were made environmental risks were usually only articulated after prompting. It is clear that the issues surrounding public understanding of chemical safety labels are highly complex and this is compounded by inconsistencies in the cognitive profiles of chemical users. Substantially different cognitive profiles are likely to contribute towards communication difficulties between different tiers of the supply chain. Further research is needed to examine the most effective ways of communicating chemical hazards information to the public. The findings demonstrate a need to improve and simplify disposal guidance to members of the public, to raise public awareness of the graphic symbols in the CHIP 3.1, 2005 regulations and to improve access to disposal guidance.

Comparison of aloe vera and omeprazole in the treatment of equine gastric ulcer syndrome.

BACKGROUND: Anecdotally, aloe vera is used to treat gastric ulceration, although no studies have yet investigated its efficacy in horses. OBJECTIVES: To test the hypothesis that aloe vera would be noninferior to omeprazole in the treatment of equine gastric ulcer syndrome. STUDY DESIGN: Randomised, blinded clinical trial. METHODS: Forty horses with grade ≥2 lesions of the squamous and/or glandular mucosa were randomly assigned to one of two groups. Horses received either aloe vera inner leaf gel (17.6 mg/kg bwt) b.i.d. or omeprazole (4 mg/kg bwt) s.i.d. for approximately 28 days, after which a repeat gastroscopic examination was performed to determine disease resolution. Horses with persistent lesions were offered a further 28 days of treatment with omeprazole (4 mg/kg bwt s.i.d.) and were re-examined on completion of treatment. RESULTS: Efficacy analyses were based on 39 horses that completed the trial. Equine squamous gastric disease (ESGD) was observed in 38 horses; improvement and healing rates in these horses were 56% and 17%, respectively, in the aloe vera group, and 85% and 75%, respectively, in the omeprazole group. Healing was less likely to occur in horses with prolonged gastric emptying. Equine glandular gastric disease (EGGD) was less common than ESGD (n = 14) and numbers were too small to perform meaningful statistical analyses. The hypothesis that aloe vera would be noninferior to omeprazole was not supported. MAIN LIMITATIONS: No placebo control group was included. Limited numbers preclude any comment on the efficacy of aloe vera in the treatment of EGGD. CONCLUSIONS: Treatment with aloe vera was inferior to treatment with omeprazole.

Optimization of the Promega PowerSeq™ Auto/Y system for efficient integration within a forensic DNA laboratory.

The application of massively parallel sequencing (MPS) is growing in the forensic DNA field, as forensic DNA laboratories are continuously seeking methods to gain information from a limited or degraded forensic sample. However, the laborious nature of current MPS methodologies required for successful library preparation and sequencing leave opportunities for improvement to make MPS a practical option for processing forensic casework. In this study, the Promega PowerSeq™ Auto/Y System Prototype, a MPS laboratory workflow that incorporates multiplex amplification, was selected for optimization with the objectives to introduce automation for relieving manual processing, and to reduce the number of steps recommended by the standard protocol. Successful changes in the optimized workflow included a switch from column-based PCR purification to automatable bead-based purification, adoption of the library preparation procedures by a liquid handling robot platform, and removal of various time-consuming quality checks. All data in this study were found to be concordant with capillary electrophoresis (CE) data and previously-generated MPS results from this workflow. Read abundance and allele balance, metrics related to sample interpretation reliability, were not significantly different when compared to samples processed with the manufacturer's protocol. All the modifications implemented resulted in increased laboratory efficiency, reduced the protocol steps associated with risk of contamination and human error events, and decreased manual processing time by approximately 12h. These findings provide forensic DNA laboratories a more streamlined option when considering implementation of a MPS workflow.

Evidence for a recycling role for Rab7 in regulating a late step in endocytosis and in retention of lysosomal enzymes in Dictyostelium discoideum.

The mammalian small molecular weight GTPase Rab7 (Ypt7 in yeast) has been implicated in regulating membrane traffic at postinternalization steps along the endosomal pathway. A cDNA encoding a protein 85% identical at the amino acid level to mammalian Rab7 has been cloned from Dictyostelium discoideum. Subcellular fractionation and immunofluorescence microscopy indicated that Rab7 was enriched in lysosomes, postlysosomes, and maturing phagosomes. Cell lines were generated that overexposed Rab7 wild-type (WT), Rab7 Q67L (constitutively active form), and Rab7 T22N (dominant negative form) proteins. The Rab7 T22N cell line internalized fluid phase markers and latex beads (phagocytosis) at one-third the rate of control cells, whereas Rab7 WT and Rab7 Q67L cell lines were normal in uptake rates but exocytosed fluid phase faster than control cells. In contrast, fluid phase markers resided in acidic compartments for longer periods of time and were more slowly exocytosed from Rab7 T22N cells as compared with control cells. Light microscopy indicated that Rab7-expressing cell lines contained morphologically altered endosomal compartments. Compared with control cells, Rab7 WT- and Rab7 Q67L-expressing cells contained a reduced number of vesicles, the size of postlysosomes (> 2.5 microns) and an increased number of smaller vesicles, many of which were nonacidic; in control cells, > 90% of the smaller vesicles were acidic. In contrast, Rab7 T22N cells contained an increased proportion of large acidic vesicles relative to nonacidic vesicles. Radiolabel pulse-chase experiments indicated that all of the cell lines processed and targeted lysosomal alpha-mannosidase normally, indicating the lack of a significant role for Rab7 in the targeting pathway; however, retention of mature lysosomal hydrolases was affected in Rab7 WT and Rab7 T22N cell lines. Contrary to the results observed for the fluid phase efflux experiments, Rab7 T22N cells oversecreted alpha-mannosidase, whereas Rab7 WT cells retained this hydrolase as compared with control cells. These data support a model that Rab7 may regulate retrograde transport of lysosomal enzymes and the V-type H(+)-ATPase from postlysosomes to lysosomes coupled with the efficient release of fluid phase from cells.

A role for a Rab4-like GTPase in endocytosis and in regulation of contractile vacuole structure and function in Dictyostelium discoideum.

The small Mr Rab4-like GTPase, RabD, localizes to the endosomal pathway and the contractile vacuole membrane system in Dictyostelium discoideum. Stably transformed cell lines overexpressing a dominant negative functioning RabD internalized fluid phase marker at 50% of the rate of wild-type cells. Mutant cells were also slower at recycling internalized fluid. Microscopic and biochemical approaches indicated that the transport of fluid to large postlysosome vacuoles was delayed in mutant cells, resulting in an accumulation in acidic smaller vesicles, probably lysosomes. Also, RabD N121I-expressing cell lines missorted a small but significant percentage of newly synthesized lysosomal alpha-mannosidase precursor polypeptides. However, the majority of the newly synthesized alpha-mannosidase was transported with normal kinetics and correctly delivered to lysosomes. Subcellular fractionation and immunofluorescent microscopy indicated that in mutant cells contractile vacuole membrane proteins were associated with compartments morphologically distinct from the normal reticular network. Osmotic tests revealed that the contractile vacuole functioned inefficiently in mutant cells. Our results suggest that RabD regulates membrane traffic along the endosomal pathway, and that this GTPase may play a role in regulating the structure and function of the contractile vacuole system by facilitating communication with the endosomal pathway.

Overexpression of a novel rho family GTPase, RacC, induces unusual actin-based structures and positively affects phagocytosis in Dictyostelium discoideum.

Rho family proteins have been implicated in regulating various cellular processes, including actin cytoskeleton organization, endocytosis, cell cycle, and gene expression. In this study, we analyzed the function of a novel Dictyostelium discoideum Rho family protein (RacC). A cell line was generated that conditionally overexpressed wild-type RacC three- to fourfold relative to endogenous RacC. Light and scanning electron microscopy indicated that the morphology of the RacC-overexpressing cells [RacC WT(+) cells] was significantly altered compared with control cells. In contrast to the cortical F-actin distribution normally observed, RacC WT(+) cells displayed unusual dorsal and peripheral F-actin-rich surface blebs (petalopodia, for flower-like). Furthermore, phagocytosis in the RacC WT(+) cells was induced threefold relative to control Ax2 cells, whereas fluid-phase pinocytosis was reduced threefold, primarily as the result of an inhibition of macropinocytosis. Efflux of fluid-phase markers was also reduced in the RacC WT(+) cells, suggesting that RacC may regulate postinternalization steps along the endolysosomal pathway. Treatment of cells with Wortmannin and LY294002 (phosphatidylinositol 3-kinase inhibitors) prevented the RacC-induced morphological changes but did not affect phagocytosis, suggesting that petalopodia are probably not required for RacC-induced phagocytosis. In contrast, inactivating diacylglycerol-binding motif-containing proteins by treating cells with the drug calphostin C completely inhibited phagocytosis in control and RacC WT(+) cells. These results suggest that RacC plays a role in actin cytoskeleton organization and phagocytosis in Dictyostelium.