In inherited prion disease, important information has accrued abo

In inherited prion disease, important information has accrued about which variants are completely penetrant, partially penetrant or simply benign polymorphisms (Figure 2). PTC124 research buy Several publications originate from groups that routinely sequence PRNP and include the distributions of inherited prion disease and new mutations from the UK, China, Japan, US, the Netherlands, and further lessons on how easily inherited prion disease, particularly that caused by truncation mutation,

can be mistaken for Alzheimer’s disease [ 10•, 11, 12, 13, 14, 15, 16, 17 and 18]. Sequencing the CEPH Human Diversity Panel and the Pakistani population showed that small insertions in the octapeptide repeat region of PRNP are probably not pathogenic as they are found in the healthy population, albeit rarely [ 10•, 13 and 19]. Sequencing of the healthy Korean population showed both the M232R and V180I variants implying that these may not be pathogenic mutations [ 11]. Finally, a study of the rare four octapeptide repeat mutation showed that penetrance of the clinical disease is determined by the genotype at codon 129. When the mutation

is linked to codon 129 methionine and the non-mutant allele is also 129 methionine, the disease appears to be penetrant, whereas it is non-penetrant when the non-mutant allele is 129 valine [ 20]. Human genetic studies provide the most direct link between susceptibility genes and patients, however, these are limited in power and inference regarding DZNeP molecular weight mechanisms may

be complex. Uniquely amongst neurodegenerative diseases mice are naturally susceptible to prion diseases thus providing an ideal model organism for both gene discovery and hypothesis testing. Previous mouse quantitative trait loci (QTL) mapping studies using simple crosses have successfully Urease identified many loci linked to prion disease incubation time [21, 22, 23, 24 and 25]. A new report has added to these data using recombinant inbred lines [26]. Many regions are implicated although only loci on Mmu11 are replicated between the experimental models. Large regions of this chromosome have also been implicated in previous studies [ 21, 22 and 23]. The main disadvantage of these studies is the limited resolution resulting in linkage to very large regions that have proved intractable for candidate gene identification. The availability of advanced crosses such as heterogeneous stocks (HS) of mice and the development of the new Collaborative Cross provide ×10–20 higher resolution and are already providing realistic prospects for identifying individual candidate genes [ 27, 28 and 29]. The Northport HS was successfully used to fine map and identify candidate genes on Mmu19 (Hectd2) and Mmu15 (Cpne8) [ 30 and 31••]. For Mmu15, the region of linkage was reduced to 3.6Mb from the previous report of 30Mb [ 24]. Haplotype analysis and genotyping representative SNPs identified Cpne8 as the most promising candidate.

The region in rmunc13-4 that is targeted by the siRNA, has 3 mism

The region in rmunc13-4 that is targeted by the siRNA, has 3 mismatches with the corresponding sequence in munc13-4, rendering the latter resistant to the siRNA. Indeed, while knock-down efficiency of rmunc13-4 is 90% using Amaxa electroporation (Fig. 3A), YFP-hmunc13-4 expression was not affected by the siRNA,

nor was knock-down of rmunc13-4 diminished when a full length hmunc13-4 construct was expressed (Fig. 3A). Transduction with the munc13-4 constructs yielded uniform expression (Fig. 1B). Over PD0325901 mouse 99% of the cells in the lentivirally transduced RBL-2H3 cell lines expressed the transfected cDNA product, ensuring that results of bulk assays are not obscured by contamination with non-transfected cells. We then determined degranulation efficiency of RBL-2H3 cells with silenced munc13-4. The cells were activated with IgE anti DNP/DNP-HSA, and β-hexosaminidase was measured colorometrically in medium and in the cells. The release of β-hexosaminidase was diminished by 80% (Fig. 3B) showing that munc13-4 is essential for degranulation in RBL-2H3 cells. In cells expressing YFP-munc13-4 we obtained a complete rescue of the β-hexosaminidase secretion defect. In contrast munc13-4 with YFP at the C-terminus was ~ 50% less effective. Cells expressing munc13-4-YFP released β-hexosaminidase to a lesser extent than cells treated with a scrambled siRNA. Thus even though there

was more munc13-4-YFP than endogenous levels in the control cells,

degranulation nevertheless was impaired (Fig. 2A), strongly suggesting BTK inhibitor that positioning of the YFP-tag at the C-terminus affected the function of the protein. The FHL3 mutant YFP-Δ608-611 failed to rescue the secretion defect since the extent of β-hexosaminidase release was statistically not different from cells with silenced munc13-4 (Fig. 3B). In summary the complementation of degranulation assay in RBL-2H3 cells faithfully recapitulated the secretion phenotype of FHL3 mutations in cytotoxic lymphocytes. RBL-2H3 cells can also be triggered to degranulate by ionomycin and PMA. This treatment elevates intracellular calcium and activates PKC, independent of FcεRI signaling pathways. We performed the degranulation assays using this experimental regimen and found that cells released more β-hexosaminidase than after triggering via FcεRI (cf siRNA bars in Fig. 3B and C). Evidently PMA/ionomycin releases β-hexosaminidase not only from stores that are regulated via FcεRI signaling and munc13-4. In agreement with this notion, the effect of munc13-4 knock down is similar as in Fig. 3B, but a substantial fraction of β-hexosaminidase can still be released from the cells by PMA/ionomycin (Fig. 3C). This has also been observed in mast cells isolated from VAMP8 knock-out mice, and suggests that munc13-4 regulates secretion of a subset of secretory granules in RBL-2H3 (Puri and Roche, 2008).

They can function in energy conservation, generating a chemiosmot

They can function in energy conservation, generating a chemiosmotic gradient for ATP production by sodium ion export. This allows energy generation buy CAL-101 over a more negative redox range than oxidative phosphorylation does. They may also function in the reverse direction to produce reduced ferredoxin, or in other as yet unknown roles; protons rather than sodium may be pumped in some cases. The six or seven Rnf genes (rnfH

is not always present) are found in different arrangements in a variety of Bacteria and Archaea, usually but not always in a cluster. At least two bacteria (Azotobacter vinelandii and Desulfobacterium autotrophicum HRM2) have two different Rnf gene clusters. The BOGUAY genome encodes two possible copies of genes for five of the seven Rnf subunits (Table S8), and one each for RnfF and RnfH. selleck compound Perhaps significantly, these are the two least-characterized subunits, and rnfH is not always found in genomes possessing the other six (putative) genes. BLASTP searches (not shown) suggest that where the BOGUAY genome has two copies of an Rnf gene, they have different phylogenies. From this analysis, the BOGUAY genome has both expected and unexpected features. Pathways for sulfide oxidation and nitrate reduction are both present, although we cannot yet explain all aspects of the possible nitrogen respiration pathways. Some experiments addressing this are

suggested in MacGregor et al. (2013b). The answer to the

question whether orange-pigmented Beggiatoaceae are autotrophs or heterotrophs is, so far, “possibly both”. Genome sequences from additional pigmented and unpigmented filaments collected in different environments may provide some insights. Experimental work will be needed to clarify Beggiatoaceae physiology, however. For example, seafloor or shipboard incubations with isotopically labeled carbon substrates could be attempted, to determine which are incorporated directly into Beggiatoaceae biomass under particular conditions. Carbon dioxide and oxygen concentrations are likely important variables, as well as sulfide, organic acid, and perhaps hydrocarbon availability. The size of the filaments L-NAME HCl might make autoradiography feasible, or phylogenetically specific RNA or lipids could be isolated for stable or radiocarbon isotopic determinations. Removal of epibionts might be attempted to minimize cross-feeding, although they may be required for nutrient supply or waste removal. Gene expression studies might be used to ask which carbon acquisition pathways are activated under a given set of conditions. As in all microbial genomes, there also remain hundreds of hypothetical proteins of unknown function, providing for any amount of future experimentation. Thanks to the Captain and crews of the RV Atlantis and HOV Alvin, and to the shipboard parties of legs AT 15-40 and AT 15-56. Genome sequencing was performed by the J.

Swabs from participants with confirmed infection were further ass

Swabs from participants with confirmed infection were further assessed in a quantitative RT-PCR assay targeted at the M gene as described previously.27 The target sequence was cloned and quantified using pico green to prepare a standard curve for quantitation. Standard curves were run in duplicate. Samples were generally tested once but RT-PCR was repeated to validate fluctuations. Results were expressed as cDNA equivalent copies of viral RNA. The limit of detection was 5 RNA copies/reaction. De novo whole genome sequencing was performed on combined nose and throat swabs with Ct values below 33. All 8 virus gene

segments were amplified in two RT-PCR reactions by using primers that target the conserved termini: (5′-GCCGGAGCTCTGCAGATATCAGCRAAAGCAGG-3′) or learn more (5′-GCCGGAGCTCTGCAGATATCAGCGAAAGCAGG-3′) HSP inhibitor cancer with (5′-CAGGAAACAGCTATGACAGTAGAAACAAGG-3′).28 454 sequencing adaptors and molecular identifier tags were ligated to combined PCR products using the SPRIworks Fragment Library System II

for Roche GS FLX* DNA Sequencer. Emulsion PCR, bead recovery and enrichment were performed manually according to the manufacturer’s protocol followed by sequencing on a Roche GS FLX+. Analysis was limited to the envelope gene sequences in the current study. Sequences will be made available in Genbank. Sera were tested in haemagglutination inhibition (HI) and microneutralization (MN) assay as previously described.26 A reference antigen supplied by WHO (A/California/7/2009(H1N1)-like) was used with turkey erythrocytes. Titres were read as the reciprocal of the highest serum dilution causing complete inhibition of agglutination. If there was no inhibition of HI at the highest serum concentration (1:10 dilution) the titre was designated as 5. Influenza infection was defined as a positive RT-PCR, regardless of the presence of symptoms. Household members with RT-PCR confirmed infection but no increase in mouth temperature

and none of the symptoms listed earlier were defined as asymptomatic infection. Serology was not routinely performed on acute sera so was not considered in the definition of secondary infection. Nevertheless, else seroconversion was reported if there was a 4-fold or greater rise in HI or MN titre between pre- and post-pandemic sera. Household secondary infection risk (SIR) was calculated as the number of household contacts infected 1–8 days after symptom onset in the index case divided by the number of household contacts, similar to other studies.6, 7, 13, 15 and 17 Serial interval was defined as the number of days between symptom onset in the index case and the first secondary case. Other secondary household cases were only included in the serial interval calculation if their symptoms started on the same day as the first secondary case. Children were defined as those up to 15 years of age. Oseltamivir treatment was considered to be timely if commenced within 2 days of symptom onset.


other environmental concerns associated with HV


other environmental concerns associated with HVHF in New York have come to the forefront of discussion. This includes a water quantity perspective, which is traditionally less critical in regions that have ample freshwater supplies in humid climates and/or large, proximate freshwater bodies (Rahm and Riha, 2012). HVHF requires large volumes of water which will ultimately increase water demand from the regions that will experience development. Increased water demand will prompt regulators to determine from where, and at what rate, this water should selleck be extracted to protect sustainable use for drinking water, agriculture, and other industry demands. Altered stream geochemistry and consequences to stream ecosystems, as a result of decreased stream discharge, are factors beyond the anthropogenic freshwater demands mentioned

above that may merit consideration. Although water budgets from the New York State Department of Environmental Conservation (NYSDEC) demonstrate that increased water demands from HVHF in New York would make up a minor fraction of total water use (NYSDEC, 2011), it is unclear how hydraulically linked groundwater–surface water systems might respond to such a development. Water budgets alone may not be sufficient in predicting the spatially variable response of these systems, particularly in identifying areas which present heightened sensitivity to withdrawals. For example, the response of aquifers and streams to increased withdrawals of water might vary as a function JAK inhibitor of valley width, thickness and depth of aquifers within the valley fill. Additionally, smaller streams might be vulnerable to induced changes in groundwater discharge during drought. The projected path of HVHF development of the Marcellus Shale in New York will most likely focus on the Southern Tier of the state, including Broome and Tioga counties (Fig. Paclitaxel solubility dmso 1). The major valleys within these counties overlie an unconsolidated glacial valley-fill aquifer

network which has been classified as a sole source aquifer since 1985 (U.S. Environmental Protection Agency, 2010). Such a designation emphasizes the importance of this groundwater source to the overlying municipalities, which receive more than half of their drinking water from the aquifer. In this region there is a high degree of hydraulic connectivity between streams and underlying unconsolidated glacial deposits (Randall, 1977, Wolcott and Coon, 2001 and Yager, 1993). High-volume withdrawals of water from groundwater may elicit a response from surface water, or vice versa, due to their physical connectivity (Winter et al., 1998). It is therefore necessary to investigate how different development scenarios might affect both the water table and stream flow.

For example, in the central TP, the dominant sandy soil would all

For example, in the central TP, the dominant sandy soil would allow infiltrated water drain quickly down to the deep soil; whereas the dominant loam soil in the eastern periphery of the TP could hold more water for soil

and vegetation evapotranspiration while desert basins in the northwest would have lower evapotranspiration than forest covered southeastern basins (FAO, 2008). There have been limited studies on the TP about hydrological processes and water balance as most studies have focused on the streamflow climatology and its relation to precipitation and temperature changes. The mechanisms for streamflow changes could be studied through complementary approaches such as modeling and analyses of field observations including hydrometeorological observations and environmental tracer collections. Hydrometeorological observations can reveal the state and fluxes RGFP966 of hydrometeorological elements such as precipitation and temperature. Environmental tracers such as isotopes and chemicals can be used to separate streamflow into surface, subsurface and baseflow components, and describe the sources of each component (Asano et al., 2002, Michel, 2004, Vache and McDonnell, 2006 and Zhang et al., 2009). Unlike the observations that are often collected over points or small-scale basins, physically based hydrological models that are vigorously evaluated can be set up for small or large domains and can be used to study

hydrological processes and water balance at various spatial and temporal scales. In other words, hydrological modeling can reveal historical trends and can project future trends of hydrological variables for larger river basins given reasonable forcings (e.g., Selleckchem Romidepsin Cuo et al., 2013a). Although there are quite a few studies that used isotopes to examine streamflow components on the TP (Nie et al., 2005, Liu et al., 2008, Pu et al., 2013 and Meng and Liu, 2013), very few, for example, Nie et al. (2005), used multiple environmental tracers including stable isotopes and chemical

tracers combined with hydrometeorological observations Protein Tyrosine Kinase inhibitor such as precipitation, streamflow and groundwater measurements to investigate the sources, components and traveling paths of the components of streamflow on the TP. Sources, components and their paths for most rivers of large or small scales are still unknown on the TP. Modeling the cryospheric processes from coherent mass and energy perspectives is another important aspect of TP hydrological research. A majority of cryospheric modeling studies on the TP focused on the specific aspects of water and energy balances for frozen soil and glacier (Fujita and Ageta, 2000, Zhang et al., 2004a, Zhang et al., 2004b, Zhang et al., 2005, Chen et al., 2010, Guo et al., 2012, Zhang et al., 2013a and Molg et al., 2013); while a few other studies looked at the integrated hydrological processes and water and energy balances for the entire basins (Yang et al., 2011, Zhang et al., 2012b, Zhang et al.

Only the outcome ‘poor response’ was studied and no significant d

Only the outcome ‘poor response’ was studied and no significant differences were found at 5-weeks follow-up. Five recent RCTs that studied interventions after an RCR were found. A low-quality RCT (Klintberg et al., 2009) compared progressive physiotherapy (i.e. early loading of the rotator cuff (active and passive motion)) to traditional physiotherapy (i.e. immobilization of 6 weeks followed

by only passive motion). Only the progressive group showed significant within group results on the pain Romidepsin cost outcomes at 12 and 24 months follow-up. However, no comparisons between the groups were made. A high-quality study (Michael et al., 2005) compared RCR and CPM plus physiotherapy with RCR and physiotherapy alone. ROM (90° active abduction of the shoulder) was managed after 31 days in the CPM plus physiotherapy group compared to 43 days in the physiotherapy alone group (p = 0.292). Another high-quality study of Hayes et al. (2004) compared individualized physiotherapy

to a standardized home exercise program after RCR and found no significant differences between the groups for any passive ROM, muscle force or overall shoulder status at 12- and 24-weeks follow-up. A low-quality study (Roddey et al., 2002) compared two instructional approaches to a home exercise program after RCR: a videotape versus personal instruction by a physiotherapist. No differences between Pyruvate dehydrogenase lipoamide kinase isozyme 1 the treatment groups were found on the Shoulder Pain and Disability Index (SPADI) and UPenn Shoulder Scale at 12-weeks, 24-weeks and 1-year follow-up. A low-quality RCT (Blum et al., 2009) studied the effectiveness of Repetitive H-Wave device stimulation (HWDS) versus placebo HWDS and

found significant within group results for both groups for external rotation (arm at slide) and internal rotation (arm at 90°) at 90 days follow-up; the HWDS group improved most. No significant within group results were found for the other ROM measurements. No comparisons were made between the groups. We found no evidence for the effectiveness of progressive compared to traditional physiotherapy, in the long-term or for the effectiveness of CPM as additive to physiotherapy after RCR. Furthermore, we found no evidence for the effectiveness of splinting in abduction versus resting the arm at the side, physiotherapy versus a standardized home exercise program, instructional approaches versus a home exercise program (videotape), or H-wave device stimulation versus placebo after RCR. This study focused on the effectiveness of non-surgical and surgical interventions for treating RotCuffTears not caused by acute traumata or systemic diseases. Neri et al.

, 1975), hyaluronidases ( Ghosh and Singh, 1974) and phospholipas

, 1975), hyaluronidases ( Ghosh and Singh, 1974) and phospholipases ( Cirino et al., 1989). As the present study did not aim at the quantification and characterization selleck compound of monoamine and other venom component, it is not possible to speculate about the precise venom components responsible

for the oedematogenic effect of S. cyanea venom. However, as indicated by previous studies, it is probably a multimediated phenomenon. Besides the significant hindpaw-induced oedema by S. cyanea venom, a slight hemorrhagic effect was observed at the assayed doses, contrary to that reported in previous studies which have shown that wasp venoms exhibit moderate to strong hemorrhagic activity ( Schmidt et al., 1986 and Tan and Ponnudurai, 1992). This hemorrhagic effect may indicate the low presence of molecules with fibrinolytic and anticoagulant activities in S. cyanea wasp venom, as already described for other wasp venoms ( Czaikoski et al., 2010). As mentioned above, a wasp sting can produce symptoms that are local, affecting only the skin, or systemic, affecting the whole body ( Ratnoff and Hymie, 1983 and Sachdev et al., 2002). The slight hemorrhagic activity from S. cyanea venom indicates that envenomation caused by this wasp may produce only local effects on mammalian skin. Studies with venom components related to hemorrhagic activity are important

for the research of new drugs for the control of diseases caused by blood clotting ( Czaikoski et al., 2010). S. cyanea venom showed a strong haemolytic activity on O positive human erythrocytes. It is worthwhile to note GDC-0068 concentration that the systemic effects induced by wasp sting include haemolysis which is associated with hematoglobinuria and hematoglobinemia

( Humblet et al., 1982). Rhabdomyolysis may also occur, leading to serum elevations of creatinine phosphatase (CPK) and lactate dehydrogenase; Cytidine deaminase CPK levels of 91,000 IU/liter have been reached within 24 h of mass stinging bees and wasp (normal < 160 IU/liter) ( Humblet et al., 1982). Other studies with wasp venoms have also demonstrated the presence of molecules with haemolytic activity, in this regard the peptides Polybia-MP-II and Polybia-MP-III, isolated from the venom of the social wasp P. paulista, showed a strong haemolytic effect ( Monson de Souza et al., 2009); this fact being consistent with our results relative to the haemolytic activity from S. cyanea venom. Experiments with P. paulista, P. occidentalis and P. ignobilis whole venoms showed haemolytic activity on human erythrocyte, the P. paulista whole venom being the most haemolytic, followed by the P. occidentalis and P. ignobilis venoms, respectively ( Mortari et al., 2005), strengthening our results with the S. cyanea wasp venom, which is even stronger than the P. paulista.

Gene isoforms are generated by alternative splicing, in which exo

Gene isoforms are generated by alternative splicing, in which exons are spliced

and joined together in different combinations. Alternative splicing is an important mechanism of gene function regulation since differences in the mRNA sequences translate into distinct protein domains with distinct roles. Alternative splicing can also affect the 5’ and 3’ UTRs that are essential for gene regulation. Therefore, identifying the transcriptional variants of a gene and the relative abundance of each of them is instrumental to dissecting the functional role of such gene. A large body Selleckchem Volasertib of evidence has identified alternative splicing differences between ESC and differentiated cell populations [30, 31 and 32•]. Pluripotency regulation by the recently identified novel isoform of FOXP1 in hESCs is a significant landmark exemplifying the importance of alternative splicing and isoform usage [33•]. The annotated FOXP1 isoform (NM_001012505) is important for differentiation, cell proliferation and development [34]. However, a novel exon (18b) was discovered to replace the annotated exon 18 in the traditional isoform NM_001012505, which produces a novel isoform of FOXP1 in hESCs. The alternative exon usage changes the protein coding sequence of the fork-head domain of FOXP1 and consequently changes the DNA-binding specificities resulting in the regulation

of a different set of target genes. This novel isoform is specifically expressed by hESCs and contribute to the regulation of pluripotency genes, such Fluorouracil purchase as OCT4, NR5A2 and

NANOG. Novel splice sites, exons and isoforms are also identified in the key pluripotency gene NANOG [35]. Novel 5’ end exons and splices result in various 5’ UTRs and N terminal domains in Nanog. As a result, two protein variants attenuate the self-renewal potential and pluripotency in ESCs. Similarly, novel splices in SALL4 and TCF3 can also change their functions in pluripotency regulation [31 and 32•]. A large body of evidence have identified alternative splicing differences between ESCs and differentiated cell populations [30, 31 and 32•]. These studies, exemplify the importance of large-scale identification of novel isoforms of annotated genes and their abundance, especially for Rebamipide pluripotency-associated genes. Au et al. reported a few novel isoforms of known pluripotency markers ( Table 1 and Figure 2). For example, in the DPPA4 locus, a RefSeq-annotated isoform is expressed but a novel isoform skipping three exons also contributes to a significant portion (∼17%) of the total gene abundance. In TERT, a novel isoform displaying cassette exon skipping junctions contributes as much as 54% of the gene abundance. Alternative splicing may be one mechanism of regulation of the telomerase activity of TERT.

for providing samples of rubber “
“The above mentioned pape

for providing samples of rubber. “
“The above mentioned paper did not include any acknowledgment to co-author Lucy Waskell’s funding source agency. learn more The funding source which was inadvertently omitted is as follows: Veterans Administration Merit Review Grant. “
“The above mentioned paper did not include any acknowledgment to co-author Lucy Waskell’s funding source agency. The funding source which was inadvertently omitted is as follows: Veterans Administration Merit Review Grant. “

imaging (DWI) and diffusion-tensor imaging (DTI) are non-invasive MRI techniques with broad clinical applications. While many clinical applications of diffusion imaging are in the brain, there is an increasing number of DWI and DTI studies in other organs [1], including the spinal cord [2], breast [3], prostate [4], liver [5], kidney [6], pancreas [7] and in the heart [8] and [9]. Bulk physiological motion has initially been a barrier to performing diffusion imaging in organs affected by motion. In cardiac selleck chemicals llc diffusion, this has been alleviated by technical advances including the use of cardiac/respiratory navigator techniques, single-shot echo planar imaging (EPI) readouts, and sequence modifications that reduce the effects of any motion that occurs

during the diffusion gradients. Such techniques have improved the robustness and reproducibility of diffusion-imaging applications in moving organs such as cardiac DTI [8] and [9]. Unfortunately, diffusion imaging suffers from substantial artifacts such as those caused by eddy currents, which are induced in conducting structures of the magnet bore by gradient switching. Diffusion selleck chemical imaging is particularly prone to eddy-current artifacts due to relatively long EPI readouts combined with strong

diffusion-sensitizing gradients. Unlike static field inhomogeneities, eddy currents do not remain constant over diffusion-encoding directions. Rather, they vary depending upon the magnitude and direction of the applied diffusion gradients. This leads to spatial misregistration and inconsistency between uncorrected images obtained with different diffusion-encoding directions or b-values. Ignoring eddy currents in the image reconstruction results in ghosting, bulk object shifts and deformations, as well as signal dropouts [10]. In DTI, this also leads to inaccuracies in estimates of the fractional anisotropy (FA). In this study, we investigate the effects of eddy currents in sequences that are suitable for performing cardiac DTI where there is substantial motion. Two sequences previously used for cardiac diffusion are compared: (i) the Stejskal-Tanner or “unipolar” spin-echo diffusion sequence [11] and (ii) a “bipolar” spin-echo sequence [12], [13] and [14]. The unipolar sequence has a shorter echo time (TE) while the bipolar sequence offers insensitivity to first-order bulk motion through its velocity-compensated nature [12], [13] and [14]. The twice-refocused sequence, described in Reese et al.