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Hematopoietic stem cells (HSCs) reside in the bone marrow (BM), can self-renew, and generate all cells of the hematopoietic system.1Most hematopoietic lineages arise through successive, increasingly lineage-committed progenitors. In contrast, megakaryocytes (MKs), hyperploid cells that generate platelets essential to hemostasis, can derive rapidly and directly from HSCs.2The underlying mechanism is unknown however. Here we show that DNA damage and subsequent arrest in the G2 phase of the cell cycle rapidly induce MK commitment specifically in HSCs, but not in progenitors, through an initially predominantly post-transcriptional mechanism. Cycling HSCs show extensive replication-induced DNA damage, which is at least in part explained by uracil misincorporation. Consistent with this notion, thymidine rescued HSC maintenancein vitroand reduced the generation of CD41+MK committed HSCs. Similarly, overexpression of the dUTP-scavenging enzyme, dUTPase, enhancedin vitromaintenance of HSCs. We conclude that a DNA damage response drives direct megakaryopoiesis and that replication stress-induced direct megakaryopoiesis, at least in part caused by uracil misincorporation, is a barrier to HSC maintenancein vitro. DNA damage-induced direct megakaryopoiesis may allow rapid generation of a lineage essential to immediate organismal survival, while simultaneously removing damaged HSCs and potentially avoiding malignant transformation of self-renewing stem cells.

Childhood interstitial lung disease (chILD) secondary to pulmonary surfactant deficiency is a devastating chronic lung disease in children. Clinical presentation includes mild to severe respiratory failure and fibrosis. There is no specific treatment, except lung transplantation, which is hampered by a severe shortage of donor organs, especially for young patients. Repair of lungs with chILD represents a longstanding therapeutic challenge but cellular therapy is a promising strategy. As surfactant is produced by alveolar type II epithelial (ATII) cells, engraftment with normal or gene-corrected ATII cells might provide an avenue to cure. Here we used a chILD disease-like model,Sftpc−/−mice, to provide proof-of-principle for this approach.Sftpc−/−mice developed chronic interstitial lung disease with age and were hypersensitive to bleomycin. We could engraft wild-type ATII cells after low dose bleomycin conditioning. Transplanted ATII cells produced mature SPC and attenuated bleomycin-induced lung injury up to four months post-transplant. This study demonstrates that partial replacement of mutant ATII cells can promote lung repair in a mouse model of chILD.

Organic anion transport proteins (OATPs) on the basolateral surface of hepatocytes mediate uptake of a number of drugs and endogenous compounds. Previous studies showed that rat OATP1A1 (rOATP1A1) has a postsynaptic density protein, drosophila disc large tumor suppressor, zonula occludens-1 protein (PDZ) consensus binding motif at its C-terminus and binds to PDZ domain containing 1 (PDZK1), which is required for its cell-surface localization. PDZK1 associates with rOATP1A1-containing endocytic vesicles within cells, mediating recruitment of motor proteins required for microtubule-based trafficking to the plasma membrane. rOATP1A4 also traffics to the plasma membrane, although it lacks a PDZ binding consensus sequence. The current study was designed to test the hypothesis that trafficking of rOATP1A4 to the plasma membrane requires its direct interaction with rOATP1A1 resulting in a complex that traffics through the cell in common subcellular vesicles in which the cytosolic tail of rOATP1A1 is bound to PDZK1. We found that 74% of rOATP1A4-containing rat liver endocytic vesicles (n = 12,044) also contained rOATP1A1. Studies in transfected HEK293 cells showed surface localization of rOATP1A1 only when coexpressed with PDZK1 whereas rOATP1A4 required coexpression with rOATP1A1 and PDZK1. Studies in stably transfected HeLa cells that constitutively expressed PDZK1 showed that coexpression of rOATP1A4 with rOATP1A1 resulted in more rapid appearance of rOATP1A4 on the plasma membrane and faster maturation to its fully glycosylated form. Similar results were observed on immunofluorescence analysis of single cells. Immunoprecipitation of rat liver or transfected HeLa cell lysates with rOATP1A1 antibody specifically co-immunoprecipitated rOATP1A4 as determined by western blotting. Conclusion: These studies indicate that optimal rOATP1A4 trafficking to the cell surface is dependent upon coexpression and interaction with rOATP1A1. As rOATP1A1 binds to the chaperone protein, PDZK1, rOATP1A4 functionally hitchhikes through the cell with this complex.

During infection of neurons by alphaherpesviruses including Pseudorabies virus (PRV) and Herpes simplex virus type 1 (HSV-1) viral nucleocapsids assemble in the cell nucleus, become enveloped in the cell body then traffic into and down axons to nerve termini for spread to adjacent epithelia. The viral membrane protein US9p and the membrane glycoprotein heterodimer gE/gI play critical roles in anterograde spread of both HSV-1 and PRV, and several models exist to explain their function. Biochemical studies suggest that PRV US9p associates with the kinesin-3 motor KIF1A in a gE/gI-stimulated manner, and the gE/gI-US9p complex has been proposed to recruit KIF1A to PRV for microtubule-mediated anterograde trafficking into or along the axon. However, as loss of gE/gI-US9p essentially abolishes delivery of alphaherpesviruses to the axon it is difficult to determine the microtubule-dependent trafficking properties and motor-composition of Δ(gE/gI−US9p) particles. Alternatively, studies in HSV-1 have suggested that gE/gI and US9p are required for the appearance of virions in the axon because they act upstream, to help assemble enveloped virions in the cell body. We prepared Δ(gE/gI-US9p) mutant, and control parental PRV particles from differentiated cultured neuronal or porcine kidney epithelial cells and quantitated the efficiency of virion assembly, the properties of microtubule-dependent transport and the ability of viral particles to recruit kinesin motors. We find that loss of gE/gI-US9p has no significant effect upon PRV particle assembly but leads to greatly diminished plus end-directed traffic, and enhanced minus end-directed and bidirectional movement along microtubules. PRV particles prepared from infected differentiated mouse CAD neurons were found to be associated with either kinesin KIF1A or kinesin KIF5C, but not both. Loss of gE/gI-US9p resulted in failure to recruit KIF1A and KF5C, but did not affect dynein binding. Unexpectedly, while KIF5C was expressed in undifferentiated and differentiated CAD neurons it was only found associated with PRV particles prepared from differentiated cells., Author summary Alphaherpesviruses including Pseudorabies virus (PRV) and Herpes simplex virus type 1 (HSV-1) are pathogens of the nervous system. They replicate in the nerve cell body then travel great distances along axons to reach nerve termini and spread to adjacent epithelial cells. Virally encoded membrane proteins US9p and the heterodimer gE/gI play critical roles in anterograde spread for both HSV-1 and PRV, however there is disagreement over their roles. Here we compared mutant PRV virions lacking gE/gI-US9p with a parental control strain, and quantitated the efficiency of virion assembly, the properties of microtubule-dependent transport and the ability of viral particles to recruit kinesins. We find that loss of gE/gI-US9p has no effect upon PRV particle assembly but that mutant virions demonstrate greatly reduced transport to the plus-ends of microtubules, enhanced minus-end traffic and greater frequency of reversal in their motion. These changes are accompanied by loss of both kinesin KIF1A and kinesin KIF5C from the mutant, motors that we find associated with distinct populations of the parental virus. We propose a model in which gE/gI-US9p binds KIF1A to ensure efficient plus end-mediated transport and delivery of PRV particles to a location at which KIF5C is subsequently recruited.

Hepatocyte transplantation is an attractive alternative to liver transplantation. Thus far, however, extensive liver repopulation by adult hepatocytes has required ongoing genetic, physical, or chemical injury to host liver. We hypothesized that providing a regulated proliferative and/or survival advantage to transplanted hepatocytes should enable repopulation in a normal liver microenvironment. Here, we repopulated livers of DPPIV− (dipeptidyl peptidase‐4) rats and Ugt1a1 (uridinediphosphoglucuronate glucuronosyltransferase 1a1)‐deficient Gunn rats (model of Crigler‐Najjar syndrome type 1), both models without underlying liver injury, for up to 1 year by transplanting lenti‐hYAP‐ERT2 (mutated estrogen receptor ligand‐binding domain 2)‐transduced hepatocytes (YAP‐Hc). Yap (yes‐associated protein) nuclear translocation/function in YAP‐Hc was regulated by tamoxifen. Repopulating YAP‐Hc and host hepatocytes were fluorescence‐activated cell sorting–purified and their transcriptomic profiles compared by RNAseq. After 1 year of liver repopulation, YAP‐Hc clusters exhibited normal morphology, integration into hepatic plates and hepatocyte‐specific gene expression, without dysplasia, dedifferentiation, or tumorigenesis. RNAseq analysis showed up‐regulation of 145 genes promoting cell proliferation and 305 genes suppressing apoptosis, including hepatocyte growth factor and connective tissue growth factor among the top 30 in each category and provided insight into the mechanism of cell competition that enabled replacement of host hepatocytes by YAP‐Hc. In Gunn rats transplanted with YAP‐Hc+tamoxifen, there was a 65%‐81% decline in serum bilirubin over 6 months versus 8%‐20% with control‐Hc, representing a 3‐4‐fold increase in therapeutic response. This correlated with liver repopulation as demonstrated by the presence of Ugt1a1‐positive hepatocyte clusters in livers and western blot analysis of tissue homogenates. Conclusion: Tamoxifen‐regulated nuclear translocation/function of hYAP‐ERT2 enabled long‐term repopulation of DPPIV−/− and Gunn rat livers by hYAP‐ERT2‐transduced hepatocytes without tumorigenesis. This cell transplantation strategy may offer a potential therapy for most of the inherited monogenic liver diseases that do not exhibit liver injury.

The seaboard extending from northern Svalbard to Scotland is the only region of the world where fjords have been comprehensively studied for their live (stained) benthic foraminiferal faunas. These modern faunas provide essential baseline data for the interpretation of the postglacial and continuing environmental changes in those fjords and this is the first biogeographic synthesis. The data come from the surface sediment assemblages (mainly sampled in the 1990's) from all the available literature. Due to limited information of shallow water assemblages in the north, only the species occurrences in deeper water from below the halocline are considered. Amongst these, only “common species” species occurring in more than one fjord are included. There is a clear pattern of distribution with five groups of taxa: 5 widespread species found throughout the region; 53 species reaching their northern limit; 13 species reaching their southern limit; 11 deep-sea species; 1 recently introduced species. Although there is an abrupt change in temperature from Tanafjorden in northern Norway to Hornsund in southern Svalbard, the faunal change from N to S is progressive throughout the investigated region. The area of overlap of the northern and southern species corresponds with the previously recognised boundary between the Barents Sea Province and the Norwegian Coast Province based on shelf and upper slope invertebrate macrofaunal benthos and plankton. Temperature is the main abiotic control on the distributions. For the fjords which have shallow sills separating them from the open shelf it is likely that most of the foraminiferal colonisers of the deeper fjord basins are sourced from the shelf or slope via propagules. One species has recently been introduced from further south into the southern region probably through the discharge of ballast water from ships. The biodiversity of the pristine Svalbard fjords extends below what is considered to reflect acceptable ecological status for mainland Norway, illustrating the need to introduce new methods to determine possible deviations from the reference conditions as defined in the EU's Water Framework Directive (WFD; 2000/60/EC). Altogether 347 species have so far been recorded in Norwegian waters: 214 in fjords (60 above and 180 below the halocline of which 26 occur both above and below the halocline) and 266 on the shelf and slope (133 of which also occur in fjords).

Depressions (known as deeps) on the Scottish continental shelf are sediment sinks and they, thus, provide important sources of information on past oceanography and climate. Muck Deep is a partially infilled relict glacial valley cut in the continental shelf. A 70 cm core shows an upper buff sandy mud layer (0–4500 BP, radiocarbon years) overlying a dark grey mud layer (4500–12 000 BP). The foraminiferal faunas suggest that the upper sandy layer formed under higher energy conditions comparable to those existing now, where storm waves from southwesterly gales are responsible for transporting material from the adjacent shelf into the deeps. The grey mud formed under lower energy conditions as a consequence of lower storm wave influence on the sea floor (therefore, reduced transport of tests from the shelf into the deep). Because of the interaction of changing sea-levels and glacial rebound, water depths over Muck Deep were 35 m greater at 14–15 ka and have never been less than now. Therefore, the likely explanation of the lower energy deposits at the base of the core is related to the impact of storm waves on sediment transport from the adjacent shelves. Either water depths over the adjacent shelves were too great to be influenced by storm waves or the wave climate (direction) differed at that time.

The major agglutinated constituents in test material of marginal marine foraminifera are identified as α-quartz and clay particles using complementary spectroscopic techniques. Electron dispersive scattering analysis, micro-laser Raman and Fourier transform infrared (FTIR) spectroscopic techniques revealed detail about elemental and mineral polymorph constituents in test walls. Additionally, FTIR identifies the existence of organic cements and lining materials in wall structures. Micro-laser Raman specifically characterized the titanium oxide mineral, anatase, as a distinctive fraction of agglutinate in Ammobaculites balkwilli Haynes. The mineral represents ≈10% of the test material, but comprises a minor component of the sediment and is identified in sediments only after heavy mineral separation. The enhanced concentration of anatase in the test of A. balkwilli suggests that there is a preferential selection for anatase. This provides further evidence that certain foraminifera can select grains specifically, which implies that there exists a selective mechanism and interaction between the organic (secreted) phases in the test walls and inorganic (grain surface) materials.

Marty Buzas is an outstanding scientist who has devoted his lifetime’s research to furthering our knowledge of statistics and its application to understanding the ecology of benthic foraminifera. Marty was born in 1934 in Bridgeport, Connecticut, USA to Hungarian parents. He has Geology degrees from the universities of Connecticut (BA 1958), Brown (MSc 1960) and Yale (PhD 1963). The influence of the statistician Hilary Seal and the ecologist G. Evelyn Hutchinson profoundly affected his career. On the completion of his PhD he was recruited by the Smithsonian Institution, Washington, DC, because of his palaeontological and quantitative skills. He has continued to work there and still does research there even in retirement and has now completed 57 years of excellent work. Throughout this long career Marty has concerned himself with the spatial and temporal distribution of foraminifera. This has been done on scales of millimetres to kilometres and time periods of days to millions of years. Statistical methods have been the key to establishing ecological relationships. For younger readers it must be pointed out that computers were not available to researchers until the mid-late 1960s and the early versions were totally unfriendly to users. Prior to this, all calculations had …

Previous studies have investigated regional distribution but this is the first attempt to investigate the global biogeographical distribution of individual morphospecies of living/stained smaller benthic foraminifera. From 8032 samples collected between 1952 and 2011 data have been gathered on the relative abundance of >120 species in five major environments ranging from marsh to deep sea. There is a spectrum of six groups of species with abundance ranging from very high (Group 1) to extremely low (Group 6). In the latter species abundance never reaches 10% of an assemblage and, in many cases, it is only 1–2%. Individual species are shown to occupy a range of environments with very few being confined to a single environment (usually either marsh or deep sea). Some species occur in several oceans while others are confined to just one. There is no correlation between species abundance and being either widely or narrowly distributed. Propagules are the most likely mechanism of dispersal but some narrowly distributed species may not produce them. Generalists may be widely or narrowly distributed but opportunists are likely to be widely distributed. The rare species of Group 6 contribute to high diversity in shelf and deep-sea assemblages. These species may be adapted to minor differences in microhabitats induced by disturbance and patchy food supply. Patterns of biogeography have application to ecology, palaeoecology and taxonomy. Supplementary Material: Taxonomy and details of types; sources of data (N1–N55) additional to that of Murray (2006); and sources of data for Figures 4–95 plus other agglutinated and hyaline taxa are available at www.geolsoc.org.uk/SUP18576

Data on modern foraminifera provide the basis for the palaeoecological interpretation of fossil forms. The validity of the use of living, dead, and total (living plus dead) assemblages is discussed. It is concluded that ecological studies can be carried out only on living assemblages and that it is important to determine the production and postmortem influences which lead to the formation of the dead assemblage. The use of total assemblages is shown to be ill-founded.

The Chagos Archipelago is a series of atolls situated in the centre of the Indian Ocean close to the equator. The area experiences small tides and periods of strong winds. The combined effects of these is to cause relatively high energy conditions to exist in the shallow waters around the reefs, therefore the bottom sediments are coarse grained and mobile. Although the coral faunas are diverse, seagrasses are rare. The total benthic foraminiferal assemblages have low to high species diversity and are dominated by hyaline taxa. On the oceanic side of the atoll reefs, the dominant foraminiferan is Amphistegina lessonii, with subsidiary miliolids. Planklonic tests form up to 20% of the combined benthic and planktonic component. In the lagoon, the assemblages are dominated by Calcarina calcar, with subsidiary miliolids. Planktonic tests are relatively uncommon. Some post-mortem transport and damage to tests has taken place but the distribution patterns are believed to be representative of the original living ones.

Summary Inability to preserve proteostasis with age contributes to the gradual loss of function that characterizes old organisms. Defective autophagy, a component of the proteostasis network for delivery and degradation of intracellular materials in lysosomes, has been described in multiple old organisms, while a robust autophagy response has been linked to longevity. The molecular mechanisms responsible for defective autophagic function with age remain, for the most part, poorly characterized. In this work, we have identified differences between young and old cells in the intracellular trafficking of the vesicular compartments that participate in autophagy. Failure to reposition autophagosomes and lysosomes toward the perinuclear region with age reduces the efficiency of their fusion and the subsequent degradation of the sequestered cargo. Hepatocytes from old mice display lower association of two microtubule‐based minus‐end‐directed motor proteins, the well‐characterized dynein, and the less‐studied KIFC3, with autophagosomes and lysosomes, respectively. Using genetic approaches to mimic the lower levels of KIFC3 observed in old cells, we confirmed that reduced content of this motor protein in fibroblasts leads to failed lysosomal repositioning and diminished autophagic flux. Our study connects defects in intracellular trafficking with insufficient autophagy in old organisms and identifies motor proteins as a novel target for future interventions aiming at correcting autophagic activity with anti‐aging purposes.

Na(+)-taurocholate cotransporting polypeptide (ntcp) mediates bile acid transport, also serving as the hepatitis B virus receptor. It traffics in vesicles along microtubules, requiring activity of protein kinase C (PKC)ζ for motility. We have now found that the epidermal growth factor receptor (EGFR) is the target of PKCζ activity and that EGFR and ntcp colocalize in vesicles. ntcp-containing vesicles that are not associated with EGFR have reduced microtubule-based motility, consistent with intracellular accumulation and reduced surface expression of ntcp in cells following EGFR knockdown.

The author is undertaking a regional study of the living (stained) benthic foraminiferal faunas of the Atlantic Ocean and adjacent seas (Barents Sea, Gulf of Mexico, Mediterranean). Quantitative and qualitative data on assemblages have been compiled on more than 2000 samples for the 0–1 cm sediment interval. Most areas have been sampled on a single occasion; replicates and repeated sampling for temporal studies are treated as individual samples. The results will be used to define patterns of distribution of standing crop and biodiversity and to relate them to potential causes. These data have come from the literature spanning the period from the introduction of the rose Bengal method of staining foraminifera (as an indicator of life at the time of collection; Walton, 1952) until December 2013. Every effort has been made to include all relevant data. A by-product of this dataset is that it is possible to recognize trends in the dates and seasons of sampling and the choice of size fraction. The three broad environments considered here are: shelf ( 200 m) and fjord (depths range down to >800 m). Dates of sampling have been grouped into decades. Seasons of sampling are defined for N and S hemispheres: winter (N: December–February; S: June–August); spring (N: March–May; S: September–November); summer (N: June–August: S: December–February) and autumn (N: September–November; S: March–May). Where authors have not given a precise date for each sample, and the dates of the sampling campaign span two seasons, the earlier …

Currently there is very little basin-scale information on patterns of standing crop in marine organisms or their structuring forces. Understanding modern patterns and controls on foraminifera is particularly critical because of their abundance and importance in benthic systems as well as their role as palaeoceanographic proxies. Here, we examine for the first time basin-scale patterns and predictors of benthic foraminiferal standing crop from shelf to abyssal deep sea in the Atlantic Ocean and adjacent seas using a large database of 967 quantitative samples. Spatial regression analyses reveal that the flux of particulate organic matter (POC) is a major control on standing crop size across all depths investigated, with increasing food supply increasing foraminiferal standing crops. Other factors also play a role. Dissolved oxygen is significant at slope depths and negatively related to standing crop. Temperature and possibly salinity are significant factors in the abyss. Dissolved silicate was important and positively related to standing crop in shelf seas, potentially indicating the importance of the nature of the organic material available. This study demonstrates that productivity is important in describing foraminiferal standing crop at a basin scale, supporting the use of paleoceanographic proxies, but also demonstrates that other environmental variables are also likely important in controlling the standing crop and should be considered in reconstruction of Earth’s past marine environment.

Previous studies identified a family of organic anion transport proteins (OATPs), many of which have C-terminal PDZ binding consensus sequences. In particular, the C-terminal four amino acids of Oatp1a1, a transporter on rat and mouse hepatocytes, comprise a consensus binding site for PDZK1. In PDZK1 knockout mice and in transfected cells where PDZK1 expression was knocked down, Oatp1a1 accumulates in intracellular vesicles. The present study tests the hypothesis that Oatp1a1 traffics to and from the cell surface in vesicles along microtubules, and that PDZK1 guides recruitment of specific motors to these vesicles. Oatp1a1-containing vesicles were prepared from wild-type and PDZK1 knockout mice. As seen by immunofluorescence, kinesin-1, a microtubule plus-end directed motor, was largely associated with vesicles from wild-type mouse liver, whereas dynein, a minus-end directed motor, was largely associated with vesicles from PDZK1 knockout mouse liver. Quantification of motility on directionally marked microtubules following addition of 50 µM ATP showed that wild-type vesicles moved equally toward the plus and minus ends whereas PDZK1 knockout vesicles moved predominantly toward the minus end, consistent with net movement toward the cell interior. These studies provide a novel mechanism by which PDZK1 regulates intracellular trafficking of Oatp1a1 by recruiting specific motors to Oatp1a1-containing vesicles. In the absence of PDZK1, Oatp1a1-containing vesicles cannot recruit kinesin-1 and associate with dynein as a predominant minus-end directed motor. Whether this is a result of direct interaction of the Oatp1a1 cytoplasmic domain with dynein or with a dynein-containing protein complex remains to be established.

Morphogroups offer a way of overcoming taxonomic differences and thereby making comparisons between assemblages of different geological ages. Relating morphogroups to environments has proved useful in palaeoecological interpretations. The model proposed by Jones and Charnock in 1985 has subsequently been adapted and still serves as the main reference for such palaeoecological interpretations. New data from > 400 samples from marsh to deep sea off NW Europe are used here to refine the model by providing the range of abundance of each morphogroup in each environment. It is shown that this range of variation is great so that there is considerable overlap between environments. Nevertheless, the mean values show patterns of distribution. Tubular forms (morphogroup A) are characteristic of slope environments, especially those under the influence of geostrophic currents. Morphogroup B is not common in marginal marine environments but is an indicator of shallow fjord to deep sea. The elongate forms of morphogroup C are widely distributed but C1 is especially common in marginal marine and shelf to upper slope settings. The quinqueloculine coiled C2 subgroup is characteristic of marsh and marginal marine environments in the temperate zone but around Antarctica this form is common down to 2000 m. The plano-convex, low trochospiral trochamminids of morphogroup D are typical of marshes. Therefore, these trends have application to palaeoecology of similar well-oxygenated temperate environments.

This is the first record of live (stained) deep-sea benthic foraminifera in the 850 m deep silled Hardangerfjord, the second deepest fjord in Western Norway. Estimates of organic carbon flux (not, vert, similar2.5 g Cm?2 y?1) show that the fjord-values are comparable to similar depths on the continental slope. Accordingly, although these first samples only provide relative abundance data, the low proportion of live to dead individuals in the top cm of the sediment suggests a low foraminifera biomass. Another similarity with the deep sea is that the abiotic environment of the deep basins is stable even though the deepest basins are isolated from the open deep sea by the continental shelf and sills in the outer parts of the fjord suggesting that the deep-sea species are introduced as propagules during deep-water renewals. There is evidence of an increase in dissolution of fragile calcareous tests (e.g., Nonionella iridea) especially in the innermost part of Hardangerfjord since the 1960s and this has led to a relative increase in dead agglutinated assemblages. The presence of larger forms with tests >1 mm provides substrata for the attachment of smaller forms and therefore an increase in species diversity. Indeed, the diversity is comparable both to that of the open deep sea and that of reported macrofauna from the same sites, reflecting similar ecological status. Holtedahl (1965) suggested that there may be some down-slope transport of sediment into the deep basins with the deposition of turbidites. Despite some evidence of transport, no major recent disturbance due to turbidite deposition seems to have occurred and hence Hardangerfjord presents a unique environment with elements of deep-sea faunas in a land-locked setting.

Although perturbation of organic anion transport protein (oatp) cell surface expression can result in drug toxicity, little is known regarding mechanisms regulating its subcellular distribution. Many members of the oatp family, including oatp1a1, have a COOH-terminal PDZ consensus binding motif that interacts with PDZK1, while serines upstream of this site (S634 and S635) can be phosphorylated. Using oatp1a1 as a prototypical member of the oatp family, we prepared plasmids in which these serines were mutated to glutamic acid [E634E635 (oatp1a1EE), phosphomimetic] or alanine [A634A635 (oatp1a1AA), nonphosphorylatable]. Distribution of oatp1a1AAand oatp1a1EEwas largely intracellular in transfected human embryonic kidney (HEK) 293T cells. Cotransfection with a plasmid encoding PDZK1 revealed that oatp1a1AAwas now expressed largely on the cell surface, while oatp1a1EEremained intracellular. To quantify these changes, studies were performed in HuH7 cells stably transfected with these oatp1a1 plasmids. These cells endogenously express PDZK1. Surface biotinylation at 4°C followed by shift to 37°C showed that oatp1a1EEinternalizes quickly compared with oatp1a1AA. To examine a physiological role for phosphorylation in oatp1a1 subcellular distribution, studies were performed in rat hepatocytes exposed to extracellular ATP, a condition that stimulates serine phosphorylation of oatp1a1 via activity of a purinergic receptor. Internalization of oatp1a1 under these conditions was rapid. Thus, although PDZK1 binding is required for optimal cell surface expression of oatp1a1, phosphorylation provides a mechanism for fast regulation of the distribution of oatp1a1 between the cell surface and intracellular vesicular pools. Identification of the proteins and motor molecules that mediate these trafficking events represents an important area for future study.

Helia Molina Milman and colleagues describe how intersectoral collaboration between health, social protection, and education sectors enabled Chile Grows with You (Chile Crece Contigo) to help all children reach their full developmental potential

Following endocytosis, internalized molecules are found within intracellular vesicles and tubules that move along the cytoskeleton and undergo fission, as demonstrated here using primary cultured rat hepatocytes. Although the use of depolymerizing drugs has shown that the cytoskeleton is not required to segregate endocytic protein, many studies suggest that the cytoskeleton is involved in the segregation of protein in normal cells. To investigate whether cytoskeletal-based movement results in the segregation of protein, we tracked the contents of vesicles during in vitro microscopy assays. These studies showed that the addition of ATP causes fission of endocytic contents along microtubules, resulting in the segregation of proteins that are targeted for different cellular compartments. The plasma membrane proteins, sodium (Na+) taurocholate cotransporting polypeptide (ntcp) and transferrin receptor, segregated from asialoorosomucoid (ASOR), an endocytic ligand that is targeted for degradation. Epidermal growth factor receptor, which is degraded, and the asialoglycoprotein receptor, which remains partially bound to ASOR, segregated less efficiently from ASOR. Vesicles containing ntcp and transferrin receptor had reduced fission in the absence of ASOR, suggesting that fission is regulated to allow proteins to segregate. A single round of fission resulted in 6.5-fold purification of ntcp from ASOR, and 25% of the resulting vesicles were completely depleted of the endocytic ligand.

Organic anion transport protein 1a1 (oatp1a1), a prototypical member of the oatp family of highly homologous transport proteins, is expressed on the basolateral (sinusoidal) surface of rat hepatocytes. The organization of oatp1a1 within the plasma membrane has not been well defined, and computer-based models have predicted possible 12- as well as 10-transmembrane domain structures. Which of oatp1a1's four potential N-linked glycosylation sites are actually glycosylated and their influence on transport function have not been investigated in a mammalian system. In the present study, topology of oatp1a1 in the rat hepatocyte plasma membrane was examined by immunofluorescence analysis using an epitope-specific antibody designed to differentiate a 10- from a 12-transmembrane domain model. To map glycosylation sites, the asparagines at the each of the four N-linked glycosylation consensus sites were mutagenized to glutamines. Mutagenized oatp1a1 constructs were expressed in HeLa cells, and effects on protein expression and transport activity were assessed. These studies revealed that oatp1a1 is a 12-transmembrane-domain protein in which the second and fifth extracellular loops are glycosylated at asparagines 124, 135, and 492, whereas the potential glycosylation site at asparagine 62 is not utilized, consistent with its position in a transmembrane domain. Constructs in which more than one glycosylation site were eliminated had reduced transport activity but not necessarily reduced transporter expression. This was in accord with the finding that fully unglycosylated oatp1a1 was well expressed but located intracellularly with limited transport ability as a consequence of its reduced cell surface expression.

Liver repopulation by transplanted hepatocytes has not been achieved previously in a normal liver microenvironment. Here we report that adult rat hepatocytes transduced ex vivo with a lentivirus expressing a human YapERT2 fusion protein (hYapERT2) under control of the hepatocyte-specific transthyretin (TTR) promoter repopulate normal rat liver in a tamoxifen-dependent manner. Transplanted hepatocytes expand very slowly but progressively to produce 10% repopulation at 6 months, showing clusters of mature hepatocytes that are fully integrated into hepatic parenchyma, with no evidence for dedifferentiation, dysplasia or malignant transformation. Thus, we have developed the first vector designed to regulate the growth control properties of Yap that renders it capable of producing effective cell therapy. The level of liver repopulation achieved has significant translational implications, as it is 2-3x the level required to cure many monogenic disorders of liver function that have no underlying hepatic pathology and is potentially applicable to diseases of other tissues and organs.

In ecological studies involving the analysis of ∼ 2.4 million living (stained) individual tests, to date ∼ 2140 species of benthic foraminifera have been recorded. Of these 602 species are agglutinated, 341 porcelaneous and 1197 hyaline. The numbers of species in the major environments are: marginal marine 701 (in ∼ 1.5 million individuals), shelf 989 (in ∼ 0.6 million individuals) and deep sea 831 (in ∼ 0.3 million individuals). 381 species occur in more than one major environment. Overall ∼ 33% have abundance of > 10% while ∼ 67% are of minor abundance (

Benthic foraminiferal assemblages in subrecent deposits are commonly used to reconstruct past sea level. Interpretations are generally made by comparison with either modern dead or total (live plus dead) assemblages. In both cases there will have been post-mortem changes that have differentially affected preservation. It is therefore important to establish the primary ecological controls by analysis of the living assemblages. We have determined the spatial and temporal variability of intertidal benthic foraminifera in the surface (0–1 cm) sediments from a time series survey of 31 sampling stations at Cowpen Marsh, for a period of 12 months. We counted 112,067 live foraminifera assigned to 28 species. The fauna was dominated by two agglutinated species (Jadammina macrescens and Trochammina inflata) on the high and middle marshes, and three calcareous species (Elphidium williamsoni, Haynesina germanica and Quinqueloculina spp.) on the low marsh and tidal flat. The standing crop of the whole intertidal zone, including the high, middle, low marsh and tidal flat habitats, and the individual species varied both temporally and spatially. The standing crop of the intertidal zone as a whole was greatest in the summer months and showed a positive correlation with elevation. The standing crops of the high and middle marshes showed similar temporal variation with peaks in summer and autumn and a trough in winter. The low marsh showed numerous peaks and troughs of standing crop during the year, whereas the tidal flat showed a single peak in summer. The standing crops of Jadammina macrescens and Trochammina inflata on the high and middle marshes peaked from April to May and August to October with troughs in winter. These agglutinated species showed a strong correlation with elevation. Haynesina germanica peaked in May to August and November to January on the low marsh, whereas on the tidal flat there was a single peak in July. The standing crops of E. williamsoni on the low marsh and tidal flat were relatively high in June and May, and July, respectively. Quinqueloculina spp. peaked in May to July on the low marsh and July on the tidal flat. The species was also found in the middle marsh from July to May and high marsh from September to November. Haynesina germanica showed a strong negative correlation with elevation, whereas the other two dominant calcareous species demonstrated weak negative correlations with both elevation and salinity. Reconstructing former sea level depends primarily on the recognition of high and middle marsh assemblages and in this study these are shown to be strongly controlled by elevation rather than salinity. Caution may be needed in interpreting low marsh and tidal flat data as salinity plays a more important role here.

Na+-taurocholate cotransporting polypeptide (ntcp) mediates uptake of bile acids as well as serving as the receptor for hepatitis B virus in human liver. Previous studies showed that ntcp traffics on microtubules between the cell surface and endocytic vesicles. Specific inhibition of protein kinase C (PKC)ζ resulted in loss of microtubule-based motility of these vesicles in vitro and in living cells. The aim of the present study was to characterize the PKCζ target. Incubation of ntcp-containing endocytic vesicles with γ-32P-ATP revealed a 180 kDa phosphoglycoprotein that was identified as the EGF receptor (EGFR). Surface biotinylation of HuH7 cells expressing GFP-ntcp revealed substantially reduced trafficking of ntcp to the cell surface with EGFR knockdown. Microtubule-based motility of ntcp-containing endocytic vesicles was also significantly reduced when they were not associated with EGFR. Ntcp was also found to undergo cellular redistribution upon stimulation of cells with EGF, consistent with a model in which ntcp and EGF-EGFR internalize into common endocytic vesicles from which they segregate, trafficking EGF-EGFR to lysosomes and recycling ntcp to the plasma membrane. EGF regulation of ntcp trafficking may play a heretofore unanticipated role in subcellular targeting of ntcp ligands such as hepatitis B.

Intracellular trafficking regulates the abundance and therefore activity of transporters present at the plasma membrane. The transporter, Na+-taurocholate co-transporting polypeptide (ntcp), is increased at the plasma membrane upon treatment of cells with cAMP, for which microtubules (MTs) are required and the PI3K pathway and PKCzeta have been implicated. However, trafficking of ntcp on MTs has not been demonstrated directly and the regulation and intracellular localization of ntcp is not well understood. Here, we utilize in vitro and whole-cell immunofluorescence microscopy assays to demonstrate that ntcp is present on intracellular vesicles that bind MTs and move bidirectionally, using kinesin-1 and dynein. These vesicles co-localize with markers for recycling endosomes and early but not late endosomes. They frequently undergo fission, providing a mechanism for the exclusion of ntcp from late endosomes. PI(3,4,5)P3 activates PKCzeta and enhances motility of the ntcp vesicles and overcomes the partial inhibition produced by a PI3-kinase inhibitor. Specific inhibition of PKCzeta blocks the motility of ntcp-containing vesicles but has no effect on late vesicles as shown both in vitro and in living cells transfected with ntcp-GFP. These data indicate that PKCzeta is required specifically for the intracellular movement of vesicles that contain the ntcp transporter.

We have collected live and dead foraminiferal times-series data at 2-weekly intervals for a 12-month period from the intertidal zone of Cowpen Marsh, Tees Estuary, UK. The data from the 689 samples show profound differences between live and dead assemblages, although assemblages are dominated by just three species, Haynesina germanica , Jadammina macrescens and Trochammina inflata , which represent over 70% of the assemblage. The cumulative increase in species of most environments approximates to a lognormal or log series. None of the datasets show a broken stick pattern. The cumulative maximum number of species, which represents the species carrying capacity of the environment, is recorded earlier in the life assemblages than the dead counterparts. The dead assemblage of Cowpen Marsh is found to have a higher abundance (435 compared to 163 individuals/10 cm 3 ) and number of species (52 compared to 28) than its live counterpart because the dead assemblage represents many generations added over a long period of time. In contrast, some species are recorded in the live dataset that were not found in the dead assemblage, indicating the dead record is either incomplete (e.g. taphonomic change) or inadequately sampled. We investigated the influence of patterns in cumulative increase on dead assemblages for sea-level reconstructions through the development of foraminiferal-based transfer functions. The cumulative transfer functions suggest that the performance improves during the first six sample intervals of the time-series dataset with reconstruction differing by 1.2 m and remains constant thereafter.

A microcosm experiment was conducted to investigate the effects of sewage-derived particulate organic matter (POM) on the food sources and diets of two species of intertidal benthic foraminifera, Ammonia beccarii and Haynesina germanica, using lipid biomarkers to determine trophic relationships. The lipid content of the sediment and associated micro-organisms was a guide to potential food sources while that of the foraminifera was a guide to what they had actually eaten. Six microcosm tanks were established, with constant salinity, temperature and oxygen content, and each with a thin layer of sediment containing living foraminifera. Three microcosms were used as controls and three were treatments to which the POM from secondary treated sewage was added. Each microcosm was treated as a single replicate (to avoid pseudoreplication). The experiment was run for 38 days. The results showed that the food sources (from the sediment) and the diet of the foraminifera did not significantly differ in the controls or the treatments, but quantities of fatty acids decreased in both the sediment system and the foraminifera over the duration of the experiment. It is concluded that sewage-POM (secondary treatment) does not have a direct effect on the food sources of the foraminifera or their diet. The foraminifera did not feed directly on the sewage-derived POM, nor did the addition of sewage stimulate growth of micro-organisms associated with the sediment system. However, recent field data collected by the authors provides evidence that season plays an important role in foraminiferal response to organic pollution (OP), and microcosm sediment might have been unknowingly collected at a time when foraminifera are now known not to respond to OP, i.e. in summer.

Terrestricythere elisabethae sp. nov. is described from a semiterrestrial coastal habitat at two sites in Hampshire, southern England. It is the first record of a living population of the genus outside the Far East (north-west Pacific). Based on extensive collections and from observations of cultures, its morphology is described (including a formal definition of the unique ‘visordont’ hinge), as well as its ontogeny and lifestyle (encompassing habitat, life cycle, mode of life and locomotion). A further new species from Somerset, south-west England is also recorded on the basis of a single specimen but left in open nomenclature. The affinities of the Terrestricytheroidea are discussed in the context of a tentative phylogeny of podocopan Ostracoda. Both carapace and appendage characters are such as to warrant maintaining it as a separate superfamily, which is more closely related to the Cytheroidea and Darwinuloidea than to the Cypridoidea. © 2004 The Linnean Society of London, Zoological Journal of the Linnean Society, 2004, 142, 253–288.

Within the past 7 years, the northern Larsen Ice Shelf has broken up so it is now possible to sample the sea floor that formerly lay beneath it. Box cores have yielded surface sediment samples (0–1 cm) that give information on living and dead foraminiferal assemblages. The living assemblages are of moderate diversity and four have >50% calcareous tests while five have >50% agglutinated tests. This is an area of high primary production and the standing crops of the benthic foraminiferal assemblages are high. All the dead assemblages are much enriched in agglutinated tests, often >90%. They give a time-averaged record of the past 7 ice-free years and several decades of ice cover. The loss of permanent ice cover (there is still seasonal ice cover) may have caused some response from the fauna, but it is likely that it was mainly changes in relative/absolute abundance of the existing fauna. The differences between the live and dead assemblages in the surface 1 cm are attributed mainly to taphonomic effects: dissolution of calcareous tests and loss of fragile agglutinated tests. Subsurface samples down to 5 cm show that dissolution of calcareous tests is widespread and there may be some loss of fragile agglutinated forms such as Reophax subdentaliniformis. For these reasons, in this area, it may be best to make palaeoecological interpretations on the agglutinated component of the fossil assemblages.

Our previous studies demonstrated that fluorescent early endocytic vesicles prepared from rat liver after injection of Texas red asialoorosomucoid contain asialoglycoprotein and its receptor and move and undergo fission along microtubules using kinesin I and KIFC2, with Rab4 regulating KIFC2 activity (J. Cell Sci. 116, 2749, 2003). In the current study, procedures to prepare fluorescent late endocytic vesicles were devised. In addition, flow cytometry was utilized to prepare highly purified fluorescent endocytic vesicles, permitting validation of microscopy-based experiments as well as direct biochemical analysis. These studies revealed that late vesicles bound to and moved along microtubules, but in contrast to early vesicles, did not undergo fission. As compared with early vesicles, late vesicles had reduced association with receptor, Rab4, and kinesin I but were highly associated with dynein, Rab7, dynactin, and KIF3A. Dynein and KIF3A antibodies inhibited late vesicle motility, whereas kinesin I and KIFC2 antibodies had no effect. Dynamitin antibodies prevented the association of late vesicles with microtubules. These results indicate that acquisition and exchange of specific motor and regulatory proteins characterizes and may regulate the transition of early to late endocytic vesicles. Flow cytometric purification should ultimately facilitate detailed proteomic analysis and mapping of endocytic vesicle-associated proteins.

After internalization, endocytic material is actively transported through the cytoplasm, predominantly by microtubule motor proteins. Microtubule-based endocytic transport facilitates sorting of endocytic contents, vesicle fusion and fission, delivery to lysosomes, cytosolic dispersal, as well as nuclear uptake and cytosolic egress of pathogens. Endosomes, like most organelles, move bidirectionally through the cytosol and regulate their cellular location by controlling the activity of motor proteins, and potentially by controlling microtubule and actin polymerization. Control of motor protein activity is manifest by increased microtubule "run lengths", and the binding of motor proteins to organelles can be regulated by motor protein receptors. A mechanistic understanding of how organelles control motor protein activity to allow for endocytic sorting presents an exciting avenue for future research.

Loch Etive, a silled 145 m deep fjord on the Scottish west coast, provides an example of modern benthic foraminiferal assemblages at intermediate depths (i.e., below the intertidal zone and above the CCD) consisting almost exclusively of organic-cemented agglutinated forms. Since such faunas from intermediate depths are rare in modern oceans but relatively common in the fossil record, the present study allows new insights into one kind of ancient environment for which there are few modern analogues. The strong dominance of agglutinated forms (both living and in some dead assemblages of foraminifera to the exclusion of calcareous taxa) is attributed to the unusual oceanographic conditions. These include a combination of restricted deep-water renewals and strong influence of freshwater which drains through large areas (relative to the size of the loch) of vegetated land. The result is calm bottom water conditions with commonly occurring oxygen depletion (although not anoxic), brackish water throughout the water column (salinity 28 in the deeper parts), and organic-rich (mostly terrestrially derived) sediments with geochemical properties, which, to a much larger degree than open marine ones, are controlled by local input. This environment supports low abundance and low diversity live assemblages, mainly restricted to the surface 1 cm of sediment. The dead assemblages show similar faunal characteristics, but the calcareous components are, due to carbonate dissolution, even more reduced. One of the calcareous species in Loch Etive is Elphidium albiumbilicatum. Its occurrence is the first record in British waters and it matches the previously suggested southern limit of its distribution. Analysis of a 90 cm long core representing sediments deposited over the past two centuries shows the presence of a calcareous dominated assemblage, including more marine species, with a higher diversity, in the lower part. This suggests that Loch Etive is in the process of going from a marine, to a more terrestrial dominated environment. The relatively high sedimentation rate (0.5 cm per yr), the apparent lack of smearing through bioturbation, and the presence of faunal changes in response to reduced marine influence over the past centuries, shows that Loch Etive has a good potential for performing high-resolution climatic studies.

We previously established conditions to reconstitute kinesin-dependent early endocytic vesicle motility and fission on microtubules in vitro. The present study examined the question whether motility and fission are regulated in this system. Screening for proteins by immunofluorescence microscopy revealed that the small G protein, Rab4, was associated with 80% of hepatocyte-derived early endocytic vesicles that contain the ligand asialoorosomucoid (ASOR). By contrast, other markers for early endocytic vesicles including clathrin, Rab5 and EEA1 were present in the preparation but did not colocalize with the ASOR vesicles. Guanine nucleotides exchanged into the Rab4 present on the vesicles as shown by solubilization of Rab4 by Rab-GDI; solubilization was inhibited by incubation with GTP-γ-S and promoted by GDP. Pre-incubation of vesicles with GDP increased the number of vesicles moving on microtubules and markedly increased vesicle fission. This increase in motility from GDP was shown to be towards the minus end of microtubules, possibly through activation of the minus-end-directed kinesin,KIFC2. Pre-incubation of vesicles with GTP-γ-S, by contrast, repressed motility. Addition of exogenous GST-Rab4- GTP-γ-S led to a further repression of motility and fission. Repression was not seen with addition of GST-Rab4-GDP. Treatment of vesicles with Rab4 antibody also repressed motility, and repression was not seen when vesicles were pre-incubated with GDP. Based on these results we hypothesize that endogenous Rab4-GTP suppresses motility of ASOR-containing vesicles in hepatocytes and that conversion of Rab4-GTP to Rab4-GDP serves as a molecular switch that activates minus-end kinesin-based motility, facilitating early endosome fission and consequent receptor-ligand segregation.

Time-series studies are essential for defining short and long-term variability in environments and without such records it is difficult to make well-founded judgments about faunal or environmental change. Datasets of intertidal benthic foraminifera from the Exe and Hamble estuaries, England, intertidal zone of Bahrain, Arabian Gulf, and subtidal Lim channel, Adriatic, have been analysed to provide information on patterns of species richness. Living assemblages comprise one or two dominant species plus two or three subsidiary species and a variable number of ephemeral rare species, the latter being the main control of species richness. The cumulative increase in species from month to month follows a pattern which approximates a log series. None of the datasets follows a broken stick or log normal pattern. The final plateau in cumulative species increase is reached in periods ranging from 5–10 months (Lim channel) to 22–23 months (estuaries). The cumulative maximum number of species gives an estimate of the carrying capacity for species, i.e. the maximum number of species for the environment. The species present in each area represent the species pool related to that environment (e.g. estuarine intertidal zone) together with introductions from a species pool in a different environment nearby (e.g. inner shelf). The time-averaged accumulation of successive live assemblages that form the dead assemblage is not identical with the cumulative total. Some live taxa are unrepresented in the dead assemblage either through taphonomic loss or the introduction of new species that have not yet left a dead record. In addition, the Exe estuary dead assemblage includes a large number of dead tests transported in from the adjacent shelf. It does not conform to any of the three possible patterns. At present the few time-series studies of benthic foraminifera that are available are either intertidal or very shallow water. There remains a need for further studies in a wide range of environments in order to understand more fully the faunal response to dynamic change.

The continental shelf west of Scotland has a veneer of medium to coarse sands and bioclastic gravels resting on basement, but in enclosed depressions (called deeps) the sediments are muddy. Sediment cores from two deeps have been studied: Muck Deep (170 and 218 m) and Stanton Deep (167 m). The live and dead foraminiferal assemblages from these shelf deeps differ in several ways. The live forms are mainly free-living taxa concentrated in the surface 0.0 to 0.5 or 1.0 cm sediment layer. The standing crop has a positive correlation with % Total organic carbon (TOC) and wt. % sediment in situ forms derived from the live assemblages plus around 50% of allochthonous taxa (without living representatives), mainly having an attached or clinging mode of life, and considered to be transported in from the surrounding shallower, higher energy shelf. The transported forms are mainly −3 for Muck and Stanton deeps, respectively) and show a logarithmic increase with increasing proportion of the 63–125 μm sediment fraction. These observations confirm that the shelf deeps form depositional sinks for finer terrigenous and biogenic material. This study demonstrates the complexity of assemblage formation in low energy depressions on a moderate to high energy continental shelf. It also demonstrates the importance of determining the in situ and transported components in order to make a reliable environmental interpretation of the preserved assemblages. The high abundance of dead tests indicates very slow accumulation rates which are typical of a maximum flooding surface starved of detrital sediment.

We describe a rapid technique for the localization and quantitation of specific proteins on organelles bound to microscope chambers. Disposable chambers are constructed from glass slides and provide a platform for the binding of organelles and subsequent immunofluorescence and biochemical assays. Several studies are presented to demonstrate the utility of this technique. Kinesin was visualized in postnuclear supernatants. Golgi and endoplasmic reticulum bound quantitatively to chambers. Endocytic vesicles prepared from rat liver that had been injected in situ with Texas red-labeled asialoorsomucoid allowed for simultaneous detection of asialoorosomucoid, asialoglycoprotein receptor, caveolin 1, and microtubules. Asialoglycoprotein receptor colocalized with asialoorosomucoid-containing vesicles, whereas many of the caveolin 1 structures had no asialoorosomucoid or asialoglycoprotein receptor. The microchambers were also used to measure the binding to endocytic vesicles of exogenously added Rab5 and to monitor the ATP-dependent acidification of endocytic vesicles using the fluorescent dye acridine orange.