TMEM165 deficiency causes a congenital disorder of glycosylation

Protein glycosylation is a complex process that depends not only on the activities of several enzymes and transporters but also on a subtle balance between vesicular Golgi trafficking, compartmental pH, and ion homeostasis. Through a combination of autozygosity mapping and expression analysis in two siblings with an abnormal serum-transferrin isoelectric focusing test (type 2) and a peculiar skeletal phenotype with epiphyseal, metaphyseal, and diaphyseal dysplasia, we identified TMEM165 (also named TPARL) as a gene involved in congenital disorders of glycosylation (CDG). The affected individuals are homozygous for a deep intronic splice mutation in TMEM165. In our cohort of unsolved CDG-II cases, we found another individual with the same mutation and two unrelated individuals with missense mutations in TMEM165. TMEM165 encodes a putative transmembrane 324 amino acid protein whose cellular functions are unknown. Using a siRNA strategy, we showed that TMEM165 deficiency causes Golgi glycosylation defects in HEK cells.     

Diel rhythmicity in amino acid uptake by Prochlorococcus

The marine cyanobacterium Prochlorococcus , the most abundant phototrophic organism on Earth, numerically dominates the phytoplankton in nitrogen (N)-depleted oceanic gyres. Alongside inorganic N sources such as nitrite and ammonium, natural populations of this genus also acquire organic N, specifically amino acids. Here, we investigated using isotopic tracer and flow cytometric cell sorting techniques whether amino acid uptake by Prochlorococcus is subject to a diel rhythmicity, and if so, whether this was linked to a specific cell cycle stage. We observed, in contrast to diurnally similar methionine uptake rates by Synechococcus cells, obvious diurnal rhythms in methionine uptake by Prochlorococcus cells in the tropical Atlantic. These rhythms were confirmed using reproducible cyclostat experiments with a light-synchronized axenic Prochlorococcus (PCC9511 strain) culture and ³⁵S-methionine and ³H-leucine tracers. Cells acquired the tracers at lower rates around dawn and higher rates around dusk despite >10⁴ times higher concentration of ammonium in the medium, presumably because amino acids can be directly incorporated into protein. Leucine uptake rates by cells in the S+G₂ cell cycle stage were consistently 2.2 times higher than those of cells at the G₁ stage. Furthermore, S+G₂ cells upregulated amino acid uptake 3.5 times from dawn to dusk to boost protein synthesis prior to cell division. Because Prochlorococcus populations can account from 13% at midday to 42% at dusk of total microbial uptake of methionine and probably of other amino acids in N-depleted oceanic waters, this genus exerts diurnally variable, strong competitive pressure on other bacterioplankton populations.     

Gene order and recombination rate in homologous chromosome regions of the chicken and a passerine bird

Genome structure has been found to be highly conserved between distantly related birds and recent data for a limited part of the genome suggest that this is true also for the gene order (synteny) within chromosomes. Here, we confirm that synteny is maintained for large chromosomal regions in chicken and a passerine bird, the great reed warbler Acrocephalus arundinaceus, with few rearrangements, but in contrast show that the recombination-based linkage map distances differ substantially between these species. We assigned a chromosomal location based on sequence similarity to the chicken genome sequence to a set of microsatellite loci mapped in a pedigree of great reed warblers. We detected homologous loci on 14 different chromosomes corresponding to chicken chromosomes Gga1-5, 7-9, 13, 19, 20, 24, 25, and Z. It is known that 2 passerine macrochromosomes correspond to the chicken chromosome Gga1. Homology of 2 different great reed warbler linkage groups (LG13 and LG5) to Gga1 allowed us to locate the split to a position between 20.8 and 84.8 Mb on Gga1. Data from the 5 chromosomal regions (on Gga1, 2, 3, 5, and Z) with 3 or more homologous loci showed that synteny was conserved with the exception of 2 large previously unreported inversions on Gga1/LG5 and Gga2/LG3, respectively. Recombination data from the 9 chromosomal regions in which we identified 2 or more homologous loci (accounting for the inversions) showed that the linkage map distances in great reed warblers were only 6.3% and 13.3% of those in chickens for males and females, respectively. This is likely to reflect the true interspecific difference in recombination rate because our markers were not located in potentially low-recombining regions: several linkage groups covered a substantial part of their corresponding chicken chromosomes and were not restricted to centromeres. We conclude that recombination rates may differ strongly between bird species with highly conserved genome structure and synteny and that the chicken linkage map may not be suitable, in terms of genetic distances, as a model for all bird species.     

Targeting IL-15 receptor-bearing cells with an antagonist mutant IL-15/Fc protein prevents disease development and progression in murine collagen-induced arthritis

It has been suggested that the inflammatory cytokine IL-15 plays an important role in the development of several autoimmune diseases, including rheumatoid arthritis. We have generated a unique lytic and antagonistic IL-15 mutant/Fcgamma2a fusion protein (CRB-15) that targets the IL-15R. In the present study we examined the effects of targeting the IL-15R on the prevention and treatment of collagen-induced arthritis (CIA) in mice and probed the possible mechanisms of action of this IL-15 mutant/Fcgamma2a protein. Upon immunization with type II collagen, DBA/1 mice develop severe articular inflammation and destruction. Treatment of DBA/1 mice with a brief course of CRB-15 at the time of type II collagen challenge markedly inhibited the incidence and severity of arthritis. Moreover, in animals with ongoing established arthritis, treatment with CRB-15 effectively blocked disease progression compared with that in control-treated animals. The therapeutic effect of CRB-15 on either disease development or disease progression is remarkably stable, because withdrawal of treatment did not lead to disease relapse. A detailed analysis revealed that treatment with CRB-15 decreased synovitis in the joints; reduced bone erosion and cartilage destruction; reduced in situ production of the proinflammatory cytokines TNF-alpha, IL-1beta, IL-6, and IL-17; and decreased the responder frequency of autoreactive T cells. Our study suggests that the effective targeting of IL-15R-triggered events with CRB-15 can be of therapeutic importance in the treatment of rheumatoid arthritis.     

Levels of extra‐pair paternity are associated with parental care in penduline tits (Remizidae)

In most passerine birds, individuals attempt to maximize their fitness by providing parental care while also mating outside their pair bond. A sex‐specific trade‐off between these two behaviours is predicted to occur, as the fitness benefits of extra‐pair mating differ between the sexes. We use nest observations and parentage analysis to reveal a negative association between male care and the incidence of extra‐pair paternity across three species of penduline tit (Remizidae). This provides evidence of a trade‐off between these two behaviours, possibly due to the devaluing of paternal care by extra‐pair offspring.     

Whole cell segmentation in solid tissue sections.

BACKGROUND: Understanding the cellular and molecular basis of tissue development and function requires analysis of individual cells while in their tissue context. METHODS: We developed software to find the optimum border around each cell (segmentation) from two-dimensional microscopic images of intact tissue. Samples were labeled with a fluorescent cell surface marker so that cell borders were brighter than elsewhere. The optimum border around each cell was defined as the border with an average intensity per unit length greater that any other possible border around that cell, and was calculated using the gray-weighted distance transform. Algorithm initiation requiring the user to mark two points per cell, one approximately in the center and the other on the border, ensured virtually 100% correct segmentation. Thereafter segmentation was automatic. RESULTS: The method was highly robust, because intermittent labeling of the cell borders, diffuse borders, and spurious signals away from the border do not significantly affect the optimum path. Computer-generated cells with increasing levels of added noise showed that the approach was accurate provided the cell could be detected visually. CONCLUSIONS: We have developed a highly robust algorithm for segmenting images of surface-labeled cells, enabling accurate and quantitative analysis of individual cells in tissue.     

Conductance Ratios and Cellular Identity

Recent experimental evidence suggests that coordinated expression of ion channels plays a role in constraining neuronal electrical activity. In particular, each neuronal cell type of the crustacean stomatogastric ganglion exhibits a unique set of positive linear correlations between ionic membrane conductances. These data suggest a causal relationship between expressed conductance correlations and features of cellular identity, namely electrical activity type. To test this idea, we used an existing database of conductance-based model neurons. We partitioned this database based on various measures of intrinsic activity, to approximate distinctions between biological cell types. We then tested individual conductance pairs for linear dependence to identify correlations. Contrary to experimental evidence, in which all conductance correlations are positive, 32% of correlations seen in this database were negative relationships. In addition, 80% of correlations seen here involved at least one calcium conductance, which have been difficult to measure experimentally. Similar to experimental results, each activity type investigated had a unique combination of correlated conductances. Finally, we found that populations of models that conform to a specific conductance correlation have a higher likelihood of exhibiting a particular feature of electrical activity. We conclude that regulating conductance ratios can support proper electrical activity of a wide range of cell types, particularly when the identity of the cell is well-defined by one or two features of its activity. Furthermore, we predict that previously unseen negative correlations and correlations involving calcium conductances are biologically plausible.