Mutations in BICD2, which Encodes a Golgin and Important Motor Adaptor,Cause Congenital Autosomal-Dominant Spinal Muscular Atrophy

Spinal muscular atrophy (SMA) is a heterogeneous group of neuromuscular disorders caused by degeneration of lower motor neurons. Although functional loss of SMN1 is associated with autosomal-recessive childhood SMA, the genetic cause for most families affected by dominantly inherited SMA is unknown. Here, we identified pathogenic variants in bicaudal D homolog 2 (Drosophila) (BICD2) in three families afflicted with autosomal-dominant SMA. Affected individuals displayed congenital slowly progressive muscle weakness mainly of the lower limbs and congenital contractures. In a large Dutch family, linkage analysis identified a 9q22.3 locus in which exome sequencing uncovered c.320C>T (p.Ser107Leu) in BICD2. Sequencing of 23 additional families affected by dominant SMA led to the identification of pathogenic variants in one family from Canada (c.2108C>T [p.Thr703Met]) and one from the Netherlands (c.563A>C [p.Asn188Thr]). BICD2 is a golgin and motor-adaptor protein involved in Golgi dynamics and vesicular and mRNA transport. Transient transfection of HeLa cells with all three mutant BICD2 cDNAs caused massive Golgi fragmentation. This observation was even more prominent in primary fibroblasts from an individual harboring c.2108C>T (p.Thr703Met) (affecting the C-terminal coiled-coil domain) and slightly less evident in individuals with c.563A>C (p.Asn188Thr) (affecting the N-terminal coiled-coil domain). Furthermore, BICD2 levels were reduced in affected individuals and trapped within the fragmented Golgi. Previous studies have shown that Drosophila mutant BicD causes reduced larvae locomotion by impaired clathrin-mediated synaptic endocytosis in neuromuscular junctions. These data emphasize the relevance of BICD2 in synaptic-vesicle recycling and support the conclusion that BICD2 mutations cause congenital slowly progressive dominant SMA.     

Nanosized Iron Oxide Colloids Strongly Enhance Microbial Iron Reduction

Microbial iron reduction is considered to be a significant subsurface process. The rate-limiting bioavailability of the insoluble iron oxyhydroxides, however, is a topic for debate. Surface area and mineral structure are recognized as crucial parameters for microbial reduction rates of bulk, macroaggregate iron minerals. However, a significant fraction of iron oxide minerals in the subsurface is supposed to be present as nanosized colloids. We therefore studied the role of colloidal iron oxides in microbial iron reduction. In batch growth experiments with Geobacter sulfurreducens, colloids of ferrihydrite (hydrodynamic diameter, 336 nm), hematite (123 nm), goethite (157 nm), and akaganeite (64 nm) were added as electron acceptors. The colloidal iron oxides were reduced up to 2 orders of magnitude more rapidly (up to 1,255 pmol h⁻¹ cell⁻¹) than bulk macroaggregates of the same iron phases (6 to 70 pmol h⁻¹ cell⁻¹). The increased reactivity was not only due to the large surface areas of the colloidal aggregates but also was due to a higher reactivity per unit surface. We hypothesize that this can be attributed to the high bioavailability of the nanosized aggregates and their colloidal suspension. Furthermore, a strong enhancement of reduction rates of bulk ferrihydrite was observed when nanosized ferrihydrite aggregates were added.Dissimilatory iron reduction is an important anaerobic respiration process in anoxic subsurface environments. However, the reactivity of ferric iron is mostly limited by the reduction kinetics of the poorly soluble, extracellular iron minerals. Electron transfer from microorganisms to iron oxides can occur via direct contact or by electron shuttling compounds (46). Transport of the electron shuttle between the redox partners is then assumed to occur via diffusion. For example, humic substances can serve as natural electron shuttles that can be reduced by microorganisms and subsequently chemically oxidized by the ferric oxide (18). Shewanella oneidensis excretes a flavin to stimulate hematite reduction, functioning in a similar manner (27). As another option, formation of conductive pili serving as nanowires was described as a possible way of transferring electrons to the oxide surface (15, 34). Nevertheless, direct attachment has been recognized as a major mode of accessing iron oxides as electron acceptors (12). Direct transfer between microbial outer membrane reductases and the ferric minerals, however, requires close contact of less than 14 Å between the terminal iron reductase on the cell surface and the iron oxide molecule at the mineral surface (19, 25), limiting the rates of electron transfer between cell and mineral.Several parameters have been discussed in this context as being decisive for the bioavailability and reactivity of iron oxides, such as, e.g., the mineral surface area (8, 41). Larger surface areas have been shown to be accompanied by higher initial reduction rates. Another parameter that might determine reactivity is the low solubility of ferric iron in water at neutral pH (20). Low solubility entails high crystallinity, which reduces reaction rates (4). Therefore, crystalline bulk iron phases such as goethite or hematite (9) are poorly reducible by microorganisms, in contrast to amorphous ferrihydrite (41). Naturally, well crystalline minerals have lower surface areas, and the effects of surface area and solubility cannot be distinguished sharply. Cell density, initial oxide and substrate concentrations, and ferrous iron adsorbed to the bulk mineral surface were also reported to control microbial reduction rates by exhibiting mutual saturation behavior in Michaelis-Menten-type kinetics (3, 22, 40).The latter studies also considered particle sizes, a parameter that has often been overlooked so far. All concepts mentioned above generally assumed a bulk state of the electron-accepting iron oxide. Indeed, iron oxides used in microbiological experiments appear mainly as coarse, flocculating macroaggregates, visible to the naked eye as sludge-like precipitates. In nature, however, nanosized iron oxides are abundant (32, 45) and play a vital role in many biogeochemical processes (2, 16, 28). Such nanoparticles may appear in stable colloidal suspension, even if aggregated as a stable cluster of multiple particles (13). Ferric oxide particles can appear in colloidal suspensions of different aggregate sizes and densities.Different particle aggregate sizes might influence the bioavailability of iron oxides in microbial reduction. Nanosized aggregates appearing in colloidal suspensions might be spatially more accessible for microorganisms than large aggregates flocculating as bulk phases. Therefore, the present study aims at assessing the reactivity and putative role of aggregate sizes of iron oxides in dissimilatory iron reduction. A set of ferrihydrite, hematite, goethite, and akaganeite colloids was compared to their respective noncolloidal bulk phases to evaluate this effect.     

α-Thalassemia in The Netherlands: a heterogeneous spectrum of both deletions and point mutations

In this article we describe the molecular characterization of 104 independent α-thalassemia patients identified by hematological analysis and family studies. During the study, another six chromosomes were identified with rearrangements of the α-cluster or point mutations in the α₂-globin gene, not associated with α-thalassemia, in healthy relatives of the patients. The molecular defects were established by Southern blot analysis and, if no deletions could be identified, the α-globin genes were investigated by denaturing gradient gel electrophoresis and single strand conformation analysis for the presence of point mutations. Following this strategy, we were able to identify the molecular basis of 131 independent α-thalassemia chromosomes. In two individuals, the α-thalassemia determinant could not be demonstrated at the molecular level. We identified eight different deletion and five non-deletion α-thalassemias, three rearrangements in the α-cluster, two α-chain variants, and a silent mutation in the α₂-globin gene not associated with α-thalassemia. The large heterogeneity of α-thalassemia mutations seen in the Dutch population might be typical for nothern European countries where, besides the more common mutations introduced by migration, a variety of sporadic mutations was also found in the autochthonous population. The screening strategy as described here, capable of identifying a wide spectrum of both deletions and point mutations, identified 98% of the α-thalassemia determinants present in 133 chromosomes. Received: 20 March 1997 / Accepted: 11 April 1997     

Herpes simplex virus type 1/adeno-associated virus hybrid vectors mediate site-specific integration at the adeno-associated virus preintegration site,AAVS1, on human chromosome 19

Herpes simplex virus type 1 (HSV-1)-based amplicon vectors have a large transgene capacity and can efficiently infect many different cell types. One disadvantage of HSV-1 vectors is their instability of transgene expression. By contrast, vectors based on adeno-associated virus (AAV) can either persist in an episomal form or integrate into the host cell genome, thereby supporting long-term gene expression. AAV expresses four rep genes, rep68, -78, -40, and -52. Of those, rep68 or rep78 are sufficient to mediate site-specific integration of the AAV DNA into the host cell genome. The major disadvantage of AAV vectors is the small transgene capacity ( approximately 4.6 kb). In this study, we constructed HSV/AAV hybrid vectors that contained, in addition to the standard HSV-1 amplicon elements, AAV rep68, rep78, both rep68 and -78, or all four rep genes and a reporter gene that was flanked by the AAV inverted terminal repeats (ITRs). Southern blots of Hirt DNA from cells transfected with the hybrid vectors and HSV-1 helper DNA demonstrated that both the AAV elements and the HSV-1 elements were functional in the context of the hybrid vector. All hybrid vectors could be packaged into HSV-1 virions, although those containing rep sequences had lower titers than vectors that did not. Site-specific integration at AAVS1 on human chromosome 19 was directly demonstrated by PCR and sequence analysis of ITR-AAVS1 junctions in hybrid vector-transduced 293 cells. Cell clones that stably expressed the transgene for at least 12 months could easily be isolated without chemical selection. In the majority of these clones, the transgene cassette was integrated at AAVS1, and no sequences outside the ITR cassette, rep in particular, were present as determined by PCR, ITR rescue/replication assays, and Southern analysis. Some of the clones contained random integrations of the transgene cassette alone or together with sequences outside the ITR cassette. These data indicate that the long-term transgene expression observed following transduction with HSV/AAV hybrid vectors is, at least in part, supported by chromosomal integration of the transgene cassette, both randomly and site specifically.     

Resting-State Neuronal Oscillatory Correlates of Working Memory Performance

Purpose

Working memory (WM) represents the brain’s ability to maintain information in a readily available state for short periods of time. This study examines the resting-state cortical activity patterns that are most associated with performance on a difficult working-memory task.

Methods

Magnetoencephalographic (MEG) band-passed (delta/theta (1–7 Hz), alpha (8–13 Hz), beta (14–30 Hz)) and sensor based regional power was collected in a population of adult men (18–28 yrs, n = 24) in both an eyes-closed and eyes-open resting state. The normalized power within each resting state condition as well as the normalized change in power between eyes closed and open (zECO) were correlated with performance on a WM task. The regional and band-limited measures that were most associated with performance were then combined using singular value decomposition (SVD) to determine the degree to which zECO power was associated with performance on the three-back verbal WM task.

Results

Changes in power from eyes closed to open revealed a significant decrease in power in all band-widths that was most pronounced in the posterior brain regions (delta/theta band). zECO right posterior frontal and parietal cortex delta/theta power were found to be inversely correlated with three-back working memory performance. The SVD evaluation of the most correlated zECO metrics then provided a singular measure that was highly correlated with three-back performance (r = −0.73, p<0.0001).

Conclusion

Our results indicate that there is an association between WM performance and changes in resting-state power (right posterior frontal and parietal delta/theta power). Moreover, an SVD of the most associated zECO measures produces a composite resting-state metric of regional neural oscillatory power that has an improved association with WM performance. To our knowledge, this is the first investigation that has found that changes in resting state electromagnetic neural patterns are highly associated with verbal working memory performance.