Dgp71WD is required for the assembly of the acentrosomal meiosis I spindle, and is not a general targeting factor for the γ-TuRC

Dgp71WD/Nedd1 proteins are essential for mitotic spindle formation. In human cells, Nedd1 targets γ-tubulin to both centrosomes and spindles, but in other organisms the function of Dgp71WD/Nedd1 is less clear. In Drosophila cells, Dgp71WD plays a major part in targeting γ-tubulin to spindles, but not centrosomes, while in Xenopus egg extracts, Nedd1 acts as a more general microtubule (MT) organiser that can function independently of γ-tubulin. The interpretation of these studies, however, is complicated by the fact that some residual Dgp71WD/Nedd1 is likely present in the cells/extracts analysed. Here we generate a Dgp71WD null mutant lacking all but the last 12 nucleotides of coding sequence. The complete loss of Dgp71WD has no quantifiable effect on γ-tubulin or Centrosomin recruitment to the centrosome in larval brain cells. The recruitment of γ-tubulin to spindle MTs, however, is severely impaired, and spindle MT density is reduced in a manner that is indistinguishable from cells lacking Augmin or γ-TuRC function. In contrast, the absence of Dgp71WD leads to defects in the assembly of the acentrosomal female Meiosis I spindle that are more severe than those seen in Augmin or γ-TuRC mutants, indicating that Dgp71WD has additional functions that are independent of these complexes in oocytes. Moreover, the localisation of bicoid RNA during oogenesis, which requires γ-TuRC function, is unperturbed in Dgp71WD(120) mutants. Thus, Dgp71WD is not simply a general cofactor required for γ-TuRC and/or Augmin targeting, and it appears to have a crucial role independent of these complexes in the acentrosomal Meiosis I spindle

Dual roles of Incenp crucial to the assembly of the acentrosomal metaphase spindle in female meiosis

Spindle formation in female meiosis differs from mitosis in many animals, as it takes place independently from centrosomes, and the molecular requirements of this pathway remain to be understood. Here we report two crucial roles of Incenp, an essential subunit of the chromosomal passenger complex (the Aurora B complex), in centrosome-independent spindle formation in Drosophila female meiosis. Firstly, the initial assembly of spindle microtubules is drastically delayed in an incenp mutant. This clearly demonstrates, for the first time, a crucial role for Incenp in chromosome-driven spindle microtubule assembly in living oocytes. Additionally, Incenp is necessary to stabilise the equatorial region of the metaphase I spindle, in contrast to mitosis, where the equivalent function becomes prominent after anaphase onset. Our analysis suggests that Subito, a kinesin-6 protein, cooperates with Incenp for this latter function, but not microtubule assembly. We propose that the two functions of Incenp are part of the mechanisms that compensate for the lack of centrosomes during meiotic spindle formation

New insights in formaldehyde-induced detoxification of the tetanus toxin: Chemical modification stoichiometry and characterization of intra- and inter-molecular cross-links

The active ingredient of the tetanus vaccine, known as tetanus toxoid (TTD, 150 kDa), is prepared by inactivation of the tetanus toxin with formaldehyde. This chemical treatment determines the efficiency and innocuity of the vaccine. The detoxification chemistry has been qualitatively studied regarding the nature and positioning of some formaldehyde-induced modifications. However, neither the stoichiometry of the chemical modifications or intra- and inter-chain cross-linking have been yet characterized, despite being of great interest for the definitive elucidation of the detoxification mechanism as well as for future manufacturing follow-up. The aim of the present study is to assess the impact of formaldehyde on TTD by identifying, mapping, and quantifying as exhaustively as possible, all formaldehyde-induced modifications over the entire three-dimensional (3D) structure of the protein. Please click Download on the upper right corner to see the full abstract

Hydroxychloroquine in a G6PD-Deficient Patient with COVID-19 Complicated by Haemolytic Anaemia: Culprit or Innocent Bystander?

Hydroxychloroquine has been used worldwide as a first-line treatment for patients hospitalized with COVID-19. Little is known about COVID-19 and its effects on patients with congenital red blood cell disorders. We report a case of haemolytic anaemia in a 32-year-old patient and a fortuitous highlighting of G6PD deficiency. We reviewed the literature to assess the risk of hydroxychloroquine use in this context

The role of frictional power dissipation (as a function of frequency) and test temperature on contact temperature and the subsequent wear behaviour in a stainless steel contact in fretting

Temperature is known to affect the fretting wear behaviour of metals; generally, a critical temperature is observed, above which there are substantial reductions in wear rate, with these being associated with the development of protective oxide beds in the fretting contact. This work has examined the gross sliding fretting behaviour of a stainless steel as a function of bulk temperature and fretting frequency(with changes in the fretting frequency altering the frictional power dissipated in the contact amongst other things). An analytical model has been developed which has suggested that at 200 Hz, an increase in the contact temperature of more than 70°C can be expected, associated with the high frictional power dissipation at this frequency (compared to that dissipated at a fretting frequency of 20 Hz). With the bulk temperature at either room temperature or 275°C, the increase in contact temperature does not result in a transition across the critical temperature (and thus fretting behaviour at these temperatures is relatively insensitive to fretting frequency). However, with a bulk temperature of 150°C, the increase in temperature associated with the increased frictional power dissipation at the higher frequency results in the critical temperature being exceeded, and in significant differences in fretting behaviour

The role of geometry changes and debris formation associated with wear on the temperature field in fretting contacts

The temperature of a fretting contact is known to be a key factor in its development. However, as a test proceeds, the wear scar changes, both geometrically and through the formation of oxide-based debris-beds. Accordingly, the effects of these on the near-surface temperature field resulting from frictional heating in fretting has been analysed via numerical modelling. Under the test conditions examined, it was predicted that (i) the development of the wear scar geometry would result in a significant (up to ~ 25%) reduction in the mean-surface temperature rise, and (ii) the formation of a typical oxide debris bed would result in a significant (up to ~ 80%) increase in the mean-surface temperature rise

The role of temperature and frequency on fretting wear of a like-on-like stainless steel contact

The influences of environmental temperature and fretting frequency on the mechanisms and rates of wear in a like-on-like 304 stainless steel contact were examined, and mainly attributed to changes in the mechanical response of the bulk material and to changes in the behaviour of the oxide debris formed in the fretting process. At low temperatures, wear proceeds by continual oxide formation and egress from the contact, whilst at high temperatures, the rate of wear is much reduced, associated with the development of oxide formed into a protective bed within the contact. The temperature at which the change between these two behaviours took place was dependent upon the fretting frequency, with evidence that, at this transition temperature, changes in behaviour can occur as the fretting test proceeds under a fixed set of conditions. An interaction diagram has been developed which provides a coherent framework by which the complex effects of these two parameters can be rationalised in terms of widely accepted physical principles

ALADIN is Required for the Production of Fertile Mouse Oocytes

Asymmetric cell divisions depend on the precise placement of the spindle apparatus. In mammalian oocytes, spindles assemble close to the cell's center, but chromosome segregation takes place at the cell periphery where half of the chromosomes are expelled into small, nondeveloping polar bodies at anaphase. By dividing so asymmetrically, most of the cytoplasmic content within the oocyte is preserved, which is critical for successful fertilization and early development. Recently we determined that the nucleoporin ALADIN participates in spindle assembly in somatic cells, and we have also shown that female mice homozygously null for ALADIN are sterile. In this study we show that this protein is involved in specific meiotic stages, including meiotic resumption, spindle assembly, and spindle positioning. In the absence of ALADIN, polar body extrusion is compromised due to problems in spindle orientation and anchoring at the first meiotic anaphase. ALADIN null oocytes that mature far enough to be fertilized in vitro are unable to support embryonic development beyond the two-cell stage. Overall, we find that ALADIN is critical for oocyte maturation and appears to be far more essential for this process than for somatic cell divisions