Mechanisms of HsSAS-6 assembly promoting centriole formation in human cells

SAS-6 proteins are thought to impart the ninefold symmetry of centrioles, but the mechanisms by which their assembly occurs within cells remain elusive. In this paper, we provide evidence that the N-terminal, coiled-coil, and C-terminal domains of HsSAS-6 are each required for procentriole formation in human cells. Moreover, the coiled coil is necessary and sufficient to mediate HsSAS-6 centrosomal targeting. High-resolution imaging reveals that GFP-tagged HsSAS-6 variants localize in a torus around the base of the parental centriole before S phase, perhaps indicative of an initial loading platform. Moreover, fluorescence recovery after photobleaching analysis demonstrates that HsSAS-6 is immobilized progressively at centrosomes during cell cycle progression. Using fluorescence correlation spectroscopy and three-dimensional stochastic optical reconstruction microscopy, we uncover that HsSAS-6 is present in the cytoplasm primarily as a homodimer and that its oligomerization into a ninefold symmetrical ring occurs at centrioles. Together, our findings lead us to propose a mechanism whereby HsSAS-6 homodimers are targeted to centrosomes where the local environment and high concentration of HsSAS-6 promote oligomerization, thus initiating procentriole formation.     

Short-term load bearing capacity of osteochondral autografts implanted by the mosaicplasty technique: an in vitro porcine model

Articular surface congruency and graft stability are considered essential factors in the success of osteochondral grafting; however, quantitative measures of short-term load bearing capacity of grafts implanted by the mosaicplasty technique have not been reported. The purpose of this study was to develop a live tissue in vitro model to examine short-term fixation strength of mosaicplasty autografts immediately after and 1 week following graft implantation. Cylindrical osteochondral autografts were implanted in vitro by the mosaicplasty technique on five pairs of porcine femoral condyles within one and a half hours of animal sacrifice. Immediately following the surgical procedure, graft push-in and pull-out strength tests as well as indentation tests to determine modulus of the surrounding cancellous bone were performed on half of the specimens from the distal femurs of each animal. The remaining specimens, matched for location in the contralateral leg, were incubated in culture medium for 7 days prior to performing the same set of mechanical tests. Averaged push-in and pull-out graft fixation strength decreased 44% from 135.7 to 75.5N over the 7-day period, while no change in modulus was detected in the surrounding cancellous bone. These in vitro results demonstrate a substantial deterioration of short-term fixation strength of mosaicplasty grafts from the immediate post-operative state. Such a reduction in short-term graft load bearing capacity may pose a threat to the surgically established articular surface congruency and blood vessels formed during the early stages of the healing response.     

Modification of different IgG1 antibodies via glutamine and lysine using bacterial and human tissue transglutaminase

The modification of proteins by chemical methods is well-established, however usually difficult to control. In this paper, we describe the posttranslational modification of different IgGs via the Lys or Gln side chains catalyzed by bacterial and human tissue transglutaminase (BTGase and TG2). For proof of concept, different IgG1s (commercial bovine IgG1, and L1CAM targeting chCE7 and chCE7 aglycosylated) were enzymatically functionalization with different fluorescent TGase substrates based on the CY3 analogue Dy547. The optimal reaction conditions were determined in order to assess the two enzymes. The efficiency of the enzymatic method was also compared with a standard chemical method employing a reactive NHS ester of Dy547. Three new TGase substrates were synthesized for this study including Lys-substrate 1 useful for BTGase and TG2 and two Gln-substrates tailor-made for BTGase (substrate 2) and TG2 (substrate 3). Of the two TGases tested, BTGase incorporated Lys-substrate 1 more efficiently than TG2. On the other hand, both enzymes reacted equally efficiently with the corresponding Gln-substrates 2 and 3. Reproducible labeling could be achieved in a broad concentration "window" of the substrates (up to 400 microM) without the risk of overlabeling of chCE7 or chCE7 aglycosylated. The biological activities of the functionalized antibodies were unaltered as shown by in vitro antigen affinity measurements and cell internalization experiments using confocal laser scanning microscopy. A maximum label-to-protein ratio of approximately 1 was achieved with chCE7 aglycosylated and substrate 1 using BTGase. It is important to recognize that the enzymatic activity of TGases enables the stable functionalization of proteins via the side chains of Gln, which is not possible by any chemical method available today. In addition, we could prove that the enzymatic modification of all antibodies occurred selectively at the heavy chain whereas the chemical method led to labeling of both the heavy and the light chains.     

Temporal genetic population structure and interannual variation in migration behavior of Pacific Lamprey <Emphasis Type="Italic">Entosphenus tridentatus</Emphasis>

In this contribution, we review our knowledge on bet-hedging strategies associated with rotifer diapause. First, we describe the ecological scenario under which bet hedging is likely to have evolved in three diapause-related traits in monogonont rotifer populations: (1) the timing of sex (because diapausing eggs are produced via sexual reproduction), (2) the sexual reproduction ratio (i.e. the fraction of sexually reproducing females) and (3) the timing of diapausing egg hatching. Then, we describe how to discriminate among bet-hedging modes and discuss which modes and mechanisms better fit the variability observed in these traits in rotifers. Finally, we evaluate the strength of the empirical evidence for bet hedging in the scarce studies available, and we call for the need of research at different levels of biological complexity to fully understand bet hedging in rotifer diapause.     

Determination of retinol and retinyl esters in human plasma by high-performance liquid chromatography with automated column switching and ultraviolet detection

A HPLC method with automated column switching and UV detection is described for the simultaneous determination of retinol and major retinyl esters (retinyl palmitate, retinyl stearate, retinyl oleate and retinyl linoleate) in human plasma. Plasma (0.2 ml) was deproteinized by adding ethanol (1.5 ml) containing the internal standard retinyl propionate. Following centrifugation the supernatant was directly injected onto the pre-column packed with LiChrospher 100 RP-18 using 1.2% ammonium acetate–acetic acid–ethanol (80:1:20, v/v) as mobile phase. The elution strength of the ethanol containing sample solution was reduced by on-line supply of 1% ammonium acetate–acetic acid–ethanol (100:2:4, v/v). The retained retinol and retinyl esters were then transferred to the analytical column (Superspher 100 RP-18, endcapped) in the backflush mode and chromatographed under isocratic conditions using acetonitrile–methanol–ethanol–2-propanol (1:1:1:1, v/v) as mobile phase. Compounds of interest were detected at 325 nm. The method was linear in the range 2.5–2000 ng/ml with a limit of quantification for retinol and retinyl esters of 2.5 ng/ml. Mean recoveries from plasma were 93.4–96.5% for retinol (range 100–1000 ng/ml) and 92.7–96.0% for retinyl palmitate (range 5–1000 ng/ml). Inter-assay precision was ≤5.1% and ≤6.3% for retinol and retinyl palmitate, respectively. The method was successfully applied to more than 2000 human plasma samples from clinical studies. Endogenous levels of retinol and retinyl esters determined in female volunteers were in good accordance with published data.     

Biparatopic sybodies neutralize SARS‐CoV‐2 variants of concern and mitigate drug resistance

The ongoing COVID‐19 pandemic represents an unprecedented global health crisis. Here, we report the identification of a synthetic nanobody (sybody) pair, Sb#15 and Sb#68, that can bind simultaneously to the SARS‐CoV‐2 spike RBD and efficiently neutralize pseudotyped and live viruses by interfering with ACE2 interaction. Cryo‐EM confirms that Sb#15 and Sb#68 engage two spatially discrete epitopes, influencing rational design of bispecific and tri‐bispecific fusion constructs that exhibit up to 100‐ and 1,000‐fold increase in neutralization potency, respectively. Cryo‐EM of the sybody‐spike complex additionally reveals a novel up‐out RBD conformation. While resistant viruses emerge rapidly in the presence of single binders, no escape variants are observed in the presence of the bispecific sybody. The multivalent bispecific constructs further increase the neutralization potency against globally circulating SARS‐CoV‐2 variants of concern. Our study illustrates the power of multivalency and biparatopic nanobody fusions for the potential development of therapeutic strategies that mitigate the emergence of new SARS‐CoV‐2 escape mutants.