Prevention,management and extent of adverse pregnancy outcomes in women with hereditary antithrombin deficiency

Antithrombin (AT) deficiency is a rare hereditary thrombophilia with a mean prevalence of 0.02 % in the general population, associated with a more than ten-fold increased risk of venous thromboembolism (VTE). Within this multicenter retrospective clinical analysis, female patients with inherited AT deficiency were evaluated concerning the type of inheritance and extent of AT deficiency, medical treatment during pregnancy and postpartally, VTE risk as well as maternal and neonatal outcome. Statistical analysis was performed with SPPS for Windows (19.0). A total of 18 pregnancies in 7 patients were evaluated, including 11 healthy newborns ≥37th gestational weeks (gw), one small for gestational age premature infant (25th gw), two late-pregnancy losses (21st and 28th gw) and four early miscarriages. Despite low molecular weight heparin (LMWH) administration, three VTE occurred during pregnancy and one postpartally. Several adverse pregnancy outcomes occurred including fetal and neonatal death, as well as severe maternal neurologic disorders occurred. Patients with substitution of AT during pregnancy in addition to LMWH showed the best maternal and neonatal outcome. Close monitoring with appropriate anticoagulant treatment including surveillance of AT levels might help to optimize maternal and fetal outcome in patients with hereditary AT deficiency.     

CD74 indicates microglial activation in experimental diabetic retinopathy and exogenous methylglyoxal mimics the response in normoglycemic retina

Diabetes induces vasoregression, neurodegeneration and glial activation in the retina. Formation of advanced glycation endoproducts (AGEs) is increased in diabetes and contributes to the pathogenesis of diabetic retinopathy. CD74 is increased in activated microglia in a rat model developing both neurodegeneration and vasoregression. In this study, we aimed at investigating whether glucose and major AGE precursor methylglyoxal induce increased CD74 expression in the retina. Expression of CD74 in retinal microglia was analyzed in streptozotocin-diabetic rats by wholemount immunofluorescence. Nondiabetic mice were intravitreally injected with methylglyoxal. Expression of CD74 was studied by retinal wholemount immunofluorescence and quantitative real-time PCR, 48 h after the injection. CD74-positive cells were increased in diabetic 4-month retinas. These cells represented a subpopulation of CD11b-labeled activated microglia and were mainly located in the superficial vascular layer (13.7-fold increase compared to nondiabetic group). Methylglyoxal induced an 9.4-fold increase of CD74-positive cells in the superficial vascular layer and elevated gene expression of CD74 in the mouse retina 2.8-fold. In summary, we identified CD74 as a microglial activation marker in the diabetic retina. Exogenous methylglyoxal mimics the response in normoglycemic retina. This suggests that methylglyoxal is important in mediating microglial activation in the diabetic retina.     

Synthesis and thermal stability of ZrO2@SiO2 core–shell submicron particles

ZrO₂@SiO₂ core–shell submicron particles are promising candidates for the development of advanced optical materials. Here, submicron zirconia particles were synthesized using a modified sol–gel method and pre-calcined at 400 °C. Silica shells were grown on these particles (average size: ∼270 nm) with well-defined thicknesses (26 to 61 nm) using a seeded-growth Stöber approach. To study the thermal stability of bare ZrO₂ cores and ZrO₂@SiO₂ core–shell particles they were calcined at 450 to 1200 °C. After heat treatments, the particles were characterized by SEM, TEM, STEM, cross-sectional EDX mapping, and XRD. The non-encapsulated, bare ZrO₂ particles predominantly transitioned to the tetragonal phase after pre-calcination at 400 °C. Increasing the temperature to 600 °C transformed them to monoclinic. Finally, grain coarsening destroyed the spheroidal particle shape after heating to 800 °C. In striking contrast, SiO₂-encapsulation significantly inhibited grain growth and the t → m transition progressed considerably only after heating to 1000 °C, whereupon the particle shape, with a smooth silica shell, remained stable. Particle disintegration was observed after heating to 1200 °C. Thus, ZrO₂@SiO₂ core–shell particles are suited for high-temperature applications up to ∼1000 °C. Different mechanisms are considered to explain the markedly enhanced stability of ZrO₂@SiO₂ core–shell particles.

Silica encapsulation dramatically enhances the thermal stability of zirconia submicron particles by grain growth inhibition and tetragonal phase stabilization.     

Bariatric surgery in severely obese adolescents improves major comorbidities including hyperuricemia

Objective

Serum uric acid (sUA) is believed to contribute to the pathogenesis of metabolic comorbidities like hypertension, insulin-resistance (IR) and endothelial dysfunction (EDF) in obese children. The present pilot study investigated the association between sUA concentrations and loss of body weight following laparoscopic sleeve gastrectomy (LSG) or laparoscopic Roux-en-Y-gastric bypass (RYGB) in severely obese adolescents.

Materials/Methods

10 severely obese adolescents underwent either LSG (n = 5) or RYGB (n = 5). 17 normal weight, healthy, age- and gender-matched adolescents served as a normal weight peer group (NWPG). Pre- and 12 months postoperatively, sUA and relevant metabolic parameters (glucose homeostasis, transaminases, lipids) were compared.

Results

Preoperatively, sUA was significantly elevated in patients with severe obesity compared to NWPG. Twelve months after LSG and RYGB, a significant decrease in sUA, BMI, CVD risk factors, hepatic transaminases, and HOMA-IR was observed. Reduction in SDS-BMI significantly correlated with changes in sUA.

Conclusions

sUA levels and metabolic comorbidities improved following bariatric surgery in severely obese adolescents. The impact of changes in sUA on long-term clinical complications of childhood obesity deserves further study.     

Crystal structure of the proteasomal deubiquitylation module Rpn8-Rpn11

The ATP-dependent degradation of polyubiquitylated proteins by the 26S proteasome is essential for the maintenance of proteome stability and the regulation of a plethora of cellular processes. Degradation of substrates is preceded by the removal of polyubiquitin moieties through the isopeptidase activity of the subunit Rpn11. Here we describe three crystal structures of the heterodimer of the Mpr1–Pad1–N-terminal domains of Rpn8 and Rpn11, crystallized as a fusion protein in complex with a nanobody. This fusion protein exhibits modest deubiquitylation activity toward a model substrate. Full activation requires incorporation of Rpn11 into the 26S proteasome and is dependent on ATP hydrolysis, suggesting that substrate processing and polyubiquitin removal are coupled. Based on our structures, we propose that premature activation is prevented by the combined effects of low intrinsic ubiquitin affinity, an insertion segment acting as a physical barrier across the substrate access channel, and a conformationally unstable catalytic loop in Rpn11. The docking of the structure into the proteasome EM density revealed contacts of Rpn11 with ATPase subunits, which likely stabilize the active conformation and boost the affinity for the proximal ubiquitin moiety. The narrow space around the Rpn11 active site at the entrance to the ATPase ring pore is likely to prevent erroneous deubiquitylation of folded proteins.In eukaryotes, the ubiquitin (Ub) proteasome system (UPS) is responsible for the regulated degradation of proteins (1–5). The UPS plays a key role in the maintenance of protein homeostasis by removing misfolded or damaged proteins, which could impair cellular functions, and by removing proteins whose functions are no longer needed. Consequently, the UPS is critically involved in numerous cellular processes, including cell cycle progression, apoptosis, and DNA damage repair, and malfunctions of the system often result in disease.The 26S proteasome executes the degradation of substrates that are marked for destruction by the covalent attachment of polyubiquitin chains. It is a molecular machine of 2.5 MDa comprising two subcomplexes, the 20S core particle (CP) and one or two 19S regulatory particles (RPs), which associate with the ends of the cylinder-shaped CP (6–8). The recognition and recruitment of polyubiquitylated substrates, their deubiquitylation, ATP-dependent unfolding, and translocation into the core particle take place in the RP. The structurally and mechanistically well-characterized CP houses the proteolytic activities and sequesters them from the environment, thereby avoiding collateral damage (9).The RPs attach to the outer α-rings of the CP, which control access to the proteolytic chamber formed by the inner β-subunit rings (10). Recently, the molecular architecture of the 26S holocomplex was established using cryo-EM–based approaches (11, 12), and a pseudoatomic model of the holocomplex was put forward (13). The RP is formed by two subcomplexes, known as the base and the lid, which assemble independently (12, 14). The base contains the hetero-hexameric AAA-ATPase ring (Rpt1–Rpt6), which drives the conformational changes required for substrate processing, including unfolding and translocation into the CP (15, 16). The base also contains the largest RP non-ATPase subunits, Rpn1 and Rpn2, and the Ub receptor Rpn13. The second resident Ub receptor, Rpn10, is not part of either the base or the lid; it binds only to the assembled 26S proteasome and is positioned close to the ATPase module.The lid scaffold is composed of the Rpn3, Rpn5, Rpn6, Rpn7, Rpn8, Rpn9, Rpn11, and Rpn12 subunits (14). These subunits can be grouped according to their domain structures. Rpn3, Rpn5, Rpn6, Rpn7, Rpn9, and Rpn12 each comprise an N-terminal helix repeat segment, a proteasome-COP9/signalosome-eIF3 (PCI) module, and a long helix at the C terminus (8). The Rpn8 and Rpn11 subunits each consist of an Mpr1–Pad1–N-terminal (MPN) domain, followed by long C-terminal helices (Fig. 1A). The PCI subunits form a horseshoe-shaped structure and the MPN domains form a heterodimer, which are connected by a large helical bundle, to which all subunits contribute (13, 17, 18). Each of these eight subunits has paralogs in the COP9/signalosome (CSN) and the elongation initiation factor 3 (eIF3), which likely adopt a similar architecture (18–21).Open in a separate windowFig. 1.Biochemical activity of the Rpn8-Rpn11 fusion protein. (A) Domain structures of Rpn8, Rpn11 and the fusion protein. (B) Ub₄ cleavage activity of 26S proteasome, WT Rpn8-Rpn11 and Rpn8-Rpn11 (E48Q). Cleavage of labeled peptide from Ub₄ was detected by the change in fluorescence polarization after 1hr incubation at 37 °C at the indicated concentrations. Values are normalized to maximum cleavage activity of 26S proteasome. The used 26S proteasome preparation contained only trace amounts of the DUB Ubp6.The lid strengthens the interaction between the CP and RP (17) and deubiquitylates substrates before their processing by the AAA-ATPase module and the CP. Cleavage of polyubiquitin chains from the substrate enables recycling of Ub into the cellular pool, and the removal of the unfolding-resistant Ub moieties promotes translocation of substrates. The MPN domain of Rpn11 contains the catalytic site for deubiquitylation (22, 23). Rpn11 belongs to the JAB1/MPN/Mov34 metalloenzyme (JAMM) family of metalloproteases, which provide the isopeptidase activities in the proteasome, CSN, and exo-deubiquitylating enzymes (DUBs), such as associated molecule with the SH3 domain of STAM-like protein (AMSH-LP). The signature motif for this family is a conserved glutamate upstream of a zinc-coordinating catalytic loop, H(S/T)HX₇SXXD, first revealed in the structure of an archaeal homolog, AfJAMM (24). The substrate-binding mode of JAMM DUBs was clarified by the crystal structure of AMSH-LP in complex with Lys63-linked diubiquitin (25). The other proteasomal MPN subunit, Rpn8, is catalytically inactive; it does not contain the JAMM motif and appears to have mainly a supporting role for Rpn11. Isolated Rpn11 is catalytically inactive, as is the isolated lid (22). Rpn11 is activated upon integration into the 26S holocomplex and is dependent on ATP hydrolysis (23). The 26S proteasome was recently shown to undergo large-scale conformational changes from a substrate-accepting conformation to a substrate-engaged conformation that may be critical for Rpn11 function (15, 26), but the mechanistic basis for the regulation of Rpn11 remains unclear. Loss-of-function mutants of the JAMM motif cause stalling of substrates above the mouth of the ATPase module and lead to clogging of the 26S proteasome (23, 26).Inhibitors of human Rpn11 (hRpn11, also known as POH1) have been proposed as potential antitumor agents working upstream of the β5 proteolytic subunits in the UPS. The β5 subunits have been clinically validated by the approval of bortezomib and carilfzomib for the treatment of hematologic malignancies. siRNA and mutagenesis studies show that expression of the zinc catalytic domain of hRpn11 is essential for cell survival (27). Inhibition of hRpn11 in combination with EGFR inhibition has been suggested to be beneficial in the treatment of nonsmall cell lung cancer (28). Overexpression of hRpn11 in cancer cells has been linked to their tumor escape from cytotoxic agents (29). Thus, hRpn11 is an attractive target for pharmacologic intervention of the UPS.Here we present three crystal structures of the catalytically active Rpn8/Rpn11 MPN heterodimer from Saccharomyces cerevisiae, revealing the details of the Rpn11 active site and the mode of interaction with other subunits. Not all structures show proper active site geometry, hinting at possible mechanisms preventing activation outside of the proteasome complex. The access path for the C-terminal peptide of the substrate-bound Ub is blocked by a highly conserved insertion specific to Rpn11. Fitting of the Rpn8-Rpn11 crystal structure into the cryo-EM density of both the substrate-accepting and substrate-engaged proteasome revealed how the subcomplex is situated between base and PCI domain subunits, which involves long insertions unique to Rpn11 and Rpn8. Contacts to the coiled coils and the oligosaccharide-binding fold (OB) domain ring of the AAA subunits appear to control active site geometry and proper access of the isopeptide bond segment. In the substrate-engaged proteasome, the catalytic center becomes situated just above the maw of the ATPase ring.