Placental Pathology Findings in Unexplained Pregnancy Losses.

There are approximately 5 million pregnancies per year in the USA, with 1 million ending in miscarriage (a loss occurring prior to 20 weeks of gestation) and over 20,000 ending in stillbirth at or beyond 20 weeks of gestation. As many as 50% of these losses are unexplained. Our objective was to evaluate the effect of expanding the placental pathology diagnostic categories to include the explicit categories of (1) dysmorphic chorionic villi and (2) small placenta in examining previously unexplained losses. Using a clinical database of 1256 previously unexplained losses at 6-43 weeks of gestation, the most prevalent abnormality associated with each loss was determined through examination of its placental pathology slides. Of 1256 cases analyzed from 922 patients, there were 878 (69.9%) miscarriages and 378 (30.1%) antepartum stillbirths. We determined the pathologic diagnoses for 1150/1256 (91.6%) of the entire series, 777/878 (88.5%) of the miscarriages (< 20 weeks' gestation), and 373/378 (98.7%) of the stillbirths (≥ 20 weeks' gestation). The most common pathologic feature observed in unexplained miscarriages was dysmorphic chorionic villi (757 cases; 86.2%), a marker associated with genetic abnormalities. The most common pathologic feature observed in unexplained stillbirths was a small placenta (128 cases; 33.9%). Our classification system reinforced the utility of placental examination for elucidating potential mechanisms behind pregnancy loss. The improved rate of diagnosis appeared to be the result of filling a gap in previous pregnancy loss classification systems via inclusion of the categories of dysmorphic chorionic villi and small placenta.

Maternal Outcomes in Subsequent Pregnancies After Classical Cesarean Delivery.

OBJECTIVE: To compare maternal outcomes in subsequent pregnancies of patients who had a prior classical cesarean delivery with those with a prior low transverse cesarean delivery. METHODS: We conducted a cross-sectional analysis of patients with live singleton births at or after 24 weeks of gestation who had a prior classical cesarean delivery or a low transverse cesarean delivery in the 2016-2019 National Inpatient Sample database. Outcome measures included mode of delivery, uterine rupture, and severe maternal morbidity (SMM), as defined by the Centers for Disease Control and Prevention. Maternal outcomes were compared using the χ2 test and the propensity score method, accounting for differences in patients' clinical risk factors. Multivariable regressions further assessed how patients' sociodemographic and hospital characteristics might influence the differences in maternal outcomes between the two groups. RESULTS: The sample included 1,671,249 patients: 25,540 with prior classical cesarean delivery and 1,645,709 with prior low transverse cesarean delivery. Cesarean delivery occurred in 95.5% of patients with prior classical cesarean compared with 91.3% of those with prior low transverse delivery (P<.001; propensity score method: odds ratio [OR] 0.99, 95% CI 0.85-1.16) and uterine rupture occurred in 1.1% and 0.3%, respectively (P<.001; propensity score method: OR 2.17, 95% CI 1.40-3.36). Among patients with prior classical cesarean delivery, uterine rupture occurred in 10.6% of those who underwent labor compared with 0.3% of those who did not (P<.001). Rates of SMM were 5.9% and 2.0% in the two groups, respectively (P<.001; propensity score method: OR 1.87, 95% CI 1.53-2.29). After adjustment of maternal sociodemographic and hospital characteristics, differences in the risk of uterine rupture and SMM between the two groups were attenuated but remained significant. CONCLUSION: Prior classical cesarean delivery was associated with a higher risk of uterine rupture and SMM in subsequent pregnancies, compared with prior low transverse cesarean delivery, even after accounting for patients' clinical, sociodemographic, and hospital characteristics.

A randomized, multi-center, prospective study comparing best medical treatment versus best medical treatment plus renal artery stenting in patients with hemodynamically relevant atherosclerotic renal artery stenosis (RADAR) - one-year results of a pre-maturely terminated study.

BACKGROUND: The indications for conservative "best medical treatment" (BMT) versus additional renal artery stenting are a matter of ongoing debate. The RADAR study aimed to evaluate the impact of percutaneous renal artery stenting on the impaired renal function in patients with hemodynamically significant atherosclerotic renal artery stenosis (RAS). METHODS: RADAR is an international, prospective, randomized (1:1) controlled study comparing BMT alone versus BMT plus renal artery stenting in patients with duplex sonographic hemodynamically relevant RAS. Follow-up assessments were at 2, 6, and 12 months and at 3 years. The primary endpoint was change in estimated glomerular filtration rate (eGFR) at 12 months. RESULTS: Due to slow enrollment, RADAR was terminated early after inclusion of 86 of the scheduled 300 patients (28.7%). Change in eGFR between baseline and 12 months was 4.3 ± 15.4 ml/min/1.73 m2 (stent group) and 3.0 ± 14.9 ml/min/1.73 m2 (BMT group), p > 0.999. Clinical event rates were low with a 12-month composite of cardiac death, stroke, myocardial infarction, and hospitalization for congestive heart failure of 2.9% in the stent and 5.3% in the BMT group, p = 0.526, and a 3-year composite of 14.8% and 12.0%, p = 0.982. At 3 years, target vessel (re-)vascularization occurred in one patient (3.0%) in the stent group and in 8 patients (29.4%) in the BMT group. CONCLUSION: In RADAR, outcomes of renal artery stenting were similar to BMT. These results have to be interpreted with the caveat that the study did not reach its statistically based sample size. TRIAL REGISTRATION: Clinicaltrials.gov, NCT00640406. Registered on 17 March 2008.

Credentialing in interventional therapy in Europe - comparison of curricula including endovascular therapy of arterial diseases.

The prevalence of peripheral artery disease (PAD) is continuously increasing. In addition, the treatment of PAD has changed substantially over the last decades. Novel treatment modalities and diagnostic tools were developed and endovascular therapy was established as a first-line therapy. In consequence, vascular medicine has become an interdisciplinary field, involving different specialties such as internal medicine/cardiology, internal medicine/angiology, vascular surgery, and (interventional) radiology. Attributed to the expanding field and to ensure high quality and standardized training, various curricula were developed. Not only within the different medical disciplines but across countries. We compared the training programmes in Germany, Switzerland, and Europe and found a great variety in these curricula and programmes. Nevertheless, we identified first attempts to harmonize training requirements within the various societies: the interdisciplinary guidelines of the German Society for Cardiology (DGK) and the German Society of Angiology, the adoption of European standards on a national level, and the Swiss initiative of the Union of Vascular Societies of Switzerland (UVSS). The standard and level of training is high in all curricula and societies. However, further harmonization is needed to optimize patient care and ensure a uniform quality level across different vascular societies.

Dynamic Behavior of Trigger Factor on the Ribosome.

Trigger factor (TF) is the only ribosome-associated chaperone in bacteria. It interacts with hydrophobic segments in nascent chain (NCs) as they emerge from the ribosome. TF binds via its N-terminal ribosome-binding domain (RBD) mainly to ribosomal protein uL23 at the tunnel exit on the large ribosomal subunit. Whereas earlier structural data suggested that TF binds as a rigid molecule to the ribosome, recent comparisons of structural data on substrate-bound, ribosome-bound, and TF in solution from different species suggest that this chaperone is a rather flexible molecule. Here, we present two cryo-electron microscopy structures of TF bound to ribosomes translating an mRNA coding for a known TF substrate from Escherichia coli of a different length. The structures reveal distinct degrees of flexibility for the different TF domains, a conformational rearrangement of the RBD upon ribosome binding, and an increase in rigidity within TF when the NC is extended. Molecular dynamics simulations agree with these data and offer a molecular basis for these observations.

alphaNAC depletion as an initiator of ER stress-induced apoptosis in hypoxia.

Accumulation of unfolded proteins triggers endoplasmic reticulum (ER) stress and is considered a part of the cellular responses to hypoxia. The nascent polypeptide-associated complex (NAC) participates in the proper maturation of newly synthesized proteins. However, thus far, there have been no comprehensive studies on NAC involvement in hypoxic stress. Here, we show that hypoxia activates glycogen synthase kinase-3beta (GSK-3beta) and that the activated GSK-3beta destabilizes alphaNAC with the subsequent apoptosis of the cell. Hypoxia of various cell types and the mouse ischemic brain was associated with rapid downregulation of alphaNAC and ER stress responses involving PERK, ATF4, gamma-taxilin, elF2alpha, Bip, and CHOP. Depletion of alphaNAC by RNA interference specifically activated ER stress responses and caused mitochondrial dysfunction, which resulted in apoptosis through caspase activation. Interestingly, we found that the hypoxic conditions activated GSK-3beta, and that GSK-3beta inhibition prevented alphaNAC protein downregulation in hypoxic cells and rescued the cells from apoptosis. In addition, alphaNAC overexpression increased the viability of hypoxic cells. Taken together, these results suggest that alphaNAC degradation triggers ER stress responses and initiates apoptotic processes in hypoxic cells, and that GSK-3beta may participate upstream in this mechanism.

The alpha and beta subunit of the nascent polypeptide-associated complex have distinct functions.

Nascent polypeptide-associated complex (NAC) is probably the first cytosolic protein to contact nascent polypeptide chains emerging from ribosomes. In this way NAC prevents inappropriate interactions with other factors. Eventually other factors involved in targeting and folding, like the Signal Recognition Particle or cytosolic chaperones, must gain access to the nascent chain. All NAC preparations to date consist of two copurifying polypeptides. Here we rigorously show that these two polypeptides, termed alpha- and betaNAC, form a very stable complex in vivo and in vitro and that a functional complex can be reconstituted from the individual subunits. A dissection of the contributions of the individual subunits to NACs function revealed that both subunits are in direct contact with nascent polypeptide chains on the ribosome and that both contribute to the prevention of inappropriate interactions. However, betaNAC alone directly binds to the ribosome and is sufficient to prevent ribosome binding to the endoplasmic reticulum membrane.

A general mechanism for regulation of access to the translocon: competition for a membrane attachment site on ribosomes.

For proteins to enter the secretory pathway, the membrane attachment site (M-site) on ribosomes must bind cotranslationally to the Sec61 complex present in the endoplasmic reticulum membrane. The signal recognition particle (SRP) and its receptor (SR) are required for targeting, and the nascent polypeptide associated complex (NAC) prevents inappropriate targeting of nonsecretory nascent chains. In the absence of NAC, any ribosome, regardless of the polypeptide being synthesized, binds to the endoplasmic reticulum membrane, and even nonsecretory proteins are translocated across the endoplasmic reticulum membrane. By occupying the M-site, NAC prevents all ribosome binding unless a signal peptide and SRP are present. The mechanism by which SRP overcomes the NAC block is unknown. We show that signal peptide-bound SRP occupies the M-site and therefore keeps it free of NAC. To expose the M-site and permit ribosome binding, SR can pull SRP away from the M-site without prior release of SRP from the signal peptide.

Unregulated exposure of the ribosomal M-site caused by NAC depletion results in delivery of non-secretory polypeptides to the Sec61 complex.

Nascent polypeptide associated complex (NAC) interacts with nascent polypeptides emerging from ribosomes. Both signal recognition particle (SRP) and NAC work together to ensure specificity in co-translational targeting by competing for binding to the ribosomal membrane attachment site. While SRP selects signal-containing ribosomes for targeting, NAC prevents targeting of signal peptide-less nascent chains to the endoplasmic reticulum membrane. Here we show that the ribosome binding that occurs in NAC's absence delivers signalless nascent chains to the Sec61 complex, underscoring the danger of unregulated exposure of the ribosomal M-site. Recently, the idea that NAC prevents ribosome binding has been challenged. By carefully examining the physiologic NAC concentration in a variety of tissues from different species we here demonstrate that the discrepancy resulted from subphysiologic NAC concentrations.

Coordination of a histidine residue of the protein-component S to the cobalt atom in coenzyme B12-dependent glutamate mutase from Clostridium cochlearium.

Electron paramagnetic resonance (EPR) spectroscopy of glutamate mutase from Clostridium cochlearium was performed in order to test the idea, that a histidine residue of component S replaces the dimethylbenzimidazole ligand of the Co-atom during binding of coenzyme B12 to the enzyme. The shapes and the superhyperfine splitting of the gz-lines of the Co(II) EPR spectra were used as indicators of the interaction of the axial base nitrogen with the Co-atom. A mixture of completely 15N-labelled component S, unlabelled component E, coenzyme B12 and glutamate gave slightly sharper gz-lines than that with unlabelled component S. A more dramatic change was observed in the Co(II) spectrum of the inactivated enzyme containing tightly bound cob(II)alamin, in which unlabelled component S caused a threefold superhyperfine-splitting of the gz-line, whereas the 15N-labelled protein only caused a twofold splitting, as expected for a direct interaction of a nitrogen of the enzyme with the Co-atom. By using a sample of 15N-labelled component S, in which only the histidines were 14N-labelled, the EPR spectra showed no difference to those with unlabelled component S. The experiments indeed demonstrate a replacement of the dimethylbenzimidazole ligand in coenzyme B12 by a histidine when bound to glutamate mutase. The most likely candidate is H16, which is conserved among the carbon skeleton rearranging mutases and methionine synthase.

Characterization of the coenzyme-B12-dependent glutamate mutase from Clostridium cochlearium produced in Escherichia coli.

The glutamate mutase dependent on adenosylcobalamin (coenzyme B12) catalyzes the carbon skeleton rearrangement of (S)-glutamate to (2S,3S)-3-methylaspartate, the first step of the glutamate fermentation pathway of the anaerobic bacterium Clostridium cochlearium. The enzyme consists of two protein components, E, a dimer epsilon 2 (epsilon, 53.5 kDa) and S, a monomer (sigma, 14.8 kDa). The corresponding genes (glmE and glmS) were cloned, sequenced and over-expressed in Escherichia coli. The genes glmS and glmE are separated by glmL encoding a protein of unknown function. The deduced amino acid sequence of GlmL contains an ATP-binding motif which is common to chaperones of the HSP70-type, actin and procaryotic cell-cycle proteins. Both components of glutamate mutase were purified with excellent yields from cell-free extracts of E. coli carrying the corresponding genes. In contrast to component E, component S was shown to bind coenzyme B12. This observation strongly supports the idea that significant similarities of the amino acid sequences of component S and several other cobamide-dependent enzymes represent a common binding motif. Incubation of pure components E and S with coenzyme B12 resulted in the formation of a fully active glutamate mutase heterotetramer (epsilon 2 sigma 2) containing one molecule of coenzyme B12. EPR spectra of recombinant glutamate mutase, now available in sufficiency large amounts, were recorded after incubation of the enzyme with coenzyme B12 and (S)-glutamate. The EPR signals (gx,y approximately 2.1, gz = 1.985) were of much better resolution than observed earlier with the clostridial enzyme. Their typical hyperfine splitting is clearly derived from Co(II), which is involved in the formation of the paramagnetic species but is different from cob(II)alamin (gx,y = 2.25). The spin concentration was 34-50% of the concentration of the enzyme (epsilon 2 sigma 2) coenzyme complex. The competitive inhibitors (2S, 4S)-4-fluoroglutamate and 2-methyleneglutarate induced similar but not identical signals with spin concentrations of 134-148% of the enzyme concentration. Even (S)-[2,3,3,4,4-2H5]glutamate induced a signal significantly different to that of (S)-glutamate with an intensity of only 7%. These data suggest an involvement of the Co(II)-containing paramagnetic species in catalysis, the concentration of which reflects a steady state between its formation and decomposition. The large difference in the spin concentrations observed with (S)-glutamate as compared to the predeuterated glutamate is probably due to a kinetic isotope effect and indicates a cleavage of a C-H bond during formation of the paramagnetic species.(ABSTRACT TRUNCATED AT 400 WORDS)

Cloning, sequencing and expression in Escherichia coli of the gene encoding component S of the coenzyme B12-dependent glutamate mutase from Clostridium cochlearium.

Adenosylcobalamin (coenzyme B12) dependent glutamate mutase catalyzes the carbon skeleton rearrangement of (S)-glutamate to (2S,3S)-methylaspartate. This is the first step of the fermentation of glutamate by the strict anaerobic bacterium Clostridium cochlearium. The enzyme consists of the two protein components E and S. The gene encoding component S (glmS) was cloned in Escherichia coli and its nucleotide sequence was determined. The nucleotide sequence and the deduced amino acid sequence showed very strong identities to the sequence of the glmS (also called mutS) gene (80%) and to component S (82%) from the related C. tetanomorphum, respectively. Cell-free extracts of E. coli carrying the glmS gene showed glutamate mutase activity which was strictly dependent on the addition of coenzyme B12 and component E purified from C. cochlearium. Enzyme activity of the recombinant protein was achieved up to 2200 nkat/g wet cells which is due to a ten-fold overexpression compared with the activities determined in cell-free extracts of C. cochlearium. This is the first report of overexpression of an active component of glutamate mutase. A rapid purification procedure consisting only of ammonium sulfate precipitation and a gel filtration step was developed to obtain large amounts of pure component S in a short time.

Cloning, sequencing and expression of the gene encoding the coenzyme B12-dependent 2-methyleneglutarate mutase from Clostridium barkeri in Escherichia coli.

The coenzyme B12 (adenosylcobalamin)-dependent 2-methyleneglutarate mutase catalyses the carbon skeleton rearrangement of 2-methyleneglutarate to (R)-3-methylitaconate in the fermentation of nicotinic acid by the strict anaerobic bacterium Clostridium barkeri. (a) The mgm gene encoding 2-methyleneglutarate mutase was cloned and its nucleotide sequence was determined. The deduced amino acid sequence revealed a 66.8-kDa protein of 614 amino acids. It shows significant similarity in its C-terminal part to that of other cobamide-dependent enzymes. Probably, this is the coenzyme-binding region. (b) The mgm gene from C. barkeri was expressed in Escherichia coli as was shown by SDS/PAGE and Western-blot analysis with rabbit antiserum directed against the native mutase. (c) Cell-free extracts from E. coli carrying the mgm gene showed 2-methyleneglutarate mutase activity that was strictly dependent on the addition of coenzyme B12. Experiments are presented which suggest that the expression product is an apoenzyme.

The biotin-dependent sodium ion pump glutaconyl-CoA decarboxylase from Fusobacterium nucleatum (subsp. nucleatum). Comparison with the glutaconyl-CoA decarboxylases from gram-positive bacteria.

Membrane preparations of Fusobacterium nucleatum grown on glutamate contain glutaconyl-CoA decarboxylase at a high specific activity (13.8 nkat/mg protein). The enzyme was solubilized with 2% Triton X-100 in 0.5 M NaCl and purified 63-fold to a specific activity of 870 nkat/mg by affinity chromatography on monomeric avidin-Sepharose. The activity of the decarboxylase was strictly dependent on Na+ (Km = 3 mM) and was stimulated up to 3-fold by phospholipids. The glutaconyl-CoA decarboxylases from the gram-positive bacteria Acidaminococcus fermentans and Clostridium symbiosum have a lower apparent Km for Na+ (1 mM) and were not stimulated by phospholipids. In addition only the fusobacterial decarboxylase required sodium ion for stability and was inactivated by potassium ion. By incorporation of this purified enzyme into phospholipids an electrogenic sodium ion pump was reconstituted. The enzyme consists of four subunits, alpha (m = 65 kDa), beta (33 kDa), gamma (19 kDa), and delta (16 kDa) with the functions of a carboxy transferase (alpha), a carboxy lyase (beta and probably delta) and a biotin carrier (gamma). The subunits are very similar to those of the glutaconyl-CoA decarboxylases from the gram-positive bacteria. With an antiserum directed against the decarboxylase from A. fermentans the alpha- and the biotin containing subunits of the three decarboxylases and that from Peptostreptococcus asaccharolyticus could be detected on Western blots.