Dipole tensor-based atomic-resolution structure determination of a nanocrystalline protein by solid-state NMR

Magic-angle spinning (MAS) solid-state NMR (SSNMR) techniques have emerged in recent years for solving complete structures of uniformly labeled proteins lacking macroscopic order. Strategies used thus far have relied primarily on semiquantitative distance restraints, analogous to the nuclear Overhauser effect (NOE) routinely used in solution NMR. Here, we present a complementary approach for using relative orientations of molecular fragments, determined from dipolar line shapes. Whereas SSNMR distance restraints typically have an uncertainty of approximately 1 A, the tensor-based experiments report on relative vector (pseudobond) angles with precision of a few degrees. By using 3D techniques of this type, vector angle (VEAN) restraints were determined for the majority of the 56-residue B1 immunoglobulin binding domain of protein G [protein GB1 (a total of 47 HN-HN, 49 HN-HC, and 12 HA-HB restraints)]. By using distance restraints alone in the structure calculations, the overall backbone root-mean-square deviation (bbRMSD) was 1.01 +/- 0.13 A (1.52 +/- 0.12 A for all heavy atoms), which improved to 0.49 +/- 0.05 A (1.19 +/- 0.07 A) on the addition of empirical chemical shift [torsion angle likelihood obtained from shift and sequence similarity (TALOS)] restraints. VEAN restraints further improved the ensemble to 0.31 +/- 0.06 A bbRMSD (1.06 +/- 0.07 A); relative to the structure with distances alone, most of the improvement remained (bbRMSD 0.64 +/- 0.09 A; 1.29 +/- 0.07 A) when TALOS restraints were removed before refinement. These results represent significant progress toward atomic-resolution protein structure determination by SSNMR, capabilities that can be applied to a large range of membrane proteins and fibrils, which are often not amenable to solution NMR or x-ray crystallography.     

Longitudinal Study of Extended-Spectrum-β-Lactamase- and AmpC-Producing Enterobacteriaceae in Household Dogs

A longitudinal study was performed to (i) investigate the continuity of shedding of extended-spectrum-beta-lactamase (ESBL)-producing Enterobacteriaceae in dogs without clinical signs, (ii) identify dominant plasmid-mediated ESBL genes, and (iii) quantify ESBL-producing Enterobacteriaceae in feces. Fecal samples from 38 dogs were collected monthly for 6 months. Additional samples were collected from 7 included dogs on a weekly basis for 6 weeks. Numbers of CFU per gram of feces for non-wild-type Enterobacteriaceae were determined by using MacConkey agar supplemented with 1 mg/liter cefotaxime (MCC), and those for total Enterobacteriaceae were determined by using MacConkey agar. Cefotaxime-resistant isolates were screened by PCR and sequence analysis for the presence of bla_CTX-M, bla_CMY, bla_SHV, bla_OXA, and bla_TEM gene families. Bacterial species were identified by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) analysis. PCR-negative isolates were tested by a double-disk synergy test for enhanced AmpC expression. A total of 259 samples were screened, and 126 samples were culture positive on MCC, resulting in 352 isolates, 327 of which were Escherichia coli. Nine dogs were continuously positive during this study, and 6 dogs were continuously negative. Monthly or weekly shifts in fecal shedding were observed for 23 dogs. Genotyping showed a large variety of ESBL genes and gene combinations at single and multiple consecutive sampling moments. The ESBL genes bla_CTX-M-1, bla_CTX-M-14, bla_CTX-M-15, bla_SHV-12, and bla_CMY-2 were most frequently found. The mean number of CFU of non-wild-type Enterobacteriaceae was 6.11 × 10⁸ CFU/g feces. This study showed an abundance of ESBL-producing Enterobacteriaceae in dogs in the Netherlands, mostly in high concentrations. Fecal shedding was shown to be highly dynamic over time, which is important to consider when studying ESBL epidemiology.     

Processing consequences of superfluous and missing prosodic breaks in auditory sentence comprehension

This ERP study investigates whether a superfluous prosodic break (i.e., a prosodic break that does not coincide with a syntactic break) has more severe processing consequences during auditory sentence comprehension than a missing prosodic break (i.e., the absence of a prosodic break at the position of a syntactic break). Participants listened to temporarily ambiguous sentences involving a prosody–syntax match or mismatch. The disambiguation of these sentences was always lexical in nature in the present experiment. This contrasts with a related study by Pauker, Itzhak, Baum, and Steinhauer (2011), where the disambiguation was of a lexical type for missing PBs and of a prosodic type for superfluous PBs. Our results converge with those of Pauker et al. (2011): superfluous prosodic breaks lead to more severe processing problems than missing prosodic breaks. Importantly, the present results extend those of Pauker et al. (2011) showing that this holds when the disambiguation is always lexical in nature. Furthermore, our results show that the way listeners use prosody can change over the course of the experiment which bears consequences for future studies.