Enterococcus faecalis is a gram-positive commensal bacterium of the gastrointestinal tract and an important opportunistic pathogen. Despite the increasing clinical significance of the enterococci, most of the genetic analysis of these organisms has focused on mobile genetic elements, and existing tools for manipulation and analysis of the core E. faecalis chromosome are limited. We are interested in a comprehensive analysis of the genetic determinants for biofilm formation encoded within the core E. faecalis genome. To identify such determinants, we developed a substantially improved system for transposon mutagenesis in E. faecalis based on a mini-mariner transposable element. Mutagenesis of wild-type E. faecalis with this element yielded predominantly mutants carrying a single copy of the transposable element, and insertions were distributed around the entire chromosome in an apparently random fashion. We constructed a library of E. faecalis transposon insertion mutants and screened this library to identify mutants exhibiting a defect in biofilm formation. Biofilm-defective mutants were found to carry transposon insertions both in genes that were previously known to play a role in biofilm formation and in new genes lacking any known function; for several genes identified in the screen, complementation analysis confirmed a direct role in biofilm formation. These results provide significant new information about the genetics of enterococcal biofilm formation and demonstrate the general utility of our transposon system for functional genomic analysis of E. faecalis.

Ultra-high field 27 Al{ 1 H} 2D correlation NMR experiments demonstrate that at least two framework Al(IV) sites with hydroxyl groups can exist in acidic zeolite catalysts in their dehydrated and catalytically active states. In addition to the known Al(IV) at the framework bridging acid site (BAS), a new site created by a second tetrahedral Al atom and its hydroxyl group protons in zeolite HZSM-5 are clearly resolved at 35.2 T field strengths, enabled by recently developed series-connected hybrid (SCH) magnet technology. Coupled with computational modeling, extensive 27 Al MQMAS experiments at multiple field strengths, and 1 H MAS NMR experiments, these data indicate that this second tetrahedrally-coordinated Al site (denoted Al(IV)-2) experiences an increased chemical shift and unique quadrupolar parameters relative to the BAS in both dehydrated and hydrated states. These new experimental data, supported by computational and catalytic reaction work, indicates that the second site arises from partiallybonded framework (SiO) 4-n -Al(OH) n species that significantly increase catalyst reactivity in benzene hydride-transfer and n-hexane cracking reactions. Al(IV)-2 sites result either from framework crystallization defects or from incomplete post-synthetic hydrolysis of a framework Al, prior to the formation of extraframework Al. Populations of this second acidic proton site created by the Al(IV)-2 species are shown to be controlled via post-synthetic catalyst treatments, should be general to different catalyst structures, and significantly enhance catalyst reactivity in the cited probe reactions when they are present. The results herein communicate the highest magnetic field strength data on active zeolite catalyst structures to date and enable for the first time the detection of Al and H association on a dry HZSM-5 catalyst, i.e., under conditions representative of typical end-use processes.

Key Points Question What are the long-term health risks of National Football League (NFL) players compared with Major League Baseball (MLB) players, another group of elite athletes? Findings In this cohort study of 3419 NFL and 2708 MLB players, NFL players had significantly higher mortality rates from all causes, cardiovascular diseases, and neurodegenerative diseases compared with MLB players. Meaning This study found that NFL players had a higher rate of mortality than MLB players, which may be associated with aspects of playing in professional American-style football.

Zeolite catalysts are solid Brønsted acids whose reactivity is typically associated with the number of protons at crystalline framework bridging acid sites (BAS’s). Postsynthetic catalyst modification, titrations with monovalent and divalent cations of varying size, quantitative spin-counting spectroscopy on all protons before and after cation exchange, amine titration, and room-temperature in situ reactions with two different probe molecules reveal that zeolite HZSM-5 reactivity strongly corresponds with the presence of acidic protons from extraframework and/or noncrystalline sites. Significantly, room-temperature hydrogen–deuterium (H/D) exchange reactions between the catalyst and the organic probe molecules reveal that reaction rates are strongly dependent on the total concentration of acidic protons from extraframework and noncrystalline proton sites. The most active catalysts in room-temperature probe reactions contain protons from both BAS’s and from noncrystalline species, including reactive extraframework aluminol species that can be removed by solvent treatments. In order to demonstrate the significance of paired framework/extraframework or noncrystalline Brønsted sites to overall catalyst activity, speciation of different protons were quantified after titration with mono- and divalent cations of varying radius (Na+, Ca2+, Cu2+, Ba2+), chemical washing with ammonium hexafluorosilicate (AHFS), and different steaming procedures for HZSM-5 catalysts with Si/Al equal to 15 and 40. Detailed manipulation of reactive Brønsted species in the Si/Al = 15 catalyst enabled direct experimental observation of H/D exchange at both the methine and methyl positions of isobutane, heretofore not reported, clarifying uncertainties surrounding that mechanism. Reaction data indicates that isolated framework BAS’s are much less important to overall catalyst reactivity than proximate framework/extraframework or noncrystalline Brønsted sites, and DFT calculations support the importance of proximate proton sites. Potential Brønsted–Brønsted synergies are unique relative to previously proposed Brønsted/Lewis synergies but do not preclude the latter’s contribution to increased reactivity.

The role of extra-framework Al (EFAL) species on industrially important reactions such as alkane cracking has been extensively discussed and debated. It has long been known that water treatments influence the framework aluminum sites and, in some cases, can modify activity. What is less understood, however, is the direct relationship between the structural modifications and reactivity of important reactions such as alkane cracking and isomerization. The collective understanding of the multiple roles that water plays in the modification of zeolites and influence on reaction rates is continuously evolving. Extra-lattice Al species in close proximity to a framework Brønsted acid sites (BAS) have been proposed to modify the energies associated with surface intermediates and kinetically relevant transition states, which results in an enhancement in the rates of alkane cracking reactions. However, the kinetic role of water on the migration of these extra-framework alumina species to generate highly active sites is less understood and is the focus of this study. Water is introduced in controlled pulses to ZSM-5 zeolites with various Si/Al ratios and EFAL densities, with responses in n-hexane cracking activity used to investigate the generation of new active sites. A pulse technique allows decoupling of water dosing, lattice rearrangement, and drying, thereby enabling the quantification of activation energies associated with the generation of new active sites without losses in crystallinity or total BAS density. Further, by subtraction of the contributions to the reaction rate associated with isolated BAS, the reaction rate associated with the newly created sites is estimated. The results show that the energy barrier required for cracking on highly active sites is much lower than that observed on traditional Brønsted sites (75 vs 110 kJ/mol). The temperature dependence for the generation of these new sites reveals a 44 kJ/mol activation energy for the kinetically relevant step associated with their generation in the presence of water vapor, which to the best of our knowledge has not been previously quantified. It is reported that, while water vapor is essential for the generation of these new active sites, it also binds to these sites and strongly inhibits the cracking rate. These findings clarify some of the conflicting reports regarding the role of water in activity enhancement.

Catalytic cleavage of strong bonds including hydrogen-hydrogen, carbon-oxygen, and carbonhydrogen bonds is a highly desired yet challenging fundamental transformation for the production of chemicals and fuels. Transition metal-containing catalysts are employed, although accompanied with poor selectivity in hydrotreatment. Here we report metal-free nitrogen-assembly carbons (NACs) with closely-placed graphitic nitrogen as active sites, achieving dihydrogen dissociation and subsequent transformation of oxygenates. NACs exhibit high selectivity towards alkylarenes for hydrogenolysis of aryl ethers as model biooxygenates without over-hydrogeneration of arenes. Activities originate from cooperating graphitic nitrogen dopants induced by the diamine precursors, as demonstrated in mechanistic and computational studies. We further show that the NAC catalyst is versatile for dehydrogenation of ethylbenzene and tetrahydroquinoline as well as for hydrogenation of common unsaturated functionalities, including ketone, alkene, alkyne, and nitro groups. The discovery of nitrogen assembly as active sites can open up broad opportunities for rational design of new metal-free catalysts for challenging chemical reactions.

Current needs for extending zeolite catalysts beyond traditional gas-phase hydrocarbon chemistry demand detailed characterization of active site structures, distributions, and hydrothermal impacts. A broad suite of homonuclear and heteronuclear NMR correlation experiments on dehydrated H-ZSM-5 catalysts with isotopically enriched 17 O frameworks reveals that at least two types of paired active sites exist, the amount of which depends on the population of fully framework-coordinated tetrahedral Al (Al(IV)-1) and partially framework-coordinated tetrahedral Al (Al(IV)-2) sites, both of which can be denoted as (SiO) 4−n −Al(OH) n . The relative amounts of Al(IV)-1 and Al(IV)-2 sites, and subsequent pairing, cannot be inferred from the catalyst Si/Al ratio, but depend on synthetic and postsynthetic modifications. Correlation experiments demonstrate that, on average, acidic hydroxyl groups from Al(IV)-1/Al(IV)-2 pairs are closer to one another than those from Al(IV)-1/Al(IV)-1 pairs, as supported by computational DFT calculations. Through-bond and through-space polarization transfer experiments exploiting 17 O nuclei reveal a number of different acidic hydroxyl groups in varying Si/Al catalysts, the relative amounts of which change following postsynthetic modifications. Using room-temperature isotopic exchange methods, it was determined that 17 O was homogeneously incorporated into the zeolite framework, while 17 O → 27 Al polarization transfer experiments demonstrated that 17 O incorporation does not occur for extra-framework Al n O m species. Data from samples exposed to controlled hydrolysis indicates that nearest neighbor Al pairs in the framework are more susceptible to hydrolytic attack. The data reported here suggest that Al(IV)-1/ Al(IV)-2 paired sites are synergistic sites leading to increased reactivity in both low-and high-temperature reactions. No evidence was found for paired framework/nonframework sites.

While noninvasive prenatal testing based on cell‐free fetal DNA has recently revolutionized the field of aneuploidy screening in pregnancy, it remains limited to aneuploidy and microdeletion screening, and is unable to reliably detect single gene disorders. A number of recent studies have demonstrated the potential of circulating trophoblastic cells in providing cell‐based noninvasive diagnosis with sequencing or array‐based assays. However, considering the extreme rarity of these cells in blood, efficient, high‐throughput, and clinically applicable enrichment technologies are yet to be developed. This study demonstrates for the first time the utility of inertial microfluidics for efficient isolation of trophoblastic cells from maternal peripheral blood. Under optimal operating conditions, high‐recovery yields (79%) are obtained using a trophoblastic cell‐line, which is subsequently confirmed with analysis of maternal blood. Feasibility of obtaining a diagnosis from cells isolated from a maternal sample is demonstrated in a case of confirmed fetal trisomy 21 in which six fetal cells are found in a 7 mL blood sample using fluorescence in situ hybridization. Finally, it is demonstrated that trophoblastic cells isolated using inertial microfluidics could be picked and subjected to a clinically validated sequencing assay, paving the way for further validation of this technology and larger clinical studies.