MONITORING FOR WASTE: EVIDENCE FROM MEDICARE AUDITS.

This paper examines the tradeoffs of monitoring for wasteful public spending. By penalizing unnecessary spending, monitoring improves the quality of public expenditure and incentivizes firms to invest in compliance technology. I study a large Medicare program that monitored for unnecessary healthcare spending and consider its effect on government savings, provider behavior, and patient health. Every dollar Medicare spent on monitoring generated $24-29 in government savings. The majority of savings stem from the deterrence of future care, rather than reclaimed payments from prior care. I do not find evidence that the health of the marginal patient is harmed, indicating that monitoring primarily deters low-value care. Monitoring does increase provider administrative costs, but these costs are mostly incurred upfront and include investments in technology to assess the medical necessity of care.

Unveiling 2,000 years of differentiation among Tungusic-speaking populations: a revised phylogeny of the paternal founder lineage C2a-M48-SK1061.

Previous studies demonstrated Y chromosome haplogroup C2a-M48-SK1061 is the only founding paternal lineage of all Tungusic-speaking populations. To infer the differentiation history of these populations, we studied more sequences and constructed downstream structure of haplogroup C2a-M48-SK1061 with better resolution. In this study, we generated 100 new sequences and co-analyzed 140 sequences of C2a-M48-SK1061 to reconstruct a highly revised phylogenetic tree with age estimates. We also performed the analysis of the geographical distribution and spatial autocorrelation of sub-branches. Dozens of new sub-branches were discovered, many sub-branches were nearly unique for Ewenki, Evens, Oroqen, Xibe, Manchu, Daur, and Mongolian. The topology of these unique sub-branches is the key evidence for understanding the complex evolutionary relationship between different Tungusic-speaking populations. The revised phylogeny provided a clear pattern for the differentiation history of haplogroup C2a-M48-SK1061 in the past 2,000 years. This study showed that the divergence pattern of founder lineage is essential to understanding the differentiation history of populations.

Edge-strand of BepA interacts with immature LptD on the ß-barrel assembly machine to direct it to on- and off-pathways.

The outer membrane (OM) of Gram-negative bacteria functions as a selective permeability barrier. Escherichia coli periplasmic Zn-metallopeptidase BepA contributes to the maintenance of OM integrity through its involvement in the biogenesis and degradation of LptD, a ß-barrel protein component of the lipopolysaccharide translocon. BepA either promotes the maturation of LptD when it is on the normal assembly pathway (on-pathway) or degrades it when its assembly is compromised (off-pathway). BepA performs these functions probably on the ß-barrel assembly machinery (BAM) complex. However, how BepA recognizes and directs an immature LptD to different pathways remains unclear. Here, we explored the interactions among BepA, LptD, and the BAM complex. We found that the interaction of the BepA edge-strand located adjacent to the active site with LptD was crucial not only for proteolysis but also, unexpectedly, for assembly promotion of LptD. Site-directed crosslinking analyses indicated that the unstructured N-terminal half of the ß-barrel-forming domain of an immature LptD contacts with the BepA edge-strand. Furthermore, the C-terminal region of the ß-barrel-forming domain of the BepA-bound LptD intermediate interacted with a 'seam' strand of BamA, suggesting that BepA recognized LptD assembling on the BAM complex. Our findings provide important insights into the functional mechanism of BepA.

Structure-Function Characterization of the Conserved Regulatory Mechanism of the Escherichia coli M48 Metalloprotease BepA.

The asymmetric Gram-negative outer membrane (OM) is the first line of defense for bacteria against environmental insults and attack by antimicrobials. The key component of the OM is lipopolysaccharide, which is transported to the surface by the essential lipopolysaccharide transport (Lpt) system. Correct folding of the Lpt system component LptD is regulated by a periplasmic metalloprotease, BepA. Here, we present the crystal structure of BepA from Escherichia coli, solved to a resolution of 2.18 Å, in which the M48 protease active site is occluded by an active-site plug. Informed by our structure, we demonstrate that free movement of the active-site plug is essential for BepA function, suggesting that the protein is autoregulated by the active-site plug, which is conserved throughout the M48 metalloprotease family. Targeted mutagenesis of conserved residues reveals that the negative pocket and the tetratricopeptide repeat (TPR) cavity are required for function and degradation of the BAM complex component BamA under conditions of stress. Last, we show that loss of BepA causes disruption of OM lipid asymmetry, leading to surface exposed phospholipid.IMPORTANCE M48 metalloproteases are widely distributed in all domains of life. E. coli possesses four members of this family located in multiple cellular compartments. The functions of these proteases are not well understood. Recent investigations revealed that one family member, BepA, has an important role in the maturation of a central component of the lipopolysaccharide (LPS) biogenesis machinery. Here, we present the structure of BepA and the results of a structure-guided mutagenesis strategy, which reveal the key residues required for activity that inform how all M48 metalloproteases function.

Reversible autoinhibitory regulation of Escherichia coli metallopeptidase BepA for selective ß-barrel protein degradation.

Escherichia coli periplasmic zinc-metallopeptidase BepA normally functions by promoting maturation of LptD, a ß-barrel outer-membrane protein involved in biogenesis of lipopolysaccharides, but degrades it when its membrane assembly is hampered. These processes should be properly regulated to ensure normal biogenesis of LptD. The underlying mechanism of regulation, however, remains to be elucidated. A recently solved BepA structure has revealed unique features: In particular, the active site is buried in the protease domain and conceivably inaccessible for substrate degradation. Additionally, the His-246 residue in the loop region containing helix α9 (α9/H246 loop), which has potential flexibility and covers the active site, coordinates the zinc ion as the fourth ligand to exclude a catalytic water molecule, thereby suggesting that the crystal structure of BepA represents a latent form. To examine the roles of the α9/H246 loop in the regulation of BepA activity, we constructed BepA mutants with a His-246 mutation or a deletion of the α9/H246 loop and analyzed their activities in vivo and in vitro. These mutants exhibited an elevated protease activity and, unlike the wild-type BepA, degraded LptD that is in the normal assembly pathway. In contrast, tethering of the α9/H246 loop repressed the LptD degradation, which suggests that the flexibility of this loop is important to the exhibition of protease activity. Based on these results, we propose that the α9/H246 loop undergoes a reversible structural change that enables His-246-mediated switching (histidine switch) of its protease activity, which is important for regulated degradation of stalled/misassembled LptD.

Protease homolog BepA (YfgC) promotes assembly and degradation of ß-barrel membrane proteins in Escherichia coli.

Gram-negative bacteria are equipped with quality-control systems for the outer membrane (OM) that sense and cope with defective biogenesis of its components. Accumulation of misfolded outer membrane proteins (OMPs) in Escherichia coli leads to activation of σ(E), an essential alternative σ factor that up-regulates transcription of multiple genes required to preserve OM structure and function. Disruption of bepA (formerly yfgC), a σ(E)-regulated gene encoding a putative periplasmic metalloprotease, sensitizes cells to multiple drugs, suggesting that it may be involved in maintaining OM integrity. However, the specific function of BepA remains unclear. Here, we show that BepA enhances biogenesis of LptD, an essential OMP involved in OM transport and assembly of lipopolysaccharide, by promoting rearrangement of intramolecular disulfide bonds of LptD. In addition, BepA possesses protease activity and is responsible for the degradation of incorrectly folded LptD. In the absence of periplasmic chaperone SurA, BepA also promotes degradation of BamA, the central OMP subunit of the ß-barrel assembly machinery (BAM) complex. Interestingly, defective oxidative folding of LptD caused by bepA disruption was partially suppressed by expression of protease-active site mutants of BepA, suggesting that BepA functions independently of its protease activity. We also show that BepA has genetic and physical interaction with components of the BAM complex. These findings raised the possibility that BepA maintains the integrity of OM both by promoting assembly of OMPs and by proteolytically eliminating OMPs when their correct assembly was compromised.

Note on the formulation of thermosensitive and mucoadhesive vaginal hydrogels containing the miniCD4 M48U1 as anti-HIV-1 microbicide.

The miniCD4 M48U1 was formulated into thermosensitive and mucoadhesive pluronic(®) hydrogels as anti-HIV-1 microbicide. The release kinetics of M48U1 from F127/HPMC (20/1 wt%) and F127/F68/HPMC (22.5/2.5/1 wt%) studied during 24h by using Franz diffusion cells showed that HEC hydrogel (1.5 wt%) used as control released 93% of the peptide, while about 25% of M48U1 remained in pluronic(®) hydrogels. The formulation of M48U1 in pluronic(®) hydrogels ensures a local delivery because no diffusion of the peptide was detected through vaginal Cynomolgus macaque mucosa using Ussing chamber. Finally, toxicological studies showed no significant difference in the HeLa cell viability of the pluronic(®) hydrogels in comparison with HEC and phosphate buffer saline.