Hospitalizations and Costs Incurred at the Facility Level after Scale-up of Malaria Control: Pre-Post Comparisons from Two Hospitals in Zambia

There is little evidence on the impact of malaria control on the health system, particularly at the facility level. Using retrospective, longitudinal facility-level and patient record data from two hospitals in Zambia, we report a pre-post comparison of hospital admissions and outpatient visits for malaria and estimated costs incurred for malaria admissions before and after malaria control scale-up. The results show a substantial reduction in inpatient admissions and outpatient visits for malaria at both hospitals after the scale-up, and malaria cases accounted for a smaller proportion of total hospital visits over time. Hospital spending on malaria admissions also decreased. In one hospital, malaria accounted for 11% of total hospital spending before large-scale malaria control compared with < 1% after malaria control. The findings demonstrate that facility-level resources are freed up as malaria is controlled, potentially making these resources available for other diseases and conditions.     

Capsid expansion mechanism of bacteriophage T7 revealed by multistate atomic models derived from cryo-EM reconstructions

Many dsDNA viruses first assemble a DNA-free procapsid, using a scaffolding protein-dependent process. The procapsid, then, undergoes dramatic conformational maturation while packaging DNA. For bacteriophage T7 we report the following four single-particle cryo-EM 3D reconstructions and the derived atomic models: procapsid (4.6-Å resolution), an early-stage DNA packaging intermediate (3.5 Å), a later-stage packaging intermediate (6.6 Å), and the final infectious phage (3.6 Å). In the procapsid, the N terminus of the major capsid protein, gp10, has a six-turn helix at the inner surface of the shell, where each skewed hexamer of gp10 interacts with two scaffolding proteins. With the exit of scaffolding proteins during maturation the gp10 N-terminal helix unfolds and swings through the capsid shell to the outer surface. The refolded N-terminal region has a hairpin that forms a novel noncovalent, joint-like, intercapsomeric interaction with a pocket formed during shell expansion. These large conformational changes also result in a new noncovalent, intracapsomeric topological linking. Both interactions further stabilize the capsids by interlocking all pentameric and hexameric capsomeres in both DNA packaging intermediate and phage. Although the final phage shell has nearly identical structure to the shell of the DNA-free intermediate, surprisingly we found that the icosahedral faces of the phage are slightly (∼4 Å) contracted relative to the faces of the intermediate, despite the internal pressure from the densely packaged DNA genome. These structures provide a basis for understanding the capsid maturation process during DNA packaging that is essential for large numbers of dsDNA viruses.Many dsDNA viruses, including tailed phages and herpes viruses, initially assemble a DNA-free procapsid with assistance of a network of scaffold proteins. Accompanying the exit of scaffolding proteins during subsequent ATP-driven DNA packaging, the icosahedral shell of the procapsid undergoes dramatic conformational changes and matures into a typically larger and more angular shell of the infectious phage (1–6). However, structural details, including those of capsid intermediates, are limited to the phage HK97 system (5, 7–9), for which recombinantly produced procapsid and nonphysiological conversion products were analyzed.The packaging of the 39.937-kbp DNA genome of the short-tail Escherichia coli bacteriophage, T7, is a model for understanding basic principles common to dsDNA tailed phages and herpes viruses. The T7 system is also of interest because it has been used for popular biotechnologies, such as recombinant protein expression (10) and protein display on the capsid surface (11). The T7 capsid contains 415 copies of the major shell protein gp10 (12) that form a T = 7L icosahedral lattice. From low-resolution cryo-EM 3D reconstructions the tertiary topology of gp10 can be divided into four regions: N-arm, E-loop, A-domain, and P-domain, which together place the gp10 protein in the HK97 fold category (2, 13, 14). The T7 procapsid, capsid I, contains 110–140 molecules of scaffolding protein, gp9 (4, 15, 16). After scaffolding protein expulsion the spherical T7 capsid I expands to more angular intermediates, which are collectively called capsid II (2, 4, 14, 16–18).Two DNA-free capsid IIs are purified in quantity sufficient for structural studies by cryo-EM (16). Both are produced during the normal process of wild-type T7 DNA packaging in vivo. One has an unusually low density during buoyant density centrifugation in a metrizamide density gradient (1.086 g/mL; metrizamide low density, or MLD, capsid II) and the other has a density as expected for hydrated proteins (1.28 g/mL; metrizamide high density, or MHD, capsid II) (16). The low density of MLD capsid II is caused by impermeability to metrizamide (789 Da) (16). The MLD capsid II particles are produced before MHD capsid II particles based on kinetic studies (16).The DNA packaging of T7 phage starts at capsid I state where the DNA is packaged by the ATPases (gp18 and gp19) to pass through the portal (gp8) apparatus (19). By analyzing kinetics of in vivo-produced capsids, MLD capsid II was found to be the first postcapsid I capsid. MLD capsid II appears with the kinetics of an intermediate (16) but is obviously no longer in the DNA packaging pathway because it has detached from the DNA molecule that it was packaging. MLD capsid II is not produced when a nonpermissive host is infected with a T7 amber mutant defective in DNA packaging (summarized in ref. 16). Thus, MLD capsid II is an intermediate that has been altered during either cellular lysis or subsequent purification. MHD capsid II also has the appearance kinetics of an intermediate of packaging, but one that occurs later (16). Whereas MLD capsid II has the internal core stack including proteins gp8, gp14, gp15, and gp16 (16), MHD capsid II does not have the internal core stack proteins, which were presumably lost when packaged DNA exited the capsid (16).The existence of these various capsids provides an opportunity to obtain a high-resolution (3–4 Å) analysis of structural dynamics that occur in vivo. Here we report cryo-EM structures of the shells of the following bacteriophage T7 capsids: capsid I (4.6 Å), MLD capsid II (3.5 Å), MHD capsid II (6.6 Å), and phage (3.6 Å). The two capsid II shells are the first postprocapsid, in vivo-generated shells (for any packaging system) to be subjected to high-resolution structural analysis, to our knowledge. The results reveal (i) an HK97-fold shell protein with an intracapsomere, noncovalent topological linking and another intercapsomere, joint interaction, neither interaction having been found for other dsDNA tailed phages; (ii) details of the interaction of gp9 scaffolding protein with the inner surface of the capsid I shell; (iii) a novel refolding and externalization of the N terminus of major capsid protein, gp10; and (iv) a subtle, surprising contraction of the gp10 shell in transit from MLD capsid II to phage. Based on these observations, we propose a general procapsid assembly and maturation pathway for dsDNA viruses.     

Transcatheter Aortic Valve Implantation in the Elderly: Who to Refer?

In recent years, experience with transcatheter aortic valve implantation has led to improved outcomes in elderly patients with severe aortic stenosis (AS) who may not have previously been considered for intervention. These patients are often frail with significant comorbid conditions.