Dental and Microbiological Risk Factors for Hospital-Acquired Pneumonia in Non-Ventilated Older Patients

Hospital acquired pneumonia (HAP) is often fatal in older patients. The mouth is the main reservoir of infection and studies have suggested that oral hygiene interventions may prevent HAP. The aim of this study was to investigate associations between HAP and preceding a) heavy dental plaque and b) oral carriage of potential respiratory pathogens in older patients with lower limb fracture to determine the target for intervention studies.

Methods

We obtained a time series of tongue/throat swabs from 90 patients with lower limb fracture, aged 65-101 in a general hospital in the North East of England between April 2009-July 2010. We used novel real-time multiplex PCR assays to detect S. aureus, MRSA, E. coli, P. aeruginosa, S. pneumoniae, H. influenza and Acinetobacter spp. We collected data on dental/denture plaque (modified Quigley-Hein index) and outcomes of clinician-diagnosed HAP.

Results

The crude incidence of HAP was 10% (n = 90), with mortality of 80% at 90 days post discharge. 50% of cases occurred within the first 25 days. HAP was not associated with being dentate, tooth number, or heavy dental/denture plaque. HAP was associated with prior oral carriage with E. coli/S. aureus/P.aeruginosa/MRSA (p = 0.002, OR 9.48 95% CI 2.28-38.78). The incidence of HAP in those with carriage was 35% (4% without), with relative risk 6.44 (95% CI 2.04-20.34, p = 0.002). HAP was associated with increased length of stay (Fishers exact test, p=0.01), with mean 30 excess days (range -11.5-115). Target organisms were first detected within 72 hours of admission in 90% participants, but HAP was significantly associated with S. aureus/MRSA/P. aeruginosa/E. coli being detected at days 5 (OR 4.39, 95%CI1.73-11.16) or 14 (OR 6.69, 95%CI 2.40-18.60).

Conclusions

Patients with lower limb fracture who were colonised orally with E. coli/ S. aureus/MRSA/P. aeruginosa after 5 days in hospital were at significantly greater risk of HAP (p = 0.002).     

Application of electron transfer dissociation (ETD) for the analysis of posttranslational modifications

Despite major advantages in the field of proteomics, the analysis of PTMs still poses a major challenge; thus far, preventing insights into the role and regulation of protein networks. Additionally, top-down sequencing of proteins is another powerful approach to reveal comprehensive information for biological function. A commonly used fragmentation technique in MS-based peptide sequencing is CID. As CID often fails in PTM-analysis and performs best on doubly-charged, short and middle-sized peptides, confident peptide identification may be hampered. A newly developed fragmentation technique, namely electron transfer dissociation (ETD), supports both, PTM- and top-down analysis, and generally results in more confident identification of long, highly charged or modified peptides. The following review presents the theoretical background of ETD and its technical implementation in mass analyzers. Furthermore, current improvements of ETD and approaches for the PTM-analysis and top-down sequencing are introduced. Alternating both fragmentation techniques, ETD and CID, increases the amount of information derived from peptide fragmentation, thereby enhancing both, peptide sequence coverage and the confidence of peptide and protein identification.     

Diminished proliferation of human immunodeficiency virus-specific CD4+ T cells is associated with diminished interleukin-2 (IL-2) production and is recovered by exogenous IL-2

Virus-specific CD4(+) T-cell function is thought to play a central role in induction and maintenance of effective CD8(+) T-cell responses in experimental animals or humans. However, the reasons that diminished proliferation of human immunodeficiency virus (HIV)-specific CD4(+) T cells is observed in the majority of infected patients and the role of these diminished responses in the loss of control of replication during the chronic phase of HIV infection remain incompletely understood. In a cohort of 15 patients that were selected for particularly strong HIV-specific CD4(+) T-cell responses, the effects of viremia on these responses were explored. Restriction of HIV replication was not observed during one to eight interruptions of antiretroviral therapy in the majority of patients (12 of 15). In each case, proliferative responses to HIV antigens were rapidly inhibited during viremia. The frequencies of cells that produce IFN-gamma in response to Gag, Pol, and Nef peptide pools were maintained during an interruption of therapy. In a subset of patients with elevated frequencies of interleukin-2 (IL-2)-producing cells, IL-2 production in response to HIV antigens was diminished during viremia. Addition of exogenous IL-2 was sufficient to rescue in vitro proliferation of DR0101 class II Gag or Pol tetramer(+) or total-Gag-specific CD4(+) T cells. These observations suggest that, during viremia, diminished in vitro proliferation of HIV-specific CD4(+) T cells is likely related to diminished IL-2 production. These results also suggest that relatively high frequencies of HIV-specific CD4(+) T cells persist in the peripheral blood during viremia, are not replicatively senescent, and proliferate when IL-2 is provided exogenously.     

Involvement of the pyrophosphate and the 2'-phosphate binding regions of ferredoxin-NADP+ reductase in coenzyme specificity

Previous studies indicated that the determinants of coenzyme specificity in ferredoxin-NADP+ reductase (FNR) from Anabaena are situated in the 2'-phosphate (2'-P) NADP+ binding region, and also suggested that other regions must undergo structural rearrangements of the protein backbone during coenzyme binding. Among the residues involved in such specificity could be those located in regions where interaction with the pyrophosphate group of the coenzyme takes place, namely loops 155-160 and 261-268 in Anabaena FNR. In order to learn more about the coenzyme specificity determinants, and to better define the structural basis of coenzyme binding, mutations in the pyrophosphate and 2'-P binding regions of FNR have been introduced. Modification of the pyrophosphate binding region, involving residues Thr-155, Ala-160, and Leu-263, indicates that this region is involved in determining coenzyme specificity and that selected alterations of these positions produce FNR enzymes that are able to bind NAD+. Thus, our results suggest that slightly different structural rearrangements of the backbone chain in the pyrophosphate binding region might determine FNR specificity for the coenzyme. Combined mutations at the 2'-P binding region, involving residues Ser-223, Arg-224, Arg-233, and Tyr-235, in combination with the residues mentioned above in the pyrophosphate binding region have also been carried out in an attempt to increase the FNR affinity for NAD+/H. However, in most cases the analyzed mutants lost the ability for NADP+/H binding and electron transfer, and no major improvements were observed with regard to the efficiency of the reactions with NAD+/H. Therefore, our results confirm that determinants for coenzyme specificity in FNR are also situated in the pyrophosphate binding region and not only in the 2'-P binding region. Such observations also suggest that other regions of the protein, yet to be identified, might also be involved in this process.     

A single Na+-Pi cotransporter in Toxoplasma plays key roles in phosphate import and control of parasite osmoregulation

Inorganic ions such as phosphate, are essential nutrients required for a broad spectrum of cellular functions and regulation. During infection, pathogens must obtain inorganic phosphate (P_i) from the host. Despite the essentiality of phosphate for all forms of life, how the intracellular parasite Toxoplasma gondii acquires P_i from the host cell is still unknown. In this study, we demonstrated that Toxoplasma actively internalizes exogenous P_i by exploiting a gradient of Na⁺ ions to drive P_i uptake across the plasma membrane. The Na⁺-dependent phosphate transport mechanism is electrogenic and functionally coupled to a cipargarmin sensitive Na⁺-H⁺-ATPase. Toxoplasma expresses one transmembrane P_i transporter harboring PHO4 binding domains that typify the PiT Family. This transporter named TgPiT, localizes to the plasma membrane, the inward buds of the endosomal organelles termed VAC, and many cytoplasmic vesicles. Upon P_i limitation in the medium, TgPiT is more abundant at the plasma membrane. We genetically ablated the PiT gene, and ΔTgPiT parasites are impaired in importing P_i and synthesizing polyphosphates. Interestingly, ΔTgPiT parasites accumulate 4-times more acidocalcisomes, storage organelles for phosphate molecules, as compared to parental parasites. In addition, these mutants have a reduced cell volume, enlarged VAC organelles, defects in calcium storage and a slightly alkaline pH. Overall, these mutants exhibit severe growth defects and have reduced acute virulence in mice. In survival mode, ΔTgPiT parasites upregulate several genes, including those encoding enzymes that cleave or transfer phosphate groups from phosphometabolites, transporters and ions exchangers localized to VAC or acidocalcisomes. Taken together, these findings point to a critical role of TgPiT for P_i supply for Toxoplasma and also for protection against osmotic stresses.     

A Lethal de Novo Mutation in the Middle Domain of the Dynamin-related GTPase Drp1 Impairs Higher Order Assembly and Mitochondrial Division

Mitochondria dynamically fuse and divide within cells, and the proper balance of fusion and fission is necessary for normal mitochondrial function, morphology, and distribution. Drp1 is a dynamin-related GTPase required for mitochondrial fission in mammalian cells. It harbors four distinct domains: GTP-binding, middle, insert B, and GTPase effector. A lethal mutation (A395D) within the Drp1 middle domain was reported in a neonate with microcephaly, abnormal brain development, optic atrophy, and lactic acidemia (Waterham, H. R., Koster, J., van Roermund, C. W., Mooyer, P. A., Wanders, R. J., and Leonard, J. V. (2007) N. Engl. J. Med. 356, 1736–1741). Mitochondria within patient-derived fibroblasts were markedly elongated, but the molecular mechanisms underlying these findings were not demonstrated. Because the middle domain is particularly important for the self-assembly of some dynamin superfamily proteins, we tested the hypothesis that this A395D mutation, and two other middle domain mutations (G350D, G363D) were important for Drp1 tetramerization, higher order assembly, and function. Although tetramerization appeared largely intact, each of these mutations compromised higher order assembly and assembly-dependent stimulation of Drp1 GTPase activity. Moreover, mutant Drp1 proteins exhibited impaired localization to mitochondria, indicating that this higher order assembly is important for mitochondrial recruitment, retention, or both. Overexpression of these middle domain mutants markedly inhibited mitochondrial division in cells. Thus, the Drp1 A395D lethal defect likely resulted in impaired higher order assembly of Drp1 at mitochondria, leading to decreased fission, elongated mitochondria, and altered cellular distribution of mitochondria.