Ganciclovir-resistant cytomegalovirus infection in solid organ transplant recipients: a single-center retrospective cohort study.

BACKGROUND: Ganciclovir-resistant cytomegalovirus (GCV-R CMV) is an emerging challenge among solid organ transplant (SOT) recipients. The literature suggests that about 1% of abdominal transplant recipients develop GCV-R CMV infection. The epidemiology and outcome of GCV-R CMV in SOT recipients who have received alemtuzumab induction is not well described. METHODS: After Institutional Review Board approval, a single-center, retrospective review of 2148 abdominal SOT recipients between January 2006 and July 2011 at our institution (n = 2148) was conducted to identify patients with proven or empirically treated GCV-R CMV. Descriptive statistics on collected demographics, clinical course, and therapeutic outcomes were performed. RESULTS: Of 116 SOT recipient treated for CMV, 14 patients (12.1% of cases; 0.65% of all SOT patients) had proven or suspected GCV-R CMV. Eight (50%) developed GCV-R CMV while receiving valganciclovir (valGCV) prophylaxis. The remainder developed late-onset disease, after having completed an average 212 days (range 83-353) of prophylaxis. Resistance was clinically suspected an average of 103 days (range 10-455) after CMV infection was initially identified; 10 patients had confirmed genotypic resistance. Foscarnet therapy was associated with resolution of CMV in 13. CONCLUSION: Suboptimal dosing of valGCV is associated with development of GCV-R CMV. Our observed rate of GCV-R CMV in alemtuzumab-induced patients is similar to rates seen to historical data for other induction agents.

Protein adhesins as vaccine antigens for Group A Streptococcus.

Group A Streptococcus (GAS) is a globally important human pathogen that causes a broad spectrum of disease ranging from mild superficial infections to severe invasive diseases with high morbidity and mortality. Currently, there is no vaccine available for human use. GAS produces a vast array of virulence factors including multiple adhesin molecules. These mediate binding of the bacteria to host tissues and are essential in the initial phases of infection. Prophylactic vaccination with adhesins is a promising vaccine strategy and many GAS adhesins are currently in development as vaccine candidates. The most advanced candidates, having entered clinical trials, are based on the M protein, while components of the pilus and a number of fibronectin-binding proteins are in pre-clinical development. Adhesin-based vaccines aim to induce protective immunity via two main mechanisms: neutralisation where adhesin-specific antibodies block the ability of the adhesin to bind to host tissue and opsonisation in which adhesin-specific antibodies tag the GAS bacteria for phagocytosis. This review summarises our current knowledge of GAS adhesins and their structural features in the context of vaccine development.

Ssp1 promotes actin depolymerization and is involved in stress response and new end take-off control in fission yeast.

The ssp1 gene encodes a protein kinase involved in alteration of cell polarity in Schizosaccharomyces pombe. ssp1 deletion causes stress sensitivity, reminiscent of defects in the stress-activated MAP kinase, Spc1; however, the two protein kinases do not act through the same pathway. Ssp1 is localized mainly in the cytoplasm, but after a rise in external osmolarity it is rapidly recruited to the plasma membrane, preferentially to active growth zones and septa. Loss of Ssp1 function inhibits actin relocalization during osmotic stress, in cdc3 and cdc8 mutant backgrounds, and in the presence of latrunculin A, implicating Ssp1 in promotion of actin depolymerization. We propose a model in which Ssp1 can be activated independently of Spc1 and can partially compensate for its loss. The ssp1 deletion mutant exhibited monopolar actin distribution, but new end take-off (NETO) could be induced in these cells by exposure to KCl or to latrunculin A pulse treatment. This treatment induced NETO in cdc10 cells arrested in G1 but not in tea1 cells. This suggests that cells that contain intact cell end markers are competent to undergo NETO throughout interphase, and Ssp1 is involved in generating the NETO stimulus by enlarging the actin monomer pool.

G2/M arrest caused by actin disruption is a manifestation of the cell size checkpoint in fission yeast.

In budding yeast, actin disruption prevents nuclear division. This has been explained as activation of a morphogenesis checkpoint monitoring the integrity of the actin cytoskeleton. The checkpoint operates through inhibitory tyrosine phosphorylation of Cdc28, the budding yeast Cdc2 homolog. Wild-type Schizosaccharomyces pombe cells also arrest before mitosis after actin depolymerization. Oversized cells, however, enter mitosis uninhibited. We carried out a careful analysis of the kinetics of mitotic initiation after actin disruption in undersized and oversized cells. We show that an inability to reach the mitotic size threshold explains the arrest in smaller cells. Among the regulators that control the level of the inhibitory Cdc2-Tyr15 phosphorylation, the Cdc25 protein tyrosine phosphatase is required to link cell size monitoring to mitotic control. This represents a novel function of the Cdc25 phosphatase. Furthermore, we demonstrate that this cell size-monitoring system fulfills the formal criteria of a cell cycle checkpoint.

The cdr2(+) gene encodes a regulator of G2/M progression and cytokinesis in Schizosaccharomyces pombe.

Schizosaccharomyces pombe cells respond to nutrient deprivation by altering G2/M cell size control. The G2/M transition is controlled by activation of the cyclin-dependent kinase Cdc2p. Cdc2p activation is regulated both positively and negatively. cdr2(+) was identified in a screen for regulators of mitotic control during nutrient deprivation. We have cloned cdr2(+) and have found that it encodes a putative serine-threonine protein kinase that is related to Saccharomyces cerevisiae Gin4p and S. pombe Cdr1p/Nim1p. cdr2(+) is not essential for viability, but cells lacking cdr2(+) are elongated relative to wild-type cells, spending a longer period of time in G2. Because of this property, upon nitrogen deprivation cdr2(+) mutants do not arrest in G1, but rather undergo another round of S phase and arrest in G2 from which they are able to enter a state of quiescence. Genetic evidence suggests that cdr2(+) acts as a mitotic inducer, functioning through wee1(+), and is also important for the completion of cytokinesis at 36 degrees C. Defects in cytokinesis are also generated by the overproduction of Cdr2p, but these defects are independent of wee1(+), suggesting that cdr2(+) encodes a second activity involved in cytokinesis.

A computational fluid dynamics study of inspiratory flow in orotracheal geometries.

Computational fluid dynamics (CFD) has been used to investigate the flow of air through the human orotracheal system. Results from an idealised geometry, and from a patient-specific geometry created from MRI scans were compared. The results showed a significant difference in the flow structures between the two geometries. Inert particles with diameters in the range 1-9 microm were tracked through the two geometries. Particle diameter has proved to be an important factor in defining the eventual destinations of inhaled particles. Results from our calculations match other experimental and computational results in the literature, and differences between the idealised and patient-specific geometries are less significant.

Controlled plastic deformation for the fastening mechanism of an internal fixation device. The new Mennen 3 PeriPro plate.

The Mennen femur plate is a fixation device used for the treatment of femoral periprosthetic fractures. It features a novel fastening method where curved prongs are plastically deformed securing the implant to the bone. Although this "clamp-on" method has been successfully used to treat fractures of long bones, there are no literature data assessing the nature of the required plastic deformation. In the present study, the parameters influencing the performance of the prongs were identified and further explored using numerical modeling. The new Mennen 3 PeriPro plate is briefly discussed focusing on the new sculpted formation of the prongs. Their design was optimized to effectively control the magnitude and position of the required plastic deformation achieving enhanced anchorage on the fractured bone with minimum effort. The work presented contains all the necessary steps in analysing a clinical problem using finite elements and illustrates how effective use of simulation techniques can accurately predict and effectively control the required plastic deformation of a structure.

Bulging brains.

Brain swelling is a serious condition associated with an accumulation of fluid inside the brain that can be caused by trauma, stroke, infection, or tumors. It increases the pressure inside the skull and reduces blood and oxygen supply. To relieve the intracranial pressure, neurosurgeons remove part of the skull and allow the swollen brain to bulge outward, a procedure known as decompressive craniectomy. Decompressive craniectomy has been preformed for more than a century; yet, its effects on the swollen brain remain poorly understood. Here we characterize the deformation, strain, and stretch in bulging brains using the nonlinear field theories of mechanics. Our study shows that even small swelling volumes of 28 to 56 ml induce maximum principal strains in excess of 30%. For radially outward-pointing axons, we observe maximal normal stretches of 1.3 deep inside the bulge and maximal tangential stretches of 1.3 around the craniectomy edge. While the stretch magnitude varies with opening site and swelling region, our study suggests that the locations of maximum stretch are universally shared amongst all bulging brains. Our model has the potential to inform neurosurgeons and rationalize the shape and position of the skull opening, with the ultimate goal to reduce brain damage and improve the structural and functional outcomes of decompressive craniectomy in trauma patients.

Harnessing ester bond chemistry for protein ligation.

The hydrolysis potential of ester bonds in covalently cross-linking proteins is captured in our novel protein ligation technology. This new type of "molecular superglue" based on the spontaneously-formed Thr-Gln ester bonds found in cell-surface adhesins, affords a unique mechanism to both rationally assemble and disassemble complex protein nanomaterials.

Purification, crystallization and preliminary X-ray analysis of Mycobacterium tuberculosisfolylpolyglutamate synthase (MtbFPGS).

The gene encoding Mycobacterium tuberculosis FPGS (MtbFPGS; Rv2447c) has been cloned and the protein (51 kDa) expressed in Escherichia coli. The purified protein was crystallized either by the batch method in the presence of adenosine diphosphate (ADP) and CoCl2 or by vapour diffusion in the presence of ADP, dihydrofolate and CaCl2. X-ray diffraction data to approximately 2.0 and 2.6 A resolution were collected at the Stanford Synchrotron Radiation Laboratory (SSRL) for crystals grown under the respective conditions. Both crystals belong to the cubic space group P2(1)3, with a unit-cell parameter of 112.6 and 111.8 A, respectively. Structure determination is proceeding.

Insertional mutagenesis based on illegitimate recombination in Schizosaccharomyces pombe.

An efficient insertional mutagenesis system has been developed for Schizosaccharomyces pombe based on linear PCR-generated cassettes containing selectable markers. It depends upon illegitimate recombination for integration into the genome. Various selectable markers of different sizes can be used to obtain sufficiently high transformation and integration frequencies. Based on Southern blotting, a single insertion is found in each strain and integration sites are broadly distributed in the genome. Sequence analysis of the insert junctions frequently reveals small regions of homology (4-10 bp) between the ends of the integrated cassette and the disrupted gene. The system has been used for simple genetic screens of various types and as a promoter trap for in-frame GFP fusions.

Expression of hsp16 in response to nucleotide depletion is regulated via the spc1 MAPK pathway in Schizosaccharomyces pombe.

A universal response to elevated temperature and other forms of physiological stress is the induction of heat shock proteins (HSPs). Hsp16 in Schizosaccharomyces pombe encodes a polypeptide of predicted molecular weight 16 kDa that belongs to the HSP20/alpha-crystallin family whose members range in size from 12 to 43 kDa. Heat shock treatment increases expression of the hsp16 gene by 64-fold in wild-type cells and 141-fold in cdc22-M45 (ribonucleotide reductase) mutant cells. Hsp16 expression is mediated by the spc1 MAPK signaling pathway through the transcription factor atf1 and in addition through the HSF pathway. Nucleotide depletion or DNA damage as occurs in cdc22-M45 mutant cells, or during hydroxyurea or camptothecin treatment, is sufficient to activate hsp16 expression through atf1. Our findings suggest a novel role for small HSPs in the stress response following nucleotide depletion and DNA damage. This extends the types of damage that are sensed by the spc1 MAPK pathway via atf1.

Development of the mennen 3 peripro fixation plate for the treatment of periprosthetic fractures of the femur.

The Mennen femur plate is an internal fixation device used for the management of femoral perisprosthetic fractures, usually after total hip replacement surgery. The implant uses a number of curved prongs that embrace the fractured bone around its circumference without interfering with the stem of the prosthesis. Although the device has been used with considerable clinical success since its first introduction, a number of negative clinical results have been reported in the literature. The failure modes of the device are described and an evaluation of its performance is briefly presented. Based on this assessment as well as comments in the open literature, modifications in the design of the device have been implemented. The new Mennen 3 PeriPro plate is presented, with all the necessary data for a coherent explanation of its improved characteristics as defined using numerical simulations and experimental tests. The new device has all the beneficial features of the previous plate with improved structural performance and fatigue life and new sculpted formation of the prongs, providing a simple implantation technique with maximum gripping and minimum effort from the surgeon. The unique mode of fixation has been further improved, providing ample anchorage on the fracture bone without compromising its biomechanical integrity. By combining the device with a cable system, the spectrum of applications will be further expanded, enabling the surgeon to treat a broader range of fracture patterns.

Development of a geometrically accurate and adaptable finite element head model for impact simulation: the Naval Research Laboratory-Simpleware Head Model.

This study demonstrates a novel model generation methodology that addresses several limitations of conventional finite element head models (FEHM). By operating chiefly in image space, new structures can be incorporated or merged, and the mesh either decimated or refined both locally and globally. This methodology is employed in the development of a highly bio-fidelic FEHM from high-resolution scan data. The model is adaptable and presented here in a form optimised for impact and blast simulations. The accuracy and feasibility of the model are successfully demonstrated against a widely used experimental benchmark in impact loading and through the investigation of potential brain injury under blast overpressure loading.

stf1: non-wee mutations epistatic to cdc25 in the fission yeast Schizosaccharomyces pombe.

In Schizosaccharomyces pombe, cdc25 is a cell cycle regulated inducer of mitosis. wee1 and phenotypically wee alleles of cdc2 are epistatic to cdc25. Mutant alleles of a new locus, stf1 (suppressor of twenty-five), identified in a reversion analysis of conditionally lethal cdr1-76 cdc25-22 and cdr2-96 cdc25-22 double mutant strains, also suppress both temperature-sensitive and gene disruption alleles of cdc25. These mutants, by themselves, are phenotypically indistinguishable from wild type strains; hence they represent the first known mutations that are epistatic to cdc25 and do not display a wee phenotype. stf1 genetically interacts with other elements of mitotic control in S. pombe. stf1-1 is additive with wee1-50, cdc2-1w and cdc2-3w for suppression of cdc25-22. Also, like wee1- and cdc2-w, stf1- suppression of cdc25 is reversed by overexpression of the putative type 1 protein phosphatase bws1+/dis2+. Interaction with various mutants and plasmid overexpression experiments suggest that stf1 does not operate either upstream or downstream of wee1. Similarly, it does not operate through cdc25 since it rescues the disruption. stf1 appears to encode an important new element of mitotic control.

Genetic and molecular analysis of cdr1/nim1 in Schizosaccharomyces pombe.

The cdr1 gene in Schizosaccharomyces pombe was identified as a mutation affecting the nutritional responsiveness of the mitotic size control. cdr1 alleles have been further analyzed for genetic interactions with elements of the mitotic control pathway and cloned by plasmid rescue of a conditional lethal cdr1-76 cdc25-22 double mutant. These analyses show that the cdr1 gene is allelic to nim1, a gene identified as a high copy number plasmid suppressor of the mitotic control gene, cdc25. The gene structure for cdr1 differs from the described nim1 gene in the carboxyl-terminal portion of the gene. The published nim1 sequence encoded a product of predicted Mr 45,000, and included 356 amino acids from the amino-terminal region of the gene and 14 amino acids from a noncontiguous carboxyl-terminal fragment. The cdr1 sequence includes an additional 237 amino acids of the contiguous fragment and encodes a product of predicted Mr 67,000. The sequence shows a high level of identity with protein kinases over the amino-terminal catalytic domain, and limited identity with yeast protein kinases SNF1, KIN2 and KIN1 over part of the carboxyl-terminal domain. The effect of overexpression of the full length gene has been examined in various genetic backgrounds. These data show that the full length gene product is required to give a normal cell cycle response to nitrogen starvation. A detailed examination of the genetic interaction of cdr1 mutants with various mutants of mitotic control genes (cdc2, cdc25, wee1, cdc13) demonstrated strong interactions with cdc25, some cdc2 alleles, and with cdc13-117. Overall, the results are interpretable within the framework of the existing model of cdr1/nim1 action in mitotic control, i.e., cdr1 functions upstream of wee1 to relieve mitotic inhibition.

Mutants of Arabidopsis thaliana Capable of Germination under Saline Conditions.

Three mutant strains of Arabidopsis thaliana var Columbia were selected for their ability to germinate in elevated concentrations of NaCl. They were not more tolerant than wild type at subsequent development stages. Wild-type strains could not germinate at concentrations > 125 mM NaCl. Two of mutant strains, RS17 and RS20, could withstand up to 225 mM, whereas RS19 was resistant to 175 mM. The RS mutants could also germinate under even lower osmotic potentials imposed by high concentrations of exogenous mannitol (550 mM), whereas the effects of elevated levels of KCl, K2SO4, and LiCl were similar among the mutants and wild type. Therefore, the mutants are primarily osmotolerant, but they also possess a degree of ionic tolerance for sodium. Sodium and potassium contents of seeds exposed to high salinities indicated that the NaCl-tolerant mutants absorbed more of these respective cations during imbibition. These higher internal concentrations of potassium and sodium could contribute to the osmotic adjustment of the germinating seeds to the low osmotic potential of the external medium. Genetic analysis of F1 and F2 progeny of outcrosses suggest that the salt-tolerant mutations are recessive and that they define three complementation groups.

On the use of a patient-specific rapid-prototyped model to simulate the response of the human head to impact and comparison with analytical and finite element models.

Every year, thousands of fatalities result from head injuries, the majority of which are sustained in automotive accidents. In this paper, an experimental study of the response of the human head to impact is presented. A rapid prototyped model of a human head was generated based on high-resolution magnetic resonance imaging (MRI) scan data. The physical model was subjected to low velocity impacts using a metallic pendulum and a sensitivity study was performed to explore the influence of various parameters, including mass and velocity of the impactor, on the response. The experimental response characteristics are compared with predictions from an analytical model as well as with numerical predictions from finite element (FE) models generated from the same MRI data set. The results from the experimental tests closely match those predicted by both the analytical and the FE models and thus provide us with substantive corroboration of all three approaches. The remarkable agreement obtained between the measured response characteristics of rapid-prototyped skulls and numerical (FE) models obtained from in vivo MRI data clearly demonstrates the potential use of rapid-prototyping to generate experimental models for head impact studies, and, more generally, for the study of the response of complex bio-structures to loading. In addition, the quantitative and qualitative accuracy of the predictions from the analytical model is clearly demonstrated by the FE and experimental corroboration. In particular, the analytical prediction that, as impact mass drops the impact duration becomes increasingly short, appears to be substantiated, which has important implications for the onset of high pressure and shear strain gradients in the brain with potentially deleterious effects.

Sequence of the Schizosaccharomyces pombe gtp1 gene and identification of a novel family of putative GTP-binding proteins.

A new gene, gtp1, has been identified by sequence analysis in Schizosaccharomyces pombe. The open reading frame was identified downstream from the stf1 locus. The deduced GTP1 protein has strong sequence similarity to a family of putative GTP-binding proteins from Halobacterium cutirubrum, Bacillus subtilis, Drosophila melanogaster and mouse. The conserved P-loop phosphate-binding motif places gtp1 in a family separate from previously described groups of such proteins.

Identification and localization of the sod2 gene product in fission yeast.

Sod2, the Na+/H+ antiporter of the fission yeast Schizosaccharomyces pombe, was identified by addition of a hemagglutinin tag to the carboxyl terminus of the protein. The tagged protein was expressed in the sod2-deficient strain of S. pombe. Transformants retained tolerance to lithium (1-10 mM) at external pH values from 3.5 to 6.5. Both Na+-dependent proton uptake and active sodium extrusion were also restored in transformed cells, suggesting that a functional antiporter was present. The protein was present in a membrane fraction. In SDS PAGE it migrated as a single 47 kDa band. The protein could be efficiently solubilized with the non-ionic detergent, dodecyl maltoside. Immunofluorescent microscopy revealed an asymmetric distribution with preferable accumulation in polar tip areas. The results are the first identification and localization of the Na+/H+ exchanger in yeast cells.