Zinc oxide nanoparticles mediate bacterial toxicity in Mueller-Hinton Broth via Zn2.

As antibiotic resistance increases and antibiotic development dwindles, new antimicrobial agents are needed. Recent advances in nanoscale engineering have increased interest in metal oxide nanoparticles, particularly zinc oxide nanoparticles, as antimicrobial agents. Zinc oxide nanoparticles are promising due to their broad-spectrum antibacterial activity and low production cost. Despite many studies demonstrating the effectiveness of zinc oxide nanoparticles, the antibacterial mechanism is still unknown. Previous work has implicated the role of reactive oxygen species such as hydrogen peroxide, physical damage of the cell envelope, and/or release of toxic Zn2+ ions as possible mechanisms of action. To evaluate the role of these proposed methods, we assessed the susceptibility of S. aureus mutant strains, ΔkatA and ΔmprF, to zinc oxide nanoparticles of approximately 50 nm in size. These assays demonstrated that hydrogen peroxide and electrostatic interactions are not crucial for mediating zinc oxide nanoparticle toxicity. Instead, we found that Zn2+ accumulates in Mueller-Hinton Broth over time and that removal of Zn2+ through chelation reverses this toxicity. Furthermore, we found that the physical separation of zinc oxide nanoparticles and bacterial cells using a semi-permeable membrane still allows for growth inhibition. We concluded that soluble Zn2+ is the primary mechanism by which zinc oxide nanoparticles mediate toxicity in Mueller-Hinton Broth. Future work investigating how factors such as particle morphology (e.g., size, polarity, surface defects) and media contribute to Zn2+ dissolution could allow for the synthesis of zinc oxide nanoparticles that possess chemical and morphological properties best suited for antibacterial efficacy.

Nanocrystalline Cubic Phase Scandium-Stabilized Zirconia Thin Films.

The cubic zirconia (ZrO2) is attractive for a broad range of applications. However, at room temperature, the cubic phase needs to be stabilized. The most studied stabilization method is the addition of the oxides of trivalent metals, such as Sc2O3. Another method is the stabilization of the cubic phase in nanostructures-nanopowders or nanocrystallites of pure zirconia. We studied the relationship between the size factor and the dopant concentration range for the formation and stabilization of the cubic phase in scandium-stabilized zirconia (ScSZ) films. The thin films of (ZrO2)1-x(Sc2O3)x, with x from 0 to 0.2, were deposited on room-temperature substrates by reactive direct current magnetron co-sputtering. The crystal structure of films with an average crystallite size of 85 Å was cubic at Sc2O3 content from 6.5 to 17.5 mol%, which is much broader than the range of 8-12 mol.% of the conventional deposition methods. The sputtering of ScSZ films on hot substrates resulted in a doubling of crystallite size and a decrease in the cubic phase range to 7.4-11 mol% of Sc2O3 content. This confirmed that the size of crystallites is one of the determining factors for expanding the concentration range for forming and stabilizing the cubic phase of ScSZ films.

Surface Photovoltage Response of ZnO to Phosphate-Buffered Saline Solution with and without Presence of Staphylococcus aureus.

Nano- and microscale zinc oxide (ZnO) exhibits significant potential as a novel antibacterial agent in biomedical applications. However, the uncertainty regarding the underlying mechanisms of the observed antimicrobial action inhibits the realization of this potential. Particularly, the nature of interactions at the free crystalline surface and the influence of the local bacterial environment remains unclear. In this investigation, we utilize ZnO particles synthesized via tunable hydrothermal growth method as a platform to elucidate the effects of interactions with phosphate-rich environments and differentiate them from those with bacteria. This is achieved using the time- and energy-dependent surface photovoltage (SPV) to monitor modifications of the surface electronic structure and surface charge dynamics of the ZnO particles due to these interactions. It is found that there exists a dramatic change in the SPV transients after exposure to phosphate-rich environments. It also presents differences in the sub-bandgap surface electronic structure after these exposures. It can be suggested that these phenomena are a consequence of phosphate adsorption at surface traps corresponding to zinc deficiency defects. This effect is shown to be suppressed in the presence of Staphylococcus aureus bacteria. Our results support the previously proposed model of the competitive nature of interactions between S. aureus and aqueous phosphates with the free surface of ZnO and bring greater clarity to the effects of phosphate-rich environments on bacterial growth inhibition of ZnO.

Estimates of fine fuel litter biomass in the northern Great Basin reveal increases during short fire-free intervals associated with invasive annual grasses.

Exotic annual grasses invasion across northern Great Basin rangelands has promoted a grass-fire cycle that threatens the sagebrush (Artemisia spp.) steppe ecosystem. In this sense, high accumulation rates and persistence of litter from annual species largely increase the amount and continuity of fine fuels. Here, we highlight the potential use and transferability of remote sensing-derived products to estimate litter biomass on sagebrush rangelands in southeastern Oregon, and link fire regime attributes (fire-free period) with litter biomass spatial patterns at the landscape scale. Every June, from 2018 to 2021, we measured litter biomass in 24 field plots (60 m × 60 m). Two remote sensing-derived datasets were used to predict litter biomass measured in the field plots. The first dataset used was the 30-m annual net primary production (NPP) product partitioned into plant functional traits (annual grass, perennial grass, shrub, and tree) from the Rangeland Analysis Platform (RAP). The second dataset included topographic variables (heat load index -HLI- and site exposure index -SEI-) computed from the USGS 30-m National Elevation Dataset. Through a frequentist model averaging approach (FMA), we determined that the NPP of annual and perennial grasses, as well as HLI and SEI, were important predictors of field-measured litter biomass in 2018, with the model featuring a high overall fit (R2 = 0.61). Model transferability based on extrapolating the FMA predictive relationships from 2018 to the following years provided similar overall fits (R2 ≈ 0.5). The fire-free period had a significant effect on the litter biomass accumulation on rangelands within the study site, with greater litter biomass in areas where the fire-free period was <10 years. Our findings suggest that the proposed remote sensing-derived products could be a key instrument to equip rangeland managers with additional information towards fuel management, fire management, and restoration efforts.

City limits: Heat tolerance is influenced by body size and hydration state in an urban ant community.

Cities are rapidly expanding, and global warming is intensified in urban environments due to the urban heat island effect. Therefore, urban animals may be particularly susceptible to warming associated with ongoing climate change. We used a comparative and manipulative approach to test three related hypotheses about the determinants of heat tolerance or critical thermal maximum (CT max) in urban ants-specifically, that (a) body size, (b) hydration status, and (c) chosen microenvironments influence CT max. We further tested a fourth hypothesis that native species are particularly physiologically vulnerable in urban environments. We manipulated water access and determined CT max for 11 species common to cities in California's Central Valley that exhibit nearly 300-fold variation in body size. There was a moderate phylogenetic signal influencing CT max, and inter (but not intra) specific variation in body size influenced CT max where larger species had higher CT max. The sensitivity of ants' CT max to water availability exhibited species-specific thresholds where short-term water limitation (8 hr) reduced CT max and body water content in some species while longer-term water limitation (32 hr) was required to reduce these traits in other species. However, CT max was not related to the temperatures chosen by ants during activity. Further, we found support for our fourth hypothesis because CT max and estimates of thermal safety margin in native species were more sensitive to water availability relative to non-native species. In sum, we provide evidence of links between heat tolerance and water availability, which will become critically important in an increasingly warm, dry, and urbanized world that others have shown may be selecting for smaller (not larger) body size.

Development and Validation of the Gene Expression Predictor of High-grade Serous Ovarian Carcinoma Molecular SubTYPE (PrOTYPE).

PURPOSE: Gene expression-based molecular subtypes of high-grade serous tubo-ovarian cancer (HGSOC), demonstrated across multiple studies, may provide improved stratification for molecularly targeted trials. However, evaluation of clinical utility has been hindered by nonstandardized methods, which are not applicable in a clinical setting. We sought to generate a clinical grade minimal gene set assay for classification of individual tumor specimens into HGSOC subtypes and confirm previously reported subtype-associated features. EXPERIMENTAL DESIGN: Adopting two independent approaches, we derived and internally validated algorithms for subtype prediction using published gene expression data from 1,650 tumors. We applied resulting models to NanoString data on 3,829 HGSOCs from the Ovarian Tumor Tissue Analysis consortium. We further developed, confirmed, and validated a reduced, minimal gene set predictor, with methods suitable for a single-patient setting. RESULTS: Gene expression data were used to derive the predictor of high-grade serous ovarian carcinoma molecular subtype (PrOTYPE) assay. We established a de facto standard as a consensus of two parallel approaches. PrOTYPE subtypes are significantly associated with age, stage, residual disease, tumor-infiltrating lymphocytes, and outcome. The locked-down clinical grade PrOTYPE test includes a model with 55 genes that predicted gene expression subtype with >95% accuracy that was maintained in all analytic and biological validations. CONCLUSIONS: We validated the PrOTYPE assay following the Institute of Medicine guidelines for the development of omics-based tests. This fully defined and locked-down clinical grade assay will enable trial design with molecular subtype stratification and allow for objective assessment of the predictive value of HGSOC molecular subtypes in precision medicine applications.See related commentary by McMullen et al., p. 5271.

To burn or not to burn: Comparing reintroducing fire with cutting an encroaching conifer for conservation of an imperiled shrub-steppe.

Woody vegetation has increased on rangelands worldwide for the past 100-200 years, often because of reduced fire frequency. However, there is a general aversion to reintroducing fire, and therefore, fire surrogates are often used in its place to reverse woody plant encroachment. Determining the conservation effectiveness of reintroducing fire compared with fire surrogates over different time scales is needed to improve conservation efforts. We evaluated the conservation effectiveness of reintroducing fire with a fire surrogate (cutting) applied over the last ~30 years to control juniper (Juniperus occidentalis Hook.) encroachment on 77 sagebrush-steppe sites. Critical to conservation of this imperiled ecosystem is to limit juniper, not encourage exotic annual grasses, and promote sagebrush dominance of the overstory. Reintroducing fire was more effective than cutting at reducing juniper abundance and extending the period of time that juniper was not dominating the plant community. Sagebrush was reduced more with burning than cutting. Sagebrush, however, was predicted to be a substantial component of the overstory longer in burned than cut areas because of more effective juniper control. Variation in exotic annual grass cover was explained by environmental variables and perennial grass abundance, but not treatment, with annual grasses being problematic on hotter and drier sites with less perennial grass. This suggests that ecological memory varies along an environmental gradient. Reintroducing fire was more effective than cutting at conserving sagebrush-steppe encroached by juniper over extended time frames; however, cutting was more effective for short-term conservation. This suggests fire and fire surrogates both have critical roles in conservation of imperiled ecosystems.

The Association Between Diabetes Mellitus and Atrial Fibrillation: Clinical and Mechanistic Insights.

A number of clinical studies have reported that diabetes mellitus (DM) is an independent risk factor for Atrial fibrillation (AF). After adjustment for other known risk factors including age, sex, and cardiovascular risk factors, DM remains a significant if modest risk factor for development of AF. The mechanisms underlying the increased susceptibility to AF in DM are incompletely understood, but are thought to involve electrical, structural, and autonomic remodeling in the atria. Electrical remodeling in DM may involve alterations in gap junction function that affect atrial conduction velocity due to changes in expression or localization of connexins. Electrical remodeling can also occur due to changes in atrial action potential morphology in association with changes in ionic currents, such as sodium or potassium currents, that can affect conduction velocity or susceptibility to triggered activity. Structural remodeling in DM results in atrial fibrosis, which can alter conduction patterns and susceptibility to re-entry in the atria. In addition, increases in atrial adipose tissue, especially in Type II DM, can lead to disruptions in atrial conduction velocity or conduction patterns that may affect arrhythmogenesis. Whether the insulin resistance in type II DM activates unique intracellular signaling pathways independent of obesity requires further investigation. In addition, the relationship between incident AF and glycemic control requires further study.

Usefulness of Released Cardiac Myosin Binding Protein-C as a Predictor of Cardiovascular Events.

Cardiac myosin binding protein-C (cMyBP-C) is a heart muscle-specific thick filament protein. Elevated level of serum cMyBP-C is an indicator of early myocardial infarction (MI), but its value as a predictor of future cardiovascular disease is unknown. Based on the presence of significant amount of cMyBP-C in the serum of previous study subjects independent of MI, we hypothesized that circulating cMyBP-C is a sensitive indicator of ongoing cardiovascular stress and disease. To test this hypothesis, 75 men and 83 women of similar ages were recruited for a prospective study. They underwent exercise stress echocardiography to provide pre- and poststress blood samples for subsequent determination of serum cMyBP-C levels. The subjects were followed for 1 to 1.5 years. Exercise stress increased serum cMyBP-C in all subjects. Twenty-seven primary events (such as death, MI, revascularization, invasive cardiovascular procedure, or cardiovascular-related hospitalization) and 7 critical events (CE; such as death, MI, stroke, or pulmonary embolism) occurred. After adjusting for sex and cardiovascular risk factors with multivariate Cox regression, a 96% sensitive prestress cMyBP-C threshold carried a hazard ratio of 8.1 with p = 0.041 for primary events. Most subjects (6 of 7) who had CE showed normal ejection fraction on echocardiography. Prestress cMyBP-C demonstrated area under receiver operating curve of 0.91 and multivariate Cox regression hazard ratio of 13.8 (p = 0.000472) for CE. Thus, basal cMyBP-C levels reflected susceptibility for a variety of cardiovascular diseases. Together with its high sensitivity, cMyBP-C holds potential as a screening biomarker for the existence of severe cardiovascular diseases.

Can dual-energy computed tomography improve visualization of hypoenhancing liver lesions in portal venous phase? Assessment of advanced image-based virtual monoenergetic images.

PURPOSE: The purpose was to assess image quality of portal-venous phase dual-energy computed tomography (DECT) for liver lesions. METHODS: We performed 120-kVp-equivalent linear-blended (LB) and monoenergetic reconstructions from 40 to 190 keV by standard (VMI) and advanced virtual monoenergetic (VMI+) methods. Diagnostic performance, and quantitative and qualitative image analyses were assessed and compared. RESULTS: Liver contrast to noise ratio peaked at 40 keV_VMI+, while image quality and reader preference peaked at 50 keV_VMI+. 50 keV_VMI+ scored overall higher diagnostic performance: lesion sensitivity 95.4% vs. 83.3% for both 75 keV_VMI and LB. CONCLUSIONS: DECT improves assessment of hypoenhancing liver lesions on portal venous phase. 50 keV_VMI+ demonstrated the highest image quality and diagnostic performance over VMI and LB.

Use of Impella 5.0 Prior to Transcatheter Aortic Valve Replacement in a Patient with Severe Aortic Stenosis and Cardiogenic Shock.

The use of transcatheter aortic valve replacement (TAVR) has risen dramatically during the past decade. Notably, the procedure can reduce symptoms and improve prognosis in patients who would otherwise be excluded from intervention due to prohibitive surgical risk. During the same time period, the use of percutaneous mechanical circulatory support devices for patients with severe left ventricular dysfunction, including the Impella System (Abiomed, Inc., Danvers, MA, USA), has also increased. These devices provide superior hemodynamic effects compared to intra-aortic balloon pumping. However, data relating to use of the Impella in patients with both severe left ventricular dysfunction and significant aortic valve disease are limited. Herein is presented the case of a 59-year-old man with severe aortic stenosis and cardiogenic shock who was treated successfully with an Impella 5.0 as a temporizing measure prior to TAVR. A brief review is also provided of the use of mechanical circulatory support devices in this subset of patients. Video 1: Transesophageal echocardiography video demonstrating stenotic aortic valve pre-intervention in side-by-side long and short axes. Video 2: Transthoracic echocardiography video, parasternal short axis, demonstrating Impella 5 in the left ventricle. Video 3: Fluoroscopy video demonstrating deployment of the Edwards SAPIEN transcatheter aortic valve. Video 4: Transesophageal echocardiography video showing the Edwards SAPIEN transcatheter aortic valve in long axis. Video 5: Transesophageal echocardiography video showing the Edwards SAPIEN transcatheter aortic valve in short axis.

Approach to MR Imaging of the Elbow and Wrist: Technical Aspects and Innovation.

Wrist and elbow MR imaging technology is advancing at a dramatic rate. Wrist and elbow MR imaging is performed at medium and higher field strengths with more specialized surface coils and more variable pulse sequences and postprocessing techniques. High field imaging and improved coils lead to an increased signal-to-noise ratio and increased variety of soft tissue contrast options. Three-dimensional imaging is improving in terms of usability and artifacts. Some of these advances have challenges in wrist and elbow imaging, such as postoperative patient imaging, cartilage mapping, and molecular imaging. This review considers technical advances in hardware and software and their clinical applications.

MRI in diagnosis of a giant prostatic utricle.

A prostatic utricle cyst is an epithelial lined diverticulum arising from the prostatic urethra and usually asymptomatic when small. When enlarged, it may be symptomatic and is typically accompanied by hypospadias. We present a case of a markedly enlarged prostatic utricle in a neonate without hypospadias, demonstrated on voiding cystourethrography (VCUG), ultrasound, and 1.5 Tesla MRI.

Perceptions of ranchers about medusahead (Taeniatherum caput-medusae (L.) Nevski) management on sagebrush steppe rangelands.

Medusahead (Taeniatherum caput-medusae (L.) Nevski) is an exotic annual grass invading rangelands in the western United States. Medusahead is a serious management concern because it decreases biodiversity, reduces livestock forage production, and degrades the ecological function of rangelands. Despite the obvious importance of ranchers as partners in preventing and managing medusahead in rangelands, little is known about their perceptions and behaviors concerning medusahead management. We present the results of a survey of ranchers operating on sagebrush steppe rangeland in a three-county area in southeast Oregon encompassing over 7.2 million ha. The primary objective of this research was to determine if the presence of medusahead on a ranch influenced its operator's perceptions and behaviors concerning invasive plant control and prevention. Ranchers operating on medusahead-infested rangeland were more likely to indicate increased awareness and concern about medusahead and the potential for its continued expansion. Ranchers operating on rangeland invaded by medusahead were also more likely to indicate use of measures to prevent the spread of medusahead and other invasive plants on rangeland, interest in educational opportunities concerning invasive annual grass management, and plans for controlling invasive annual grasses in the future. This study revealed an alarming trend in which individuals are less likely to implement important prevention measures and participate in education opportunities to improve their knowledge of invasive plants until they directly experience the negative consequences of invasion. Information campaigns on invasive plants and their impacts may rectify this problem; however, appropriate delivery methods are critical for success. Web- or computer-based invasive plant information and tools were largely unpopular among ranchers, whereas traditional forms of information delivery including brochures/pamphlets and face-to-face interaction were preferred. However, in the future web- or computer-based information may become more popular as ranchers become more familiar with them.

A novel inhibitor of Chlamydophila pneumoniae protein kinase D (PknD) inhibits phosphorylation of CdsD and suppresses bacterial replication.

BACKGROUND: We have shown previously that Chlamydophila pneumoniae contains a dual-specific Ser/Thr protein kinase that phosphorylates CdsD, a structural component of the type III secretion apparatus. To further study the role of PknD in growth and development we sought to identify a PknD inhibitor to determine whether PknD activity is required for replication. RESULTS: Using an in vitro kinase assay we screened 80 known eukaryotic protein kinase inhibitors for activity against PknD and identified a 3'-pyridyl oxindole compound that inhibited PknD autophosphorylation and phosphorylation of CdsD. The PknD inhibitor significantly retarded the growth rate of C. pneumoniae as evidenced by the presence of very small inclusions with a reduced number of bacteria as seen by electron microscopy. These inclusions contained the normal replicative forms including elementary bodies (EB), intermediate bodies (IB) and reticulate bodies (RB), but lacked persistent bodies (PB), indicating that induction of persistence was not the cause of reduced chlamydial growth. Blind passage of C. pneumoniae grown in the presence of this PknD inhibitor for 72 or 84 hr failed to produce inclusions, suggesting this compound blocks an essential step in the production of infectious chlamydial EB. The compound was not toxic to HeLa cells, did not block activation of the MEK/ERK pathway required for chlamydial invasion and did not block intracellular replication of either Chlamydia trachomatis serovar D or Salmonella enterica sv. Typhimurium suggesting that the inhibitory effect of the compound is specific for C. pneumoniae. CONCLUSION: We have identified a 3'-pyridyl oxindole compound that inhibits the in vitro kinase activity of C. pneumoniae PknD and inhibits the growth and production of infectious C. pneumoniae progeny in HeLa cells. Together, these results suggest that PknD may play a key role in the developmental cycle of C. pneumoniae.

Influence of mowing Artemisia tridentata ssp. wyomingensis on winter habitat for wildlife.

Mowing is commonly implemented to Artemisia tridentata ssp. wyomingensis (Beetle & A. Young) S.L. Welsh (Wyoming big sagebrush) plant communities to improve wildlife habitat, increase forage production for livestock, and create fuel breaks for fire suppression. However, information detailing the influence of mowing on winter habitat for wildlife is lacking. This information is crucial because many wildlife species depended on A. tridentata spp. wyomingensis plant communities for winter habitat and consume significant quantities of Artemisia during this time. Furthermore, information is generally limited describing the recovery of A. tridentata spp. wyomingensis to mowing and the impacts of mowing on stand structure. Stand characteristics and Artemisia leaf tissue crude protein (CP), acid detergent fiber (ADF), and neutral detergent fiber (NDF) concentrations were measured in midwinter on 0-, 2-, 4-, and 6-year-old fall-applied mechanical (mowed at 20 cm height) treatments and compared to adjacent untreated (control) areas. Mowing compared to the control decreased Artemisia cover, density, canopy volume, canopy elliptical area, and height (P < 0.05), but all characteristics were recovering (P < 0.05). Mowing A. tridentata spp. wyomingensis plant communities slightly increases the nutritional quality of Artemisia leaves (P < 0.05), but it simultaneously results in up to 20 years of decrease in Artemisia structural characteristics. Because of the large reduction in A. tridentata spp. wyomingensis for potentially 20 years following mowing, mowing should not be applied in Artemisia facultative and obligate wildlife winter habitat. Considering the decline in A. tridentata spp. wyomingensis-dominated landscapes, we caution against mowing these communities.

Characterization of the putative type III secretion ATPase CdsN (Cpn0707) of Chlamydophila pneumoniae.

Type III secretion (T3S) is utilized by a wide range of gram-negative bacterial pathogens to allow the efficient delivery of effector proteins into the host cell cytoplasm through the use of a syringe-like injectisome. Chlamydophila pneumoniae is a gram-negative, obligate intracellular pathogen that has the structural genes coding for a T3S system, but the functionality of the system has not yet been demonstrated. T3S is dependent on ATPase activity, which catalyzes the unfolding of proteins and the secretion of effector proteins through the injectisome. CdsN (Cpn0707) is predicted to be the T3S ATPase of C. pneumoniae based on sequence similarity to other T3S ATPases. Full-length CdsN and a C-terminal truncation of CdsN were cloned as glutathione S-transferase (GST)-tagged constructs and expressed in Escherichia coli. The GST-tagged C-terminal truncation of CdsN possessed ATPase activity, catalyzing the release of ADP and P(i) from ATP at a rate of 0.55 +/- 0.07 micromol min(-1) mg(-1) in a time- and dose-dependent manner. CdsN formed oligomers and high-molecular-weight multimers, as assessed by formaldehyde fixation and nondenaturing polyacrylamide gel electrophoresis. Using bacterial two-hybrid and GST pull-down assays, CdsN was shown to interact with CdsD, CdsL, CdsQ, and CopN, four putative structural components of the C. pneumoniae T3S system. CdsN also interacted with an unannotated protein, Cpn0706, a putative CdsN chaperone. Interactions between CdsN, CdsD, and CopN represent novel interactions not previously reported for other bacterial T3S systems and may be important in the localization and/or function of the ATPase at the inner membrane of C. pneumoniae.

Comparison of hand-traced and computerized cephalograms: landmark identification, measurement, and superimposition accuracy.

INTRODUCTION: The purposes of this study were (1) to investigate the variations of landmark identification between film and digital cephalometric tracings, (2) to compare the ability of Quick Ceph 2000 (Quick Ceph Systems, Inc, San Diego, Calif) to measure the linear and angular measurements with the hand-traced method, and (3) to compare Quick Ceph 2000 superimpositions to the hand-traced method of superimpositions that are currently accepted by the American Board of Orthodontics (ABO). MATERIALS: We used 30 sets of serial cephalometric radiographs of growing patients from 1 orthodontic office. Fiduciary x- and y-axes were drawn in pencil on the T1 radiographs in the regions of the cranial base, the maxilla, and the mandible. The fiduciary lines were transferred to the digital and film serial cephalograms by regionally superimposing the tracings as described in the ABO Phase III examination handbook. A Mann-Whitney test was done to compare the median and Delta of the T1 and T2 values for each measurement acquired by hand and by Quick Ceph. RESULTS AND CONCLUSIONS: There was no difference in the identification of cephalometric landmarks made manually vs digitally with Quick Ceph 2000. There was no difference in acquiring consistent cephalometric values for the measurements required by the ABO for the Phase III clinical examination manually vs digitally by using Quick Ceph 2000. There was no difference in the regional superimpositions of the mandible, the maxilla, and the cranial base, manually vs digitally with Quick Ceph 2000.

Interactions between CdsD, CdsQ, and CdsL, three putative Chlamydophila pneumoniae type III secretion proteins.

Chlamydophila pneumoniae is a gram-negative obligate intracellular bacterial pathogen that causes pneumonia and bronchitis and may contribute to atherosclerosis. The developmental cycle of C. pneumoniae includes a morphological transition from an infectious extracellular elementary body (EB) to a noninfectious intracellular reticulate body (RB) that divides by binary fission. The C. pneumoniae genome encodes a type III secretion (T3S) apparatus that may be used to infect eukaryotic cells and to evade the host immune response. In the present study, Cpn0712 (CdsD), Cpn0704 (CdsQ), and Cpn0826 (CdsL), three C. pneumoniae genes encoding yersiniae T3S YscD, YscQ, and YscL homologs, respectively, were cloned and expressed as histidine- and glutathione S-transferase (GST)-tagged proteins in Escherichia coli. Purified recombinant proteins were used to raise hyper-immune polyclonal antiserum and were used in GST pull-down and copurification assays to identify protein-protein interactions. CdsD was detected in both EB and RB lysates by Western blot analyses, and immunofluorescent staining demonstrated the presence of CdsD within inclusions. Triton X-114 solubilization and phase separation of chlamydial EB proteins indicated that CdsD partitions with cytoplasmic proteins, suggesting it is not an integral membrane protein. GST pull-down assays indicated that recombinant CdsD interacts with CdsQ and CdsL, and copurification assays with chlamydial lysates confirmed that native CdsD interacts with CdsQ and CdsL. To the best of our knowledge, this is the first report demonstrating interactions between YscD, YscQ, and YscL homologs of bacterial T3S systems. These novel protein interactions may play important roles in the assembly or function of the chlamydial T3S apparatus.

Chlamydophila pneumoniae PknD exhibits dual amino acid specificity and phosphorylates Cpn0712, a putative type III secretion YscD homolog.

Chlamydophila pneumoniae is an obligate intracellular bacterium that causes bronchitis, pharyngitis, and pneumonia and may be involved in atherogenesis and Alzheimer's disease. Genome sequencing has identified three eukaryote-type serine/threonine protein kinases, Pkn1, Pkn5, and PknD, that may be important signaling molecules in Chlamydia. Full-length PknD was cloned and expressed as a histidine-tagged protein in Escherichia coli. Differential centrifugation followed by sodium carbonate treatment of E. coli membranes demonstrated that His-PknD is an integral membrane protein. Fusions of overlapping PknD fragments to alkaline phosphatase revealed that PknD contains a single transmembrane domain and that the kinase domain is in the cytoplasm. To facilitate solubility, the kinase domain was cloned and expressed as a glutathione S-transferase (GST) fusion protein in E. coli. Purified GST-PknD kinase domain autophosphorylated, and catalytic mutants (K33G, D156G, and K33G-D156G mutants) and activation loop mutants (T185A and T193A) were inactive. PknD phosphorylated recombinant Cpn0712, a type III secretion YscD homolog that has two forkhead-associated domains. Thin-layer chromatography revealed that the PknD kinase domain autophosphorylated on threonine and tyrosine and phosphorylated the FHA-2 domain of Cpn0712 on serine and tyrosine. To our knowledge, this is the first demonstration of a bacterial protein kinase with amino acid specificity for both serine/threonine and tyrosine residues and this is the first study to show phosphorylation of a predicted type III secretion structural protein.