Mechanics of lung cancer: A finite element model shows strain amplification during early tumorigenesis.

Early lung cancer lesions develop within a unique microenvironment that undergoes constant cyclic stretch from respiration. While tumor stiffening is an established driver of tumor progression, the contribution of stress and strain to lung cancer is unknown. We developed tissue scale finite element models of lung tissue to test how early lesions alter respiration-induced strain. We found that an early tumor, represented as alveolar filling, amplified the strain experienced in the adjacent alveolar walls. Tumor stiffening further increased the amplitude of the strain in the adjacent alveolar walls and extended the strain amplification deeper into the normal lung. In contrast, the strain experienced in the tumor proper was less than the applied strain, although regions of amplification appeared at the tumor edge. Measurements of the alveolar wall thickness in clinical and mouse model samples of lung adenocarcinoma (LUAD) showed wall thickening adjacent to the tumors, consistent with cellular response to strain. Modeling alveolar wall thickening by encircling the tumor with thickened walls moved the strain amplification radially outward, to the next adjacent alveolus. Simulating iterative thickening in response to amplified strain produced tracks of thickened walls. We observed such tracks in early-stage clinical samples. The tracks were populated with invading tumor cells, suggesting that strain amplification in very early lung lesions could guide pro-invasive remodeling of the tumor microenvironment. The simulation results and tumor measurements suggest that cells at the edge of a lung tumor and in surrounding alveolar walls experience increased strain during respiration that could promote tumor progression.

Disruption of VirB6 Paralogs in Anaplasma phagocytophilum Attenuates Its Growth.

Many pathogenic bacteria translocate virulence factors into their eukaryotic hosts by means of type IV secretion systems (T4SS) spanning the inner and outer membranes. Genes encoding components of these systems have been identified within the order Rickettsiales based upon their sequence similarities to other prototypical systems. Anaplasma phagocytophilum strains are obligate intracellular, tick-borne bacteria that are members of this order. The organization of these components at the genomic level was determined in several Anaplasma phagocytophilum strains, showing overall conservation, with the exceptions of the virB2 and virB6 genes. The virB6 loci are characterized by the presence of four virB6 copies (virB6-1 through virB6-4) arranged in tandem within a gene cluster known as the sodB-virB operon. Interestingly, the virB6-4 gene varies significantly in length among different strains due to extensive tandem repeats at the 3' end. To gain an understanding of how these enigmatic virB6 genes function in A. phagocytophilum, we investigated their expression in infected human and tick cells. Our results show that these genes are expressed by A. phagocytophilum replicating in both cell types and that VirB6-3 and VirB6-4 proteins are surface exposed. Analysis of an A. phagocytophilum mutant carrying the Himar1 transposon within the virB6-4 gene demonstrated that the insertion not only disrupted its expression but also exerted a polar effect on the sodB-virB operon. Moreover, the altered expression of genes within this operon was associated with the attenuated in vitro growth of A. phagocytophilum in human and tick cells, indicating the importance of these genes in the physiology of this obligate intracellular bacterium in such different environments.IMPORTANCE Knowledge of the T4SS is derived from model systems, such as Agrobacterium tumefaciens The structure of the T4SS in Rickettsiales differs from the classical arrangement. These differences include missing and duplicated components with structural alterations. Particularly, two sequenced virB6-4 genes encode unusual C-terminal structural extensions resulting in proteins of 4,322 (GenBank accession number AGR79286.1) and 9,935 (GenBank accession number ANC34101.1) amino acids. To understand how the T4SS is used in A. phagocytophilum, we describe the expression of the virB6 paralogs and explore their role as the bacteria replicate within its host cell. Conclusions about the importance of these paralogs for colonization of human and tick cells are supported by the deficient phenotype of an A. phagocytophilum mutant isolated from a sequence-defined transposon insertion library.

An O-Methyltransferase Is Required for Infection of Tick Cells by Anaplasma phagocytophilum.

Anaplasma phagocytophilum, the causative agent of Human Granulocytic Anaplasmosis (HGA), is an obligately intracellular α-proteobacterium that is transmitted by Ixodes spp ticks. However, the pathogen is not transovarially transmitted between tick generations and therefore needs to survive in both a mammalian host and the arthropod vector to complete its life cycle. To adapt to different environments, pathogens rely on differential gene expression as well as the modification of proteins and other molecules. Random transposon mutagenesis of A. phagocytophilum resulted in an insertion within the coding region of an o-methyltransferase (omt) family 3 gene. In wild-type bacteria, expression of omt was up-regulated during binding to tick cells (ISE6) at 2 hr post-inoculation, but nearly absent by 4 hr p.i. Gene disruption reduced bacterial binding to ISE6 cells, and the mutant bacteria that were able to enter the cells were arrested in their replication and development. Analyses of the proteomes of wild-type versus mutant bacteria during binding to ISE6 cells identified Major Surface Protein 4 (Msp4), but also hypothetical protein APH_0406, as the most differentially methylated. Importantly, two glutamic acid residues (the targets of the OMT) were methyl-modified in wild-type Msp4, whereas a single asparagine (not a target of the OMT) was methylated in APH_0406. In vitro methylation assays demonstrated that recombinant OMT specifically methylated Msp4. Towards a greater understanding of the overall structure and catalytic activity of the OMT, we solved the apo (PDB_ID:4OA8), the S-adenosine homocystein-bound (PDB_ID:4OA5), the SAH-Mn2+ bound (PDB_ID:4PCA), and SAM- Mn2+ bound (PDB_ID:4PCL) X-ray crystal structures of the enzyme. Here, we characterized a mutation in A. phagocytophilum that affected the ability of the bacteria to productively infect cells from its natural vector. Nevertheless, due to the lack of complementation, we cannot rule out secondary mutations.

Prevalence and types of strabismus in cerebral palsy: A global and historical perspective based on a systematic review and meta-analysis.

Purpose: Strabismus is more frequent in cerebral palsy (CP) than in the normal population, but reports differ how much it is increased. We here examined the global prevalence and types of strabismus in CP, whether esotropia or exotropia is more frequent, and whether the prevalence differs between ethnicities and/or country income levels, and between generations. Methods: We compiled in a systematic review and meta-analysis the results of 147 CP studies that report the prevalence of strabismus or the ratio of esotropia to exotropia, and we conducted subgroup analyses for region (income level) and ethnicity. We performed a pooled analysis for the CP strabismus prevalence, and estimated the global number of CP cases with strabismus. Results: The pooled prevalence of strabismus in CP is 49.8% in high-income countries and 39.8% in lower-income countries. We estimate the global number of strabismus cases in CP as 12.2 million, with 7.6 million males and 4.6 million females, based on current estimates of 29.6 million global CP cases. Esotropia is more frequent than exotropia in Caucasians, while exotropia is more frequent than esotropia in Hispanic and in some Asian and African populations. The strabismus prevalence in CP increases with increasing country income levels. Conclusion: Generational changes in strabismus prevalence appear to reflect a transition of CP types and an increase in prevalence as countries attain higher income and more effective maternal health care. The distribution of esotropia and exotropia in CP patients largely reflects the horizontal strabismus type that is predominant in the subject's ethnicity.

Prevalence and Types of Strabismus in Cerebral Palsy: A Global and Historical Perspective Based on a Systematic Review and Meta-Analysis.

PURPOSE: Strabismus is more frequent in cerebral palsy (CP) than in the normal population, but reports differ how much it is increased. We here examined the global prevalence and types of strabismus in CP, whether esotropia or exotropia is more frequent, and whether the prevalence differs between ethnicities and/or country income levels, and between generations. METHODS: We compiled in a systematic review and meta-analysis the results of 147 CP studies that report the prevalence of strabismus or the ratio of esotropia to exotropia, and we conducted subgroup analyses for region (income level) and ethnicity. We performed a pooled analysis for the CP strabismus prevalence, and estimated the global number of CP cases with strabismus. RESULTS: The pooled prevalence of strabismus in CP is 49.8% in high-income countries and 39.8% in lower-income countries. We estimate the global number of strabismus cases in CP as 12.2 million, with 7.6 million males and 4.6 million females, based on current estimates of 29.6 million global CP cases. Esotropia is more frequent than exotropia in Caucasians, while exotropia is more frequent than esotropia in Hispanic and in some Asian and African populations. The strabismus prevalence in CP increases with increasing country income levels. CONCLUSION: Generational changes in strabismus prevalence appear to reflect a transition of CP types and an increase in prevalence as countries attain higher income and more effective maternal health care. The distribution of esotropia and exotropia in CP patients largely reflects the horizontal strabismus type that is predominant in the subject's ethnicity.

Changes in Posterior Cornea and Posterior-To-Anterior Curvature Radii Ratio 1 Year After LASIK, PRK, and SMILE Treatment of Myopia.

PURPOSE: The purpose of this study was to compare changes in the posterior curvature and the posterior-anterior radii ratio of the cornea, 1 year postoperatively in laser in situ keratomileusis (LASIK), photorefractive keratectomy (PRK), and small incision lenticule extraction (SMILE). METHODS: This retrospective study was performed at a single surgical center. 199 eyes were included in the study from 119 patients with manifest refraction spherical equivalents from -7.61 to -2.54 D. 67 eyes underwent LASIK, 89 underwent PRK, and 43 underwent SMILE. Both preoperative and 1-year postoperative front and back sagittal keratometry were measured at 4- to 6-mm zones around the corneal vertex. Corneal asphericity (Q-value) was measured at an 8-mm zone around the corneal vertex. RESULTS: The average change in the posterior-anterior radii ratio after LASIK, PRK, and SMILE did not differ between surgery groups at 4 mm (LASIK: -0.075, PRK: -0.073, SMILE: -0.072, P = 0.720), 5 mm (LASIK: -0.072, PRK: -0.068, SMILE: -0.068, P = 0.531), or 6 mm (LASIK: -0.075, PRK: -0.071, SMILE: -0.072, P = 0.456) zones. Anterior Q-value significantly positively increased after all 3 surgeries ( P < 0.001). The posterior Q-value also significantly positively increased after LASIK ( P < 0.001) and SMILE ( P < 0.001), but not after PRK ( P = 0.227). Both anterior and posterior keratometric power decreased significantly after LASIK, PRK, and SMILE for all diameters. CONCLUSIONS: The change in the posterior-anterior radii ratio was not influenced by the type of refractive surgery performed, as indicated by statistically identical preoperative, postoperative, and delta values. In addition, the posterior cornea exhibited paracentral flattening after LASIK, SMILE, and PRK and increased oblateness after LASIK and SMILE.

Correlational Analysis of the Effective Optical Zone with Myopia, Myopic Astigmatism, and Spherical Equivalent in LASIK, PRK, and SMILE.

Purpose: We assess the relationship between preoperative myopic sphere, astigmatism, and spherical equivalent and effective optical zone (EOZ) size, shape, and decentration within individual populations of post-LASIK, PRK, and SMILE patients. Patients and Methods: A retrospective chart review was conducted with 118 LASIK, 144 PRK, and 41 SMILE eyes from 179 total patients that underwent compound myopic ablation. One-year postoperative Pentacam tangential difference maps were used for EOZ data measurements. Correlational analysis between compound myopic measures [sphere, cylinder, manifest refractive spherical equivalent (MRSE)] and EOZ parameters was performed, and differences between groups of myopic sphere and cylinder within each surgery type were assessed. Results: An increase in absolute myopic sphere (and subsequent MRSE) is associated with a smaller EOZ area in SMILE (r=0.454, p=0.003) and a more circular EOZ shape in LASIK (r=0.396, p<0.001) and PRK (r=0.563, p<0.001). An increase in absolute myopic cylinder is associated with an increased EOZ area in all three surgery types [LASIK (r=-0.459, p<0.001), PRK (r=-0.716, p<0.001), SMILE (r=-0.429, p=0.005)] and a more elliptical EOZ in LASIK (r=-0.491, p<0.001) and PRK (r=-0.538, p<0.001). Conclusion: While astigmatism may be correlated to EOZ size within all three refractive surgery types, myopic sphere alone is insufficient to estimate EOZ size differences for procedures with a large blend zone of ablation like LASIK or PRK. Shape is just as important a factor as size to consider when examining corneal EOZ differences; reported correlative findings likely result from inherent differences in surgical technique and abruptness of planned surgical ablation borders.

Prediction of Posterior-to-Anterior Corneal Curvature Radii Ratio in Myopic Patients after LASIK, SMILE, and PRK Using Multivariate Regression Analysis.

The ratio of posterior-to-anterior curvature radii of the cornea (P/A ratio) is an important element in determining corneal refractive power. P/A ratio has been well studied in patients prior to undergoing refractive surgery, but its postoperative value remains less so. We aimed to examine the value of preoperative characteristics of refractive surgery patients in predicting the 1-year postoperative P/A ratio in LASIK, PRK, and SMILE using both linear and multivariate regression analyses. This was a retrospective study that included patients with manifest refraction spherical equivalents (MRSE) from -7.71D to -0.25D. In total, 164 eyes underwent LASIK, 183 underwent PRK, and 46 underwent SMILE. All patients had preoperative and 1-year postoperative front sagittal and back sagittal keratometry measurements at 4, 5, and 6 mm around the corneal vertex. Postoperative P/A after LASIK, PRK, and SMILE was found to be significantly correlated with MRSE and preoperative P/A. Stepwise variable selection in multivariate regression revealed that spherical equivalent was the most significant predictor of postoperative P/A. When coupled with other preoperative characteristics, including P/A, age, asphericity, and keratometry, the multivariate regressions were able to produce models with high predictive value in LASIK (adjusted R2: 0.957), PRK (adjusted R2: 0.934), and SMILE (adjusted R2: 0.894).

Comparing Effective Optical Zones After Myopic Ablation Between LASIK, PRK, and SMILE With Correlation to Higher Order Aberrations.

PURPOSE: To explore size, decentration, and eccentricity of effective optical zones (EOZs) in laser in situ keratomileusis (LASIK), photorefractive keratectomy (PRK), and small incision lenticule extraction (SMILE) and correlate them to higher order aberrations (HOAs). METHODS: This was a retrospective chart review of 188 eyes that underwent refractive surgery for compound myopia (61 LASIK, 84 PRK, 43 SMILE). EOZ measurements were determined using 1-year postoperative Pentacam (Oculus Optikgeräte GmbH) tangential difference maps. HOA data were measured using Pentacam wavefront aberration Zernike polynomials. Correlations between EOZs and HOAs were analyzed. RESULTS: The EOZs of LASIK and PRK are smaller than SMILE at 19.54 ± 1.44, 19.39 ± 1.66, and 22.18 ± 2.61 mm2, respectively (P < .001). No difference existed in absolute decentration from corneal vertex (P = .078) or pupil center (P = .131), but horizontal and vertical components differed significantly (P < .001). Smaller EOZ areas were correlated with greater spherical aberration induction (rLASIK = -0.378, rPRK = -0.555, rSMILE = -0.501) and total HOA induction in all groups. Absolute decentration from corneal vertex positively correlated with total HOA (rLASIK = 0.396, rPRK = 0.463, rSMILE = 0.399) and directional vertical coma induction negatively correlated with vertical decentration from the corneal vertex (rLASIK = -0.776, rPRK = -0.665, rSMILE = -0.576) in all groups. CONCLUSIONS: SMILE results in a larger EOZ than LASIK and PRK, and absolute decentration remains comparable regardless of surgical reference center, despite horizontal/vertical differences. Surgical planning to ensure adequate EOZ size and centration may reduce induction of HOAs, including spherical aberrations and vertical coma. [J Refract Surg. 2023;39(11):741-750.].

Exploring Nomograms for Implantable Collamer Lens Size Selection in Myopia: A Literature-based Compilation.

Purpose: To provide a comprehensive guide of all implantable collamer lens (ICL) sizing nomograms and the respective preoperative diagnostic devices that are required. This guide would help clinicians in choosing the appropriate ICL size for myopic patients to optimize postoperative vault height. Methods: A literature search of peer-reviewed journals describing methods and postoperative outcomes of ICL sizing was conducted. Research articles containing ICL nomograms or formulas were identified from this search. Preoperative variables necessary for these nomograms and the required diagnostic devices to measure these parameters such as topography, biometry, or ultrasound biomicroscopy (UBM) were noted. An additional search was conducted to identify artificial intelligence (AI) or machine learning (ML)-derived nomograms. Results: Eighteen ICL sizing nomograms were identified through literature search. Five of these nomograms are available for use and require topography or biometry devices. Of these, four include the manufacturer's, optimized white-to-white (WTW), Kang, Kim, and Rocamora Nomograms. Eight of the 18 nomograms available for use require UBM. Eight of these include the Kojima, Nakamura, KS, ZZ, Dougherty, Parkhurst, Russo, and Reinstein Nomograms. Four of the 18 nomograms are ML-derived including Shen, Rocamora, Russo, and Kang Nomograms. Conclusion: ICL nomograms are a vital tool in helping clinicians select the right ICL size for myopic patients to optimize postoperative vault reducing risk of postoperative complications. Based on available diagnostic devices such as topography, biometry, or UBM clinicians can integrate specific nomograms into practice.

Anaplasma phagocytophilum dihydrolipoamide dehydrogenase 1 affects host-derived immunopathology during microbial colonization.

Anaplasma phagocytophilum is a tick-borne rickettsial pathogen that provokes an acute inflammatory response during mammalian infection. The illness caused by A. phagocytophilum, human granulocytic anaplasmosis, occurs irrespective of pathogen load and results instead from host-derived immunopathology. Thus, characterizing A. phagocytophilum genes that affect the inflammatory process is critical for understanding disease etiology. By using an A. phagocytophilum Himar1 transposon mutant library, we showed that a single transposon insertion into the A. phagocytophilum dihydrolipoamide dehydrogenase 1 gene (lpda1 [APH_0065]) affects inflammation during infection. A. phagocytophilum lacking lpda1 revealed enlargement of the spleen, increased splenic extramedullary hematopoiesis, and altered clinicopathological abnormalities during mammalian colonization. Furthermore, LPDA1-derived immunopathology was independent of neutrophil infection and correlated with enhanced reactive oxygen species from NADPH oxidase and nuclear factor (NF)-κB signaling in macrophages. Taken together, these findings suggest the presence of different signaling pathways in neutrophils and macrophages during A. phagocytophilum invasion and highlight the importance of LPDA1 as an immunopathological molecule.

Biostatistical prediction of genes essential for growth of Anaplasma phagocytophilum in a human promyelocytic cell line using a random transposon mutant library.

Anaplasma phagocytophilum (Ap), agent of human anaplasmosis, is an intracellular bacterium that causes the second most common tick-borne illness in North America. To address the lack of a genetic system for these pathogens, we used random Himar1 transposon mutagenesis to generate a library of Ap mutants capable of replicating in human promyelocytes (HL-60 cells). Illumina sequencing identified 1195 non-randomly distributed insertions. As the density of mutants was non-saturating, genes without insertions were either essential for Ap, or spared randomly. To resolve this question, we applied a biostatistical method for prediction of essential genes. Since the chances that a transposon was inserted into genomic TA dinucleotide sites should be the same for all loci, we used a Markov chain Monte Carlo model to estimate the probability that a non-mutated gene was essential for Ap. Predicted essential genes included those coding for structural ribosomal proteins, enzymes involved in metabolism, components of the type IV secretion system, antioxidant defense molecules and hypothetical proteins. We have used an in silico post-genomic approach to predict genes with high probability of being essential for replication of Ap in HL-60 cells. These results will help target genes to investigate their role in the pathogenesis of human anaplasmosis.

Febio finite element models of the human cervical spine.

Finite element (FE) analysis has proven to be useful when studying the biomechanics of the cervical spine. Although many FE studies of the cervical spine have been published, they typically develop their models using commercial software, making the sharing of models between researchers difficult. They also often model only one part of the cervical spine. The goal of this study was to develop and evaluate three FE models of the adult cervical spine using open-source software and to freely provide these models to the scientific community. The models were created from computed tomography scans of 26-, 59-, and 64-year old female subjects. These models were evaluated against previously published experimental and FE data. Despite the fact that all three models were assigned identical material properties and boundary conditions, there was notable variation in their biomechanical behavior. It was therefore apparent that these differences were the result of morphological differences between the models.

Global Transcription Profiles of Anaplasma phagocytophilum at Key Stages of Infection in Tick and Human Cell Lines and Granulocytes.

The incidence of human diseases caused by tick-borne pathogens is increasing but little is known about the molecular interactions between the agents and their vectors and hosts. Anaplasma phagocytophilum (Ap) is an obligate intracellular, tick-borne bacterium that causes granulocytic anaplasmosis in humans, dogs, sheep, and horses. In mammals, neutrophil granulocytes are a primary target of infection, and in ticks, Ap has been found in gut and salivary gland cells. To identify bacterial genes that enable Ap to invade and proliferate in human and tick cells, labeled mRNA from Ap bound to or replicating within human and tick cells (lines HL-60 and ISE6), and replicating in primary human granulocytes ex vivo, was hybridized to a custom tiling microarray containing probes representing the entire Ap genome. Probe signal values plotted over a map of the Ap genome revealed antisense transcripts and unannotated genes. Comparisons of transcript levels from each annotated gene between test conditions (e.g., Ap replicating in HL-60 vs. ISE6) identified those that were differentially transcribed, thereby highlighting genes associated with each condition. Bacteria replicating in HL-60 cells upregulated 122 genes compared to those in ISE6, including numerous p44 paralogs, five HGE-14 paralogs, and 32 hypothetical protein genes, of which 47% were predicted to be secreted or localized to the membrane. By comparison, 60% of genes upregulated in ISE6 encoded hypothetical proteins, 60% of which were predicted to be secreted or membrane associated. In granulocytes, Ap upregulated 120 genes compared to HL-60, 33% of them hypothetical and 43% of those predicted to encode secreted or membrane associated proteins. HL-60-grown bacteria binding to HL-60 cells barely responded transcriptionally, while ISE6-grown bacteria binding to ISE6 cells upregulated 48 genes. HL-60-grown bacteria, when incubated with ISE6 cells, upregulated the same genes that were upregulated by ISE6-grown bacteria exposed to uninfected ISE6. Hypothetical genes (constituting about 29% of Ap genes) played a disproportionate role in most infection scenarios, and particular sets of them were consistently upregulated in bacteria binding/entering both ISE6 and HL-60 cells. This suggested that the encoded proteins played central roles in establishing infection in ticks and humans.

Microbial mineral weathering for nutrient acquisition releases arsenic.

Tens of millions of people in Southeast Asia drink groundwater contaminated with naturally occurring arsenic. How arsenic is released from the sediment into the water remains poorly understood. Here, we show in laboratory experiments that phosphate-limited cells of Burkholderia fungorum mobilize ancillary arsenic from apatite. We hypothesize that arsenic mobilization is a by-product of mineral weathering for nutrient acquisition. The released arsenic does not undergo a redox transformation but appears to be solubilized from the apatite mineral lattice during weathering. Analysis of apatite from the source area in the Himalayan basin indicates the presence of elevated levels of arsenic, with an average concentration of 210 mg/kg. The rate of arsenic release is independent of the initial dissolved arsenic concentration and occurs at phosphate levels observed in Bangladesh aquifers. We also demonstrate the presence of the microbial phenotype that releases arsenic from apatite in Bangladesh aquifer sediments and groundwater. These results suggest that microbial mineral weathering for nutrient acquisition could be an important mechanism for arsenic mobilization.

The occipitoatlantal capsular ligaments are the primary stabilizers of the occipitoatlantal joint in the craniocervical junction: a finite element analysis.

OBJECTIVEThere is contradictory evidence regarding the relative contribution of the key stabilizing ligaments of the occipitoatlantal (OA) joint. Cadaveric studies are limited by the nature and the number of injury scenarios that can be tested to identify OA stabilizing ligaments. Finite element (FE) analysis can overcome these limitations and provide valuable data in this area. The authors completed an FE analysis of 5 subject-specific craniocervical junction (CCJ) models to investigate the biomechanics of the OA joint and identify the ligamentous structures essential for stability.METHODSIsolated and combined injury scenarios were simulated under physiological loads for 5 validated CCJ FE models to assess the relative role of key ligamentous structures on OA joint stability. Each model was tested in flexion-extension, axial rotation, and lateral bending in various injury scenarios. Isolated ligamentous injury scenarios consisted of either decreasing the stiffness of the OA capsular ligaments (OACLs) or completely removing the transverse ligament (TL), tectorial membrane (TM), or alar ligaments (ALs). Combination scenarios were also evaluated.RESULTSAn isolated OACL injury resulted in the largest percentage increase in all ranges of motion (ROMs) at the OA joint compared with the other isolated injuries. Flexion, extension, lateral bending, and axial rotation significantly increased by 12.4% ± 7.4%, 11.1% ± 10.3%, 83.6% ± 14.4%, and 81.9% ± 9.4%, respectively (p ≤ 0.05 for all). Among combination injuries, OACL+TM+TL injury resulted in the most consistent significant increases in ROM for both the OA joint and the CCJ during all loading scenarios. OACL+AL injury caused the most significant percentage increase for OA joint axial rotation.CONCLUSIONSThese results demonstrate that the OACLs are the key stabilizing ligamentous structures of the OA joint. Injury of these primary stabilizing ligaments is necessary to cause OA instability. Isolated injuries of TL, TM, or AL are unlikely to result in appreciable instability at the OA joint.

Mutational analysis of gene function in the Anaplasmataceae: Challenges and perspectives.

Mutational analysis is an efficient approach to identifying microbial gene function. Until recently, lack of an effective tool for Anaplasmataceae yielding reproducible results has created an obstacle to functional genomics, because surrogate systems, e.g., ectopic gene expression and analysis in E. coli, may not provide accurate answers. We chose to focus on a method for high-throughput generation of mutants via random mutagenesis as opposed to targeted gene inactivation. In our search for a suitable mutagenesis tool, we considered attributes of the Himar1 transposase system, i.e., random insertion into AT dinucleotide sites, which are abundant in Anaplasmataceae, and lack of requirement for specific host factors. We chose the Anaplasma marginale tr promoter, and the clinically irrelevant antibiotic spectinomycin for selection, and in addition successfully implemented non-antibiotic selection using an herbicide resistance gene. These constructs function reasonably well in Anaplasma phagocytophilum harvested from human promyelocyte HL-60 cells or Ixodes scapularis tick cells. We describe protocols developed in our laboratory, and discuss what likely makes them successful. What makes Anaplasmataceae electroporation competent is unknown and manipulating electroporation conditions has not improved mutational efficiency. A concerted effort is needed to resolve remaining problems that are inherent to the obligate intracellular bacteria. Finally, using this approach, we describe the discovery and characterization of a putative secreted effector necessary for Ap survival in HL-60 cells.

Whole genome transcription profiling of Anaplasma phagocytophilum in human and tick host cells by tiling array analysis.

BACKGROUND: Anaplasma phagocytophilum (Ap) is an obligate intracellular bacterium and the agent of human granulocytic anaplasmosis, an emerging tick-borne disease. Ap alternately infects ticks and mammals and a variety of cell types within each. Understanding the biology behind such versatile cellular parasitism may be derived through the use of tiling microarrays to establish high resolution, genome-wide transcription profiles of the organism as it infects cell lines representative of its life cycle (tick; ISE6) and pathogenesis (human; HL-60 and HMEC-1). RESULTS: Detailed, host cell specific transcriptional behavior was revealed. There was extensive differential Ap gene transcription between the tick (ISE6) and the human (HL-60 and HMEC-1) cell lines, with far fewer differentially transcribed genes between the human cell lines, and all disproportionately represented by membrane or surface proteins. There were Ap genes exclusively transcribed in each cell line, apparent human- and tick-specific operons and paralogs, and anti-sense transcripts that suggest novel expression regulation processes. Seven virB2 paralogs (of the bacterial type IV secretion system) showed human or tick cell dependent transcription. Previously unrecognized genes and coding sequences were identified, as were the expressed p44/msp2 (major surface proteins) paralogs (of 114 total), through elevated signal produced to the unique hypervariable region of each - 2/114 in HL-60, 3/114 in HMEC-1, and none in ISE6. CONCLUSION: Using these methods, whole genome transcription profiles can likely be generated for Ap, as well as other obligate intracellular organisms, in any host cells and for all stages of the cell infection process. Visual representation of comprehensive transcription data alongside an annotated map of the genome renders complex transcription into discernable patterns.

Transgene expression and silencing in a tick cell line: A model system for functional tick genomics.

The genome project of the black legged tick, Ixodes scapularis, provides sequence data for testing gene function and regulation in this important pathogen vector. We tested Sleeping Beauty (SB), a Tc1/mariner group transposable element, and cationic lipid-based transfection reagents for delivery and genomic integration of transgenes into I. scapularis cell line ISE6. Plasmid DNA and dsRNA were effectively transfected into ISE6 cells and they were successfully transformed to express a red fluorescent protein (DsRed2) and a selectable marker, neomycin phosphotransferase (NEO). Frequency of transformation was estimated as 1 transformant per 5000-10,000 cells and cultures were incubated for 2-3 months in medium containing the neomycin analog G418 in order to isolate transformants. Genomic integration of the DsRed2 transgene was confirmed by inverse PCR and sequencing that demonstrated a TA nucleotide pair inserted between SB inverted/direct repeat sequences and tick genomic sequences, indicating that insertion of the DsRed2 gene into the tick cell genome occurred through the activity of SB transposase. RNAi using dsRNA transcribed from the DsRed2 gene silenced expression of red fluorescent protein in transformed ISE6 cells. SB transposition in cell line ISE6 provides an effective means to explore the functional genomics of I. scapularis.

Transformation of Anaplasma phagocytophilum.

BACKGROUND: Tick-borne pathogens cause emerging zoonoses, and include fastidious organisms such as Anaplasma phagocytophilum. Because of their obligate intracellular nature, methods for mutagenesis and transformation have not been available. RESULTS: To facilitate genetic manipulation, we transformed A. phagocytophilum (Ap) to express a green fluorescent protein (GFP) with the Himar1 transposase system and selection with the clinically irrelevant antibiotic spectinomycin. CONCLUSION: These transformed bacteria (GFP/Ap) grow at normal rates and are brightly fluorescent in human, monkey, and tick cell culture. Molecular characterization of the GFP/Ap genomic DNA confirmed transposition and the flanking genomic insertion locations were sequenced. Three mice inoculated with GFP/Ap by intraperitoneal injection became infected as demonstrated by the appearance of morulae in a peripheral blood neutrophil and re-isolation of the bacteria in culture.