Train-of-four ratio, counts and post tetanic counts with the Tetragraph electromyograph in comparison to mechanomyography.

Automated EMG devices to detect compound muscle action potentials from the adductor pollicis muscle in response to ulnar nerve stimulation, regardless of hand and thumb position, may serve as a better reference ("gold standard") for clinical assessment of neuromuscular function than traditional mechanomyography (MMG) systems that need custom design and validation in lab settings. This evaluation compared the TetraGraph EMG system against a validated MMG device to investigate the accuracy and repeatability of this quantitative EMG monitor for detecting onset, offset and deep neuromuscular block. Simultaneous muscle action potential recordings from the EMG neuromuscular monitor and muscle contractions from an in-house developed MMG monitor in response to ulnar nerve stimulation were obtained from patients having elective surgery requiring neuromuscular block. Train-of-four (TOF) ratios, TOF counts, and post-tetanic counts (PTCs) were recorded simultaneously from the same hand muscle and compared. In total, 685 pairs of simultaneous TOF ratios were evaluated. The mean difference (bias) of TOF ratios between devices was small (- 2.1%). TOF counts from 285 data pairs were within a count of 2 or less 96% of the time. During deep block, PTC comparisons from 215 data pairs were within a count of 2 or less 95% of the time. These findings, along with prior EMG device evaluations, indicate that real-time EMG neuromuscular monitoring technology to detect muscle action potentials from the adductor pollicis in the clinical setting is closely aligned with the force of thumb contraction determined from MMG. The accuracy of quantitative EMG technology of the TetraGraph EMG system lends strong support for this monitor, along with other similarly validated EMG monitors, to become a clinical standard for all phases (onset, depth and reversal) of neuromuscular block in clinical practice.

Investigating the contribution of hyaluronan to the breast tumour microenvironment using multiparametric MRI and MR elastography.

Hyaluronan (HA) is a key component of the dense extracellular matrix in breast cancer, and its accumulation is associated with poor prognosis and metastasis. Pegvorhyaluronidase alfa (PEGPH20) enzymatically degrades HA and can enhance drug delivery and treatment response in preclinical tumour models. Clinical development of stromal-targeted therapies would be accelerated by imaging biomarkers that inform on therapeutic efficacy in vivo. Here, PEGPH20 response was assessed by multiparametric magnetic resonance imaging (MRI) in three orthotopic breast tumour models. Treatment of 4T1/HAS3 tumours, the model with the highest HA accumulation, reduced T1 and T2 relaxation times and the apparent diffusion coefficient (ADC), and increased the magnetisation transfer ratio, consistent with lower tissue water content and collapse of the extracellular space. The transverse relaxation rate R2 * increased, consistent with greater erythrocyte accessibility following vascular decompression. Treatment of MDA-MB-231 LM2-4 tumours reduced ADC and dramatically increased tumour viscoelasticity measured by MR elastography. Correlation matrix analyses of data from all models identified ADC as having the strongest correlation with HA accumulation, suggesting that ADC is the most sensitive imaging biomarker of tumour response to PEGPH20.

Probabilistic Mixture Models Improve Calibration of Panel-derived Tumor Mutational Burden in the Context of both Tumor-normal and Tumor-only Sequencing.

Background: Tumor mutational burden (TMB) has been investigated as a biomarker for immune checkpoint blockade (ICB) therapy. Increasingly, TMB is being estimated with gene panel-based assays (as opposed to full exome sequencing) and different gene panels cover overlapping but distinct genomic coordinates, making comparisons across panels difficult. Previous studies have suggested that standardization and calibration to exome-derived TMB be done for each panel to ensure comparability. With TMB cutoffs being developed from panel-based assays, there is a need to understand how to properly estimate exomic TMB values from different panel-based assays. Design: Our approach to calibration of panel-derived TMB to exomic TMB proposes the use of probabilistic mixture models that allow for nonlinear relationships along with heteroscedastic error. We examined various inputs including nonsynonymous, synonymous, and hotspot counts along with genetic ancestry. Using The Cancer Genome Atlas cohort, we generated a tumor-only version of the panel-restricted data by reintroducing private germline variants. Results: We were able to model more accurately the distribution of both tumor-normal and tumor-only data using the proposed probabilistic mixture models as compared with linear regression. Applying a model trained on tumor-normal data to tumor-only input results in biased TMB predictions. Including synonymous mutations resulted in better regression metrics across both data types, but ultimately a model able to dynamically weight the various input mutation types exhibited optimal performance. Including genetic ancestry improved model performance only in the context of tumor-only data, wherein private germline variants are observed. Significance: A probabilistic mixture model better models the nonlinearity and heteroscedasticity of the data as compared with linear regression. Tumor-only panel data are needed to properly calibrate tumor-only panels to exomic TMB. Leveraging the uncertainty of point estimates from these models better informs cohort stratification in terms of TMB.

A longitudinal circulating tumor DNA-based model associated with survival in metastatic non-small-cell lung cancer.

One of the great challenges in therapeutic oncology is determining who might achieve survival benefits from a particular therapy. Studies on longitudinal circulating tumor DNA (ctDNA) dynamics for the prediction of survival have generally been small or nonrandomized. We assessed ctDNA across 5 time points in 466 non-small-cell lung cancer (NSCLC) patients from the randomized phase 3 IMpower150 study comparing chemotherapy-immune checkpoint inhibitor (chemo-ICI) combinations and used machine learning to jointly model multiple ctDNA metrics to predict overall survival (OS). ctDNA assessments through cycle 3 day 1 of treatment enabled risk stratification of patients with stable disease (hazard ratio (HR) = 3.2 (2.0-5.3), P < 0.001; median 7.1 versus 22.3 months for high- versus low-intermediate risk) and with partial response (HR = 3.3 (1.7-6.4), P < 0.001; median 8.8 versus 28.6 months). The model also identified high-risk patients in an external validation cohort from the randomized phase 3 OAK study of ICI versus chemo in NSCLC (OS HR = 3.73 (1.83-7.60), P = 0.00012). Simulations of clinical trial scenarios employing our ctDNA model suggested that early ctDNA testing outperforms early radiographic imaging for predicting trial outcomes. Overall, measuring ctDNA dynamics during treatment can improve patient risk stratification and may allow early differentiation between competing therapies during clinical trials.

Developing and testing a robotic MRI/CT fusion biopsy technique using a purpose-built interventional phantom.

BACKGROUND: Magnetic resonance imaging (MRI) can be used to target tumour components in biopsy procedures, while the ability to precisely correlate histology and MRI signal is crucial for imaging biomarker validation. Robotic MRI/computed tomography (CT) fusion biopsy offers the potential for this without in-gantry biopsy, although requires development. METHODS: Test-retest T1 and T2 relaxation times, attenuation (Hounsfield units, HU), and biopsy core quality were prospectively assessed (January-December 2021) in a range of gelatin, agar, and mixed gelatin/agar solutions of differing concentrations on days 1 and 8 after manufacture. Suitable materials were chosen, and four biopsy phantoms were constructed with twelve spherical 1-3-cm diameter targets visible on MRI, but not on CT. A technical pipeline was developed, and intraoperator and interoperator reliability was tested in four operators performing a total of 96 biopsies. Statistical analysis included T1, T2, and HU repeatability using Bland-Altman analysis, Dice similarity coefficient (DSC), and intraoperator and interoperator reliability. RESULTS: T1, T2, and HU repeatability had 95% limits-of-agreement of 8.3%, 3.4%, and 17.9%, respectively. The phantom was highly reproducible, with DSC of 0.93 versus 0.92 for scanning the same or two different phantoms, respectively. Hit rate was 100% (96/96 targets), and all operators performed robotic biopsies using a single volumetric acquisition. The fastest procedure time was 32 min for all 12 targets. CONCLUSIONS: A reproducible biopsy phantom was developed, validated, and used to test robotic MRI/CT-fusion biopsy. The technique was highly accurate, reliable, and achievable in clinically acceptable timescales meaning it is suitable for clinical application.

New Opioid Persistence in Veterans Following Major and Minor Surgery.

INTRODUCTION: Opioids are often a mainstay of managing postsurgical pain. Persistent use of opioids for more than 90 days after surgery is problematic, and the incidence of this adverse outcome has been reported in the civilian population ranging from 0.4% to 7%. Veterans compose a special population exposed to trauma and stressful situations and consequently face increased risk for habit-forming behavior and drug overdose. This evaluation determined the prevalence of opioid persistence after surgery and its relationship to patient characteristics in a military veteran population. METHODS: A retrospective chart review was completed on 1,257 veterans who were opioid naive and had undergone a surgical procedure between January 2017 and May 2018. Patient characteristics, health conditions, and discharge opioid medications were recorded, and the incidence of persistent opioid use beyond 90 days was determined. RESULTS: The incidence of opioid persistence following major (3.3%) and minor (3.4%) procedures was similar. The incidence in patients younger than 45 years (3.3%), between 45 and 64 years (4.3%), and 65 years and older (2.2%) was also determined to be similar. Univariate patient factors associated with an increased risk for persistent opioid use include cancer (odds ratio [OR], 2.13; 95% CI, 1.11-4.09), mental health disorders (OR, 2.32; 95% CI, 1.17-4.60), and substance use disorders (OR, 2.09; 95% CI, 1.09-4.00). CONCLUSIONS: Among a cohort of over 1,200 opioid-naïve veterans undergoing surgery at a VA Medical Center, just over 3% went on to develop persistent opioid use beyond 3 months following their procedure. Persistent use was not found to be related to the type of procedure (major or minor) or patient age. Significant patient-level risk factors for opioid persistence were cancer and a history of mental health and substance use disorders.

Molecular determinants of response to PD-L1 blockade across tumor types.

Immune checkpoint inhibitors targeting the PD-1/PD-L1 axis lead to durable clinical responses in subsets of cancer patients across multiple indications, including non-small cell lung cancer (NSCLC), urothelial carcinoma (UC) and renal cell carcinoma (RCC). Herein, we complement PD-L1 immunohistochemistry (IHC) and tumor mutation burden (TMB) with RNA-seq in 366 patients to identify unifying and indication-specific molecular profiles that can predict response to checkpoint blockade across these tumor types. Multiple machine learning approaches failed to identify a baseline transcriptional signature highly predictive of response across these indications. Signatures described previously for immune checkpoint inhibitors also failed to validate. At the pathway level, significant heterogeneity is observed between indications, in particular within the PD-L1+ tumors. mUC and NSCLC are molecularly aligned, with cell cycle and DNA damage repair genes associated with response in PD-L1- tumors. At the gene level, the CDK4/6 inhibitor CDKN2A is identified as a significant transcriptional correlate of response, highlighting the association of non-immune pathways to the outcome of checkpoint blockade. This cross-indication analysis reveals molecular heterogeneity between mUC, NSCLC and RCC tumors, suggesting that indication-specific molecular approaches should be prioritized to formulate treatment strategies.

ALK Mutation Status Before and After Alectinib Treatment in Locally Advanced or Metastatic ALK-Positive NSCLC: Pooled Analysis of Two Prospective Trials.

INTRODUCTION: The effectiveness of ALK receptor tyrosine kinase (ALK) inhibitors can be limited by the development of ALK resistance mutations. This exploratory analysis assessed the efficacy of alectinib in patients with NSCLC and ALK point mutations using pooled data from two single-arm phase II studies. METHODS: Studies NP28673 and NP28761 enrolled adults with locally advanced/metastatic ALK-positive NSCLC who had progressed on crizotinib. ALK mutation analysis was conducted on cell-free DNA from 187 patients post-crizotinib/pre-alectinib, and from 49 of these patients who subsequently progressed on alectinib. RESULTS: Baseline characteristics were generally balanced across patient subgroups. At baseline, 34 distinct ALK mutations were identified in 48 of 187 patients (25.7%). Median investigator-assessed progression-free survival was longer in patients without ALK single-nucleotide variants (n = 138) versus those with (n = 48): 10.2 months (95% confidence interval [CI]: 8.1-14.3) versus 5.6 months (95% CI: 4.5-10.9), respectively. Sixteen of 32 patients (50%) with ALK resistance mutations to crizotinib achieved an investigator-assessed response to alectinib (all partial responses); most of these ALK mutations were known to be sensitive to alectinib. Analysis of plasma samples obtained post-progression on alectinib revealed that 26 of 49 (53%) samples harbored 16 distinct ALK mutations, with known alectinib-resistance mutations, I1171 T/N/S, G1202R, and V1180L, observed in 15 of 49 (31%) tumors. CONCLUSIONS: Alectinib appears clinically active against ALK rearrangements and mutations, as well as several ALK variants that can cause resistance to crizotinib. The use of cell-free DNA in plasma samples may be an alternative noninvasive method for monitoring resistance mutations during therapy.

Investigating the Contribution of Collagen to the Tumor Biomechanical Phenotype with Noninvasive Magnetic Resonance Elastography.

Increased stiffness in the extracellular matrix (ECM) contributes to tumor progression and metastasis. Therefore, stromal modulating therapies and accompanying biomarkers are being developed to target ECM stiffness. Magnetic resonance (MR) elastography can noninvasively and quantitatively map the viscoelastic properties of tumors in vivo and thus has clear clinical applications. Herein, we used MR elastography, coupled with computational histopathology, to interrogate the contribution of collagen to the tumor biomechanical phenotype and to evaluate its sensitivity to collagenase-induced stromal modulation. Elasticity (G d) and viscosity (G l) were significantly greater for orthotopic BT-474 (G d = 5.9 ± 0.2 kPa, G l = 4.7 ± 0.2 kPa, n = 7) and luc-MDA-MB-231-LM2-4 (G d = 7.9 ± 0.4 kPa, G l = 6.0 ± 0.2 kPa, n = 6) breast cancer xenografts, and luc-PANC1 (G d = 6.9 ± 0.3 kPa, G l = 6.2 ± 0.2 kPa, n = 7) pancreatic cancer xenografts, compared with tumors associated with the nervous system, including GTML/Trp53KI/KI medulloblastoma (G d = 3.5 ± 0.2 kPa, G l = 2.3 ± 0.2 kPa, n = 7), orthotopic luc-D-212-MG (G d = 3.5 ± 0.2 kPa, G l = 2.3 ± 0.2 kPa, n = 7), luc-RG2 (G d = 3.5 ± 0.2 kPa, G l = 2.3 ± 0.2 kPa, n = 5), and luc-U-87-MG (G d = 3.5 ± 0.2 kPa, G l = 2.3 ± 0.2 kPa, n = 8) glioblastoma xenografts, intracranially propagated luc-MDA-MB-231-LM2-4 (G d = 3.7 ± 0.2 kPa, G l = 2.2 ± 0.1 kPa, n = 7) breast cancer xenografts, and Th-MYCN neuroblastomas (G d = 3.5 ± 0.2 kPa, G l = 2.3 ± 0.2 kPa, n = 5). Positive correlations between both elasticity (r = 0.72, P < 0.0001) and viscosity (r = 0.78, P < 0.0001) were determined with collagen fraction, but not with cellular or vascular density. Treatment with collagenase significantly reduced G d (P = 0.002) and G l (P = 0.0006) in orthotopic breast tumors. Texture analysis of extracted images of picrosirius red staining revealed significant negative correlations of entropy with G d (r = -0.69, P < 0.0001) and G l (r = -0.76, P < 0.0001), and positive correlations of fractal dimension with G d (r = 0.75, P < 0.0001) and G l (r = 0.78, P < 0.0001). MR elastography can thus provide sensitive imaging biomarkers of tumor collagen deposition and its therapeutic modulation. SIGNIFICANCE: MR elastography enables noninvasive detection of tumor stiffness and will aid in the development of ECM-targeting therapies.

Low-pass Whole-genome Sequencing of Circulating Cell-free DNA Demonstrates Dynamic Changes in Genomic Copy Number in a Squamous Lung Cancer Clinical Cohort.

PURPOSE: We developed a method to monitor copy number variations (CNV) in plasma cell-free DNA (cfDNA) from patients with metastatic squamous non-small cell lung cancer (NSCLC). We aimed to explore the association between tumor-derived cfDNA and clinical outcomes, and sought CNVs that may suggest potential resistance mechanisms. EXPERIMENTAL DESIGN: Sensitivity and specificity of low-pass whole-genome sequencing (LP-WGS) were first determined using cell line DNA and cfDNA. LP-WGS was performed on baseline and longitudinal cfDNA of 152 patients with squamous NSCLC treated with chemotherapy, or in combination with pictilisib, a pan-PI3K inhibitor. cfDNA tumor fraction and detected CNVs were analyzed in association with clinical outcomes. RESULTS: LP-WGS successfully detected CNVs in cfDNA with tumor fraction ≥10%, which represented approximately 30% of the first-line NSCLC patients in this study. The most frequent CNVs were gains in chromosome 3q, which harbors the PIK3CA and SOX2 oncogenes. The CNV landscape in cfDNA with a high tumor fraction generally matched that of corresponding tumor tissue. Tumor fraction in cfDNA was dynamic during treatment, and increases in tumor fraction and corresponding CNVs could be detected before radiographic progression in 7 of 12 patients. Recurrent CNVs, such as MYC amplification, were enriched in cfDNA from posttreatment samples compared with the baseline, suggesting a potential resistance mechanism to pictilisib. CONCLUSIONS: LP-WGS offers an unbiased and high-throughput way to investigate CNVs and tumor fraction in cfDNA of patients with cancer. It may also be valuable for monitoring treatment response, detecting disease progression early, and identifying emergent clones associated with therapeutic resistance.

Blood-based tumor mutational burden as a predictor of clinical benefit in non-small-cell lung cancer patients treated with atezolizumab.

Although programmed death-ligand 1-programmed death 1 (PD-L1-PD-1) inhibitors are broadly efficacious, improved outcomes have been observed in patients with high PD-L1 expression or high tumor mutational burden (TMB). PD-L1 testing is required for checkpoint inhibitor monotherapy in front-line non-small-cell lung cancer (NSCLC). However, obtaining adequate tumor tissue for molecular testing in patients with advanced disease can be challenging. Thus, an unmet medical need exists for diagnostic approaches that do not require tissue to identify patients who may benefit from immunotherapy. Here, we describe a novel, technically robust, blood-based assay to measure TMB in plasma (bTMB) that is distinct from tissue-based approaches. Using a retrospective analysis of two large randomized trials as test and validation studies, we show that bTMB reproducibly identifies patients who derive clinically significant improvements in progression-free survival from atezolizumab (an anti-PD-L1) in second-line and higher NSCLC. Collectively, our data show that high bTMB is a clinically actionable biomarker for atezolizumab in NSCLC.

A preclinical microbeam facility with a conventional x-ray tube.

PURPOSE: Microbeam radiation therapy is an innovative treatment approach in radiation therapy that uses arrays of a few tens of micrometer wide and a few hundreds of micrometer spaced planar x-ray beams as treatment fields. In preclinical studies these fields efficiently eradicated tumors while normal tissue could effectively be spared. However, development and clinical application of microbeam radiation therapy is impeded by a lack of suitable small scale sources. Until now, only large synchrotrons provide appropriate beam properties for the production of microbeams. METHODS: In this work, a conventional x-ray tube with a small focal spot and a specially designed collimator are used to produce microbeams for preclinical research. The applicability of the developed source is demonstrated in a pilot in vitro experiment. The properties of the produced radiation field are characterized by radiochromic film dosimetry. RESULTS: 50 µm wide and 400 µm spaced microbeams were produced in a 20 × 20 mm2 sized microbeam field. The peak to valley dose ratio ranged from 15.5 to 30, which is comparable to values obtained at synchrotrons. A dose rate of up to 300 mGy/s was achieved in the microbeam peaks. Analysis of DNA double strand repair and cell cycle distribution after in vitro exposures of pancreatic cancer cells (Panc1) at the x-ray tube and the European Synchrotron leads to similar results. In particular, a reduced G2 cell cycle arrest is observed in cells in the microbeam peak region. CONCLUSIONS: At its current stage, the source is restricted to in vitro applications. However, moderate modifications of the setup may soon allow in vivo research in mice and rats.

Abdo-Man: a 3D-printed anthropomorphic phantom for validating quantitative SIRT.

BACKGROUND: The use of selective internal radiation therapy (SIRT) is rapidly increasing, and the need for quantification and dosimetry is becoming more widespread to facilitate treatment planning and verification. The aim of this project was to develop an anthropomorphic phantom that can be used as a validation tool for post-SIRT imaging and its application to dosimetry. METHOD: The phantom design was based on anatomical data obtained from a T1-weighted volume-interpolated breath-hold examination (VIBE) on a Siemens Aera 1.5 T MRI scanner. The liver, lungs and abdominal trunk were segmented using the Hermes image processing workstation. Organ volumes were then uploaded to the Delft Visualization and Image processing Development Environment for smoothing and surface rendering. Triangular meshes defining the iso-surfaces were saved as stereo lithography (STL) files and imported into the Autodesk® Meshmixer software. Organ volumes were subtracted from the abdomen and a removable base designed to allow access to the liver cavity. Connection points for placing lesion inserts and filling holes were also included. The phantom was manufactured using a Stratasys Connex3 PolyJet 3D printer. The printer uses stereolithography technology combined with ink jet printing. Print material is a solid acrylic plastic, with similar properties to polymethylmethacrylate (PMMA). RESULTS: Measured Hounsfield units and calculated attenuation coefficients of the material were shown to also be similar to PMMA. Total print time for the phantom was approximately 5 days. Initial scans of the phantom have been performed with Y-90 bremsstrahlung SPECT/CT, Y-90 PET/CT and Tc-99m SPECT/CT. The CT component of these images compared well with the original anatomical reference, and measurements of volume agreed to within 9 %. Quantitative analysis of the phantom was performed using all three imaging techniques. Lesion and normal liver absorbed doses were calculated from the quantitative images in three dimensions using the local deposition method. CONCLUSIONS: 3D printing is a flexible and cost-efficient technology for manufacture of anthropomorphic phantom. Application of such phantoms will enable quantitative imaging and dosimetry methodologies to be evaluated, which with optimisation could help improve outcome for patients.

Exploring the biomechanical properties of brain malignancies and their pathologic determinants in vivo with magnetic resonance elastography.

Malignant tumors are typically associated with altered rigidity relative to normal host tissue. Magnetic resonance elastography (MRE) enables the noninvasive quantitation of the mechanical properties of deep-seated tissue following application of an external vibrational mechanical stress to that tissue. In this preclinical study, we used MRE to quantify (kPa) the elasticity modulus Gd and viscosity modulus Gl of three intracranially implanted glioma and breast metastatic tumor models. In all these brain tumors, we found a notable softness characterized by lower elasticity and viscosity than normal brain parenchyma, enabling their detection on Gd and Gl parametric maps. The most circumscribed tumor (U-87 MG glioma) was the stiffest, whereas the most infiltrative tumor (MDA-MB-231 metastatic breast carcinoma) was the softest. Tumor cell density and microvessel density correlated significantly and positively with elasticity and viscosity, whereas there was no association with the extent of collagen deposition or myelin fiber entrapment. In conclusion, although malignant tumors tend to exhibit increased rigidity, intracranial tumors presented as remarkably softer than normal brain parenchyma. Our findings reinforce the case for MRE use in diagnosing and staging brain malignancies, based on the association of different tumor phenotypes with different mechanical properties.

Improved survival with T cell clonotype stability after anti-CTLA-4 treatment in cancer patients.

Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) blockade can promote antitumor T cell immunity and clinical responses. The mechanism by which anti-CTLA-4 antibodies induces antitumor responses is controversial. To determine the effects of CTLA-4 blockade on the T cell repertoire, we used next-generation deep sequencing to measure the frequency of individual rearranged T cell receptor ß (TCRß) genes, thereby characterizing the diversity of rearrangements, known as T cell clonotypes. CTLA-4 blockade in patients with metastatic castration-resistant prostate cancer and metastatic melanoma resulted in both expansion and loss of T cell clonotypes, consistent with a global turnover of the T cell repertoire. Overall, this treatment increased TCR diversity as reflected in the number of unique TCR clonotypes. The repertoire of clonotypes continued to evolve over subsequent months of treatment. Whereas the number of clonotypes that increased with treatment was not associated with clinical outcome, improved overall survival was associated with maintenance of high-frequency clones at baseline. In contrast, the highest-frequency clonotypes fell with treatment in patients with short overall survival. Stably maintained clonotypes included T cells having high-avidity TCR such as virus-reactive T cells. Together, these results suggest that CTLA-4 blockade induces T cell repertoire evolution and diversification. Moreover, improved clinical outcomes are associated with less clonotype loss, consistent with the maintenance of high-frequency TCR clonotypes during treatment. These clones may represent the presence of preexisting high-avidity T cells that may be relevant in the antitumor response.

Transcriptional profiling of the iron starvation response in Bordetella pertussis provides new insights into siderophore utilization and virulence gene expression.

Serological studies of patients with pertussis and the identification of antigenic Bordetella pertussis proteins support the hypothesis that B. pertussis perceives an iron starvation cue and expresses multiple iron source utilization systems in its natural human host environment. Furthermore, previous studies using a murine respiratory tract infection model showed that several of these B. pertussis iron systems are required for colonization and persistence and are differentially expressed over the course of infection. The present study examined genome-wide changes in B. pertussis gene transcript abundance in response to iron starvation in vitro. In addition to known iron source utilization genes, we identified a previously uncharacterized iron-repressed cytoplasmic membrane transporter system, fbpABC, that is required for the utilization of multiple structurally distinct siderophores including alcaligin, enterobactin, ferrichrome, and desferrioxamine B. Expression of type III secretion system genes was also found to be upregulated during iron starvation in both B. pertussis strain Tohama I and Bordetella bronchiseptica strain RB50. In a survey of type III secretion system protein production by an assortment of B. pertussis laboratory-adapted and low-passage clinical isolate strains, iron limitation increased the production and secretion of the type III secretion system-specific translocation apparatus tip protein Bsp22 in all Bvg-proficient strains. These results indicate that iron starvation in the infected host is an important environmental cue influencing not only Bordetella iron transport gene expression but also the expression of other important virulence-associated genes.

A novel rhoptry protein in Toxoplasma gondii bradyzoites and merozoites.

The secretory organelles of Toxoplasma gondii orchestrate invasion of the host cell and establish the parasitophorous vacuole. Although much has been learned about the roles played by these organelles in invasion by the tachyzoite stage, little is known about the contents or functions of these organelles during bradyzoite development or pathogenesis. We identified a novel protein that localizes to the rhoptries of the bradyzoite stage, but is absent from the tachyzoite stage. This protein, BRP1, first appears in the nascent rhoptries during the first division of bradyzoite stage development. We observed secretion of BRP1 and other rhoptry proteins into the parasitophorous vacuole during bradyzoite development in vitro, but there was no evidence that this occurs in vivo. Brp1 knockout parasites did not appear to have any developmental or growth defects in vitro, and were able to establish infections in mice both as tachyzoites (via intraperitoneal injection of in vitro-derived tachyzoites) or bradyzoites (via oral gavage using cysts harvested from mouse brain). Mice infected using brain cysts from the brp1 knockout or the control strain developed similar numbers and sizes of brain cysts. Thus BRP1 does not appear to play an essential role in development of the bradyzoite stage, development of brain cysts, or oral infection of new hosts, at least in the mouse model used here. Since we also observed that BRP1 is expressed in the merozoite stages in the gut of infected cats, the coccidian phase of the life cycle may be where BRP1 plays its most important role.

Daughterless coordinates somatic cell proliferation, differentiation and germline cyst survival during follicle formation in Drosophila.

During Drosophila oogenesis two distinct stem cell populations produce either germline cysts or the somatic cells that surround each cyst and separate each formed follicle. From analyzing daughterless (da) loss-of-function, overexpression and genetic interaction phenotypes, we have identified several specific requirements for da(+) in somatic cells during follicle formation. First, da is a critical regulator of somatic cell proliferation. Also, da is required for the complete differentiation of polar and stalk cells, and elevated da levels can even drive the convergence and extension that is characteristic of interfollicular stalks. Finally, da is a genetic regulator of an early checkpoint for germline cyst progression: Loss of da function inhibits normally occurring apoptosis of germline cysts at the region 2a/2b boundary of the germarium, while da overexpression leads to postmitotic cyst degradation. Collectively, these da functions govern the abundance and diversity of somatic cells as they coordinate with germline cysts to form functional follicles.