Maintenance of bacterial outer membrane lipid asymmetry: insight into MlaA.

The outer membrane (OM) of Gram-negative bacteria acts as an effective barrier to protect against toxic compounds. By nature, the OM is asymmetric with the highly packed lipopolysaccharide (LPS) at the outer leaflet and glycerophospholipids at the inner leaflet. OM asymmetry is maintained by the Mla system, in which is responsible for the retrograde transport of glycerophospholipids from the OM to the inner membrane. This system is comprised of six Mla proteins, including MlaA, an OM lipoprotein involved in the removal of glycerophospholipids that are mis-localized at the outer leaflet of the OM. Interestingly, MlaA was initially identified - and called VacJ - based on its role in the intracellular spreading of Shigella flexneri.Many open questions remain with respect to the Mla system and the mechanism involved in the translocation of mislocated glycerophospholipids at the outer leaflet of the OM, by MlaA. After summarizing the current knowledge on MlaA, we focus on the impact of mlaA deletion on OM lipid composition and biophysical properties of the OM. How changes in OM lipid composition and biophysical properties can impact the generation of membrane vesicles and membrane permeability is discussed. Finally, we explore whether and how MlaA might be a candidate for improving the activity of antibiotics and as a vaccine candidate.Efforts dedicated to understanding the relationship between the OM lipid composition and the mechanical strength of the bacterial envelope and, in turn, how such properties act against external stress, are needed for the design of new targets or drugs for Gram-negative infections.

Super-resolution reconstruction of γ-ray CT images for PET-enabled dual-energy CT imaging.

Dual-energy computed tomography (DECT) enables material decomposition for tissues and produces additional information for PET/CT imaging to potentially improve the characterization of diseases. PET-enabled DECT (PDECT) allows the generation of PET and DECT images simultaneously with a conventional PET/CT scanner without the need for a second x-ray CT scan. In PDECT, high-energy γ-ray CT (GCT) images at 511 keV are obtained from time-of-flight (TOF) PET data and are combined with the existing x-ray CT images to form DECT imaging. We have developed a kernel-based maximum-likelihood attenuation and activity (MLAA) method that uses x-ray CT images as a priori information for noise suppression. However, our previous studies focused on GCT image reconstruction at the PET image resolution which is coarser than the image resolution of the x-ray CT. In this work, we explored the feasibility of generating super-resolution GCT images at the corresponding CT resolution. The study was conducted using both phantom and patient scans acquired with the uEXPLORER total-body PET/CT system. GCT images at the PET resolution with a pixel size of 4.0 mm × 4.0 mm and at the CT resolution with a pixel size of 1.2 mm × 1.2 mm were reconstructed using both the standard MLAA and kernel MLAA methods. The results indicated that the GCT images at the CT resolution had sharper edges and revealed more structural details compared to the images reconstructed at the PET resolution. Furthermore, images from the kernel MLAA method showed substantially improved image quality compared to those obtained with the standard MLAA method.

Spatio-Temporal Positron Emission Tomography Reconstruction with Attenuation and Motion Correction.

The detection of cancer lesions of a comparable size to that of the typical system resolution of modern scanners is a long-standing problem in Positron Emission Tomography. In this paper, the effect of composing an image-registering convolutional neural network with the modeling of the static data acquisition (i.e., the forward model) is investigated. Two algorithms for Positron Emission Tomography reconstruction with motion and attenuation correction are proposed and their performance is evaluated in the detectability of small pulmonary lesions. The evaluation is performed on synthetic data with respect to chosen figures of merit, visual inspection, and an ideal observer. The commonly used figures of merit-Peak Signal-to-Noise Ratio, Recovery Coefficient, and Signal Difference-to-Noise Ration-give inconclusive responses, whereas visual inspection and the Channelised Hotelling Observer suggest that the proposed algorithms outperform current clinical practice.

A review of PET attenuation correction methods for PET-MR.

Despite being thirteen years since the installation of the first PET-MR system, the scanners constitute a very small proportion of the total hybrid PET systems installed. This is in stark contrast to the rapid expansion of the PET-CT scanner, which quickly established its importance in patient diagnosis within a similar timeframe. One of the main hurdles is the development of an accurate, reproducible and easy-to-use method for attenuation correction. Quantitative discrepancies in PET images between the manufacturer-provided MR methods and the more established CT- or transmission-based attenuation correction methods have led the scientific community in a continuous effort to develop a robust and accurate alternative. These can be divided into four broad categories: (i) MR-based, (ii) emission-based, (iii) atlas-based and the (iv) machine learning-based attenuation correction, which is rapidly gaining momentum. The first is based on segmenting the MR images in various tissues and allocating a predefined attenuation coefficient for each tissue. Emission-based attenuation correction methods aim in utilising the PET emission data by simultaneously reconstructing the radioactivity distribution and the attenuation image. Atlas-based attenuation correction methods aim to predict a CT or transmission image given an MR image of a new patient, by using databases containing CT or transmission images from the general population. Finally, in machine learning methods, a model that could predict the required image given the acquired MR or non-attenuation-corrected PET image is developed by exploiting the underlying features of the images. Deep learning methods are the dominant approach in this category. Compared to the more traditional machine learning, which uses structured data for building a model, deep learning makes direct use of the acquired images to identify underlying features. This up-to-date review goes through the literature of attenuation correction approaches in PET-MR after categorising them. The various approaches in each category are described and discussed. After exploring each category separately, a general overview is given of the current status and potential future approaches along with a comparison of the four outlined categories.

Deficient Pseudomonas aeruginosa in MlaA/VacJ outer membrane lipoprotein shows decrease in rhamnolipids secretion, motility, and biofilm formation, and increase in fluoroquinolones susceptibility and innate immune response.

Pseudomonas aeruginosa, a Gram-negative bacterium that causes severe hospital acquired infections poses threat by its ability for adaptation to various growth modes and environmental conditions and by its intrinsic resistance to antibiotics. The latter is mainly due to the outer membrane (OM) asymmetry which is maintained by the Mla pathway resulting in the retrograde transport of glycerophospholipids from the OM to the inner membrane. It comprises six Mla proteins, including MlaA, an OM lipoprotein involved in the removal of glycerophospholipids mislocalized at the outer leaflet of OM. To investigate the role of P. aeruginosa OM asymmetry especially MlaA, this study investigated the effect of mlaA deletion on (i) the susceptibility to antibiotics, (ii) the secretion of virulence factors, the motility, biofilm formation, and (iii) the inflammatory response. mlaA deletion in P. aeruginosa ATCC27853 results in phenotypic changes including, an increase in fluoroquinolones susceptibility and in PQS (Pseudomonas Quinolone Signal) and TNF-α release and a decrease in rhamnolipids secretion, motility and biofilm formation. Investigating how the mlaA knockout impacts on antibiotic susceptibility, bacterial virulence and innate immune response will help to elucidate the biological significance of the Mla system and contribute to the understanding of MlaA in P. aeruginosa OM asymmetry.

A quantitative clinical evaluation of simultaneous reconstruction of attenuation and activity in time-of-flight PET.

BACKGROUND: The maximum likelihood activity and attenuation (MLAA) reconstruction algorithm has been proposed to jointly estimate tracer activity and attenuation at the same time, and proven to be a promising solution to the CT attenuation correction (CT-AC) artifacts in PET images. This study aimed to perform a quantitative evaluation and clinical validation of the MLAA method. METHODS: A uniform cylinder phantom filled with 18F-FDG solution was scanned to optimize the reconstruction parameters for the implemented MLAA algorithm. 67 patients who underwent whole-body 18F-FDG PET/CT scan were retrospectively recruited. PET images were reconstructed using MLAA and clinical standard OSEM algorithm with CT-AC (CT-OSEM). The mean and maximum standardized uptake values (SUVmean and SUVmax) in regions of interest (ROIs) of organs, high uptake lesions and areas affected by metal implants and respiration motion artifacts were quantitatively analyzed. RESULTS: In quantitative analysis, SUVs in patient's organ ROIs between two methods showed R2 ranging from 0.91 to 0.98 and k ranging from 0.90 to 1.06, and the average SUVmax and SUVmean differences between two methods were within 10% range, except for the lung ROI, which was 10.5% and 16.73% respectively. The average SUVmax and SUVmean differences of a total of 117 high uptake lesions were 7.25% and 7.10% respectively. 20 patients were identified to have apparent respiration motion artifacts in the liver in CT-OSEM images, and the SUVs differences between two methods measured at dome of the liver were significantly larger than measured at middle part of the liver. 10 regions with obvious metal artifacts were identified in CT-OSEM images and the average SUVmean and SUVmax differences in metal implants affected regions were reported to be 52.90% and 56.20% respectively. CONCLUSIONS: PET images reconstructed using MLAA are clinically acceptable in terms of image quality as well as quantification and it is a useful tool in clinical practice, especially when CT-AC may cause respiration motion and metal artifacts. Moreover, this study also provides technical reference and data support for the future iteration and development of PET reconstruction technology of SUV accurate quantification.

Deep learning-based attenuation map generation with simultaneously reconstructed PET activity and attenuation and low-dose application.

Objective. In PET/CT imaging, CT is used for positron emission tomography (PET) attenuation correction (AC). CT artifacts or misalignment between PET and CT can cause AC artifacts and quantification errors in PET. Simultaneous reconstruction (MLAA) of PET activity (λ-MLAA) and attenuation (µ-MLAA) maps was proposed to solve those issues using the time-of-flight PET raw data only. However,λ-MLAA still suffers from quantification error as compared to reconstruction using the gold-standard CT-based attenuation map (µ-CT). Recently, a deep learning (DL)-based framework was proposed to improve MLAA by predictingµ-DL fromλ-MLAA andµ-MLAA using an image domain loss function (IM-loss). However, IM-loss does not directly measure the AC errors according to the PET attenuation physics. Our preliminary studies showed that an additional physics-based loss function can lead to more accurate PET AC. The main objective of this study is to optimize the attenuation map generation framework for clinical full-dose18F-FDG studies. We also investigate the effectiveness of the optimized network on predicting attenuation maps for synthetic low-dose oncological PET studies.Approach. We optimized the proposed DL framework by applying different preprocessing steps and hyperparameter optimization, including patch size, weights of the loss terms and number of angles in the projection-domain loss term. The optimization was performed based on 100 skull-to-toe18F-FDG PET/CT scans with minimal misalignment. The optimized framework was further evaluated on 85 clinical full-dose neck-to-thigh18F-FDG cancer datasets as well as synthetic low-dose studies with only 10% of the full-dose raw data.Main results. Clinical evaluation of tumor quantification as well as physics-based figure-of-merit metric evaluation validated the promising performance of our proposed method. For both full-dose and low-dose studies, the proposed framework achieved <1% error in tumor standardized uptake value measures.Significance. It is of great clinical interest to achieve CT-less PET reconstruction, especially for low-dose PET studies.

Deep learning-based attenuation correction for whole-body PET - a multi-tracer study with 18F-FDG, 68 Ga-DOTATATE, and 18F-Fluciclovine.

A novel deep learning (DL)-based attenuation correction (AC) framework was applied to clinical whole-body oncology studies using 18F-FDG, 68 Ga-DOTATATE, and 18F-Fluciclovine. The framework used activity (λ-MLAA) and attenuation (µ-MLAA) maps estimated by the maximum likelihood reconstruction of activity and attenuation (MLAA) algorithm as inputs to a modified U-net neural network with a novel imaging physics-based loss function to learn a CT-derived attenuation map (µ-CT). METHODS: Clinical whole-body PET/CT datasets of 18F-FDG (N = 113), 68 Ga-DOTATATE (N = 76), and 18F-Fluciclovine (N = 90) were used to train and test tracer-specific neural networks. For each tracer, forty subjects were used to train the neural network to predict attenuation maps (µ-DL). µ-DL and µ-MLAA were compared to the gold-standard µ-CT. PET images reconstructed using the OSEM algorithm with µ-DL (OSEMDL) and µ-MLAA (OSEMMLAA) were compared to the CT-based reconstruction (OSEMCT). Tumor regions of interest were segmented by two radiologists and tumor SUV and volume measures were reported, as well as evaluation using conventional image analysis metrics. RESULTS: µ-DL yielded high resolution and fine detail recovery of the attenuation map, which was superior in quality as compared to µ-MLAA in all metrics for all tracers. Using OSEMCT as the gold-standard, OSEMDL provided more accurate tumor quantification than OSEMMLAA for all three tracers, e.g., error in SUVmax for OSEMMLAA vs. OSEMDL: - 3.6 ± 4.4% vs. - 1.7 ± 4.5% for 18F-FDG (N = 152), - 4.3 ± 5.1% vs. 0.4 ± 2.8% for 68 Ga-DOTATATE (N = 70), and - 7.3 ± 2.9% vs. - 2.8 ± 2.3% for 18F-Fluciclovine (N = 44). OSEMDL also yielded more accurate tumor volume measures than OSEMMLAA, i.e., - 8.4 ± 14.5% (OSEMMLAA) vs. - 3.0 ± 15.0% for 18F-FDG, - 14.1 ± 19.7% vs. 1.8 ± 11.6% for 68 Ga-DOTATATE, and - 15.9 ± 9.1% vs. - 6.4 ± 6.4% for 18F-Fluciclovine. CONCLUSIONS: The proposed framework provides accurate and robust attenuation correction for whole-body 18F-FDG, 68 Ga-DOTATATE and 18F-Fluciclovine in tumor SUV measures as well as tumor volume estimation. The proposed method provides clinically equivalent quality as compared to CT in attenuation correction for the three tracers.

A CT-less approach to quantitative PET imaging using the LSO intrinsic radiation for long-axial FOV PET scanners.

PURPOSE: Long-axial field-of-view (FOV) positron emission tomography (PET) scanners have gained a lot of interest in the recent years. Such scanners provide increased sensitivity and enable unique imaging opportunities that were not previously feasible. Benefiting from the high sensitivity of a long-axial FOV PET scanner, we studied a computed tomography (CT)-less reconstruction algorithm for the Siemens Biograph Vision Quadra with an axial FOV of 106 cm. METHODS: In this work, the background radiation from radioisotope lutetium-176 in the scintillators was used to create an initial estimate of the attenuation maps. Then, joint activity and attenuation reconstruction algorithms were used to create an improved attenuation map of the object. The final attenuation maps were then used to reconstruct quantitative PET images, which were compared against CT-based PET images. The proposed method was evaluated on data from three patients who underwent a flurodeoxyglucouse PET scan. RESULTS: Segmentation of the PET images of the three studied patients showed an average quantitative error of 6.5%-8.3% across all studied organs when using attenuation maps from maximum likelihood estimation of attenuation and activity and 5.3%-6.6% when using attenuation maps from maximum likelihood estimation of activity and attenuation correction coefficients. CONCLUSIONS: Benefiting from the background radiation of lutetium-based scintillators, a quantitative CT-less PET imaging technique was evaluated in this work.

Attenuation Coefficient Estimation for PET/MRI With Bayesian Deep Learning Pseudo-CT and Maximum-Likelihood Estimation of Activity and Attenuation.

A major remaining challenge for magnetic resonance-based attenuation correction methods (MRAC) is their susceptibility to sources of magnetic resonance imaging (MRI) artifacts (e.g., implants and motion) and uncertainties due to the limitations of MRI contrast (e.g., accurate bone delineation and density, and separation of air/bone). We propose using a Bayesian deep convolutional neural network that in addition to generating an initial pseudo-CT from MR data, it also produces uncertainty estimates of the pseudo-CT to quantify the limitations of the MR data. These outputs are combined with the maximum-likelihood estimation of activity and attenuation (MLAA) reconstruction that uses the PET emission data to improve the attenuation maps. With the proposed approach uncertainty estimation and pseudo-CT prior for robust MLAA (UpCT-MLAA), we demonstrate accurate estimation of PET uptake in pelvic lesions and show recovery of metal implants. In patients without implants, UpCT-MLAA had acceptable but slightly higher root-mean-squared-error (RMSE) than Zero-echotime and Dixon Deep pseudo-CT when compared to CTAC. In patients with metal implants, MLAA recovered the metal implant; however, anatomy outside the implant region was obscured by noise and crosstalk artifacts. Attenuation coefficients from the pseudo-CT from Dixon MRI were accurate in normal anatomy; however, the metal implant region was estimated to have attenuation coefficients of air. UpCT-MLAA estimated attenuation coefficients of metal implants alongside accurate anatomic depiction outside of implant regions.

Current mechanistic understanding of intermembrane lipid trafficking important for maintenance of bacterial outer membrane lipid asymmetry.

The outer membrane (OM) of Gram-negative bacteria exhibits unique lipid asymmetry that makes it an effective permeability barrier against toxic molecules, including antibiotics. Central to the maintenance of OM lipid asymmetry is the OmpC-Mla (maintenance of lipid asymmetry) system, which mediates the retrograde transport of phospholipids from the outer leaflet of the OM to the inner membrane. The molecular mechanism(s) of this lipid trafficking process is not fully understood; however, recent advances in structural elucidations and biochemical reconstitutions have provided detailed new insights. Here, we present an integrated understanding of how the OmpC-Mla system transports mislocalized phospholipids across the bacterial cell envelope.

Can a ToF-PET photon attenuation reconstruction test stopping-power estimations in proton therapy? A phantom study.

Objective. The aim of the phantom study was to validate and to improve the computed tomography (CT) images used for the dose computation in proton therapy. It was tested, if the joint reconstruction of activity and attenuation images of time-of-flight PET (ToF-PET) scans could improve the estimation of the proton stopping-power.Approach. The attenuation images, i.e. CT images with 511 keV gamma-rays (γCTs), were jointly reconstructed with activity maps from ToF-PET scans. Theß+activity was produced with FDG and in a separate experiment with proton-induced radioactivation. The phantoms contained slabs of tissue substitutes. The use of theγCTs for the prediction of the beam stopping in proton therapy was based on a linear relationship between theγ-ray attenuation, the electron density, and the stopping-power of fast protons.Main results. The FDG based experiment showed sufficient linearity to detect a bias of bony tissue in the heuristic look-up table, which maps between x-ray CT images and proton stopping-power.γCTs can be used for dose computation, if the electron density of one type of tissue is provided as a scaling factor. A possible limitation is imposed by the spatial resolution, which is inferior by a factor of 2.5 compared to the one of the x-ray CT.γCTs can also be derived from off-line, ToF-PET scans subsequent to the application of a proton field with a hypofractionated dose level.Significance. γCTs are a viable tool to support the estimation of proton stopping with radiotracer-based ToF-PET data from diagnosis or staging. This could be of higher potential relevance in MRI-guided proton therapy.γCTs could form an alternative approach to make use of in-beam or off-line PET scans of proton-inducedß+activity with possible clinical limitations due to the low number of coincidence counts.

Phenotypic and Multi-Omics Characterization of Escherichia coli K-12 Adapted to Chlorhexidine Identifies the Role of MlaA and Other Cell Envelope Alterations Regulated by Stress Inducible Pathways in CHX Resistance.

Chlorhexidine (CHX) is an essential medicine used as a topical antiseptic in skin and oral healthcare treatments. The widespread use of CHX has increased concerns regarding the development of antiseptic resistance in Enterobacteria and its potential impact on cross-resistance to other antimicrobials. Similar to other cationic antiseptics, resistance to CHX is believed to be driven by three membrane-based mechanisms: lipid synthesis/transport, altered porin expression, and increased efflux pump activity; however, specific gene and protein alterations associated with CHX resistance remain unclear. Here, we adapted Escherichia coli K-12 BW25113 to increasing concentrations of CHX to determine what phenotypic, morphological, genomic, transcriptomic, and proteomic changes occurred. We found that CHX-adapted E. coli isolates possessed no cross-resistance to any other antimicrobials we tested. Scanning electron microscopy imaging revealed that CHX adaptation significantly altered mean cell widths and lengths. Proteomic analyses identified changes in the abundance of porin OmpF, lipid synthesis/transporter MlaA, and efflux pump MdfA. Proteomic and transcriptomic analyses identified that CHX adaptation altered E. coli transcripts and proteins controlling acid resistance (gadE, cdaR) and antimicrobial stress-inducible pathways Mar-Sox-Rob, stringent response systems. Whole genome sequencing analyses revealed that all CHX-resistant isolates had single nucleotide variants in the retrograde lipid transporter gene mlaA as well as the yghQ gene associated with lipid A transport and synthesis. CHX resistant phenotypes were reversible only when complemented with a functional copy of the mlaA gene. Our results highlight the importance of retrograde phospholipid transport and stress response systems in CHX resistance and the consequences of prolonged CHX exposure.

Peptides Affecting the Outer Membrane Lipid Asymmetry System (MlaA-OmpC/F) Reduce Avian Pathogenic Escherichia coli (APEC) Colonization in Chickens.

Avian pathogenic Escherichia coli (APEC), an extraintestinal pathogenic E. coli (ExPEC), causes colibacillosis in chickens and is reportedly associated with urinary tract infections and meningitis in humans. Development of resistance is a major limitation of current ExPEC antibiotic therapy. New antibacterials that can circumvent resistance problem such as antimicrobial peptides (AMPs) are critically needed. Here, we evaluated the efficacy of Lactobacillus rhamnosus GG (LGG)-derived peptides against APEC and uncovered their potential antibacterial targets. Three peptides (NPSRQERR [P1], PDENK [P2], and VHTAPK [P3]) displayed inhibitory activity against APEC. These peptides were effective against APEC in biofilm and chicken macrophage HD11 cells. Treatment with these peptides reduced the cecum colonization (0.5 to 1.3 log) of APEC in chickens. Microbiota analysis revealed two peptides (P1 and P2) decreased Enterobacteriaceae abundance with minimal impact on overall cecal microbiota of chickens. Bacterial cytological profiling showed peptides disrupt APEC membranes either by causing membrane shedding, rupturing, or flaccidity. Furthermore, gene expression analysis revealed that peptides downregulated the expression of ompC (>13.0-fold), ompF (>11.3-fold), and mlaA (>4.9-fold), genes responsible for the maintenance of outer membrane (OM) lipid asymmetry. Consistently, immunoblot analysis also showed decreased levels of OmpC and MlaA proteins in APEC treated with peptides. Alanine scanning studies revealed residues crucial (P1, N, E, R and P; P2, D and E; P3, T, P, and K) for their activity. Overall, our study identified peptides with a new antibacterial target that can be developed to control APEC infections in chickens, thereby curtailing poultry-originated human ExPEC infections. IMPORTANCE Avian pathogenic Escherichia coli (APEC) is a subgroup of extraintestinal pathogenic E. coli (ExPEC) and considered a foodborne zoonotic pathogen transmitted through consumption of contaminated poultry products. APEC shares genetic similarities with human ExPECs, including uropathogenic E. coli (UPEC) and neonatal meningitis E. coli (NMEC). Our study identified Lactobacillus rhamnosus GG (LGG)-derived peptides (P1 [NPSRQERR], P2 [PDENK], and P3 [VHTAPK]) effective in reducing APEC infection in chickens. Antimicrobial peptides (AMPs) are regarded as ideal candidates for antibacterial development because of their low propensity for resistance development and ability to kill resistant bacteria. Mechanistic studies showed peptides disrupt the APEC membrane by affecting the MlaA-OmpC/F system responsible for the maintenance of outer membrane (OM) lipid asymmetry, a promising new druggable target to overcome resistance problems in Gram-negative bacteria. Altogether, these peptides can provide a valuable approach for development of novel anti-ExPEC therapies, including APEC, human ExPECs, and other related Gram-negative pathogens. Furthermore, effective control of APEC infections in chickens can curb poultry-originated ExPEC infections in humans.

Neural MLAA for PET-enabled Dual-Energy CT Imaging.

The PET-enabled dual-energy CT method allows dual-energy CT imaging on PET/CT scanners without the need for a second x-ray CT scan. A 511 keV γ-ray attenuation image can be reconstructed from time-of-flight PET emission data using the maximum-likelihood attenuation and activity (MLAA) algorithm. However, the attenuation image reconstructed by standard MLAA is commonly noisy. To suppress noise, we propose a neural-network approach for MLAA reconstruction. The PET attenuation image is described as a function of kernelized neural networks with the flexibility of incorporating the available x-ray CT image as anatomical prior. By applying optimization transfer, the complex optimization problem of the proposed neural MLAA reconstruction is solved by a modularized iterative algorithm with each iteration decomposed into three steps: PET activity image update, attenuation image update, and neural-network learning using a weighed mean squared-error loss. The optimization algorithm is guaranteed to monotonically increase the data likelihood. The results from computer simulations demonstrated the neural MLAA algorithm can achieve a significant improvement on the γ-ray CT image quality as compared to other algorithms.

MLAA-34 knockdown shows enhanced antitumor activity via JAK2/STAT3 signaling pathway in acute monocytic leukemia.

MLAA-34 is a novel leukemia-associated gene closely related to the carcinogenesis of acute monocytic leukemia (AML). MLAA-34 over expression has been observed to inhibit apoptosis in vitro. JAK2/STAT3 pathway plays an important role in cell proliferation, differentiation and inhibition of apoptosis in number of cancers. However, the relationship and interaction between MLAA-34 and JAK2/STAT3 has never been investigated in AML. This study investigates and reports a novel relationship between MLAA-34 and JAK2/STAT3 pathway in AML both in vitro and in vivo. We constructed MLAA-34 knockdown vector and transfected U937 cells to observe its apoptotic activities in relation to JAK2/STAT3 signaling pathway in vitro and then in vivo in mouse model. Levels of expression of MLAA-34 and JAK2/STAT3 and its downstream targets were also measured in AML patients and a few volunteers. We found that MLAA-34 knockdown increased U937 apoptosis in vitro and inhibited tumor growth in vivo. Components of the canonical JAK2/STAT3 pathway or its downstream targets, including c-myc, bcl-2, Bax, and caspase-3, were shown to be involved in the carcinogenesis of AML. We also found that the JAK2/STAT3 pathway positively regulated MLAA-34 expression. We additionally identified a STAT3 binding site in the MLAA-34 promoter where STAT3 binds directly and activates MLAA-34 expression. In addition, MLAA-34 was found to form a complex with JAK2 and was enhanced by JAK2 activation. Correlation of MLAA-34 and JAK2/STAT3 was further confirmed in AML patients. In conclusion, MLAA-34 is a novel regulator for JAK2/STAT3 signaling, and in turn, is regulated by this interaction in a positive feedback loop. Thus we report a novel model of interaction mechanism between MLAA-34 and JAK2/STAT3 which can be utilized as a potential target for a novel therapeutic approach in AML.

The inner membrane protein YhdP modulates the rate of anterograde phospholipid flow in Escherichia coli.

The outer membrane (OM) of Gram-negative bacteria is a selective permeability barrier that allows uptake of nutrients while simultaneously protecting the cell from harmful compounds. The basic pathways and molecular machinery responsible for transporting lipopolysaccharides (LPS), lipoproteins, and ß-barrel proteins to the OM have been identified, but very little is known about phospholipid (PL) transport. To identify genes capable of affecting PL transport, we screened for genetic interactions with mlaA*, a mutant in which anterograde PL transport causes the inner membrane (IM) to shrink and eventually rupture; characterization of mlaA*-mediated lysis suggested that PL transport can occur via a high-flux diffusive flow mechanism. We found that YhdP, an IM protein involved in maintaining the OM permeability barrier, modulates the rate of PL transport during mlaA*-mediated lysis. Deletion of yhdP from mlaA* reduced the rate of IM transport to the OM by 50%, slowing shrinkage of the IM and delaying lysis. As a result, the weakened OM of ∆yhdP cells was further compromised and ruptured before the IM during mlaA*-mediated death. These findings demonstrate the existence of a high-flux diffusive pathway for PL flow in Escherichia coli that is modulated by YhdP.

Evaluation of improved attenuation correction in whole-body PET/MR on patients with bone metastasis using various radiotracers.

PURPOSE: This study evaluates the quantitative effect of improved MR-based attenuation correction (AC), including bone segmentation and the HUGE method for truncation correction in PET/MR whole-body hybrid imaging specifically of oncologic patients with bone metastasis and using various radiotracers. METHODS: Twenty-three patients that underwent altogether 28 whole-body PET/MR examinations with findings of bone metastasis were included in this study. Different radiotracers (18F-FDG, 68Ga-PSMA, 68Ga-DOTATOC, 124I-MIBG) were injected according to appropriate clinical indications. Each of the 28 whole-body PET datasets was reconstructed three times using AC with (1) standard four-compartment µ-maps (background air, lung, muscle, and soft tissue), (2) five-compartment µ-maps (adding bone), and (3) six-compartment µ-maps (adding bone and HUGE truncation correction). The SUVmax of each detected bone lesion was measured in each reconstruction to evaluate the quantitative impact of improved MR-based AC. Relative difference images between four- and six-compartment µ-maps were calculated. MR-based HUGE truncation correction was compared with the PET-based MLAA truncation correction method in all patients. RESULTS: Overall, 69 bone lesions were detected and evaluated. The mean increase in relative difference over all 69 lesions in SUVmax was 5.4 ± 6.4% when comparing the improved six-compartment AC with the standard four-compartment AC. Maximal relative difference of 28.4% was measured in one lesion. Truncation correction with HUGE worked robust and resulted in realistic body contouring in all 28 exams and for all 4 different radiotracers. Truncation correction with MLAA revealed overestimations of arm tissue volume in all PET/MR exams with 18F-FDG radiotracer and failed in all other exams with radiotracers 68Ga-PSMA, 68Ga-DOTATOC, and 124I- MIBG due to limitations in body contour detection. CONCLUSION: Improved MR-based AC, including bone segmentation and HUGE truncation correction in whole-body PET/MR on patients with bone lesions and using various radiotracers, is important to ensure best possible diagnostic image quality and accurate PET quantification. The HUGE method for truncation correction based on MR worked robust and results in realistic body contouring, independent of the radiotracers used.

Targeted silencing of genes related to acute monocytic leukaemia by CpG(B)-MLAA-34 siRNA conjugates.

Acute monocytic leukaemia (AML-M5) associated antigen-34 (MLAA-34) is a novel antigen overexpressed in patients with acute monocytic leukaemia. RNA interference is a promising therapy in oncology, especially for refractory acute leukaemia. In this study, we delivered MLAA-34 siRNA into AML-M5 THP-1 cells using CpG(B)-MLAA-34 siRNA conjugates, in the absence of any other transfection reagent. The uptake efficiency and the rate of apoptosis were measured by using flow cytometry. The level of relevant mRNAs was measured by quantitative PCR. THP-1 cell invasion was assessed by transwell assay. Protein expression was analysed by western blotting. The spleen and liver of AML-M5 nude mice were measured and weighted after euthanisation. Spleen sections were analysed by immunohistochemistry. We found that MLAA-34 siRNA was successfully delivered into THP-1 cells and induced MLAA-34 gene silencing via the blockade of JAK2/STAT3 and Wnt/-catenin signalling pathways. In addition, CpG(B)-MLAA-34 siRNA upregulated Gsk3ß protein expression, resulting in retraining of the JAK2/STAT3 and Wnt/ß-catenin signalling pathways. Importantly, CpG(B)-MLAA-34 siRNA reduced the survival and invasiveness of THP-1 cells. We further demonstrated that CAB39L was effectively downregulated by CpG(B)-MLAA-34 siRNA in vivo. These findings suggested CpG(B)-MLAA-34 siRNA conjugates may provide a novel therapeutic strategy for acute monocytic leukaemia.

Sodium Taurocholate Stimulates Campylobacter jejuni Outer Membrane Vesicle Production via Down-Regulation of the Maintenance of Lipid Asymmetry Pathway.

Campylobacter jejuni outer membrane vesicles (OMVs) contain numerous virulence-associated proteins including the cytolethal distending toxin and three serine proteases. As C. jejuni lacks the classical virulence-associated secretion systems of other enteric pathogens that deliver effectors directly into target cells, OMVs may have a particularly important role in virulence. C. jejuni OMV production is stimulated by the presence of physiological concentrations of the bile salt sodium taurocholate (ST) through an unknown mechanism. The maintenance of lipid asymmetry (MLA) pathway has been implicated in a novel mechanism for OMV biogenesis, open to regulation by host signals. In this study we investigated the role of the MLA pathway in C. jejuni OMV biogenesis with ST as a potential regulator. OMV production was quantified by analyzing protein and lipid concentrations of OMV preparations and OMV particle counts produced by nanoparticle tracking analysis. Mutation of mlaA which encodes the outer membrane component of the MLA pathway significantly increased OMV production compared to the wild-type strain. Detergent sensitivity and membrane permeability assays confirmed the increased OMV production was not due to changes in membrane stability. The presence of 0.2% (w/v) ST increased wild-type OMV production and reduced OMV size, but did not further stimulate mlaA mutant OMV production or significantly alter mlaA mutant OMV size. qRT-PCR analysis demonstrated that the presence of ST decreased expression of both mlaA and mlaC in C. jejuni wild-type strains 11168 and 488. Collectively the data in this study suggests C. jejuni can regulate OMV production in response to host gut signals through changes in expression of the MLA pathway. As the gut bile composition is dependent on both diet and the microbiota, this study highlights the potential importance of diet and lifestyle factors on the varying disease presentations associated with gut pathogen infection.