Acute Lymphoblastic Leukemia Cell Lines in Immunology Research.

A considerable portion of our knowledge on T and B cell biology is acquired from research using acute lymphoblastic leukemia (ALL) cell lines, which are invaluable tools used in many immunology and leukemia studies. Here, we discuss the advantages and limitations of ALL cell lines and provide guidelines on their proper usage.

Polyphenolic Characterization and Anti-Inflammatory Effect of In Vitro Digested Extracts of Echinacea purpurea L. Plant Parts in an Inflammatory Model of Human Colon Cells.

Echinacea purpurea L. (EP) preparations are globally popular herbal supplements known for their medicinal benefits, including anti-inflammatory activities, partly related to their phenolic composition. However, regarding their use for the management of inflammation-related intestinal diseases, the knowledge about the fate of orally ingested constituents throughout the human gastrointestinal tract and the exposition of in vitro digested extracts in relevant inflammatory models are unknown. This study investigated for the first time the impact of in vitro gastrointestinal digestion (INFOGEST) on the phenolic composition and anti-inflammatory properties of EP extracts from flowers (EF), leaves (EL), and roots (ER) on IL-1ß-treated human colon-derived CCD-18Co cells. Among the seven hydroxycinnamic acids identified using HPLC-UV-MS/MS, chicoric and caftaric acids showed the highest concentrations in EL, followed by EF and ER, and all extracts exerted significant reductions in IL-6, IL-8, and PGE2 levels. After digestion, despite reducing the bioaccessibility of their phenolics, the anti-inflammatory effects were preserved for digested EL and, to a lesser extent, for EF, but not for digested ER. The lower phenolic content in digested EF and ER could explain these findings. Overall, this study emphasizes the potential of EP in alleviating intestinal inflammatory conditions and related disorders.

Main Determinants Affecting the Antiproliferative Activity of Stilbenes and Their Gut Microbiota Metabolites in Colon Cancer Cells: A Structure-Activity Relationship Study.

trans-Resveratrol can be catabolized by the gut microbiota to dihydroresveratrol, 3,4'-dihydroxy-trans-stilbene, lunularin, and 4-hydroxydibenzyl. These metabolites can reach relevant concentrations in the colon. However, not all individuals metabolize RSV equally, as it depends on their RSV gut microbiota metabotype (i.e., lunularin producers vs. non-producers). However, how this microbial metabolism affects the cancer chemopreventive activity of stilbenes and their microbial metabolites is poorly known. We investigated the structure-antiproliferative activity relationship of dietary stilbenes, their gut microbial metabolites, and various analogs in human cancer (Caco-2 and HT-29) and non-tumorigenic (CCD18-Co) colon cells. The antiproliferative IC50 values of pterostilbene, oxy-resveratrol, piceatannol, resveratrol, dihydroresveratrol, lunularin, 3,4'-dihydroxy-trans-stilbene, pinosylvin, dihydropinosylvin, 4-hydroxy-trans-stilbene, 4-hydroxydibenzyl, 3-hydroxydibenzyl, and 4-trans-stilbenemethanol were calculated. IC50 values were correlated with 34 molecular characteristics by bi- and multivariate analysis. Little or no activity on CCD18-Co was observed, while Caco-2 was more sensitive than HT-29, which was explained by their different capacities to metabolize the compounds. Caco-2 IC50 values ranged from 11.4 ± 10.1 µM (4-hydroxy-trans-stilbene) to 73.9 ± 13.8 µM (dihydropinosylvin). In HT-29, the values ranged from 24.4 ± 11.3 µM (4-hydroxy-trans-stilbene) to 96.7 ± 6.7 µM (4-hydroxydibenzyl). At their IC50, most compounds induced apoptosis and arrested the cell cycle at the S phase, pterostilbene at G2/M, while 4-hydroxy-trans-stilbene and 3,4'-dihydroxy-trans-stilbene arrested at both phases. Higher Connolly values (larger size) hindered the antiproliferative activity, while a lower pKa1 enhanced the activity in Caco-2, and higher LogP values (more hydrophobicity) increased the activity in HT-29. Reducing the styrene double bond in stilbenes was the most critical feature in decreasing the antiproliferative activity. These results (i) suggest that gut microbiota metabolism determines the antiproliferative effects of dietary stilbenes. Therefore, RSV consumption might exert different effects in individuals depending on their gut microbiota metabotypes associated with RSV metabolism, and (ii) could help design customized drugs with a stilbenoid and (or) dibenzyl core against colorectal cancer.

Nα-Acetylation of the virulence factor EsxA is required for mycobacterial cytosolic translocation and virulence.

The Mycobacterium tuberculosis virulence factor EsxA and its chaperone EsxB are secreted as a heterodimer (EsxA:B) and are crucial for mycobacterial escape from phagosomes and cytosolic translocation. Current findings support the idea that for EsxA to interact with host membranes, EsxA must dissociate from EsxB at low pH. However, the molecular mechanism by which the EsxA:B heterodimer separates is not clear. In the present study, using liposome-leakage and cytotoxicity assays, LC-MS/MS-based proteomics, and CCF-4 FRET analysis, we obtained evidence that the Nα-acetylation of the Thr-2 residue on EsxA, a post-translational modification that is present in mycobacteria but absent in Escherichia coli, is required for the EsxA:B separation. Substitutions at Thr-2 that precluded Nα-acetylation inhibited the heterodimer separation and hence prevented EsxA from interacting with the host membrane, resulting in attenuated mycobacterial cytosolic translocation and virulence. Molecular dynamics simulations revealed that at low pH, the Nα-acetylated Thr-2 makes direct and frequent "bind-and-release" contacts with EsxB, which generates a force that pulls EsxB away from EsxA. In summary, our findings provide evidence that the Nα-acetylation at Thr-2 of EsxA facilitates dissociation of the EsxA:B heterodimer required for EsxA membrane permeabilization and mycobacterial cytosolic translocation and virulence.

Kv1.3 Current Voltage Dependence in Lymphocytes is Modulated by Co-Culture with Bone Marrow-Derived Stromal Cells: B and T Cells Respond Differentially.

BACKGROUND/AIMS: Kv1.3 channel is the only voltage-dependent potassium channel in plasma membrane of human lymphocytes. Bearing in mind a rather steep voltage-dependence of Kv1.3 activation and inactivation, its modulation by B and T cells activation and by co-culture with stromal bone-marrow cells was addressed. METHODS: Patch-clamp technique in the whole cell mode was applied to human resting and activated human B and T cells, in monoculture and co-culture with stromal OP9 cells. RESULTS: Polyclonal activation of B and T cells in monoculture caused Kv1.3 current in B cells to activate at more negative and in T cells at more positive potentials, whereas the inactivation of Kv1.3 current in resting T cells occurred at more negative voltages. Co-culture with OP9 cells abolished the shift of voltage dependence upon the polyclonal activation but fixed the substantial difference between B and T cells, resting or activated, with both activation and inactivation negatively shifted by 15 mV for T lymphocytes. However, activated B cells displayed an incomplete inactivation, which was augmented by the co-culture. Neither activation nor co-culture caused substantial changes in the Kv1.3 current density. CONCLUSION: The combination of activation and inactivation processes yields the fraction of steady-state Kv1.3 current (window current), which was higher in activated B cells, partly due to an incomplete inactivation. A relatively smaller window current in resting B cells and resting T cells in co-culture correlated with a more depolarized resting membrane potential. Rather than insignificant changes in the Kv1.3 channels functional expression, the modulation of their voltage dependence by activation and co-culture with bone-marrow stromal cells was essential for the control of membrane potential.

Wavefronts and caustics associated with Mathieu beams.

In this work we compute the wavefronts and the caustics associated with the solutions to the scalar wave equation introduced by Durnin in elliptical cylindrical coordinates generated by the function A(ϕ)=ceν(ϕ,q)+iseν(ϕ,q), with ν being an integral or nonintegral number. We show that the wavefronts and the caustic are invariant under translations along the direction of evolution of the beam. We remark that the wavefronts of the separable Mathieu beams generated by A(ϕ)=ceν(ϕ,q) and A(ϕ)=seν(ϕ,q) are cones and their caustic is the z axis; thus, they are not structurally stable. However, in general, the Mathieu beam generated by A(ϕ)=ceν(ϕ,q)+iseν(ϕ,q) is stable because locally its caustic has singularities of the fold and cusp types. To show this property, we present the wavefronts and the caustics for the Mathieu beams with characteristic value aν=0 and q=0,0.2,0.3,0.5. For q=0, we obtain the Bessel beam of order zero; in this case, the wavefronts are cones and the caustic coincides with the z axis. For q≠0, the wavefronts are deformations of conical ones, and the caustic surface, for some values of q, has singularities of the cusp ridge type. Furthermore, we remark that the set of Mathieu beams with characteristic value aν=0 and 0≤q<1 has associated a caustic with singularities of the swallowtail type, which is structurally stable. Therefore, we conclude that this type of Mathieu beam is more stable than plane waves, Bessel beams, parabolic beams, and those generated by A(ϕ)=ceν(ϕ,q) and A(ϕ)=seν(ϕ,q). To support this conclusion, we present experimental results showing the pattern obtained after obstructing a plane wave, the Bessel beam of order m=5, and the Mathieu beam of order m=5 and q=50 with complex transversal amplitude given by Ce5(ξ,50)ce5(η,50)+iSe5(ξ,50)se5(η,50), where (ξ, η) are the elliptical coordinates on the plane.

Wavefronts, caustic, and intensity of a plane wave refracted by an arbitrary surface: the axicon and the plano spherical lenses.

The aim of the present work is to obtain an integral representation of the field associated with the refraction of a plane wave by an arbitrary surface. To this end, in the first part we consider two optical media with refraction indexes n1 and n2 separated by an arbitrary interface, and we show that the optical path length, ϕ, associated with the evolution of the plane wave is a complete integral of the eikonal equation in the optical medium with refraction index n2. Then by using the k function procedure introduced by Stavroudis, we define a new complete integral, S, of the eikonal equation. We remark that both complete integrals in general do not provide the same information; however, they give the geometrical wavefronts, light rays, and the caustic associated with the refraction of the plane wave. In the second part, using the Fresnel-Kirchhoff diffraction formula and the complete integral, S, we obtain an integral representation for the field associated only with the refraction phenomena, the geometric field approximation, in terms of secondary plane waves and the k-function introduced by Stavroudis in solving the problem from the geometrical optics point of view. We use the general results to compute: the wavefronts, light rays, caustic, and the intensity associated with the refraction of a plane wave by an axicon and plano-spherical lenses.

Internal structure of an optical null Ronchi grating test for a plano-parabolic lens.

In this work we use geometrical optics and the caustic touching theorem, introduced by Berry, to describe the internal structure of the null Ronchi grating for a plano-parabolic lens illuminated by a point light source placed on the optical axis. The aim of this work is to explain the role of the caustic region in the process of morphology change between image and object in computing the null Ronchi grating. To this end, we obtain the analytic expression of the null Ronchi grating, and after that we deeply study the change in morphology between a single straight fringe image at the Ronchigram and the multiple curve rulings that can generate it (one open and one closed). We analyze exactly how multiple rulings generate the same straight image fringe, or how an entire ruling collapses into a single point image. For this analysis, we take different observation planes at different positions with respect to the caustic region. Finally, we characterize this topological change as one of two possible kinds depending on the relative position between the observation plane and the caustic region.

Curvatures of the refracted wavefronts and Ronchigrams for a plano arbitrary lens.

In this work we obtain the equations for curvatures of refracted wavefronts for a plano arbitrary lens. The functions H0, H1, and H2 that determine the caustic also determine the curvature of these wavefronts. The analysis performed in these calculations allows us to study the behavior of the Ronchigrams for the case of plane incident wavefronts. We apply this procedure for a plano-spherical lens, and we discover that it is possible to describe the behavior of the Ronchigrams based on the τ function, which labels the refracted wavefronts of the optical system.

Wavefronts, caustic, ronchigram, and null ronchigrating of a plane wave refracted by an axicon lens.

The aim of this work is threefold: first we obtain analytical expressions for the wavefront train and the caustic associated with the refraction of a plane wavefront by an axicon lens, second we describe the structure of the ronchigram when the ronchiruling is placed at the flat surface of the axicon and the screen is placed at different relative positions to the caustic region, and third we describe in detail the structure of the null ronchigrating for this system; that is, we obtain the grating such that when it is placed at the flat surface of the axicon its associated pattern, at a given plane perpendicular to the optical axis, is a set of parallel fringes. We find that the caustic has only one branch, which is a segment of a line along the optical axis; the ronchigram exhibits self-intersecting fringes when the screen is placed at the caustic region, and the null ronchigrating exhibits closed loop rulings if we want to obtain its associated pattern at the caustic region.

Reliable multihop broadcast protocol with a low-overhead link quality assessment for ITS based on VANETs in highway scenarios.

Vehicular ad hoc networks (VANETs) have been identified as a key technology to enable intelligent transport systems (ITS), which are aimed to radically improve the safety, comfort, and greenness of the vehicles in the road. However, in order to fully exploit VANETs potential, several issues must be addressed. Because of the high dynamic of VANETs and the impairments in the wireless channel, one key issue arising when working with VANETs is the multihop dissemination of broadcast packets for safety and infotainment applications. In this paper a reliable low-overhead multihop broadcast (RLMB) protocol is proposed to address the well-known broadcast storm problem. The proposed RLMB takes advantage of the hello messages exchanged between the vehicles and it processes such information to intelligently select a relay set and reduce the redundant broadcast. Additionally, to reduce the hello messages rate dependency, RLMB uses a point-to-zone link evaluation approach. RLMB performance is compared with one of the leading multihop broadcast protocols existing to date. Performance metrics show that our RLMB solution outperforms the leading protocol in terms of important metrics such as packet dissemination ratio, overhead, and delay.

Diagnosis and Characterization of Plant Viruses Using HTS to Support Virus Management and Tomato Breeding.

Viral diseases pose a significant threat to tomato crops (Solanum lycopersicum L.), one of the world's most economically important vegetable crops. The limited genetic diversity of cultivated tomatoes contributes to their high susceptibility to viral infections. To address this challenge, tomato breeding programs must harness the genetic resources found in native populations and wild relatives. Breeding efforts may aim to develop broad-spectrum resistance against the virome. To identify the viruses naturally infecting 19 advanced lines, derived from native tomatoes, high-throughput sequencing (HTS) of small RNAs and confirmation with PCR and RT-PCR were used. Single and mixed infections with tomato mosaic virus (ToMV), tomato golden mosaic virus (ToGMoV), and pepper huasteco yellow vein virus (PHYVV) were detected. The complete consensus genomes of three variants of Mexican ToMV isolates were reconstructed, potentially forming a new ToMV clade with a distinct 3' UTR. The absence of reported mutations associated with resistance-breaking to ToMV suggests that the Tm-1, Tm-2, and Tm-22 genes could theoretically be used to confer resistance. However, the high mutation rates and a 63 nucleotide insertion in the 3' UTR, as well as amino acid mutations in the ORFs encoding 126 KDa, 183 KDa, and MP of Mexican ToMV isolates, suggest that it is necessary to evaluate the capacity of these variants to overcome Tm-1, Tm-2, and Tm-22 resistance genes. This evaluation, along with the characterization of advanced lines using molecular markers linked to these resistant genes, will be addressed in future studies as part of the breeding strategy. This study emphasizes the importance of using HTS for accurate identification and characterization of plant viruses that naturally infect tomato germplasm based on the consensus genome sequences. This study provides crucial insights to select appropriate disease management strategies and resistance genes and guide breeding efforts toward the development of virus-resistant tomato varieties.

Conventional office blood pressure measurements and unattended automated office blood pressure compared with home self-measurement and 24-h ambulatory blood pressure monitoring.

OBJECTIVE: To assess whether automated office blood pressure (BP) (AOBP) measurement is a better method for measuring BP in the office than conventional techniques and an alternative to out-of-office BP measurements: home-self BP (HSBP) or ambulatory BP monitoring (ABPM). METHODS: We conducted a cross-sectional study of 74 patients and compared AOBP with the conventional technique using a mercury sphygmomanometer and with both out-to-office BP measurements: HSBP of 7 days (three measurements in the morning, afternoon, and night) and daytime ABPM. In addition, we compared BP values obtained using HSBP and ABPM to determine their level of agreement. We used ANOVA to compare means, Bland-Altman, and intraclass correlation coefficients (ICC) for concordance. RESULTS: BP values obtained by the two office methods were similar: conventional 147.2/85.0 mmHg and AOBP 146.0/85.5 mmHg ( P > 0.05) with good agreement (ICC 0.85). The mean SBP differences between AOBP and HSBP ( P < 0.001) and between AOBP and ABPM ( P < 0.001) were 8.6/13.0 mmHg with limits of agreement of -21.2 to 38.5 and -18.4 to 44.3 mmHg, respectively. The average SBP values obtained by HSBP were 4.3 mmHg higher than those obtained by ABPM ( P < 0.01). CONCLUSION: Our study showed good agreement and concordance between the two office methods as well between the two out-to-office methods, although there was a significant difference in the mean SBP between the HSBP and ABPM. Moreover, AOBP was not comparable to either HSBP or ABPM; therefore, the estimation of out-to-office BP using AOBP is not supported.

Reliable freestanding position-based routing in highway scenarios.

Vehicular Ad Hoc Networks (VANETs) are considered by car manufacturers and the research community as the enabling technology to radically improve the safety, efficiency and comfort of everyday driving. However, before VANET technology can fulfill all its expected potential, several difficulties must be addressed. One key issue arising when working with VANETs is the complexity of the networking protocols compared to those used by traditional infrastructure networks. Therefore, proper design of the routing strategy becomes a main issue for the effective deployment of VANETs. In this paper, a reliable freestanding position-based routing algorithm (FPBR) for highway scenarios is proposed. For this scenario, several important issues such as the high mobility of vehicles and the propagation conditions may affect the performance of the routing strategy. These constraints have only been partially addressed in previous proposals. In contrast, the design approach used for developing FPBR considered the constraints imposed by a highway scenario and implements mechanisms to overcome them. FPBR performance is compared to one of the leading protocols for highway scenarios. Performance metrics show that FPBR yields similar results when considering freespace propagation conditions, and outperforms the leading protocol when considering a realistic highway path loss model.

ADAM17/MMP inhibition prevents neutrophilia and lung injury in a mouse model of COVID-19.

Severe coronavirus disease 2019 (COVID-19) is characterized by lung injury, cytokine storm, and increased neutrophil-to-lymphocyte ratio (NLR). Current therapies focus on reducing viral replication and inflammatory responses, but no specific treatment exists to prevent the development of severe COVID-19 in infected individuals. Angiotensin-converting enzyme-2 (ACE2) is the receptor for SARS-CoV-2, the virus causing COVID-19, but it is also critical for maintaining the correct functionality of lung epithelium and endothelium. Coronaviruses induce activation of a disintegrin and metalloprotease 17 (ADAM17) and shedding of ACE2 from the cell surface resulting in exacerbated inflammatory responses. Thus, we hypothesized that ADAM17 inhibition ameliorates COVID-19-related lung inflammation. We employed a preclinical mouse model using intratracheal instillation of a combination of polyinosinic:polycytidylic acid (poly(I:C)) and the receptor-binding domain of the SARS-CoV-2 spike protein (RBD-S) to mimic lung damage associated with COVID-19. Histologic analysis of inflamed mice confirmed the expected signs of lung injury including edema, fibrosis, vascular congestion, and leukocyte infiltration. Moreover, inflamed mice also showed an increased NLR as observed in critically ill COVID-19 patients. Administration of the ADAM17/MMP inhibitors apratastat and TMI-1 significantly improved lung histology and prevented leukocyte infiltration. Reduced leukocyte recruitment could be explained by reduced production of proinflammatory cytokines and lower levels of the endothelial adhesion molecules ICAM-1 and VCAM-1. Additionally, the NLR was significantly reduced by ADAM17/MMP inhibition. Thus, we propose inhibition of ADAM17/MMP as a novel promising treatment strategy in SARS-CoV-2-infected individuals to prevent the progression toward severe COVID-19.

A Fluorescence Dequenching-based Liposome Leakage Assay to Measure Membrane Permeabilization by Pore-forming Proteins.

Pore-forming toxins (PFTs) have been discovered in a wide range of organisms. Their functions are essential to the survival or virulence of many species. PFTs often interact with lipid membranes. Large unilamellar vesicles (LUV), also known as liposomes, have been commonly used as reliable membrane models for testing PFTs activity. Liposomes have great adaptability in size, lipid composition, and loading cargo. Incorporating the fluorescent dye/quencher pair, 8-Aminonaphthalene-1,3,6-Trisulfonic Acid (ANTS) and p-Xylene-Bis-Pyridinium Bromide (DPX), in liposomes is an effective approach for measuring membrane leakage. When ANTS and DPX are encapsulated in a liposome, the fluorescence of ANTS is quenched by DPX. However, disruption of liposome integrity and subsequent leakage result in measurable fluorescence emitted by ANTS. Here, we report our protocol for optimal liposome preparation for measuring liposome leakage by fluorescence dequenching.

Deep-learning based detection of COVID-19 using lung ultrasound imagery.

BACKGROUND: The COVID-19 pandemic has exposed the vulnerability of healthcare services worldwide, especially in underdeveloped countries. There is a clear need to develop novel computer-assisted diagnosis tools to provide rapid and cost-effective screening in places where massive traditional testing is not feasible. Lung ultrasound is a portable, easy to disinfect, low cost and non-invasive tool that can be used to identify lung diseases. Computer-assisted analysis of lung ultrasound imagery is a relatively recent approach that has shown great potential for diagnosing pulmonary conditions, being a viable alternative for screening and diagnosing COVID-19. OBJECTIVE: To evaluate and compare the performance of deep-learning techniques for detecting COVID-19 infections from lung ultrasound imagery. METHODS: We adapted different pre-trained deep learning architectures, including VGG19, InceptionV3, Xception, and ResNet50. We used the publicly available POCUS dataset comprising 3326 lung ultrasound frames of healthy, COVID-19, and pneumonia patients for training and fine-tuning. We conducted two experiments considering three classes (COVID-19, pneumonia, and healthy) and two classes (COVID-19 versus pneumonia and COVID-19 versus non-COVID-19) of predictive models. The obtained results were also compared with the POCOVID-net model. For performance evaluation, we calculated per-class classification metrics (Precision, Recall, and F1-score) and overall metrics (Accuracy, Balanced Accuracy, and Area Under the Receiver Operating Characteristic Curve). Lastly, we performed a statistical analysis of performance results using ANOVA and Friedman tests followed by post-hoc analysis using the Wilcoxon signed-rank test with the Holm's step-down correction. RESULTS: InceptionV3 network achieved the best average accuracy (89.1%), balanced accuracy (89.3%), and area under the receiver operating curve (97.1%) for COVID-19 detection from bacterial pneumonia and healthy lung ultrasound data. The ANOVA and Friedman tests found statistically significant performance differences between models for accuracy, balanced accuracy and area under the receiver operating curve. Post-hoc analysis showed statistically significant differences between the performance obtained with the InceptionV3-based model and POCOVID-net, VGG19-, and ResNet50-based models. No statistically significant differences were found in the performance obtained with InceptionV3- and Xception-based models. CONCLUSIONS: Deep learning techniques for computer-assisted analysis of lung ultrasound imagery provide a promising avenue for COVID-19 screening and diagnosis. Particularly, we found that the InceptionV3 network provides the most promising predictive results from all AI-based techniques evaluated in this work. InceptionV3- and Xception-based models can be used to further develop a viable computer-assisted screening tool for COVID-19 based on ultrasound imagery.

Development of an In Vitro Membrane Model to Study the Function of EsxAB Heterodimer and Establish the Role of EsxB in Membrane Permeabilizing Activity of Mycobacterium tuberculosis.

EsxA and EsxB are secreted as a heterodimer and have been shown to play critical roles in phagosome rupture and translocation of Mycobacterium tuberculosis into the cytosol. Recent in vitro studies have suggested that the EsxAB heterodimer is dissociated upon acidification, which might allow EsxA insertion into lipid membranes. While the membrane permeabilizing activity (MPA) of EsxA has been well characterized in liposomes composed of di-oleoyl-phosphatidylcholine (DOPC), the MPA of EsxAB heterodimer has not been detected through in vitro assays due to its negligible activity with DOPC liposomes. In this study, we established a new in vitro membrane assay to test the MPA activity of N-terminal acetylated EsxA (N-EsxA). We established that a dose-dependent increase in anionic charged lipids enhances the MPA of N-EsxA. The MPA of both N-EsxA and EsxAB were significantly increased with this new liposome system and made it possible to characterize the MPA of EsxAB in more physiologically-relevant conditions. We tested, for the first time, the effect of temperature on the MPA of N-EsxA and EsxAB in this new system. Interestingly, the MPA of N-EsxA was lower at 37 °C than at RT, and on the contrary, the MPA of EsxAB was higher at 37 °C than at RT. Surprisingly, after incubation at 37 °C, the MPA of N-EsxA continuously decreased over time, while MPA of EsxAB remained stable, suggesting EsxB plays a key role in stabilizing N-EsxA to preserve its MPA at 37 °C. In summary, this study established a new in vitro model system that characterizes the MPA of EsxAB and the role of EsxB at physiological-relevant conditions.

Phase III Trial of Adjuvant Capecitabine After Standard Neo-/Adjuvant Chemotherapy in Patients With Early Triple-Negative Breast Cancer (GEICAM/2003-11_CIBOMA/2004-01).

PURPOSE: Operable triple-negative breast cancers (TNBCs) have a higher risk of relapse than non-TNBCs with standard therapy. The GEICAM/2003-11_CIBOMA/2004-01 trial explored extended adjuvant capecitabine after completion of standard chemotherapy in patients with early TNBC. PATIENTS AND METHODS: Eligible patients were those with operable, node-positive-or node negative with tumor 1 cm or greater-TNBC, with prior anthracycline- and/or taxane-containing chemotherapy. After central confirmation of TNBC status by immunohistochemistry, patients were randomly assigned to either capecitabine or observation. Stratification factors included institution, prior taxane-based therapy, involved axillary lymph nodes, and centrally determined phenotype (basal v nonbasal, according to cytokeratins 5/6 and/or epidermal growth factor receptor positivity by immunohistochemistry). The primary objective was to compare disease-free survival (DFS) between both arms. RESULTS: Eight hundred seventy-six patients were randomly assigned to capecitabine (n = 448) or observation (n = 428). Median age was 49 years, 55.9% were lymph node negative, 73.9% had a basal phenotype, and 67.5% received previous anthracyclines plus taxanes. Median length of follow-up was 7.3 years. DFS was not significantly prolonged with capecitabine versus observation [hazard ratio (HR), 0.82; 95% CI, 0.63 to 1.06; P = .136]. In a preplanned subgroup analysis, nonbasal patients seemed to derive benefit from the addition of capecitabine with a DFS HR of 0.53 versus 0.94 in those with basal phenotype (interaction test P = .0694) and an HR for overall survival of 0.42 versus 1.23 in basal phenotype (interaction test P = .0052). Tolerance of capecitabine was as expected, with 75.2% of patients completing the planned 8 cycles. CONCLUSION: This study failed to show a statistically significant increase in DFS by adding extended capecitabine to standard chemotherapy in patients with early TNBC. In a preplanned subset analysis, patients with nonbasal phenotype seemed to obtain benefit with capecitabine, although this will require additional validation.

Effects of membrane lipid composition on Mycobacterium tuberculosis EsxA membrane insertion: A dual play of fluidity and charge.

As a key virulence factor of Mycobacterium tuberculosis, EsxA or 6-kDa early secreted antigenic target (ESAT-6) has been implicated in phagosome rupture and mycobacterial translocation from the phagosome to the cytosol within macrophages. Our previous studies have shown that EsxA permeabilizes liposomal membrane at acidic pH and a membrane-permeabilization defective mutant Q5K attenuates mycobacterial cytosolic translocation and virulence in macrophages. To further probe the mechanism of EsxA membrane permeabilization, here we characterized the effects of various lipid compositions, including biologically relevant phagosome-mimicking lipids and lipid rafts, on the structural stability and membrane insertion of EsxA WT and Q5K. We have found a complex dual play of membrane fluidity and charge in regulating EsxA membrane insertion. Moreover, Q5K affects the membrane insertion through a structure- and lipid composition-independent mechanism. The results of this study provide a novel insights into the mechanism of EsxA membrane interaction.