Phenotypic and functional analysis in HER2+ targeted therapy of human NK cell subpopulation according to the expression of FcεRIγ and NKG2C in breast cancer patients.

Adaptive NK cells constitute an NK cell subpopulation, which expands after human cytomegalovirus (HCMV) infection. This subpopulation has stronger production of cytokines after CD16 stimulation, longer life and persistence than conventional NK cells and are, therefore, interesting tools for cancer immunotherapy. Since there is limited information on adaptive NK cells in cancer patients, we described this population phenotypically and functionally, by flow cytometry, in the context of HER2 + breast cancer (BC) directed therapy. We assessed HCMV status in 78 patients with BC. We found that, similarly to healthy donors (HD), a high proportion of BC patients were HCMV-positive, and nearly 72% of them had an adaptive NK cell subpopulation characterized by the loss of FcεRIγ intracellular adaptor protein or the presence of NKG2C receptor. However, in BC patients, FcεRIγ- and NKG2C + NK cell populations overlapped to a lesser extent than in HD. Otherwise, no profound phenotypic differences were found between BC patients and HD. Although FcεRIγ- or NKG2C + NK cell subsets from BC patients produced more IFN-γ than their FcεRIγ + or NKG2C- NK cell counterparts, IFN-γ production increased only when NK cells simultaneously expressed FcεRIγ- and NKG2C + , whereas in HD the presence of NKG2C marker was sufficient to display greater functionality. Furthermore, in a group of patients treated with chemotherapy and Trastuzumab plus Pertuzumab, FcεRIγ-NKG2C + and FcεRIγ-NKG2C- NK cells retained greater functionality after treatment than FcεRIγ + NKG2C- NK cells. These results suggest that the presence or magnitude of adaptive NK cell subsets might serve as a key determinant for therapeutic approaches based on antibodies directed against tumor antigens.

Multidrug efflux pumps as main players in intrinsic and acquired resistance to antimicrobials.

Multidrug efflux pumps constitute a group of transporters that are ubiquitously found in any organism. In addition to other functions with relevance for the cell physiology, efflux pumps contribute to the resistance to compounds used for treating different diseases, including resistance to anticancer drugs, antibiotics or antifungal compounds. In the case of antimicrobials, efflux pumps are major players in both intrinsic and acquired resistance to drugs currently in use for the treatment of infectious diseases. One important aspect not fully explored of efflux pumps consists on the identification of effectors able to induce their expression. Indeed, whereas the analysis of clinical isolates have shown that mutants overexpressing these resistance elements are frequently found, less is known on the conditions that may trigger expression of efflux pumps, hence leading to transient induction of resistance in vivo, a situation that is barely detectable using classical susceptibility tests. In the current article we review the structure and mechanisms of regulation of the expression of bacterial and fungal efflux pumps, with a particular focus in those for which a role in clinically relevant resistance has been reported.

Whole-genome sequence of Stenotrophomonas maltophilia D457, a clinical isolate and a model strain.

Stenotrophomonas maltophilia is an opportunistic pathogen with an environmental origin, and it is an increasingly relevant cause of nosocomial infections. Here we present the whole-genome sequence of S. maltophilia strain D457, a clinical isolate that is being used as a model for studying antibiotic resistance in this bacterial species.

The Inactivation of intrinsic antibiotic resistance determinants widens the mutant selection window for quinolones in Stenotrophomonas maltophilia.

We have determined that the mutational inactivation of the SmeDEF efflux pump and the SmQnr quinolone resistance protein widens the mutant selection windows for ofloxacin and ciprofloxacin of Stenotrophomonas maltophilia by reducing their MICs. Resistant mutants arising from a strain lacking SmeDEF and SmQnr presented levels of susceptibility similar to those of the wild-type strain. This indicates that inactivation of intrinsic resistance determinants might increase the chances for selecting resistant mutants at low antibiotic concentrations.

The prevalence of patellofemoral pain in the Rugby League World Cup (RLWC) 2021 spectators: A protocol of a cross-sectional study.

Patellofemoral pain (PFP) can cause significant pain leading to limitations in societal participation and physical activity. PFP is usually associated with athletes undergoing intensive physical training, or military recruits; but recent evidence shows that PFP is common in the general population. The relationship of PFP with physical activity is not entirely clear. Our aim is to provide a better estimate of the general population prevalence of PFP and to relate this to the level of physical activity, and demographic characteristics. The Survey instrument for Natural history, Aetiology and Prevalence of Patellofemoral pain Studies (SNAPPS) was developed as a PFP screening tool to be used in the community. The electronic version of the SNAPPS (eSNAPPS) has recently been validated and was used to survey attendees at mass-participation running events. We will use an electronic survey to collect data from a sample of 1100 Rugby League World Cup spectators. The survey will have four sections: i) general and demographic; ii) knee pain (eSNAPPS); iii) level of physical activity; and iv) quality of life in relation to knee pain. The primary analytic approach will be descriptive of PFP prevalence. Secondary analyses will explore the relationships of the presence of PFP and the other variables. We will disseminate this work by publication of peer-reviewed papers in scientific journals, presentations at scientific conferences, and on the dedicated SNAPPS website https://www.snappspfp.com/.

Objective Analysis of Neck Muscle Boundaries for Cervical Dystonia Using Ultrasound Imaging and Deep Learning.

OBJECTIVE: To provide objective visualization and pattern analysis of neck muscle boundaries to inform and monitor treatment of cervical dystonia. METHODS: We recorded transverse cervical ultrasound (US) images and whole-body motion analysis of sixty-one standing participants (35 cervical dystonia, 26 age matched controls). We manually annotated 3,272 US images sampling posture and the functional range of pitch, yaw, and roll head movements. Using previously validated methods, we used 60-fold cross validation to train, validate and test a deep neural network (U-net) to classify pixels to 13 categories (five paired neck muscles, skin, ligamentum nuchae, vertebra). For all participants for their normal standing posture, we segmented US images and classified condition (Dystonia/Control), sex and age (higher/lower) from segment boundaries. We performed an explanatory, visualization analysis of dystonia muscle-boundaries. RESULTS: For all segments, agreement with manual labels was Dice Coefficient (64 ± 21%) and Hausdorff Distance (5.7 ± 4 mm). For deep muscle layers, boundaries predicted central injection sites with average precision 94 ± 3%. Using leave-one-out cross-validation, a support-vector-machine classified condition, sex, and age from predicted muscle boundaries at accuracy 70.5%, 67.2%, 52.4% respectively, exceeding classification by manual labels. From muscle boundaries, Dystonia clustered optimally into three sub-groups. These sub-groups are visualized and explained by three eigen-patterns which correlate significantly with truncal and head posture. CONCLUSION: Using US, neck muscle shape alone discriminates dystonia from healthy controls. SIGNIFICANCE: Using deep learning, US imaging allows online, automated visualization, and diagnostic analysis of cervical dystonia and segmentation of individual muscles for targeted injection.

Fully automated image-based estimation of postural point-features in children with cerebral palsy using deep learning.

The aim of this study was to provide automated identification of postural point-features required to estimate the location and orientation of the head, multi-segmented trunk and arms from videos of the clinical test 'Segmental Assessment of Trunk Control' (SATCo). Three expert operators manually annotated 13 point-features in every fourth image of 177 short (5-10 s) videos (25 Hz) of 12 children with cerebral palsy (aged: 4.52 ± 2.4 years), participating in SATCo testing. Linear interpolation for the remaining images resulted in 30 825 annotated images. Convolutional neural networks were trained with cross-validation, giving held-out test results for all children. The point-features were estimated with error 4.4 ± 3.8 pixels at approximately 100 images per second. Truncal segment angles (head, neck and six thoraco-lumbar-pelvic segments) were estimated with error 6.4 ± 2.8°, allowing accurate classification (F 1 > 80%) of deviation from a reference posture at thresholds up to 3°, 3° and 2°, respectively. Contact between arm point-features (elbow and wrist) and supporting surface was classified at F 1 = 80.5%. This study demonstrates, for the first time, technical feasibility to automate the identification of (i) a sitting segmental posture including individual trunk segments, (ii) changes away from that posture, and (iii) support from the upper limb, required for the clinical SATCo.

The intrinsic resistome of Klebsiella pneumoniae.

Molecular epidemiology studies aiming at understanding the acquisition of antimicrobial resistance by clinical isolates of Klebsiella pneumoniae are regularly published; however, information on the genes that contribute to its characteristic phenotype of resistance to antibiotics (intrinsic resistome) is scarce. To fill this gap, a K. pneumoniae transposon mutant library was screened and 171 mutants presenting changes in their susceptibility to antibiotics were selected, in which the transposon insertion site was determined in 75. Twenty-seven mutants for which insertion points had been previously identified were included in the analysis. A total of 102 mutants were selected for further studies. In 70 mutants the transposon was inserted in a gene with a known function, whilst in 19 the insertion occurred in genes encoding proteins with unknown functions and 13 insertions occurred in intergenic regions. Moreover, 87 of the insertions were localised in the chromosome, with 15 insertions located in the two plasmids carried by this strain. Whereas some of the mutated genes are already known to be involved in antimicrobial resistance (ampG, acrB, tolC), several of them are involved in regular processes of bacterial physiology, including K. pneumoniae virulence. Together with results published for other organisms, these results support that determinants involved in basic processes of bacterial physiology may contribute to antimicrobial resistance. These findings also indicate that, besides acquired resistance genes, plasmids may harbour other genes belonging to their backbone that can also be involved in resistance.

Predictive analysis of transmissible quinolone resistance indicates Stenotrophomonas maltophilia as a potential source of a novel family of Qnr determinants.

BACKGROUND: Predicting antibiotic resistance before it emerges at clinical settings constitutes a novel approach for preventing and fighting resistance of bacterial pathogens. To analyse the possibility that novel plasmid-encoded quinolone resistance determinants (Qnr) can emerge and disseminate among bacterial pathogens, we searched the presence of those elements in nearly 1000 bacterial genomes and metagenomes. RESULTS: We have found a number of novel potential qnr genes in the chromosomes of aquatic bacteria and in metagenomes from marine organisms. Functional studies of the Stenotrophomonas maltophilia Smqnr gene show that plasmid-encoded SmQnr confers quinolone resistance upon its expression in a heterologous host. CONCLUSION: Altogether, the data presented in our work support the notion that predictive studies on antibiotic resistance are feasible, using currently available information on bacterial genomes and with the aid of bioinformatic and functional tools. Our results confirm that aquatic bacteria can be the origin of plasmid-encoded Qnr, and highlight the potential role of S. maltophilia as a source of novel Qnr determinants.

Working towards an objective segmental assessment of trunk control in children with cerebral palsy.

BACKGROUND: Physical therapy evaluations of motor control are currently based on subjective clinical assessments. Despite validation, these can still be inconsistent between therapists and between clinics, compromising the process of validating a therapeutic intervention and the subsequent generation of evidence-based practice (EBP) guidelines. EBP benefits from well-defined objective measurements that complement existing subjective assessments. RESEARCH QUESTION: The aim of this study was to develop an objective measure of head/trunk control in children with Cerebral Palsy (CP) using previously developed video-based methods of head/trunk alignment and absence of external support and compare these with the existing subjective Segmental Assessment of Trunk Control (SATCo). METHODS: Twelve children with CP were recruited and an average of 3 (±1.1) SATCo tests performed per child. The full SATCo was concurrently video-recorded from a sagittal view; markers were placed on specific landmarks of the head, trunk and pelvis to track and estimate head/trunk segment position. A simplified objective rule was created for control and used on videos showing no external support. This replicated the clinical parameters and enabled identification of the segmental-loss-of-control. The subjectively and objectively identified segmental-loss-of-control were compared using a Pearson Correlation Coefficient. RESULTS: An angular-threshold of 17° from alignment showed the minimum bias between the subjectively and the objectively measured segmental-loss-of-control (mean error =-0.11 and RMSE = 1.5) and a significant correlation (r = 0.78, r2 = 0.61, p < .01). SIGNIFICANCE: This study showed that simple objective video-based measurements can be used to reconstruct the subjective assessment of segmental head/trunk control. This suggests that a clinically-friendly video-based objective measure has future potential to complement subjective assessments and to assist in the generation of EBP guidelines. Further development will increase the information that can be extracted from video images and enable generation of a fully automated objective measure.

The potential of an automated system to identify the upper limb component of a controlled sitting posture.

Full trunk control in sitting is demonstrated only when the head-trunk are aligned and upper limbs remain free of contact from mechanical support. These components represent a Controlled Kinetic Chain and can be evaluated in people with neuromotor disability using the Segmental Assessment of Trunk Control (SATCo) when a therapist provides manual trunk support at different segmental levels. However, the SATCo, as with other clinical assessments of control, is subjective. The SATCo was translated to objective rules relating the position of the hands and elbows to the head-trunk and then tested to determine the extent to which this automated objective method replicated the clinical judgement. Clinical evaluation used video to determine whether the upper limb was free of mechanical support while the objective evaluation used 3D motion capture of the trunk and upper limbs with a classification rule. The agreement between clinical and objective classification was calculated for three conditions of a distance-from-support-surface threshold parameter in five healthy adults and five children with cerebral palsy. The unfitted (zero-threshold values) method replicated the clinical judgement in part (68.26%±15.7, adults, 48.3%±33.9 children). The fitted (level-of-support determined) agreement showed that the process could be refined using trial specific parameters (88.32%±5.3 adults, 89.84%±10.2 children). The fixed-values agreement showed high values when using general group parameters (80.80%±3.1 adults, 74.31%±21.5 children). This objective classification of the upper limb component of trunk control largely captures the clinical evaluation. It provides the first stages in development of a clinically-friendly fully automated method.

A video based method to quantify posture of the head and trunk in sitting.

Maintenance of a vertically aligned posture of the head and trunk in sitting is a fundamental skill that demonstrates the presence of neuromotor control. Clinical assessments of posture are generally subjective. Studies have quantified posture using different technologies, but the application of such technologies in a clinical environment remains difficult. Video recordings, however, are easily used clinically and have potential for quantitative analysis of movement. This study used a video-based method to generate a numerical measure of postural alignment of the head and trunk in sitting. Static and dynamic trials of 12 healthy seated adults were simultaneously recorded with a sagittal video camera and a 3D motion capture system. Segmental angles were calculated for the Head, Neck and six Trunk segments. An agreed definition of aligned static sitting posture agreed was used by five clinically experienced experts to identify video frames where the participants' posture was aligned. The five subsets of frames that defined the aligned posture were combined to give aligned segments (mean±SD) for each participant. Agreement between experts in the definition (mean) of aligned segmental angles was excellent (ICC=0.99) and intra-assessor reliability (SD) lay within 2.1°-11.6°. Agreement between the video-based method and the 3D system was below 3.8° and 8.4° for static and dynamic trials respectively. This video-based method allowed the quantification of sitting posture and provided greater detail of the trunk/spinal profile than previous methods. It has potential as a complementary tool, alongside subjective assessments, for patients with a wide variety of pathologies.

Gene expression changes in colon tissues from colorectal cancer patients following the intake of an ellagitannin-containing pomegranate extract: a randomized clinical trial.

The clinical evidence of dietary polyphenols as colorectal cancer (CRC) chemopreventive compounds is very weak. Verification in humans of tissue-specific molecular regulation by the intake of polyphenols requires complex clinical trials that allow for the procurement of sufficient pre- and postsupplementation tissue samples. Ellagitannins (ETs), ellagic acid (EA) and their gut microbiota-derived metabolites, the urolithins, modify gene expression in colon normal and cancer cultured cells. We conducted here the first clinical trial with 35 CRC patients daily supplemented with 900 mg of an ET-containing pomegranate extract (PE) and evaluated the expression of various CRC-related genes in normal and cancerous colon tissues before (biopsies) and after (surgical specimens) 5-35 days of supplementation. Tissues were also obtained from 10 control patients (no supplementation) that confirmed a large, gene- and tissue-specific interindividual variability and impact of the experimental protocol on gene expression, with some genes induced (MYC, CD44, CDKN1A, CTNNB1), some repressed (CASP3) and others not affected (KRAS). Despite these issues, the consumption of the PE was significantly associated with a counterbalance effect in the expression of CD44, CTNNB1, CDKN1A, EGFR and TYMs, suggesting that the intake of this PE modulated the impact of the protocol on gene expression in a gene- and tissue-specific manner. These effects were not associated with the individuals' capacity to produce specific urolithins (i.e., metabotypes) or the levels of urolithins and EA in the colon tissues and did not reproduce in vitro effects evidencing the difficulty of demonstrating in vivo the in vitro results.

An evaluation of 3D head pose estimation using the Microsoft Kinect v2.

The Kinect v2 sensor supports real-time non-invasive 3D head pose estimation. Because the sensor is small, widely available and relatively cheap it has great potential as a tool for groups interested in measuring head posture. In this paper we compare the Kinect's head pose estimates with a marker-based record of ground truth in order to establish its accuracy. During movement of the head and neck alone (with static torso), we find average errors in absolute yaw, pitch and roll angles of 2.0±1.2°, 7.3±3.2° and 2.6±0.7°, and in rotations relative to the rest pose of 1.4±0.5°, 2.1±0.4° and 2.0±0.8°. Larger head rotations where it becomes difficult to see facial features can cause estimation to fail (10.2±6.1% of all poses in our static torso range of motion tests) but we found no significant changes in performance with the participant standing further away from Kinect - additionally enabling full-body pose estimation - or without performing face shape calibration, something which is not always possible for younger or disabled participants. Where facial features remain visible, the sensor has applications in the non-invasive assessment of postural control, e.g. during a programme of physical therapy. In particular, a multi-Kinect setup covering the full range of head (and body) movement would appear to be a promising way forward.

The analysis of the antibiotic resistome offers new opportunities for therapeutic intervention.

Most efforts in the development of antimicrobials have focused on the screening of lethal targets. Nevertheless, the constant expansion of antimicrobial resistance makes the antibiotic resistance determinants themselves suitable targets for finding inhibitors to be used in combination with antibiotics. Among them, inhibitors of antibiotic inactivating enzymes and of multidrug efflux pumps are suitable candidates for improving the efficacy of antibiotics. In addition, the application of systems biology tools is helping to understand the changes in bacterial physiology associated to the acquisition of resistance, including the increased susceptibility to other antibiotics displayed by some antibiotic-resistant mutants. This information is useful for implementing novel strategies based in metabolic interventions or combination of antibiotics for improving the efficacy of antibacterial therapy.

Use of phenotype microarrays to study the effect of acquisition of resistance to antimicrobials in bacterial physiology.

It is widely accepted that the acquisition of resistance to antimicrobials confers a fitness cost. Different works have shown that the effect of acquiring resistance in bacterial physiology may be more specific than previously thought. Study of these specific changes may help to predict the outcome of resistant organisms in different ecosystems. In addition to changing bacterial physiology, acquisition of resistance either increases or reduces susceptibility to other antimicrobials. In the current article, we review recent information on the effect of acquiring resistance upon bacterial physiology, with a specific focus on studies using phenotype microarray technology.

Antibiotic resistance in the opportunistic pathogen Stenotrophomonas maltophilia.

Stenotrophomonas maltophilia is an environmental bacterium found in the soil, associated with plants and animals, and in aquatic environments. It is also an opportunistic pathogen now causing an increasing number of nosocomial infections. The treatment of S. maltophilia is quite difficult given its intrinsic resistance to a number of antibiotics, and because it is able to acquire new resistances via horizontal gene transfer and mutations. Certainly, strains resistant to quinolones, cotrimoxale and/or cephalosporins-antibiotics commonly used to treat S. maltophilia infections-have emerged. The increasing number of available S. maltophilia genomes has allowed the identification and annotation of a large number of antimicrobial resistance genes. Most encode inactivating enzymes and efflux pumps, but information on their role in intrinsic and acquired resistance is limited. Non-typical antibiotic resistance mechanisms that also form part of the intrinsic resistome have been identified via mutant library screening. These include non-typical antibiotic resistance genes, such as bacterial metabolism genes, and non-inheritable resistant phenotypes, such as biofilm formation and persistence. Their relationships with resistance are complex and require further study.

The inactivation of RNase G reduces the Stenotrophomonas maltophilia susceptibility to quinolones by triggering the heat shock response.

Quinolone resistance is usually due to mutations in the genes encoding bacterial topoisomerases. However, different reports have shown that neither clinical quinolone resistant isolates nor in vitro obtained Stenotrophomonas maltophilia mutants present mutations in such genes. The mechanisms so far described consist on efflux pumps' overexpression. Our objective is to get information on novel mechanisms of S. maltophilia quinolone resistance. For this purpose, a transposon-insertion mutant library was obtained in S. maltophilia D457. One mutant presenting reduced susceptibility to nalidixic acid was selected. Inverse PCR showed that the inactivated gene encodes RNase G. Complementation of the mutant with wild-type RNase G allele restored the susceptibility to quinolones. Transcriptomic and real-time RT-PCR analyses showed that several genes encoding heat-shock response proteins were expressed at higher levels in the RNase defective mutant than in the wild-type strain. In agreement with this situation, heat-shock reduces the S. maltophilia susceptibility to quinolone. We can then conclude that the inactivation of the RNase G reduces the susceptibility of S. maltophilia to quinolones, most likely by regulating the expression of heat-shock response genes. Heat-shock induces a transient phenotype of quinolone resistance in S. maltophilia.

MicroRNAs expression in normal and malignant colon tissues as biomarkers of colorectal cancer and in response to pomegranate extracts consumption: Critical issues to discern between modulatory effects and potential artefacts.

SCOPE: MicroRNAs (miRs) are proposed as colorectal cancer (CRC) biomarkers. Pomegranate ellagic acid and their microbiota metabolites urolithins exert anticancer effects in preclinical CRC models, and target normal and malignant colon tissues in CRC patients. Herein, we investigated whether the intake of pomegranate extract (PE) modified miRs expression in surgical colon tissues versus biopsies from CRC patients. METHODS AND RESULTS: We conducted a randomized, double-blind, controlled trial. Thirty-five CRC patients consumed 900 mg PE daily before surgery. Control CRC patients (no PE intake, n = 10) were included. Our results revealed: (1) significant differences for specific miRs between malignant and normal tissues modifiable by the surgical protocols; (2) opposed trends between -5p and -3p isomolecules; (3) general induction of miRs attributable to the surgery; (4) moderate modulation of various miRs following the PE intake, and (5) no association between tissue urolithins and the observed miRs changes. CONCLUSION: PE consumption appears to affect specific colon tissue miRs but surgery critically alters miRs levels hindering the discrimination of significant changes caused by dietary factors and the establishment of genuine differences between malignant and normal tissues as biomarkers. The components responsible for the PE effects and the clinical relevance of these observations deserve further research.