Light Modulates Important Pathogenic Determinants and Virulence in ESKAPE Pathogens Acinetobacter baumannii, Pseudomonas aeruginosa, and Staphylococcus aureus.

Light sensing has been extensively characterized in the human pathogen Acinetobacter baumannii at environmental temperatures. However, the influence of light on the physiology and pathogenicity of human bacterial pathogens at temperatures found in warm-blooded hosts is still poorly understand. In this work, we show that Staphylococcus aureus, Acinetobacter baumannii, and Pseudomonas aeruginosa (ESKAPE) priority pathogens, which have been recognized by the WHO and the CDC as critical, can also sense and respond to light at temperatures found in human hosts. Most interestingly, in these pathogens, light modulates important pathogenicity determinants as well as virulence in an epithelial infection model, which could have implications in human infections. In fact, we found that alpha-toxin-dependent hemolysis, motility, and growth under iron-deprived conditions are modulated by light in S. aureus Light also regulates persistence, metabolism, and the ability to kill competitors in some of these microorganisms. Finally, light exerts a profound effect on the virulence of these pathogens in an epithelial infection model, although the response is not the same in the different species; virulence was enhanced by light in A. baumannii and S. aureus, while in A. nosocomialis and P. aeruginosa it was reduced. Neither the BlsA photoreceptor nor the type VI secretion system (T6SS) is involved in virulence modulation by light in A. baumannii Overall, this fundamental knowledge highlights the potential use of light to control pathogen virulence, either directly or by manipulating the light regulatory switch toward the lowest virulence/persistence configuration.IMPORTANCE Pathogenic bacteria are microorganisms capable of producing disease. Dangerous bacterial pathogens, such as Staphylococcus aureus, Pseudomonas aeruginosa, and Acinetobacter baumannii, are responsible for serious intrahospital and community infections in humans. Therapeutics is often complicated due to resistance to multiple antibiotics, rendering them ineffective. In this work, we show that these pathogens sense natural light and respond to it by modulating aspects related to their ability to cause disease; in the presence of light, some of them become more aggressive, while others show an opposite response. Overall, we provide new understanding on the behavior of these pathogens, which could contribute to the control of infections caused by them. Since the response is distributed in diverse pathogens, this notion could prove a general concept.

Exchange-bias phenomenon: the role of the ferromagnetic spin structure.

The exchange bias of antiferromagnetic-ferromagnetic (AFM-FM) bilayers is found to be strongly dependent on the ferromagnetic spin configuration. The widely accepted inverse proportionality of the exchange bias field with the ferromagnetic thickness is broken in FM layers thinner than the FM correlation length. Moreover, an anomalous thermal dependence of both exchange bias field and coercivity is also found. A model based on springlike domain walls parallel to the AFM-FM interface quantitatively accounts for the experimental results and, in particular, for the deviation from the inverse proportionality law. These results reveal the active role the ferromagnetic spin structure plays in AFM-FM hybrids which leads to a new paradigm of the exchange bias phenomenon.

A brain-enriched circular RNA controls excitatory neurotransmission and restricts sensitivity to aversive stimuli.

Circular RNAs (circRNAs) are a large class of noncoding RNAs. Despite the identification of thousands of circular transcripts, the biological significance of most of them remains unexplored, partly because of the lack of effective methods for generating loss-of-function animal models. In this study, we focused on circTulp4, an abundant circRNA derived from the Tulp4 gene that is enriched in the brain and synaptic compartments. By creating a circTulp4-deficient mouse model, in which we mutated the splice acceptor site responsible for generating circTulp4 without affecting the linear mRNA or protein levels, we were able to conduct a comprehensive phenotypic analysis. Our results demonstrate that circTulp4 is critical in regulating neuronal and brain physiology, modulating the strength of excitatory neurotransmission and sensitivity to aversive stimuli. This study provides evidence that circRNAs can regulate biologically relevant functions in neurons, with modulatory effects at multiple levels of the phenotype, establishing a proof of principle for the regulatory role of circRNAs in neural processes.

Probing inter-areal computations with a cellular resolution two-photon holographic mesoscope.

Brain computation depends on intricately connected yet highly distributed neural networks. Due to the absence of the requisite technologies, causally testing fundamental hypotheses on the nature of inter-areal processing have remained largely out-of-each. Here we developed the first two photon holographic mesoscope, a system capable of simultaneously reading and writing neural activity patterns with single cell resolution across large regions of the brain. We demonstrate the precise photo-activation of spatial and temporal sequences of neurons in one brain area while reading out the downstream effect in several other regions. Investigators can use this new platform to understand feed-forward and feed-back processing in distributed neural circuits with single cell precision for the first time.

Recurrent pattern completion drives the neocortical representation of sensory inference.

When sensory information is incomplete or ambiguous, the brain relies on prior expectations to infer perceptual objects. Despite the centrality of this process to perception, the neural mechanism of sensory inference is not known. Illusory contours (ICs) are key tools to study sensory inference because they contain edges or objects that are implied only by their spatial context. Using cellular resolution, mesoscale two-photon calcium imaging and multi-Neuropixels recordings in the mouse visual cortex, we identified a sparse subset of neurons in the primary visual cortex (V1) and higher visual areas that respond emergently to ICs. We found that these highly selective 'IC-encoders' mediate the neural representation of IC inference. Strikingly, selective activation of these neurons using two-photon holographic optogenetics was sufficient to recreate IC representation in the rest of the V1 network, in the absence of any visual stimulus. This outlines a model in which primary sensory cortex facilitates sensory inference by selectively strengthening input patterns that match prior expectations through local, recurrent circuitry. Our data thus suggest a clear computational purpose for recurrence in the generation of holistic percepts under sensory ambiguity. More generally, selective reinforcement of top-down predictions by pattern-completing recurrent circuits in lower sensory cortices may constitute a key step in sensory inference.

Coeliac disease in the COVID-19 pandemic: does HLA have a protective effect?

BACKGROUND: The coronavirus disease 2019 (COVID-19), an acute respiratory disease caused by a novel coronavirus (SARS-CoV-2), is emerging as a worldwide public health emergency. Several scientific contributions reported the potential relevance of human leukocyte antigen (HLA) polymorphism and susceptibility to viruses, such as SARS-CoV. In our study, we examined a population of coeliac subjects presenting the HLA haplotype DQ2 and/or DQ8. Our aim was to evaluate whether HLA DQ2 and/or DQ8 haplotype play a role in SARS-CoV-2-infection. The aim was also to evaluate the difficulty in following the gluten-free diet due to all the adversities produced by the pandemic, such as the food supply disruption, and the difficulties in managing the clinical follow-up. METHODS: 191 consecutive coeliac patients completed a questionnaire on their current clinical status, psychological effects, and management of the gluten-free diet experienced during the COVID-19 pandemic and questions regarding possible SARS-CoV-2 infection. RESULTS: Out of the 191 patients who participated in the study, 42 were full-blown coeliac and 149 were in remission. From the answers provided, 84.8% of patients declared that they no longer consider themselves vulnerable to COVID-19 as they suffer from coeliac disease; 94.2% of patients did not encounter any difficulties in managing the gluten-free diet or in acquiring specific foods and 64.9% of patients in our study underwent diagnostic testing for SARS-CoV-2. Out of this number, 31.5% did so due to contacts with subjects affected by COVID-19, 26.6% for work related reasons, 11.3% due to flu-like symptoms and 30.6% for other reasons. Only 5.8% of the enrolled patients received a diagnosis of COVID-19. Out of all the patients in our population who were diagnosed with COVID-19, 94.8% developed no symptoms and none of them needed hospitalization or intensive care. CONCLUSION: The hypothesis that the HLADQ2 and/or DQ8 haplotype plays a protective role against SARS-CoV-2 infection, as against other viral infections, is intriguingly suggestive.KEY MESSAGESCOVID-19 as a public health emergency;SARS-CoV-2 and possible complications in coeliac disease;Role of HLA DQ2 and/or DQ8 in SARS-CoV-2 infection.

High-performance microbial opsins for spatially and temporally precise perturbations of large neuronal networks.

The biophysical properties of existing optogenetic tools constrain the scale, speed, and fidelity of precise optogenetic control. Here, we use structure-guided mutagenesis to engineer opsins that exhibit very high potency while retaining fast kinetics. These new opsins enable large-scale, temporally and spatially precise control of population neural activity. We extensively benchmark these new opsins against existing optogenetic tools and provide a detailed biophysical characterization of a diverse family of opsins under two-photon illumination. This establishes a resource for matching the optimal opsin to the goals and constraints of patterned optogenetics experiments. Finally, by combining these new opsins with optimized procedures for holographic photostimulation, we demonstrate the simultaneous coactivation of several hundred spatially defined neurons with a single hologram and nearly double that number by temporally interleaving holograms at fast rates. These newly engineered opsins substantially extend the capabilities of patterned illumination optogenetic paradigms for addressing neural circuits and behavior.

Genetic association of HLA-DQB1 and HLA-DRB1 polymorphisms with alopecia areata in the Italian population.

BACKGROUND: Alopecia areata (AA) is a multifactorial disease characterized by hair loss especially from the scalp. As for other autoimmune conditions, the major histocompatibility complex (HLA) region is associated with AA susceptibility. OBJECTIVE: To provide evidence for the association of specific HLA-DQB1 and HLA-DRB1 alleles with AA in an Italian population, using a case-control approach. METHODS: We performed a case-control study to investigate whether HLA-DQB1 and -DRB1 alleles predispose to AA in the Italian population. HLA class II typing was performed in 85 patients with AA and 210 healthy controls from the same ethnic group. RESULTS: An increased frequency of DQB1*03, coding for DQ7 heterodimers, and a decreased rate of the DQB1*06 allele were observed in patients when compared with controls; the greatest and significant difference was in the group of cases with a more severe phenotype [AA>50% patients (more than 50% hair loss) vs. controls, P=4·5×10(-3) , P(c)=0·031, odds ratio (OR) 2·01, 95% confidence interval (CI) 1·22-3·31 and P=2·5×10(-3) , P(c)=0·017, OR 0·22, 95% CI 0·07-0·72, respectively]. DQB1*03, serologically related to DQ8 or coding for DQ9 molecules, was not associated with AA susceptibility. Out of all patients, 65·9% carried DQ7 heterodimers compared with 49·5% of the controls (P=7·3×10(-3) , OR 1·97, 95% CI 1·17-3·32) and DQ7 prevalence rose to 76·3% in patients with AA>50% (P=1·7×10(-3) , OR 3·28, 95% CI 1·48-7·27). No significant difference was found in the distribution of DRB1 variants or phenotypes among cases and controls. CONCLUSION: Our data show a correlation between the HLA-DQB1 locus and the occurrence of AA in Italy supporting DQB1*03(DQ7) as a predisposing allele for the disease and the relevance of the HLA genetic test in the clinical management of AA.

Validation of cardiac output measurement with the LiDCO™ pulse contour system in patients with impaired left ventricular function after cardiac surgery.

After cardiac surgery, patients with low left ventricular ejection fraction probably benefit the most from accurate monitoring of continuous cardiac output. Thirty patients with impaired ventricular function were studied, and intermittent bolus thermodilution and continuous pulse contour (LiDCO plus™) cardiac output compared. Following lithium dilution calibration, a total of 220 paired results were recorded. Thermodilution and LiDCO measurements ranged from 2.3 to 11.0 and 2.6 to 10.8 l.min(-1), respectively. Corresponding means (SD) were 6.1 (1.6) and 6.2 (1.9) l.min(-1), with coefficients of variance of 26 and 31%, respectively. The correlation coefficient was 0.82, bias 0.28 l.min(-1) with upper and lower limits of agreement 1.96 and -1.41 l.min(-1); the percentage error was 27%. LiDCO showed good correlation, marginal bias and acceptable limits of agreement and percentage error. It could therefore potentially replace thermodilution as a means of measuring cardiac output in the ICU, particularly when determination of pulmonary artery pressure is not required.

Cholinergic modulation of dentate gyrus processing through dynamic reconfiguration of inhibitory circuits.

The dentate gyrus (DG) of the hippocampus plays a key role in memory formation, and it is known to be modulated by septal projections. By performing electrophysiology and optogenetics, we evaluated the role of cholinergic modulation in the processing of afferent inputs in the DG. We show that mature granule cells (GCs), but not adult-born immature neurons, have increased responses to afferent perforant path stimuli upon cholinergic modulation. This is due to a highly precise reconfiguration of inhibitory circuits, differentially affecting Parvalbumin and Somatostatin interneurons, resulting in a nicotinic-dependent perisomatic disinhibition of GCs. This circuit reorganization provides a mechanism by which mature GCs could escape the strong inhibition they receive, creating a window of opportunity for plasticity. Indeed, coincident activation of perforant path inputs with optogenetic release of acetylcholine produces a long-term potentiated response in GCs, essential for memory formation.

Effects of highly active antiretroviral therapy on vaccine-induced humoral immunity in HIV-infected adults.

OBJECTIVES: The acquisition of adequate vaccine-induced humoral immunity is especially important in HIV-infected individuals, who are at increased risk of infections. The aim of the study was to assess the safety of administering a complete vaccination programme to successfully treated HIV-infected adults and to evaluate specific humoral responses and the effect of highly active antiretroviral therapy (HAART) interruption on these responses. METHODS: A placebo-controlled, double-blind clinical trial was designed and 26 HIV-infected adults enrolled. Study participants were randomized to receive either a complete immunization schedule with commercial vaccines or placebo for 12 months. HAART was then discontinued for 6 months. Specific humoral responses were evaluated at baseline, at month 12 and after HAART interruption and compared between groups. RESULTS: There were neither local nor systemic secondary effects related to vaccination. Specific humoral responses to vaccines were adequate, but a loss of immunoglobulin G titres was observed after HAART interruption in 12 study participants. CONCLUSIONS: HAART interruption may cause impairment of previously acquired vaccine-induced immunity in HIV-infected adults.

Multiband negative refraction in one-dimensional photonic crystals.

We simulate a lossless one-dimensional photonic crystals (1DPC) structure and show that negative refraction could be present near the low frequency edge of at least the second, fourth and sixth bandgaps. We experimentally demonstrate for the first time negative refraction in strongly modulated porous silicon 1D-PC in the visible and near infrared regions. This 1D-PC structure may allow the realization of short-focus Veselago lenses in different optical bands. An advantage of our structure is its simplicity allowing for cheap and rapid fabrication of samples.

Emergency ambulance transport induces stress in patients with acute coronary syndrome.

BACKGROUND: Trials with healthy volunteers have shown that emergency ambulance transportation induces stress, which becomes evident by an increase in heart rate, blood pressure and plasma levels of stress hormones such as adrenaline, noradrenaline, cortisol and prolactin. A study was undertaken to test the hypothesis that emergency ambulance transportation may also lead to stress in patients with acute coronary syndrome. METHODS: Venous plasma levels of epinephrine, norepinephrine and lactate as well as visual analogue scale (VAS) scores for pain and anxiety were measured in 32 patients with defined clinical signs of acute coronary syndrome before and after transportation. Heart rate, blood pressure and transcutaneous oxygen saturation levels were recorded every 3 min. RESULTS: Mean (SD) plasma levels of epinephrine and norepinephrine increased significantly (p<0.01) during transportation (159.29 (55.34) ng/l and 632.53 (156.32) ng/l before transportation vs 211.03 (70.12) ng/l and 782.93 (173.95) ng/l after transportation), while lactate levels, heart rate and mean blood pressure remained almost stable. There was no significant change in mean (SD) VAS scores for pain and anxiety (3.79 (3.70) and 2.89 (3.01) vs 2.13 (3.30) and 1.57 (2.78)). CONCLUSION: Emergency ambulance transportation induces a rise in plasma catecholamine levels and therefore stress in patients with acute coronary syndrome, but does not result in cardiac shock as lactate levels and haemodynamic parameters remain normal.

Involvement of MAF/SPP1 axis in the development of bone marrow fibrosis in PMF patients.

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by hyperplastic megakaryopoiesis and myelofibrosis. We recently described the upregulation of MAF (v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog) in PMF CD34+ hematopoietic progenitor cells (HPCs) compared to healthy donor. Here we demonstrated that MAF is also upregulated in PMF compared with the essential thrombocytemia (ET) and polycytemia vera (PV) HPCs. MAF overexpression and knockdown experiments shed some light into the role of MAF in PMF pathogenesis, by demonstrating that MAF favors the megakaryocyte and monocyte/macrophage commitment of HPCs and leads to the increased expression of proinflammatory and profibrotic mediators. Among them, we focused our further studies on SPP1 and LGALS3. We assessed SPP1 and LGALS3 protein levels in 115 PMF, 47 ET and 24 PV patients plasma samples and we found that SPP1 plasma levels are significantly higher in PMF compared with ET and PV patients. Furthermore, in vitro assays demonstrated that SPP1 promotes fibroblasts and mesenchymal stromal cells proliferation and collagen production. Strikingly, clinical correlation analyses uncovered that higher SPP1 plasma levels in PMF patients correlate with a more severe fibrosis degree and a shorter overall survival. Collectively our data unveil that MAF overexpression contributes to PMF pathogenesis by driving the deranged production of the profibrotic mediator SPP1.

A clinical-molecular prognostic model to predict survival in patients with post polycythemia vera and post essential thrombocythemia myelofibrosis.

Polycythemia vera (PV) and essential thrombocythemia (ET) are myeloproliferative neoplasms with variable risk of evolution into post-PV and post-ET myelofibrosis, from now on referred to as secondary myelofibrosis (SMF). No specific tools have been defined for risk stratification in SMF. To develop a prognostic model for predicting survival, we studied 685 JAK2, CALR, and MPL annotated patients with SMF. Median survival of the whole cohort was 9.3 years (95% CI: 8-not reached-NR-). Through penalized Cox regressions we identified negative predictors of survival and according to beta risk coefficients we assigned 2 points to hemoglobin level <11 g/dl, to circulating blasts ⩾3%, and to CALR-unmutated genotype, 1 point to platelet count <150 × 109/l and to constitutional symptoms, and 0.15 points to any year of age. Myelofibrosis Secondary to PV and ET-Prognostic Model (MYSEC-PM) allocated SMF patients into four risk categories with different survival (P<0.0001): low (median survival NR; 133 patients), intermediate-1 (9.3 years, 95% CI: 8.1-NR; 245 patients), intermediate-2 (4.4 years, 95% CI: 3.2-7.9; 126 patients), and high risk (2 years, 95% CI: 1.7-3.9; 75 patients). Finally, we found that the MYSEC-PM represents the most appropriate tool for SMF decision-making to be used in clinical and trial settings.

In vivo functional localization of the human visual cortex using positron emission tomography and magnetic resonance imaging.

Positron emission tomography (PET) and magnetic resonance imaging (MRI) are two recently developed methods for imaging the human brain in vivo. One application of PET measures stimulus-evoked changes in cerebral blood flow while MRI provides a detailed anatomical map of the brain. Here we report the combined application of these two techniques in the same human subject. Subtracted PET scans of a brain receiving visual stimulation were superimposed upon MRI images of the same brain. The PET scans were converted into the MRI coordinate space before superposition, which allowed for a more precise correlation between MRI anatomical data and PET physiological data. Responses were localized in striate and extrastriate visual areas as well as in the posterior thalamus.

Reinterventions after repair of common arterial trunk in neonates and young infants.

OBJECTIVES: To determine rates of reintervention after repair of common arterial trunk in the neonatal and early infant periods. BACKGROUND: With improving success in the early treatment of common arterial trunk, the need for reinterventional procedures in older children, adolescents and adults will become an increasingly widespread concern in the treatment of these patients. METHODS: We reviewed our experience with 159 infants younger than four months of age who underwent complete primary repair of common arterial trunk at our institution from 1975 to 1998, with a focus on postoperative reinterventions. RESULTS: Of 128 early survivors, 40 underwent early reinterventions for persistent mediastinal bleeding or other reasons. During a median follow-up of 98 months (range, 2 to 235 months), 121 reinterventions were performed in 81 patients. Actuarial freedom from reintervention was 50% at four years, and freedom from a second reintervention was 75% at 11 years. A total of 92 conduit reinterventions were performed in 75 patients, with a single reintervention in 61 patients, 2 reinterventions in 11 patients and 3 reinterventions in 3 patients. Freedom from a first conduit reintervention was 45% at five years. The only independent variable predictive of a longer time to first conduit replacement was use of an allograft conduit at the original repair (p = 0.05), despite the significantly younger age of patients receiving an allograft conduit (p < 0.001). Reintervention on the truncal valve was performed on 22 occasions in 19 patients, including 21 valve replacements in 18 patients and repair in 1, with a freedom from truncal valve reintervention of 83% at 10 years. Surgical (n = 29) or balloon (n = 12) reintervention for pulmonary artery stenosis was performed 41 times in 32 patients. Closure of a residual ventricular septal defect was required in 13 patients, all of whom underwent closure originally with a continuous suture technique. Eight of 16 late deaths were related to reintervention. CONCLUSIONS: The burden of reintervention after repair of common arterial trunk in early infancy is high. Although conduit reintervention is inevitable, efforts should be made at the time of the initial repair to minimize factors leading to reintervention, including prevention of branch pulmonary artery stenosis and residual interventricular communications.

Toxicity of cationic liposome-DNA complex in lung isografts.

BACKGROUND: Cationic lipids have been successfully employed as vectors for gene transfer in lung grafts, yet those lipid vectors have potential toxicity. Furthermore, the optimal concentration of cationic lipids for gene transfection to lung grafts has not been determined. We evaluated liposome concentration/toxicity relationships in an in vivo rat lung transplantation model. METHODS: Left lungs were harvested and infused via the pulmonary artery with chloramphenicol acetyl-transferase (CAT)-DNA/lipid 67 (cationic lipid)/dioleoylphosphatidylethanolamine complex (4:1:2 in a final concentration ratio). Donor lungs were allocated into six groups according to lipid 67 concentration: group 1, 0 microM (control); group 2, 10 microM; group 3, 50 microM; group 4, 100 microM; group 5, 250 microM; group 6, 500 microM. Forty-eight hours after orthotopic transplantation, the recipient contralateral right main pulmonary artery and bronchus were ligated. The graft was ventilated with 100% oxygen for 5 min. Arterial blood gas analysis (PaO2, PaCO2), peak airway pressure (PAP), and CAT activity of the grafts were measured. RESULTS: Recipient survival, and PaO2, PAP, and CAT levels correlated with the lipid-DNA complex concentration. The grafts in groups 4-6 were more injured as evidenced by decreased PaO2 and increased PAP levels in comparison to the control group. CAT level was significantly lower in group 2 than in groups 3-6. CONCLUSIONS: The pulmonary toxicity of cationic lipid is dose-dependent. The balance between lung graft function and transgene expression is optimal at a lipid 67 concentration of 50 microM.

Absence of in vivo DNA-protein interactions in the DQA2 and DQB2 promoter regions.

The DQ subregion of the human major histocompatibility complex (HLA) contains two pairs of loci: the DQA1/B1 genes (hereafter called DQ1), coding for the DQ molecules, and the DQA2/B2 pseudogenes (hereafter called DQ2). These pseudogenes are highly homologous to the functional DQ1 genes and they have no apparent abnormal features in their sequences that could prevent their activity. Only recently a low expression of the DQA2 gene has been observed whereas the DQB2 transcript was never found. The comparison between the DQ1 and DQ2 regulatory sequences revealed several differences in their W, X, and Y cis-acting elements. To examine the DNA/protein interactions in the DQ promoter regions, we performed in vivo footprinting experiments. Whereas the functional DQ1 loci showed a series of DNA-protein contact points in the X and Y boxes, the promoters of the DQ2 pseudogenes displayed an unoccupied phenotype. These findings suggest that the very low level of DQA2 expression and the apparent lack of DQB2 activity are caused by the reduced binding affinity of specific transcription factors.