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Bacteria and many non-metazoan Eukaryotes respond to stresses and threats using two-component systems (TCSs) comprising sensor kinases (SKs) and response regulators (RRs). Multikinase networks, where multiple SKs work together, detect and integrate different signals to control important lifestyle decisions such as sporulation and virulence. Here, we study interactions between two SKs from Pseudomonas aeruginosa , GacS and RetS, which control the switch between acute and chronic virulence. We demonstrate three mechanisms by which RetS attenuates GacS signalling: RetS takes phosphoryl groups from GacS-P; RetS has transmitter phosphatase activity against the receiver domain of GacS-P; and RetS inhibits GacS autophosphorylation. These mechanisms play important roles in vivo and during infection, and exemplify an unprecedented degree of signal processing by SKs that may be exploited in other multikinase networks. Bacteria respond to stresses using two-component systems consisting of sensor kinases (SKs) and response regulators. Here, Francis et al . reveal three specific interaction mechanisms between a pair of SKs that are important for regulation of virulence in the pathogen Pseudomonas aeruginosa .

Cryptococcal meningitis causes 10%–15% of AIDS-associated deaths globally. Still, brain-specific immunity to cryptococci is a conundrum. By employing innovative imaging, this study reveals what occurs during the first days of infection in brain and in airways. We found that titan cells predominate in upper airways and that cryptococci breach the upper airway mucosa, which implies that, at least in mice, the upper airways are a site for fungal dissemination. This would signify that mucosal immunity of the upper airway needs to be better understood. Importantly, we also show that microglia, the brain-resident macrophages, are the first responders to infection, and microglia clusters are formed surrounding cryptococci. This study opens the field to detailed molecular investigations on airway immune response, how fungus traverses the blood-brain barrier, how microglia respond to infection, and ultimately how microglia monitor the blood-brain barrier to preserve brain function.

Abstract Pseudomonas aeruginosa is a versatile opportunistic pathogen capable of infecting a broad range of hosts, in addition to thriving in a broad range of environmental conditions outside of hosts. With this versatility comes the need to tightly regulate its genome to optimise its gene expression and behaviour to the prevailing conditions. Two-component systems (TCSs) comprising sensor kinases and response regulators play a major role in this regulation. This minireview discusses the growing number of TCSs that have been implicated in the virulence of P. aeruginosa, with a special focus on the emerging theme of multikinase networks, which are networks comprising multiple sensor kinases working together, sensing and integrating multiple signals to decide upon the best response. The networks covered in depth regulate processes such as the switch between acute and chronic virulence (GacS network), the Cup fimbriae (Roc network and Rcs/Pvr network), the aminoarabinose modification of lipopolysaccharide (a network involving the PhoQP and PmrBA TCSs), twitching motility and virulence (a network formed from the Chp chemosensory pathway and the FimS/AlgR TCS), and biofilm formation (Wsp chemosensory pathway). In addition, we highlight the important interfaces between these systems and secondary messenger signals such as cAMP and c-di-GMP. Two-component systems controlling the virulence of the opportunistic pathogen, Pseudomonas aeruginosa.

Bdellovibrio bacteriovorus is a predatory deltaproteobacterium that encounters individual Gram-negative prey bacteria with gliding or swimming motility, and then is able to invade such prey cells via type IVa pilus-dependent mechanisms. Movement control (pili or gliding) in other deltaproteobacteria, such as the pack hunting Myxococcus xanthus , uses a response regulator protein, RomR Mx (which dynamically relocalises between the cell poles) and a GTPase, MglA Mx , previously postulated as an interface between the Frz Mx chemosensory system and gliding or pilus-motility apparatus, to produce regulated bidirectional motility. In contrast, B. bacteriovorus predation is a more singular encounter between a lone predator and prey; contact is always via the piliated, non-flagellar pole of the predator, involving MglA Bd , but no Frz system. In this new study, tracking fluorescent RomR Bd microscopically during predatory growth shows that it does not dynamically relocalise, in contrast to the M. xanthus protein; instead having possible roles in growth events. Furthermore, transcriptional start analysis, site-directed mutagenesis and bacterial two-hybrid interaction studies, indicate an evolutionary loss of RomR Bd activation (via receiver domain phosphorylation) in this lone hunting bacterium, demonstrating divergence from its bipolar role in motility in pack-hunting M. xanthus and further evolution that may differentiate lone from pack predators.

The fungus causes lethal meningitis in humans with weakened immune systems and is estimated to account for 10-15% of AIDS-associated deaths worldwide. There are major gaps in our understanding of how this environmental fungus evades the immune system and invades the mammalian brain before the onset of overt symptoms. To investigate the dynamics of C. neoformans tissue invasion, we mapped early fungal localisation and host cell interactions at early times in infected brain, lung, and upper airways using mouse models of systemic and airway infection. To enable this, we developed an in situ imaging pipeline capable of measuring large volumes of tissue while preserving anatomical and cellular information by combining thick tissue sections, tissue clarification, and confocal imaging. Made possible by these techniques, we confirm high fungal burden in mouse upper airway turbinates after nasal inoculation. Surprisingly, most yeasts in turbinates were titan cells, indicating this microenvironment enables titan cell formation with faster kinetics than reported in mouse lungs. Importantly, we observed one instance of fungal cells enmeshed in lamina propria of upper airways, suggesting penetration of airway mucosa as a possible route of tissue invasion and dissemination to the bloodstream. We extend previous literature positing bloodstream dissemination of , via imaging within blood vessels of mouse lungs and finding viable fungi in the bloodstream of mice a few days after intranasal infection, suggesting that bloodstream access can occur via lung alveoli. In a model of systemic cryptococcosis, we show that as early as 24 h post infection, majority of cells traversed the blood-brain barrier, and are engulfed or in close proximity to microglia. Our work establishes that can breach multiple tissue barriers within the first days of infection. This work presents a new method for investigating cryptococcal invasion mechanisms and demonstrates microglia as the primary cells responding to C. neoformans invasion.

causes lethal meningitis and accounts for approximately 10%-15% of AIDS-associated deaths worldwide. There are major gaps in our understanding of how this fungus invades the mammalian brain. To investigate the dynamics of tissue invasion, we mapped fungal localization and host cell interactions in infected brain, lung, and upper airways using mouse models of systemic and airway infection. To enable this, we developed an imaging pipeline capable of measuring large volumes of tissue while preserving anatomical and cellular information by combining thick tissue sections, tissue clarification, and confocal imaging. We confirm high fungal burden in mouse upper airway after nasal inoculation. Yeast in turbinates were frequently titan cells, with faster kinetics than reported in mouse lungs. Importantly, we observed one instance of fungal cells enmeshed in lamina propria of the upper airways, suggesting penetration of airway mucosa as a possible route of tissue invasion and dissemination to the bloodstream. We extend previous literature positing bloodstream dissemination of , by finding viable fungi in the bloodstream of mice a few days after intranasal infection. As early as 24 h post systemic infection, the majority of cells traversed the blood-brain barrier, and were engulfed or in close proximity to microglia. Our work presents a new method for investigating microbial invasion, establishes that can breach multiple tissue barriers within the first days of infection, and demonstrates microglia as the first cells responding to invasion of the brain.IMPORTANCECryptococcal meningitis causes 10%-15% of AIDS-associated deaths globally. Still, brain-specific immunity to cryptococci is a conundrum. By employing innovative imaging, this study reveals what occurs during the first days of infection in brain and in airways. We found that titan cells predominate in upper airways and that cryptococci breach the upper airway mucosa, which implies that, at least in mice, the upper airways are a site for fungal dissemination. This would signify that mucosal immunity of the upper airway needs to be better understood. Importantly, we also show that microglia, the brain-resident macrophages, are the first responders to infection, and microglia clusters are formed surrounding cryptococci. This study opens the field to detailed molecular investigations on airway immune response, how fungus traverses the blood-brain barrier, how microglia respond to infection, and ultimately how microglia monitor the blood-brain barrier to preserve brain function.

Pluripotent stem cell‐derived kidney organoids offer a promising solution to renal failure, yet current organoid protocols often lead to off‐target cells and phenotypic alterations, preventing maturity. Here, various dynamic hydrogel architectures are created, conferring a controlled and biomimetic environment for organoid encapsulation. How hydrogel stiffness and stress relaxation affect renal phenotype and undesired fibrotic markers are investigated. The authors observe that stiff hydrogel encapsulation leads to an absence of certain renal cell types and signs of an epithelial–mesenchymal transition (EMT), whereas encapsulation in soft, stress‐relaxing hydrogels leads to all major renal segments, fewer fibrosis or EMT associated proteins, apical proximal tubule polarization, and primary cilia formation, representing a significant improvement over current approaches to culture kidney organoids. The findings show that engineering hydrogel mechanics and dynamics have a decided benefit for organoid culture. These structure–property–function relationships can enable the rational design of materials, bringing us closer to functional engraftments and disease‐modeling applications. Current kidney organoid protocols often lead to off‐target cells and phenotypic alterations. Various dynamic hydrogel architectures are investigated for organoid encapsulation. Encapsulation in soft, fast stress‐relaxing hydrogels leads to all major renal segments, fewer fibrosis or epithelial‐mesenchymal transition (EMT) associated proteins, apical proximal tubule polarization, and primary cilia formation, representing a significant improvement over current approaches to culture kidney organoids.

Current cardiovascular magnetic resonance (CMR) methods, such as late gadolinium enhancement (LGE) and oedema imaging (T2W) used to depict myocardial ischemia, have limitations. Novel quantitative T1-mapping techniques have the potential to further characterize the components of ischemic injury. In patients with myocardial infarction (MI) we sought to investigate whether state-of the art pre-contrast T1-mapping (1) detects acute myocardial injury, (2) allows for quantification of the severity of damage when compared to standard techniques such as LGE and T2W, and (3) has the ability to predict long term functional recovery. 3T CMR including T2W, T1-mapping and LGE was performed in 41 patients [of these, 78% were ST elevation MI (STEMI)] with acute MI at 12-48 hour after chest pain onset and at 6 months (6M). Patients with STEMI underwent primary PCI prior to CMR. Assessment of acute regional wall motion abnormalities, acute segmental damaged fraction by T2W and LGE and mean segmental T1 values was performed on matching short axis slices. LGE and improvement in regional wall motion at 6M were also obtained. We found that the variability of T1 measurements was significantly lower compared to T2W and that, while the diagnostic performance of acute T1-mapping for detecting myocardial injury was at least as good as that of T2W-CMR in STEMI patients, it was superior to T2W imaging in NSTEMI. There was a significant relationship between the segmental damaged fraction assessed by either by LGE or T2W, and mean segmental T1 values (P < 0.01). The index of salvaged myocardium derived by acute T1-mapping and 6M LGE was not different to the one derived from T2W (P = 0.88). Furthermore, the likelihood of improvement of segmental function at 6M decreased progressively as acute T1 values increased (P < 0.0004). In acute MI, pre-contrast T1-mapping allows assessment of the extent of myocardial damage. T1-mapping might become an important complementary technique to LGE and T2W for identification of reversible myocardial injury and prediction of functional recovery in acute MI.

OBJECTIVE:--Type 2 diabetes is the leading cause of end-stage renal disease worldwide. Aside from hyperglycemia and hypertension, other metabolic factors may determine renal outcome. We examined risk associations of metabolic syndrome with new onset of chronic kidney disease (CKD) in 5,829 Chinese patients with type 2 diabetes enrolled between 1995 and 2005. RESEARCH DESIGN AND METHODS--Metabolic syndrome was defined by National Cholesterol Education Program Adult Treatment Panel III criteria with the Asian definition of obesity. Estimated glomerular filtration rate (eGFR) was calculated using the abbreviated Modification of Diet in Renal Disease formula modified for the Chinese population. New onset of CKD was defined as eGFR <60 ml/min per 1.73 m² at the time of censor. Subjects with CKD at baseline were excluded from the analysis. RESULTS:--After a median follow-up duration of 4.6 years (interquartile range: 1.9-7.3 years), 741 patients developed CKD. The multivariable-adjusted hazard ratio (HR) of CKD was 1.31 (95% CI 1.12-1.54, P = 0.001) for subjects with metabolic syndrome compared with those without metabolic syndrome. Relative to subjects with no other components of metabolic syndrome except for diabetes, those with two, three, four, and five metabolic syndrome components had HRs of an increased risk of CKD of 1.15 (0.83-1.60, P = 0.407) 1.32 (0.94-1.86, P = 0.112), 1.64 (1.17-2.32, P = 0.004), and 2.34 (1.54-3.54, P < 0.001), respectively. The metabolic syndrome traits of central obesity, hypertriglyceridemia, hypertension, and low BMI were independent predictors for CKD. CONCLUSIONS:--The presence of metabolic syndrome independently predicts the development of CKD in subjects with type 2 diabetes.

T2w-CMR is used widely to assess myocardial edema. Quantitative T1-mapping is also sensitive to changes in free water content. We hypothesized that T1-mapping would have a higher diagnostic performance in detecting acute edema than dark-blood and bright-blood T2w-CMR. We investigated 21 controls (55 ± 13 years) and 21 patients (61 ± 10 years) with Takotsubo cardiomyopathy or acute regional myocardial edema without infarction. CMR performed within 7 days included cine, T1-mapping using ShMOLLI, dark-blood T2-STIR, bright-blood ACUT2E and LGE imaging. We analyzed wall motion, myocardial T1 values and T2 signal intensity (SI) ratio relative to both skeletal muscle and remote myocardium. All patients had acute cardiac symptoms, increased Troponin I (0.15-36.80 ug/L) and acute wall motion abnormalities but no LGE. T1 was increased in patient segments with abnormal and normal wall motion compared to controls (1113 ± 94 ms, 1029 ± 59 ms and 944 ± 17 ms, respectively; p < 0.001). T2 SI ratio using STIR and ACUT2E was also increased in patient segments with abnormal and normal wall motion compared to controls (all p < 0.02). Receiver operator characteristics analysis showed that T1-mapping had a significantly larger area-under-the-curve (AUC = 0.94) compared to T2-weighted methods, whether the reference ROI was skeletal muscle or remote myocardium (AUC = 0.58-0.89; p < 0.03). A T1 value of greater than 990 ms most optimally differentiated segments affected by edema from normal segments at 1.5 T, with a sensitivity and specificity of 92 %. Non-contrast T1-mapping using ShMOLLI is a novel method for objectively detecting myocardial edema with a high diagnostic performance. T1-mapping may serve as a complementary technique to T2-weighted imaging for assessing myocardial edema in ischemic and non-ischemic heart disease, such as quantifying area-at-risk and diagnosing myocarditis.