Long QT syndrome-associated calmodulin variants disrupt the activity of the slowly activating delayed rectifier potassium channel.

Calmodulin (CaM) is a highly conserved mediator of calcium (Ca2+ )-dependent signalling and modulates various cardiac ion channels. Genotyping has revealed several CaM mutations associated with long QT syndrome (LQTS). LQTS patients display prolonged ventricular recovery times (QT interval), increasing their risk of incurring life-threatening arrhythmic events. Loss-of-function mutations to Kv7.1 (which drives the slow delayed rectifier potassium current, IKs, a key ventricular repolarising current) are the largest contributor to congenital LQTS (>50% of cases). CaM modulates Kv7.1 to produce a Ca2+ -sensitive IKs, but little is known about the consequences of LQTS-associated CaM mutations on Kv7.1 function. Here, we present novel data characterising the biophysical and modulatory properties of three LQTS-associated CaM variants (D95V, N97I and D131H). We showed that mutations induced structural alterations in CaM and reduced affinity for Kv7.1, when compared with wild-type (WT). Using HEK293T cells expressing Kv7.1 channel subunits (KCNQ1/KCNE1) and patch-clamp electrophysiology, we demonstrated that LQTS-associated CaM variants reduced current density at systolic Ca2+ concentrations (1 µm), revealing a direct QT-prolonging modulatory effect. Our data highlight for the first time that LQTS-associated perturbations to CaM's structure impede complex formation with Kv7.1 and subsequently result in reduced IKs. This provides a novel mechanistic insight into how the perturbed structure-function relationship of CaM variants contributes to the LQTS phenotype. KEY POINTS: Calmodulin (CaM) is a ubiquitous, highly conserved calcium (Ca2+ ) sensor playing a key role in cardiac muscle contraction. Genotyping has revealed several CaM mutations associated with long QT syndrome (LQTS), a life-threatening cardiac arrhythmia syndrome. LQTS-associated CaM variants (D95V, N97I and D131H) induced structural alterations, altered binding to Kv7.1 and reduced IKs. Our data provide a novel mechanistic insight into how the perturbed structure-function relationship of CaM variants contributes to the LQTS phenotype.

Suppressor Mutants: History and Today's Applications.

For decades, biologist have exploited the near boundless advantages that molecular and genetic tools and analysis provide for our ability to understand biological systems. One of these genetic tools, suppressor analysis, has proven invaluable in furthering our understanding of biological processes and pathways and in discovering unknown interactions between genes and gene products. The power of suppressor analysis lies in its ability to discover genetic interactions in an unbiased manner, often leading to surprising discoveries. With advancements in technology, high-throughput approaches have aided in large-scale identification of suppressors and have helped provide insight into the core functional mechanisms through which suppressors act. In this review, we examine some of the fundamental discoveries that have been made possible through analysis of suppressor mutations. In addition, we cover the different types of suppressor mutants that can be isolated and the biological insights afforded by each type. Moreover, we provide considerations for the design of experiments to isolate suppressor mutants and for strategies to identify intergenic suppressor mutations. Finally, we provide guidance and example protocols for the isolation and mapping of suppressor mutants.

ElyC and Cyclic Enterobacterial Common Antigen Regulate Synthesis of Phosphoglyceride-Linked Enterobacterial Common Antigen.

The Gram-negative cell envelope is a complex structure delineating the cell from its environment. Recently, we found that enterobacterial common antigen (ECA) plays a role maintaining the outer membrane (OM) permeability barrier, which excludes toxic molecules including many antibiotics. ECA is a conserved carbohydrate found throughout Enterobacterales (e.g., Salmonella, Klebsiella, and Yersinia). There are two OM forms of ECA (phosphoglyceride-linked ECAPG and lipopolysaccharide-linked ECALPS) and one periplasmic form of ECA (cyclic ECACYC). ECAPG, found in the outer leaflet of the OM, consists of a linear ECA oligomer attached to phosphoglyceride through a phosphodiester linkage. The process through which ECAPG is produced from polymerized ECA is unknown. Therefore, we set out to identify genes interacting genetically with ECAPG biosynthesis in Escherichia coli K-12 using the competition between ECA and peptidoglycan biosynthesis. Through transposon-directed insertion sequencing, we identified an interaction between elyC and ECAPG biosynthesis. ElyC is an inner membrane protein previously shown to alter peptidoglycan biosynthesis rates. We found ΔelyC was lethal specifically in strains producing ECAPG without other ECA forms, suggesting ECAPG biosynthesis impairment or dysregulation. Further characterization suggested ElyC inhibits ECAPG synthesis in a posttranscriptional manner. Moreover, the full impact of ElyC on ECA levels requires the presence of ECACYC. Our data demonstrate ECACYC can regulate ECAPG synthesis in strains wild type for elyC. Overall, our data demonstrate ElyC and ECACYC act in a novel pathway that regulates the production of ECAPG, supporting a model in which ElyC provides feedback regulation of ECAPG production based on the periplasmic levels of ECACYC. IMPORTANCE Enterobacterial common antigen (ECA) is a conserved polysaccharide present on the surface of the outer membrane (OM) and in the periplasm of the many pathogenic bacteria belonging to Enterobacterales, including Klebsiella pneumoniae, Salmonella enterica, and Yersinia pestis. As the OM is a permeability barrier that excludes many antibiotics, synthesis pathways for OM molecules are promising targets for antimicrobial discovery. Here, we elucidated, in E. coli K-12, a new pathway for the regulation of biosynthesis of one cell surface form of ECA, ECAPG. In this pathway, an inner membrane protein, ElyC, and the periplasmic form of ECA, ECACYC, genetically interact to inhibit the synthesis of ECAPG, potentially through feedback regulation based on ECACYC levels. This is the first insight into the pathway responsible for synthesis of ECAPG and represents a potential target for weakening the OM permeability barrier. Furthermore, this pathway provides a tool for experimental manipulation of ECAPG levels.

Polyphosphate is an extracellular signal that can facilitate bacterial survival in eukaryotic cells.

Polyphosphate is a linear chain of phosphate residues and is present in organisms ranging from bacteria to humans. Pathogens such as Mycobacterium tuberculosis accumulate polyphosphate, and reduced expression of the polyphosphate kinase that synthesizes polyphosphate decreases their survival. How polyphosphate potentiates pathogenicity is poorly understood. Escherichia coli K-12 do not accumulate detectable levels of extracellular polyphosphate and have poor survival after phagocytosis by Dictyostelium discoideum or human macrophages. In contrast, Mycobacterium smegmatis and Mycobacterium tuberculosis accumulate detectable levels of extracellular polyphosphate, and have relatively better survival after phagocytosis by D. discoideum or macrophages. Adding extracellular polyphosphate increased E. coli survival after phagocytosis by D. discoideum and macrophages. Reducing expression of polyphosphate kinase 1 in M. smegmatis reduced extracellular polyphosphate and reduced survival in D. discoideum and macrophages, and this was reversed by the addition of extracellular polyphosphate. Conversely, treatment of D. discoideum and macrophages with recombinant yeast exopolyphosphatase reduced the survival of phagocytosed M. smegmatis or M. tuberculosisD. discoideum cells lacking the putative polyphosphate receptor GrlD had reduced sensitivity to polyphosphate and, compared to wild-type cells, showed increased killing of phagocytosed E. coli and M. smegmatis Polyphosphate inhibited phagosome acidification and lysosome activity in D. discoideum and macrophages and reduced early endosomal markers in macrophages. Together, these results suggest that bacterial polyphosphate potentiates pathogenicity by acting as an extracellular signal that inhibits phagosome maturation.

Reductions in Retrobulbar and Retinal Capillary Blood Flow Strongly Correlate With Changes in Optic Nerve Head and Retinal Morphology Over 4 Years in Open-angle Glaucoma Patients of African Descent Compared With Patients of European Descent.

PURPOSE: To investigate the relationship of changes in ocular blood flow with optic nerve head (ONH) and retinal morphology in open-angle glaucoma (OAG) patients of African versus European descent (ED) over 4 years. MATERIALS AND METHODS: In this study, 112 patients with OAG were examined at baseline, 79 [59 ED, 20 African descent (AD)] of which were followed for 4 years. Retinal capillary blood flow was assessed with Heidelberg retinal flowmetry. Retrobulbar blood flow was measured by color Doppler imaging. Retinal structural changes were examined with optical coherence tomography and Heidelberg retinal tomography-III. Mixed-model analysis of covariance was used to test for the significance of change from baseline to 4-year follow-up, and Pearson correlation coefficients were calculated to evaluate linear associations. RESULTS: In OAG patients of AD, structural changes of the ONH demonstrated a strong association with the end diastolic velocities and resistive indices of the short posterior ciliary arteries over 4 years. In addition, there was a significantly larger increase in the avascular area of the inferior retina in patients of AD, and this reduction in retinal capillaries strongly correlated with a reduction in macular thickness. CONCLUSIONS: Reductions in retinal capillary and retrobulbar blood flow strongly correlated with changes in the ONH and macular thickness over 4 years in OAG patients of AD compared with ED. These data suggest that ocular vascular health may be a more influential contributing factor in the pathophysiology of OAG in patients of AD compared with ED.

The neural mechanisms of hallucinations: A quantitative meta-analysis of neuroimaging studies.

Activation likelihood estimation meta-analysis of functional neuroimaging data was used to investigate the neural mechanisms underlying auditory-verbal and visual hallucinations (AVHs and VHs). Consistent activation across studies during AVHs, but not VHs, in Wernicke's and Broca's areas is consistent with involvement of speech and language processes in the experience of hearing voices when none are present. Similarly, greater activity in auditory cortex during AVHs and in visual cortex during VHs supports models proposing over-stimulation of sensory cortices in the generation of these perceptual anomalies. Activation across studies in the medial temporal lobe highlights a role for memory intrusions in the provision of content for AVHs, whereas insula activation may relate to the involvement of awareness and self-representation. Finally, activation in the paracingulate region of medial prefrontal cortex during AVHs is consistent with models implicating reality monitoring impairment in the misattribution of self-generated information as externally perceived. In the light of the results, the need for unified theoretical frameworks that account for the full range of hallucinatory experiences is discussed.