Role of CAMK2D in neurodevelopment and associated conditions.

The calcium/calmodulin-dependent protein kinase type 2 (CAMK2) family consists of four different isozymes, encoded by four different genes-CAMK2A, CAMK2B, CAMK2G, and CAMK2D-of which the first three have been associated recently with neurodevelopmental disorders. CAMK2D is one of the major CAMK2 proteins expressed in the heart and has been associated with cardiac anomalies. Although this CAMK2 isoform is also known to be one of the major CAMK2 subtypes expressed during early brain development, it has never been linked with neurodevelopmental disorders until now. Here we show that CAMK2D plays an important role in neurodevelopment not only in mice but also in humans. We identified eight individuals harboring heterozygous variants in CAMK2D who display symptoms of intellectual disability, delayed speech, behavioral problems, and dilated cardiomyopathy. The majority of the variants tested lead to a gain of function (GoF), which appears to cause both neurological problems and dilated cardiomyopathy. In contrast, loss-of-function (LoF) variants appear to induce only neurological symptoms. Together, we describe a cohort of individuals with neurodevelopmental disorders and cardiac anomalies, harboring pathogenic variants in CAMK2D, confirming an important role for the CAMK2D isozyme in both heart and brain function.

Architectural groups of a subtelomeric gene family evolve along distinct paths in Candida albicans.

Subtelomeres are dynamic genomic regions shaped by elevated rates of recombination, mutation, and gene birth/death. These processes contribute to formation of lineage-specific gene family expansions that commonly occupy subtelomeres across eukaryotes. Investigating the evolution of subtelomeric gene families is complicated by the presence of repetitive DNA and high sequence similarity among gene family members that prevents accurate assembly from whole genome sequences. Here, we investigated the evolution of the telomere-associated (TLO) gene family in Candida albicans using 189 complete coding sequences retrieved from 23 genetically diverse strains across the species. Tlo genes conformed to the 3 major architectural groups (α/ß/γ) previously defined in the genome reference strain but significantly differed in the degree of within-group diversity. One group, Tloß, was always found at the same chromosome arm with strong sequence similarity among all strains. In contrast, diverse Tloα sequences have proliferated among chromosome arms. Tloγ genes formed 7 primary clades that included each of the previously identified Tloγ genes from the genome reference strain with 3 Tloγ genes always found on the same chromosome arm among strains. Architectural groups displayed regions of high conservation that resolved newly identified functional motifs, providing insight into potential regulatory mechanisms that distinguish groups. Thus, by resolving intraspecies subtelomeric gene variation, it is possible to identify previously unknown gene family complexity that may underpin adaptive functional variation.

Measured effects of workload and auditory feedback on remote pilot task performance.

Absent or reduced sensory cueing can deprive pilots operating remotely piloted aircraft beyond visual line of sight (BVLOS) of vital information necessary for safe flight. The present study tested the effects of real-time auditory feedback on remote pilot perception and decision-making task performance in an automated BVLOS flight, under three levels of workload (Low, Moderate and High). Results from 36 participants revealed workload and auditory feedback influenced perception task performance in terms of error type count, with misses more frequent than wrong identifications. In terms of performance in the decision-making task, under low and moderate levels of workload, auditory feedback was found to improve performance. Conversely, under high workloads, an inflexion or tipping point occurred whereby auditory feedback became detrimental to task performance. These results correspond with the expected behavioural responses to external stressors as predicted by the Arousal and Maximal Adaptability theory, and build upon previous findings related to workload, auditory feedback and remote pilot task performance. Practitioner summary: This study tested the effect of real-time auditory feedback and dynamic workloads on remote pilots' task performance. Auditory feedback and workload each influenced the perception tasks in terms of error types committed. Auditory feedback improved decision-making task performance under low and moderate workloads, and reduced performance under high workloads. These results may benefit practitioners by considering the nuanced effects of auditory feedback on human task performance within sensory deprived working environments, including those utilising teleoperated systems.

Intraspecies Transcriptional Profiling Reveals Key Regulators of Candida albicans Pathogenic Traits.

The human commensal and opportunistic fungal pathogen Candida albicans displays extensive genetic and phenotypic variation across clinical isolates. Here, we performed RNA sequencing on 21 well-characterized isolates to examine how genetic variation contributes to gene expression differences and to link these differences to phenotypic traits. C. albicans adapts primarily through clonal evolution, and yet hierarchical clustering of gene expression profiles in this set of isolates did not reproduce their phylogenetic relationship. Strikingly, strain-specific gene expression was prevalent in some strain backgrounds. Association of gene expression with phenotypic data by differential analysis, linear correlation, and assembly of gene networks connected both previously characterized and novel genes with 23 C. albicans traits. Construction of de novo gene modules produced a gene atlas incorporating 67% of C. albicans genes and revealed correlations between expression modules and important phenotypes such as systemic virulence. Furthermore, targeted investigation of two modules that have novel roles in growth and filamentation supported our bioinformatic predictions. Together, these studies reveal widespread transcriptional variation across C. albicans isolates and identify genetic and epigenetic links to phenotypic variation based on coexpression network analysis.IMPORTANCE Infectious fungal species are often treated uniformly despite clear evidence of genotypic and phenotypic heterogeneity being widespread across strains. Identifying the genetic basis for this phenotypic diversity is extremely challenging because of the tens or hundreds of thousands of variants that may distinguish two strains. Here, we use transcriptional profiling to determine differences in gene expression that can be linked to phenotypic variation among a set of 21 Candida albicans isolates. Analysis of this transcriptional data set uncovered clear trends in gene expression characteristics for this species and new genes and pathways that were associated with variation in pathogenic processes. Direct investigation confirmed functional predictions for a number of new regulators associated with growth and filamentation, demonstrating the utility of these approaches in linking genes to important phenotypes.

Automated quantification of Candida albicans biofilm-related phenotypes reveals additive contributions to biofilm production.

Biofilms are organized communities of microbial cells that promote persistence among bacterial and fungal species. Biofilm formation by host-associated Candida species of fungi occurs on both tissue surfaces and implanted devices, contributing to host colonization and disease. In C. albicans, biofilms are built sequentially by adherence of yeast to a surface, invasion into the substrate, the formation of aerial hyphal projections, and the secretion of extracellular matrix. Measurement of these biofilm-related phenotypes remains highly qualitative and often subjective. Here, we designed an informatics pipeline for quantifying filamentation, adhesion, and invasion of Candida species on solid agar media and utilized this approach to determine the importance of these component phenotypes to C. albicans biofilm production. Characterization of 23 C. albicans clinical isolates across three media and two temperatures revealed a wide range of phenotypic responses among isolates in any single condition. Media profoundly altered all biofilm-related phenotypes among these isolates, whereas temperature minimally impacted these traits. Importantly, the extent of biofilm formation correlated significantly with the additive score for its component phenotypes under some conditions, experimentally linking the strength of each component to biofilm mass. In addition, the response of the genome reference strain, SC5314, across these conditions was an extreme outlier compared to all other strains, suggesting it may not be representative of the species. Taken together, development of a high-throughput, unbiased approach to quantifying Candida biofilm-related phenotypes linked variability in these phenotypes to biofilm production and can facilitate genetic dissection of these critical processes to pathogenesis in the host.

Heterogeneity in human hippocampal CaMKII transcripts reveals allosteric hub-dependent regulation.

Calcium/calmodulin-dependent protein kinase II (CaMKII) plays a central role in Ca2+ signaling throughout the body. In the hippocampus, CaMKII is required for learning and memory. Vertebrate genomes encode four CaMKII homologs: CaMKIIα, CaMKIIß, CaMKIIγ, and CaMKIIδ. All CaMKIIs consist of a kinase domain, a regulatory segment, a variable linker region, and a hub domain, which is responsible for oligomerization. The four proteins differ primarily in linker length and composition because of extensive alternative splicing. Here, we report the heterogeneity of CaMKII transcripts in three complex samples of human hippocampus using deep sequencing. We showed that hippocampal cells contain a diverse collection of over 70 CaMKII transcripts from all four CaMKII-encoding genes. We characterized the Ca2+/CaM sensitivity of hippocampal CaMKII variants spanning a broad range of linker lengths and compositions. The effect of the variable linker on Ca2+/CaM sensitivity depended on the kinase and hub domains. Moreover, we revealed a previously uncharacterized role for the hub domain as an allosteric regulator of kinase activity, which may provide a pharmacological target for modulating CaMKII activity. Using small-angle x-ray scattering and single-particle cryo-electron microscopy (cryo-EM), we present evidence for extensive interactions between the kinase and the hub domains, even in the presence of a 30-residue linker. Together, these data suggest that Ca2+/CaM sensitivity in CaMKII is homolog dependent and includes substantial contributions from the hub domain. Our sequencing approach, combined with biochemistry, provides insights into understanding the complex pool of endogenous CaMKII splice variants.

Effects of auditory and visual feedback on remote pilot manual flying performance.

Remotely Piloted Aircraft Systems (RPAS) have facilitated new growth in civil aviation. Unlike manned aircraft, however, they are operated without auditory feedback and normally flown under two visual conditions: in direct visual-line-of-sight to the remote pilot (VLOS) and beyond VLOS with first-person-view imagery transmitted via onboard cameras (BVLOS). The present research examined the effectiveness of audiovisual cueing on remote pilot manual flying performance. Eighteen pilots (three female) completed six navigation and 12 spotting tasks. Their flying performance (horizontal accuracy, vertical accuracy and timeliness) was examined under three different visual display types (VLOS (Control), BVLOS-Monitor & BVLOS-Goggles), with and without real-time auditory feedback, and two wind component (no wind and wind) conditions. Horizontal deviation and timeliness improved in the BVLOS-Monitor condition navigation task, while auditory feedback produced nuanced examples of improved and degraded pilot performance. These results indicate how the specificity of the task, combined with different levels of audiovisual feedback influences remote pilot performance. These findings support the rationalisation for the provision of multimodal dynamic sensory cueing in future RPAS. Practitioner summary: Accuracy and timeliness of remote pilot manual flying performance was measured under a combination of audiovisual feedback in calm and wind shear conditions. The inclusion of real-time auditory feedback as an additional sensory cue is uncommon; this study demonstrated nuanced examples of improved and degraded manual flying performance. The provision of dynamic sensory cueing made available to remote pilots in future RPAS should be considered.

To Repeat or Not to Repeat: Repetitive Sequences Regulate Genome Stability in Candida albicans.

Genome instability often leads to cell death but can also give rise to innovative genotypic and phenotypic variation through mutation and structural rearrangements. Repetitive sequences and chromatin architecture in particular are critical modulators of recombination and mutability. In Candida albicans, four major classes of repeats exist in the genome: telomeres, subtelomeres, the major repeat sequence (MRS), and the ribosomal DNA (rDNA) locus. Characterization of these loci has revealed how their structure contributes to recombination and either promotes or restricts sequence evolution. The mechanisms of recombination that give rise to genome instability are known for some of these regions, whereas others are generally unexplored. More recent work has revealed additional repetitive elements, including expanded gene families and centromeric repeats that facilitate recombination and genetic innovation. Together, the repeats facilitate C. albicans evolution through construction of novel genotypes that underlie C. albicans adaptive potential and promote persistence across its human host.

Utilization of Point-of-Care Ultrasonography (POCUS) for Small Bowel Obstruction.

Small bowel obstruction is a frequently encountered condition throughout emergency departments and accounts for a large number of surgical hospital admissions. Point-of-care ultrasonography (POCUS) by emergency providers can serve as a valuable tool to assist in prompt and accurate diagnosis, which could potentially reduce the quantity of undesirable effects such as radiation and cost seen with other advanced imaging modalities (e.g., computed tomography).

The Galleria mellonella Waxworm Infection Model for Disseminated Candidiasis.

Candida species are common fungal commensals of humans colonizing the skin, mucosal surfaces, and gastrointestinal tract. Under certain conditions, Candida can overgrow their natural niches resulting in debilitating mucosal infections as well as life-threatening systemic infections, which are a major focus of investigation due to their associated high mortality rates. Animal models of disseminated infection exist for studying disease progression and dissecting the characteristics of Candida pathogenicity. Of these, the Galleria mellonella waxworm infection model provides a cost-effective experimental tool for high-throughput investigations of systemic virulence. Many other bacterial and eukaryotic infectious agents have been effectively studied in G. mellonella to understand pathogenicity, making it a widely accepted model system. Yet, variation in the method used to infect G. mellonella can alter phenotypic outcomes and complicate interpretation of the results. Here, we outline the benefits and drawbacks of the waxworm model to study systemic Candida pathogenesis and detail an approach to improve reproducibility. Our results highlight the range of mortality kinetics in G. mellonella and describe the variables which can modulate these kinetics. Ultimately, this method stands as an ethical, rapid, and cost-effective approach to study virulence in a model of disseminated candidiasis.

Candida albicans Dispersed Cells Are Developmentally Distinct from Biofilm and Planktonic Cells.

Candida albicans surface-attached biofilms such as those formed on intravenous catheters with direct access to the bloodstream often serve as a nidus for continuous release of cells capable of initiating new infectious foci. We previously reported that cells dispersed from a biofilm are yeast cells that originate from the top-most hyphal layers of the biofilm. Compared to their planktonic counterparts, these biofilm dispersal yeast cells displayed enhanced virulence-associated characteristics and drug resistance. However, little is known about their molecular properties. To address that issue, in this study we aimed to define the molecular characteristics of these biofilm dispersal cells. We found that the inducer of dispersal, PES1, genetically interacts with the repressor of filamentation, NRG1, in a manner consistent with the definition of dispersed cells as yeast cells. Further, using a flow biofilm model, we performed comprehensive comparative RNA sequencing on freshly dispersed cells in order to identify unique transcriptomic characteristics. Gene expression analysis demonstrated that dispersed cells largely inherit a biofilm-like mRNA profile. Strikingly, however, dispersed cells seemed transcriptionally reprogrammed to acquire nutrients such as zinc and amino acids and to metabolize alternative carbon sources, while their biofilm-associated parent cells did not induce the same high-affinity transporters or express gluconeogenetic genes, despite exposure to the same nutritional signals. Collectively, the findings from this study characterize cell dispersal as an intrinsic step of biofilm development which generates propagules more adept at colonizing distant host sites. This developmental step anticipates the need for virulence-associated gene expression before the cells experience the associated external signals.IMPORTANCECandida albicans surface-attached biofilms serve as a reservoir of cells to perpetuate and expand an infection; cells released from biofilms on catheters have direct access to the bloodstream. Biofilm dispersal yeast cells exhibit enhanced adhesion, invasion, and biofilm formation compared to their planktonic counterparts. Here, we show using transcriptome sequencing (RNA-seq) that dispersed yeast cells are developmentally distinct from the cells in their parent biofilms as well as from planktonic yeast cells. Dispersal cells possess an anticipatory expression pattern that primes them to infect new sites in the host, to survive in nutrient-starved niches, and to invade new sites. These studies identified dispersal cells as a unique proliferative cell type of the biofilm and showed that they could serve as targets for antibiofilm drug development in the future.

Functional diversification accompanies gene family expansion of MED2 homologs in Candida albicans.

Gene duplication facilitates functional diversification and provides greater phenotypic flexibility to an organism. Expanded gene families arise through repeated gene duplication but the extent of functional divergence that accompanies each paralogous gene is generally unexplored because of the difficulty in isolating the effects of single family members. The telomere-associated (TLO) gene family is a remarkable example of gene family expansion, with 14 members in the more pathogenic Candida albicans relative to two TLO genes in the closely-related species C. dubliniensis. TLO genes encode interchangeable Med2 subunits of the major transcriptional regulatory complex Mediator. To identify biological functions associated with each C. albicans TLO, expression of individual family members was regulated using a Tet-ON system and the strains were assessed across a range of phenotypes involved in growth and virulence traits. All TLOs affected multiple phenotypes and a single phenotype was often affected by multiple TLOs, including simple phenotypes such as cell aggregation and complex phenotypes such as virulence in a Galleria mellonella model of infection. No phenotype was regulated by all TLOs, suggesting neofunctionalization or subfunctionalization of ancestral properties among different family members. Importantly, regulation of three phenotypes could be mapped to individual polymorphic sites among the TLO genes, including an indel correlated with two phenotypes, growth in sucrose and macrophage killing. Different selective pressures have operated on the TLO sequence, with the 5' conserved Med2 domain experiencing purifying selection and the gene/clade-specific 3' end undergoing extensive positive selection that may contribute to the impact of individual TLOs on phenotypic variability. Therefore, expansion of the TLO gene family has conferred unique regulatory properties to each paralog such that it influences a range of phenotypes. We posit that the genetic diversity associated with this expansion contributed to C. albicans success as a commensal and opportunistic pathogen.