Anders Olsen

The increasing world population necessitates new sustainable nutrient sources, making microalgae like Chlorella sorokiniana interesting due to its rich nutrient profile and sustainable cultivation methods. With genetic optimization tools like CRISPR/Cas9, microalgae as a nutrient source can be improved even further. However, degradation of the rigid cell wall of microalgae, and thereby developing protoplasts, is often necessary prior to transformation, but monitoring protoplast development in spherical, single-celled organisms like C. sorokiniana is challenging using bright-field microscopy. Carbotrace 480 and 630 were tested as fluorescent markers of the cell wall of a C. sorokiniana mutant for protoplast detection, and Carbotrace 480 was successfully used to distinguish protoplast from normal cells in a cell suspension. The enzymes Driselase, Glucanex, Snailase, and Saczyme were tested in different combinations to degrade the cell wall of the mutant, with Snailase as the most effective yielding ∼60 % protoplasts. This study provides a quick and easy tool for monitoring protoplast development in the microalgae C. sorokiniana , the first step to improve C. sorokiniana as a sustainable nutrient source using genetic optimization tools like CRISPR/Cas9.

Calmodulin is an intracellular Ca2+ sensor that regulates numerous cellular processes through binding effector proteins and changing their activity. Humans have three calmodulin paralogs, CALM1, CALM2 and CALM3, encoding identical proteins, and missense variants in all three are included in the ACMG recommendations for reporting of secondary findings related to early onset sudden cardiac death caused by arrhythmias long QT syndrome and CPVT. Recently, a subset of individuals with de novo variants in calmodulin genes were described who also presented with neurologic phenotypes. Here we report two individuals with the same de novo variant c.419A > T in CALM1 or CALM2 that may generate a 5′ splice donor gain and/or a missense change p.E140V. These individuals share hypotonia, motor delay, intellectual disability, and abnormal electroencephalograms but lack cardiac arrhythmia/electrocardiogram abnormalities of previously described CALM-associated disease, suggesting the variant causes phenotypic expansion beyond the known Mendelian phenotypes. RNA-seq of the CALM1 proband blood’s sample revealed that most transcripts from the variant allele showed usage of the new splice site or intron retention, without NMD, resulting in frameshifted C-terminal truncations, while a minority resulted in production of the p.E140V missense protein. We modeled the CALM1/2 p.E140V variant as well as a known arrhythmia variant, CALM1 p.E141G, using the C. elegans ortholog cmd-1. We found that cmd-1 E140V displayed both qualitative and quantitative differences in phenotype from E141G, indicating distinct genetic mechanisms. Together, these findings support CALM1/2 phenotypic expansion, with c.419A > T p.E140V resulting in neurologic, but not arrhythmia phenotypes.

Calmodulin is a vital cellular calcium sensor expressed by the genes CALM1-3 . Ultra-rare loss-of-function missense variants in the C-terminal lobe (C-lobe) of calmodulin are linked to cardiac arrhythmia, primarily long QT syndrome. Emerging evidence suggests calmodulin variants also contribute to neurological disorders. This study explores the allelic spectrum and functional consequences of calmodulin variants in schizophrenia. In a large-scale sequencing effort involving 24,248 schizophrenia patients and 97,322 control individuals, 25 unique calmodulin variants were found in 27 carriers, all ultra-rare. Seven carriers were schizophrenia patients, and 20 were controls. All patient variants affected the C-lobe, while only five of 20 control variants were in the C-lobe, linking C-lobe variants to increased schizophrenia risk. Functional analyses identified two variant classes in patients: Loss-of-function variants, reducing calcium affinity and impairing Ca _V 1.2 interaction, and gain-of-function variants, enhancing calcium affinity without affecting Ca _V 1.2 gating. Together, this study suggests that consequences of calmodulin variants include increased schizophrenia risk.

CED-1 (cell death abnormal) is a transmembrane receptor involved in the recognition of “eat-me” signals displayed on the surface of apoptotic cells and thus central for the subsequent engulfment of the cell corpse in Caenorhabditis elegans. The roles of CED-1 in engulfment are well established, as are its downstream effectors. The latter include the adapter protein CED-6/GULP and the ATP-binding cassette family homolog CED-7. However, how CED-1 is maintained on the plasma membrane in the absence of engulfment is currently unknown. Here, we show that CED-6 and CED-7 have a novel role in maintaining CED-1 correctly on the plasma membrane. We propose that the underlying mechanism is via endocytosis as CED-6 and CED-7 act redundantly with clathrin and its adaptor, the Adaptor protein 2 complex, in ensuring correct CED-1 localization. In conclusion, CED-6 and CED-7 impact other cellular processes than engulfment of apoptotic cells.

The role of the microbiota in health and disease is a research area receiving much attention in academia and industry. A person’s microbiota refers to a community of microorganisms found mainly in the gut. It is estimated that around 39 trillion bacteria can be found on and inside the human body and there is increasing evidence that they influence human health. Advances in sequencing techniques are revolutionizing characterization of the human microbiome. However, causality and underlying molecular mechanisms are still largely unknown due to the complexity of the human microbiome and its interaction with the host. Turning towards simpler host organisms and using well-defined microbiomes are two ways to strengthen studies of causality and mechanism. Here, we show that the nematode Caenorhabditis elegans can be used as host to study sub-microbiomes derived from human feces samples prepared for fecal microbiota transplantation following a simple feeding protocol. Approximately 200 amplicon sequence variants were identified in the worm gut following transplantation with human fecal microbiota samples. We find that the gut microbiome does not simply reflect the bacterial community initially fed to the worms. Hence, our experimental setup can be used to identify and characterize host genetic factors shaping the microbiota and improving our understanding of host–human microbiome interactions.

In humans, mutations in calmodulin cause cardiac arrhythmia. These mutations disrupt the ability of calmodulin to sense calcium concentrations and correctly regulate two central calcium channels, together obstructing heart rhythm. This correlation is well established, but also surprising since calmodulin is expressed in all tissues and interacts with hundreds of proteins. Until now, most studies have focused on cardiac cell function and regulation of specific cardiac targets, and thus, potential other effects of these mutations have largely been unexplored. Here, we introduce the nematode Caenorhabditis elegans as an in vivo model to study effects of three human calmodulin mutations with different impairment on calcium binding. We find that arrhythmic effects of the calmodulin mutations N54I and D96V can be recapitulated in disruption of two rhythmic behaviors, pharynx pumping and defecation motor program. Interestingly, we also find that these mutations affect neuronal function, but in different ways. Whereas D96V sensitizes signaling at the neuromuscular junction, N54I has a protective effect. The mutation N98S did not affect rhythmic behavior, but impaired chemosensing. Therefore, pathogenic calmodulin mutations act through different mechanisms in rhythmic behavior and neuronal function in C. elegans, emphasizing the strength of using live multicellular models. Finally, our results support the hypothesis that human calmodulin mutations could also contribute to neurological diseases.

Probiotic bacteria are increasingly popular as dietary supplements and have the potential as alternatives to traditional antibiotics. We have recently shown that pretreatment with Lactobacillus spp. Lb21 increases the life span of C. elegans and results in resistance toward pathogenic methicillin-resistant Staphylococcus aureus (MRSA). The Lb21-mediated MRSA resistance is dependent on the DBL-1 ligand of the TGF-β signaling pathway. However, the underlying changes at the metabolite level are not understood which limits the application of probiotic bacteria as timely alternatives to traditional antibiotics. In this study, we have performed untargeted nuclear magnetic resonance-based metabolic profiling. We report the metabolomes of Lactobacillus spp. Lb21 and control E. coli OP50 bacteria as well as the nematode-host metabolomes after feeding with these diets. We identify 48 metabolites in the bacteria samples and 51 metabolites in the nematode samples and 63 across all samples. Compared to the control diet, the Lactobacilli pretreatment significantly alters the metabolic profile of the worms. Through sparse Partial Least Squares discriminant analyses, we identify the 20 most important metabolites distinguishing probiotics from the regular OP50 food and worms fed the two different bacterial diets, respectively. Among the changed metabolites, we find lower levels of essential amino acids as well as increased levels of the antioxidants, ascorbate, and glutathione. Since the probiotic diet offers significant protection against MRSA, these metabolites could provide novel ways of combatting MRSA infections.

Worldwide the increase in multi-resistant bacteria due to misuse of traditional antibiotics is a growing threat for our health. Finding alternatives to traditional antibiotics is thus timely. Probiotic bacteria have numerous beneficial effects and could offer safer alternatives to traditional antibiotics. Here, we use the nematodeCaenorhabditis elegans(C. elegans) to screen a library of different lactobacilli to identify potential probiotic bacteria and characterize their mechanisms of action. We show that pretreatment with theLactobacillusspp. Lb21 increases lifespan ofC. elegansand results in resistance towards pathogenic methicillin-resistantStaphylococcus aureus(MRSA).Using genetic analysis, we find that Lb21-mediated MRSA resistance is dependent on the DBL-1 ligand of the TGF-β signaling pathway inC. elegans. This response is evolutionarily conserved as we find that Lb21 also induces the TGF-β pathway in porcine epithelial cells. We further characterize the host responses in an unbiased proteome analysis and identify 474 proteins regulated in worms fed Lb21 compared to control food. These include fatty acid CoA synthetase ACS-22, aspartic protease ASP-6 and vitellogenin VIT-2 which are important for Lb21-mediated MRSA resistance. Thus, Lb21 exerts its probiotic effect onC. elegansin a multifactorial manner. In summary, our study establishes a mechanistic basis for the antimicrobial potential of lactobacilli.