HiLIFE – Elämäntieteiden Instituutti

MOTIVATION: Protein structure comparison plays a fundamental role in understanding the evolutionary relationships between proteins. Here, we release a new version of the DaliLite standalone software. The novelties are hierarchical search of the structure database organized into sequence based clusters, and remote access to our knowledge base of structural neighbors. The detection of fold, superfamily and family level similarities by DaliLite and state-of-the-art competitors was benchmarked against a manually curated structural classification. RESULTS: Database search strategies were evaluated using Fmax with query-specific thresholds. DaliLite and DeepAlign outperformed TM-score based methods at all levels of the benchmark, and DaliLite outperformed DeepAlign at fold level. Hierarchical and knowledge-based searches got close to the performance of systematic pairwise comparison. The knowledge-based search was four times as efficient as the hierarchical search. The knowledge-based search dynamically adjusts the depth of the search, enabling a trade-off between speed and recall. AVAILABILITY AND IMPLEMENTATION: http://ekhidna2.biocenter.helsinki.fi/dali/README.v5.html. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

We review the theory and algorithms of electrophysiological brain connectivity analysis. This tutorial is aimed at providing an introduction to brain functional connectivity from electrophysiological signals, including electroencephalography (EEG), magnetoencephalography (MEG), electrocorticography (ECoG), stereoelectroencephalography (SEEG). Various connectivity estimators are discussed, and algorithms introduced. Important issues for estimating and mapping brain functional connectivity with electrophysiology are discussed.

The locus coeruleus (LC) is a seemingly singular and compact neuromodulatory nucleus that is a prominent component of disparate theories of brain function due to its broad noradrenergic projections throughout the CNS. As a diffuse neuromodulatory system, noradrenaline affects learning and decision making, control of sleep and wakefulness, sensory salience including pain, and the physiology of correlated forebrain activity (ensembles and networks) and brain hemodynamic responses. However, our understanding of the LC is undergoing a dramatic shift due to the application of state-of-the-art methods that reveal a nucleus of many modules that provide targeted neuromodulation. Here, we review the evidence supporting a modular LC based on multiple levels of observation (developmental, genetic, molecular, anatomical, and neurophysiological). We suggest that the concept of the LC as a singular nucleus and, alongside it, the role of the LC in diverse theories of brain function must be reconsidered.

Gestational diabetes mellitus (GDM) is associated with increased risk of pregnancy complications and adverse perinatal outcomes. GDM often reoccurs and is associated with increased risk of subsequent diagnosis of type 2 diabetes (T2D). To improve our understanding of the aetiological factors and molecular processes driving the occurrence of GDM, including the extent to which these overlap with T2D pathophysiology, the GENetics of Diabetes In Pregnancy Consortium assembled genome-wide association studies of diverse ancestry in a total of 5485 women with GDM and 347 856 without GDM. Through multi-ancestry meta-analysis, we identified five loci with genome-wide significant association (P < 5 × 10-8) with GDM, mapping to/near MTNR1B (P = 4.3 × 10-54), TCF7L2 (P = 4.0 × 10-16), CDKAL1 (P = 1.6 × 10-14), CDKN2A-CDKN2B (P = 4.1 × 10-9) and HKDC1 (P = 2.9 × 10-8). Multiple lines of evidence pointed to the shared pathophysiology of GDM and T2D: (i) four of the five GDM loci (not HKDC1) have been previously reported at genome-wide significance for T2D; (ii) significant enrichment for associations with GDM at previously reported T2D loci; (iii) strong genetic correlation between GDM and T2D and (iv) enrichment of GDM associations mapping to genomic annotations in diabetes-relevant tissues and transcription factor binding sites. Mendelian randomization analyses demonstrated significant causal association (5% false discovery rate) of higher body mass index on increased GDM risk. Our results provide support for the hypothesis that GDM and T2D are part of the same underlying pathology but that, as exemplified by the HKDC1 locus, there are genetic determinants of GDM that are specific to glucose regulation in pregnancy.

Background The molecular mechanisms mediating postnatal loss of cardiac regeneration in mammals are not fully understood. We aimed to provide an integrated resource of mRNA , protein, and metabolite changes in the neonatal heart for identification of metabolism-related mechanisms associated with cardiac regeneration. Methods and Results Mouse ventricular tissue samples taken on postnatal day 1 (P01), P04, P09, and P23 were analyzed with RNA sequencing and global proteomics and metabolomics. Gene ontology analysis, KEGG pathway analysis, and fuzzy c-means clustering were used to identify up- or downregulated biological processes and metabolic pathways on all 3 levels, and Ingenuity pathway analysis (Qiagen) was used to identify upstream regulators. Differential expression was observed for 8547 mRNA s and for 1199 of 2285 quantified proteins. Furthermore, 151 metabolites with significant changes were identified. Differentially regulated metabolic pathways include branched chain amino acid degradation (upregulated at P23), fatty acid metabolism (upregulated at P04 and P09; downregulated at P23) as well as the HMGCS ( HMG -CoA [hydroxymethylglutaryl-coenzyme A] synthase)-mediated mevalonate pathway and ketogenesis (transiently activated). Pharmacological inhibition of HMGCS in primary neonatal cardiomyocytes reduced the percentage of BrdU-positive cardiomyocytes, providing evidence that the mevalonate and ketogenesis routes may participate in regulating the cardiomyocyte cell cycle. Conclusions This study is the first systems-level resource combining data from genomewide transcriptomics with global quantitative proteomics and untargeted metabolomics analyses in the mouse heart throughout the early postnatal period. These integrated data of molecular changes associated with the loss of cardiac regeneration may open up new possibilities for the development of regenerative therapies.

Abstract Biochar has been widely recognized for its potential to increase carbon (C) sequestration and mitigate climate change. This potential is affected by how biochar interacts with native soil organic carbon (SOC) and fresh organic substrates added to soil. However, only a few studies have been conducted to understand this interaction. To fill this knowledge gap, we conducted a 13 C-glucose labelling soil incubation for 6 months using fine-textured agricultural soil (Stagnosol) with two different biochar amounts. Biochar addition reduced the mineralization of SOC and 13 C-glucose and increased soil microbial biomass carbon (MBC) and microbial carbon use efficiency (CUE). The effects were found to be additive i.e., higher biochar application rate resulted in lower mineralization of SOC and 13 C-glucose. Additionally, soil density fractionation after 6 months revealed that most of the added biochar particles were recovered in free particulate organic matter (POM) fraction. Biochar also increased the retention of 13 C in free POM fraction, indicating that added 13 C-glucose was preserved within the biochar particles. The measurement of 13 C from the total amino sugar fraction extracted from the biochar particles suggested that biochar increased the microbial uptake of added 13 C-glucose and after they died, the dead microbial residues (necromass) accumulated inside biochar pores. Biochar also increased the proportion of occluded POM, demonstrating that increased soil occlusion following biochar addition reduced SOC mineralization. Overall, the study demonstrates the additional C sequestering potential of biochar by inducing negative priming of native SOC as well as increasing CUE, resulting in the formation and stabilization of microbial necromass. Graphical Abstract

Soil microbial carbon-use efficiency (CUE), described as the ratio of growth over total carbon (C) uptake, i.e. the sum of growth and respiration, is a key variable in all soil organic matter (SOM) models and critical to ecosystem C cycling. However, there is still a lack of consensus on microbial CUE when estimated using different methods. Furthermore, the significance of many fundamental drivers of CUE remains largely unknown and inconclusive, especially for tropical ecosystems. For these reasons, we determined CUE and microbial indicators of soil nutrient availability in seven tropical forest soils along an altitudinal gradient (circa 900–2200 m a.s.l) occurring at Taita Hills, Kenya. We used this gradient to study the soil nutrient (N and P) availability and its relation to microbial CUE estimates. For assessing the soil nutrient availability, we determined both the soil bulk stoichiometric nutrient ratios (soil C:N, C:P and N:P), as well as SOM degradation related enzyme activities. We estimated soil microbial CUE using two methods: substrate independent 18O-water tracing and 13C-glucose tracing method. Based on these two approaches, we estimated the microbial uptake efficiency of added glucose versus native SOM, with the latter defined by 18O-water tracing method. Based on the bulk soil C:N stoichiometry, the studied soils did not reveal N limitation. However, soil bulk P limitation increased slightly with elevation. Additionally, based on extracellular enzyme activities, the SOM nutrient availability decreased with elevation. The 13C-CUE did not change with altitude indicating that glucose was efficiently taken up and used by the microbes. On the other hand, 18O-CUE, which reflects the growth efficiency of microbes growing on native SOM, clearly declined with increasing altitude and was associated with SOM nutrient availability indicators. Based on our results, microbes at higher elevations invested more energy to scavenge for nutrients and energy from complex SOM whereas at lower elevations the soil nutrients may have been more readily available.

Abstract Background Cognitive dysfunction is common in haemodialysis patients but whether poor kidney function in the general population is also associated with higher risk of dementia remains unclear. Objective To examine the association of kidney function with incident dementia in community dwelling older adults. Design Whitehall II prospective study. Setting Population-based study on 6,050 adults, mean age 65.8 in 2007–2009. Methods Poor kidney function, defined as estimated Glomerular Filtration Rate (eGFR) &amp;lt;60 ml/min/1.73 m2 in 2007–2009, and adverse change in eGFR was defined as decrease ≥4 ml/min/1.73 m2 between 2007–2009 and 2012–2013. Incident dementia was ascertained through linkage to electronic health records, and Cox regression was used to examine associations with dementia. Results A total of 306 cases of dementia were recorded over a mean follow-up of 10 years. Baseline eGFR &amp;lt;60 was associated with a hazard ratio (HR) for dementia of 1.37 (95% CI 1.02, 1.85) in analysis adjusted for sociodemographic factors, hypertension, obesity, stroke, diabetes and cardiovascular disease/medication. Removing stroke cases at baseline and censoring them over the follow-up yielded an HR of 1.42 (95% CI 1.00, 2.00) for the association between CKD and dementia. Decline of eGFR ≥4 between 2007–2009 and 2012–2013 was associated with incidence of dementia over a 6.3 year mean follow-up (HR: 1.37; 95% CI 1.02, 1.85), with somewhat stronger associations when analyses were restricted to those with eGFR ≥60 in 2007–2009 (1.56; 95% CI: 1.12, 2.19). Conclusion Poor and declining kidney function in older adults is associated with a higher risk of dementia that is not attributable to stroke and persists after accounting for major cardiometabolic conditions.

Natural biopolymer nanoparticles (NPs), including nanocrystalline cellulose (CNC) and lignin, have shown potential as scaffolds for targeted drug delivery systems due to their wide availability, cost-efficient preparation, and anticipated biocompatibility. As both CNC and lignin can potentially cause complications in cell viability assays because of their ability to scatter the emitted light and absorb the assay reagents, we investigated the response of bioluminescent (CellTiter-Glo®), colorimetric (MTT® and AlamarBlue®), and fluorometric (LIVE/DEAD®) assays for the determination of the biocompatibility of the multimodal CNC and lignin constructs in murine RAW 264.7 macrophages and 4T1 breast adenocarcinoma cell lines. Here, we have developed multimodal CNC and lignin NPs harboring the radiometal chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid and the fluorescent dye cyanine 5 for the investigation of nanomaterial biodistribution in vivo with nuclear and optical imaging, which were then used as the model CNC and lignin nanosystems in the cell viability assay comparison. CellTiter-Glo® based on the detection of ATP-dependent luminescence in viable cells revealed to be the best assay for both nanoconstructs for its robust linear response to increasing NP concentration and lack of interference from either of the NP types. Both multimodal CNC and lignin NPs displayed low cytotoxicity and favorable interactions with the cell lines, suggesting that they are good candidates for nanosystem development for targeted drug delivery in breast cancer and for theranostic applications. Our results provide useful guidance for cell viability assay compatibility for CNC and lignin NPs and facilitate the future translation of the materials for in vivo applications.

Abstract Metastatic melanoma can be difficult to detect until at the advanced state that decreases the survival rate of patients. Several FDA‐approved BRAF inhibitors have been used for treatment of metastatic melanoma, but overall therapeutic efficacy has been limited. Lutetium‐177 ( 177 Lu) enables simultaneous tracking of tracer accumulation with single‐photon emission computed tomography and radiotherapy. Therefore, the codelivery of 177 Lu alongside chemotherapeutic agents using nanoparticles (NPs) might improve the therapeutic outcome in metastatic melanoma. Cellulose nanocrystals (CNC NPs) can particularly deliver payloads to lung capillaries in vivo. Herein, 177 Lu‐labeled CNC NPs loaded with vemurafenib ([ 177 Lu]Lu‐CNC‐V NPs) is developed and the therapeutic effect in BRAF V600E mutation‐harboring YUMM1.G1 murine model of lung metastatic melanoma is investigated. The [ 177 Lu]Lu‐CNC‐V NPs demonstrate favorable radiolabel stability, drug release profile, cellular uptake, and cell growth inhibition in vitro. In vivo biodistribution reveals significant retention of the [ 177 Lu]Lu‐CNC‐V NPs in the lung, liver, and spleen. Ultimately, the median survival time of animals is doubly increased after treatment with [ 177 Lu]Lu‐CNC‐V NPs compared to control groups. The enhanced therapeutic efficacy of [ 177 Lu]Lu‐CNC‐V NPs in the lung metastatic melanoma animal model provides convincing evidence for the potential of clinical translation for theranostic CNC NP‐based drug delivery systems after intravenous administration.

Cellulose nanocrystals (CNCs) have remarkable potential to improve the delivery of diagnostic and therapeutic agents to tumors; however, the in vivo studies on CNC biodistribution are still limited. We developed CNC-based imaging probes for the in vitro and in vivo evaluation using two labeling strategies: site-specific hydrazone linkage to the terminal aldehyde of the CNC and nonsite-specific activation using 1,1′-carbonyldiimidazole (CDI). The in vivo behavior of unmodified CNC, DOTA-CNC (ald.), and DOTA-CNC (OH) was investigated in healthy and 4T1 breast cancer mouse models. They displayed good biocompatibility in cell models. Moreover, the biodistribution profile and SPECT/CT imaging confirmed that the accumulation of 111In-labeled DOTA-CNC (ald.) and 111In-DOTA-CNC (OH) was primarily in hepatic, splenic, and pulmonary ducts in accordance with the clearance of nontargeted nanoparticles. The developed CNC imaging probes can be used to obtain information with noninvasive imaging on the behavior in vivo to guide structural optimization for targeted delivery.

Relapse and refractory T-cell acute lymphoblastic leukemia (T-ALL) has a poor prognosis, and new combination therapies are sorely needed. Here, we used an ex vivo high-throughput screening platform to identify drug combinations that kill zebrafish T-ALL and then validated top drug combinations for preclinical efficacy in human disease. This work uncovered potent drug synergies between AKT/mTORC1 (mammalian target of rapamycin complex 1) inhibitors and the general tyrosine kinase inhibitor dasatinib. Importantly, these same drug combinations effectively killed a subset of relapse and dexamethasone-resistant zebrafish T-ALL. Clinical trials are currently underway using the combination of mTORC1 inhibitor temsirolimus and dasatinib in other pediatric cancer indications, leading us to prioritize this therapy for preclinical testing. This combination effectively curbed T-ALL growth in human cell lines and primary human T-ALL and was well tolerated and effective in suppressing leukemia growth in patient-derived xenografts (PDX) grown in mice. Mechanistically, dasatinib inhibited phosphorylation and activation of the lymphocyte-specific protein tyrosine kinase (LCK) to blunt the T-cell receptor (TCR) signaling pathway, and when complexed with mTORC1 inhibition, induced potent T-ALL cell killing through reducing MCL-1 protein expression. In total, our work uncovered unexpected roles for the LCK kinase and its regulation of downstream TCR signaling in suppressing apoptosis and driving continued leukemia growth. Analysis of a wide array of primary human T-ALLs and PDXs grown in mice suggest that combination of temsirolimus and dasatinib treatment will be efficacious for a large fraction of human T-ALLs.

Collaboration and diversity are increasingly promoted in science. Yet how collaborations influence academic career progression, and whether this differs by gender, remains largely unknown. Here, we use co-authorship ego networks to quantify collaboration behaviour and career progression of a cohort of contributors to biennial International Society of Behavioral Ecology meetings (1992, 1994, 1996). Among this cohort, women were slower and less likely to become a principal investigator (PI; approximated by having at least three last-author publications) and published fewer papers over fewer years (i.e. had shorter academic careers) than men. After adjusting for publication number, women also had fewer collaborators (lower adjusted network size) and published fewer times with each co-author (lower adjusted tie strength), albeit more often with the same group of collaborators (higher adjusted clustering coefficient). Authors with stronger networks were more likely to become a PI, and those with less clustered networks did so more quickly. Women, however, showed a stronger positive relationship with adjusted network size (increased career length) and adjusted tie strength (increased likelihood to become a PI). Finally, early-career network characteristics correlated with career length. Our results suggest that large and varied collaboration networks are positively correlated with career progression, especially for women.

Abstract Mechanisms controlling ureter lenght and the position of the kidney are poorly understood. Glial cell-line derived neurotrophic factor (GDNF) induced RET signaling is critical for ureteric bud outgrowth, but the function of endogenous GDNF in further renal differentiation and urogenital system development remains discursive. Here we analyzed mice where 3′ untranslated region (UTR) of GDNF is replaced with sequence less responsive to microRNA-mediated regulation, leading to increased GDNF expression specifically in cells naturally transcribing Gdnf . We demonstrate that increased Gdnf leads to short ureters in kidneys located in an abnormally caudal position thus resembling human pelvic kidneys. High GDNF levels expand collecting ductal progenitors at the expense of ureteric trunk elongation and result in expanded tip and short trunk phenotype due to changes in cell cycle length and progenitor motility. MEK-inhibition rescues these defects suggesting that MAPK-activity mediates GDNF’s effects on progenitors. Moreover, Gdnf hyper mice are infertile likely due to effects of excess GDNF on distal ureter remodeling. Our findings suggest that dysregulation of GDNF levels, for example via alterations in 3′UTR, may account for a subset of congenital anomalies of the kidney and urinary tract (CAKUT) and/or congenital infertility cases in humans and pave way to future studies.

Abstract Age at maturity is a key life history trait involving a trade-off between survival risk and reproductive investment, and is an important factor for population structures. In ectotherms, a warming environment may have a dramatic influence on development and life history, but this influence may differ between populations. While an increasing number of studies have examined population-dependent reactions with temperature, few have investigated this in the context of maturation timing. Atlantic salmon, a species of high conservation relevance, is a good study species for this topic as it displays considerable variation in age at maturity, of which a large proportion has been associated with a genomic region including the strong candidate gene vgll3. Until now, the effect of this gene in the context of different environments and populations has not been studied. Using a large-scale common-garden experiment, we find strong effects of temperature, population-of-origin, and vgll3 genotype on maturation in 2-year-old male Atlantic salmon (Salmo salar). With a temperature difference of 1.8°C, maturation probability was 4.8 times higher in the warm treatment than the cold treatment. This temperature effect was population-specific and was higher in the southern (60.48°N) compared to the northern (65.01°N) population. The early maturation vgll3*E allele was associated with a significantly higher maturation probability, but there was no vgll3 interaction with temperature or population. Both body condition and body mass associated with maturation. The body mass association was only present in the warm treatment. Our findings demonstrate that (i) populations can vary in their response to temperature change in terms of age at maturity, (ii) high intrinsic growth could be associated with higher thermal sensitivity for life history variation and (iii) vgll3 effects on age at maturity might be similar between populations and different thermal environments.

We tested the possibility that vgll3, a gene linked with maturation age in Atlantic salmon (Salmo salar), may be associated with behaviour by measuring aggressiveness and feeding activity in 380 juveniles with different vgll3 genotypes. Contrary to our prediction, individuals with the genotype associated with later maturation (vgll3*LL) were significantly more aggressive than individuals with the genotype associated with earlier maturation (vgll3*EE). Individuals with higher aggression were also significantly lighter in colour and had higher feeding activity. Although higher aggression was associated with higher feeding activity, there was no association between feeding activity and vgll3 genotype. Increased aggression of vgll3*LL individuals was independent of their sex and size, and genotypes did not differ in their condition factor. These results imply that aggressive behaviour may have an energetic cost impairing growth and condition, especially when food cannot be monopolized. This may have implications for individual fitness and aquaculture practices.

Multiple protocols have been published for generation of iMGLs from hESCs/iPSCs. To date, there are no guides to assist researchers to determine the most appropriate methodology for microglial studies. To establish a framework to facilitate future microglial studies, we first performed a comparative transcriptional analysis between iMGLs derived using three published datasets, which allowed us to establish the baseline protocol that is most representative of bona fide human microglia. Secondly, using CRISPR to tag the classic microglial marker CX3CR1 with nanoluciferase and tdTomato, we generated and functionally validated a reporter ESC line. Finally, using this cell line, we demonstrated that co-culture of iMGL precursors with human glia and neurons enhanced transcriptional resemblance of iMGLs to ex vivo microglia. Together, our comprehensive molecular analysis and reporter cell line are a useful resource for neurobiologists seeking to use iMGLs for disease modeling and drug screening studies.

Abstract Type-A γ-aminobutyric acid (GABA A ) receptors are pentameric ligand-gated ion channels (pLGICs), typically consisting of α/β/γ subunit combinations. They are the principal mediators of inhibitory neurotransmission throughout the central nervous system and targets of major clinical drugs, such as benzodiazepines (BZDs) used to treat epilepsy, insomnia, anxiety, panic disorder and muscle spasm. However, the structures of heteromeric receptors and the molecular basis of BZD operation remain unknown. Here we report the cryo-EM structure of a human α1β3γ2 GABA A R in complex with GABA and a nanobody that acts as a novel positive allosteric modulator (PAM). The receptor subunits assume a unified quaternary activated conformation around an open pore. We also present crystal structures of engineered α5 and α5γ2 GABA A R constructs, revealing the interfacial site for allosteric modulation by BZDs, including the binding modes and the conformational impact of the potent anxiolytic and partial PAM, bretazenil, and the BZD antagonist, flumazenil. These findings provide the foundation for understanding the mechanistic basis of GABA A R activation.

Mutations in Leucine-rich repeat kinase 2 (LRRK2) are responsible for late-onset autosomal dominant Parkinson's disease. LRRK2 has been implicated in a wide range of physiological processes including membrane repair in the endolysosomal system. Here, using cell-free systems, we report that purified LRRK2 directly binds acidic lipid bilayers with a preference for highly curved bilayers. While this binding is nucleotide independent, LRRK2 can also deform low-curvature liposomes into narrow tubules in a guanylnucleotide-dependent but Adenosine 5'-triphosphate-independent way. Moreover, assembly of LRRK2 into scaffolds at the surface of lipid tubules can constrict them. We suggest that an interplay between the membrane remodeling and signaling properties of LRRK2 may be key to its physiological function. LRRK2, via its kinase activity, may achieve its signaling role at sites where membrane remodeling occurs.

The number of deaths resulting from aneurysmal subarachnoid haemorrhage (aSAH) is commonly considered negligible in comparison with other causes of death. In fact, it has been estimated that subarachnoid haemorrhage accounts for only 4% of stroke deaths.1 However, addressing the aSAH death toll is challenging since many aSAH deaths happen outside of hospitals and are commonly classified as cardiac deaths when autopsies are not done.2 3 Middle-aged people are affected most frequently by aSAH. Since cancer-, traffic-, alcohol- and cardiovascular-related deaths are decreasing in middle-aged people in high-income countries,4 we hypothesise that the proportion of aSAH deaths may be increasing in this age group. Therefore, we aimed to determine the proportion of aSAH deaths relative to other causes of death in middle-aged people. ### Ethical considerations Statistics Finland and the National Institute of Health and Welfare approved the data extractions and analysis from the nationwide Cause of Death (CDR) and Hospital Discharge Registers (HDR). According to Finnish legislation, no separate informed consent was required, as we did not access personally identifiable information. ### Study period Since 1936, all deaths in Finland have been archived to the nationwide CDR governed by Statistics Finland. The causes of death have been classified using the 10th version of the International Statistical Classification of Diseases and Related Health Problems (ICD-10) coding from 1996 onwards. To avoid possible misclassification immediately after the transition to ICD-10, we included death diagnoses between 1998 and 2017. ### Causes of death We extracted aSAH deaths registered to the CDR with the four-character ICD-10 classification codes (I60.0–I60.6). We used the nationwide …