Browsing by Subject "RNA sequencing"

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  • Mehine, Miika; Khamaiseh, Sara; Ahvenainen, Terhi; Heikkinen, Tuomas; Äyräväinen, Anna; Pakarinen, Päivi; Härkki, Päivi; Pasanen, Annukka; Bützow, Ralf; Vahteristo, Pia (2020)
    Simple Summary Uterine leiomyomas are benign smooth muscle tumors affecting millions of women globally. On a molecular level, leiomyomas can be classified into three main subtypes, each characterized by mutations affecting either MED12, HMGA2, or FH. Leiomyomas are still widely regarded as a single entity, although early observations suggest that different subtypes behave differently, in terms of both clinical outcomes and therapeutic requirements. The majority of classification studies on leiomyomas have been performed using fresh frozen tissue. Archival formalin-fixed paraffin-embedded (FFPE) tissue represents an invaluable source of biological material that can be studied retrospectively. Methods capable of generating high-quality data from FFPE material are in high demand. Here, we show that 3 ' RNA sequencing can accurately classify leiomyomas that have been stored as FFPE tissue in hospital archives for years. A targeted 3 ' RNA sequencing panel could provide researchers and clinicians with a cost-effective and scalable diagnostic tool for classifying smooth muscle tumors. Uterine leiomyomas are benign smooth muscle tumors occurring in 70% of women of reproductive age. The majority of leiomyomas harbor one of three well-established genetic changes: a hotspot mutation in MED12, overexpression of HMGA2, or biallelic loss of FH. The majority of studies have classified leiomyomas by complex and costly methods, such as whole-genome sequencing, or by combining multiple traditional methods, such as immunohistochemistry and Sanger sequencing. The type of specimens and the amount of resources available often determine the choice. A more universal, cost-effective, and scalable method for classifying leiomyomas is needed. The aim of this study was to evaluate whether RNA sequencing can accurately classify formalin-fixed paraffin-embedded (FFPE) leiomyomas. We performed 3 ' RNA sequencing with 44 leiomyoma and 5 myometrium FFPE samples, revealing that the samples clustered according to the mutation status of MED12, HMGA2, and FH. Furthermore, we confirmed each subtype in a publicly available fresh frozen dataset. These results indicate that a targeted 3 ' RNA sequencing panel could serve as a cost-effective and robust tool for stratifying both fresh frozen and FFPE leiomyomas. This study also highlights 3 ' RNA sequencing as a promising method for studying the abundance of unexploited tissue material that is routinely stored in hospital archives.
  • Harjuhaahto, Sandra; Rasila, Tiina S.; Molchanova, Svetlana M.; Woldegebriel, Rosa; Kvist, Jouni; Konovalova, Svetlana; Sainio, Markus T.; Pennonen, Jana; Torregrosa-Munumer, Ruben; Ibrahim, Hazem; Otonkoski, Timo; Taira, Tomi; Ylikallio, Emil; Tyynismaa, Henna (2020)
    Mitochondrial intermembrane space proteins CHCHD2 and CHCHD10 have roles in motor neuron diseases such as amyotrophic lateral sclerosis, spinal muscular atrophy and axonal neuropathy and in Parkinson's disease. They form a complex of unknown function. Here we address the importance of these two proteins in human motor neurons. We show that gene edited human induced pluripotent stem cells (iPSC) lacking either CHCHD2 or CHCHD10 are viable and can be differentiated into functional motor neurons that fire spontaneous and evoked action potentials. Mitochondria in knockout iPSC and motor neurons sustain ultrastructure but show increased proton leakage and respiration, and reciprocal compensatory increases in CHCHD2 or CHCHD10. Knockout motor neurons have largely overlapping transcriptome profiles compared to isogenic control line, in particular for synaptic gene expression. Our results show that the absence of either CHCHD2 or CHCHD10 alters mitochondrial respiration in human motor neurons, inducing similar compensatory responses. Thus, pathogenic mechanisms may involve loss of synaptic function resulting from defective energy metabolism.
  • Kentala, Henriikka; Koponen, Annika; Kivelä, Annukka M.; Andrews, Robert; Li, ChunHei; Zhou, You; Olkkonen, Vesa M. (2018)
    ORP2 is implicated in cholesterol transport, triglyceride metabolism, and adrenocortical steroid hormone production. We addressed ORP2 function in hepatocytes by generating ORP2-knockout (KO) HuH7 cells by CRISPR-Cas9 gene editing, followed by analyses of transcriptome, F-actin morphology, migration, adhesion, and proliferation. RNA sequencing of ORP2-KO cells revealed >2-fold changes in 579 mRNAs. The Ingenuity Pathway Analysis (IPA) uncovered alterations in the following functional categories: cellular movement, cell-cell signaling and interaction, cellular development, cellular function and maintenance, cellular growth and proliferation, and cell morphology. Many pathways in these categories involved actin cytoskeleton, cell migration, adhesion, or proliferation. Analysis of the ORP2 interactome uncovered 109 putative new partners. Their IPA analysis revealed Ras homolog A (RhoA) signaling as the most significant pathway. Interactions of ORP2 with SEPT9, MLC12, and ARHGAP12 were validated by independent assays. ORP2-KO resulted in abnormal F-actin morphology characterized by impaired capacity to form lamellipodia, migration defect, and impaired adhesion and proliferation. Rescue of the migration phenotype and generation of typical cell surface morphology required an intact ORP2 phosphoinositide binding site, suggesting that ORP2 function involves phosphoinositide binding and transport. The results point at a novel function of ORP2 as a lipid-sensing regulator of the actin cytoskeleton, with impacts on hepatocellular migration, adhesion, and proliferation.-Kentala, H., Koponen, A., Kivela, A. M., Andrews, R., Li, C., Zhou, Y., Olkkonen, V. M. Analysis of ORP2-knockout hepatocytes uncovers a novel function in actin cytoskeletal regulation.
  • Cui, Fuqiang; Wenwu, Wu; Wang, Kai; Zhang, Yuan; Zhubing, Hu; Brosche, Mikael; Liu, Shenkui; Overmyer, Kirk (2019)
    Prevailing evidence indicates that abscisic acid (ABA) negatively influences immunity to the fungal pathogen Botrytis cinerea in most but not all cases. ABA is required for cuticle biosynthesis, and cuticle permeability enhances immunity to Botrytis via unknown mechanisms. This complex web of responses obscures the role of ABA in Botrytis immunity. Here, we addressed the relationships between ABA sensitivity, cuticle permeability, and Botrytis immunity in the Arabidopsis thaliana ABA-hypersensitive mutants protein phosphatase2c quadruple mutant (pp2c-q) and enhanced response to aba1 (era1-2). Neither pp2c-q nor era1-2 exhibited phenotypes predicted by the known roles of ABA; conversely, era1-2 had a permeable cuticle and was Botrytis resistant. We employed RNA-seq analysis in cuticle-permeable mutants of differing ABA sensitivities and identified a core set of constitutively activated genes involved in Botrytis immunity and susceptibility to biotrophs, independent of ABA signaling. Furthermore, botrytis susceptible1 (bos1), a mutant with deregulated cell death and enhanced ABA sensitivity, suppressed the Botrytis immunity of cuticle permeable mutants, and this effect was linearly correlated with the extent of spread of wound-induced cell death in bos1. Overall, our data demonstrate that Botrytis immunity conferred by cuticle permeability can be genetically uncoupled from PP2C-regulated ABA sensitivity, but requires negative regulation of a parallel ABA-dependent cell-death pathway.
  • Joo, SeoJeong (Helsingin yliopisto, 2019)
    Although lung transplantation has become a routine procedure and is optimal therapy for patients with end-stage pulmonary diseases, the lifespan of lung allografts is still shorter than that of other organ transplants. As acute allograft rejection is one of the main risk factors for the development of chronic lung allograft dysfunction (CLAD) which threatens the long-term survival rate of the recipients, it is crucial to predict and diagnose acute lung allograft rejection. However, there are no specific methods established so far to predict acute rejection (AR). Even though the histopathological evaluation of transbronchial biopsies (TBBs) is used as the gold standard to ensure the diagnosis of AR, it is essential to discover novel biomarkers for AR to overcome the limitations of the TBB-based invasive diagnostics. Recently extracellular vesicles (EVs) got noticed as potential biomarkers in various fields of medicine based on the findings that they exist in high concentration in body fluids and deliver functional genetic molecules which can modulate gene expression in target cells. In that regard, this preliminary study was designed with two different approaches; a time-point analysis and a case analysis of rejection and non-rejection episodes to validate their potentials as diagnostic and predictive biomarkers for acute lung allograft rejection. To discover biomarkers, EV RNA was isolated from the plasma of four patients that was collected at different time points, and whole EV mRNA transcriptome sequencing was performed on the Illumina platform to obtain at least 15 million reads. The time-point analysis showed that the mRNA contents of EVs changed according to the time points and clinical presentations of the patients. At the same time, gene expression profiles showed that mRNA molecules inside the EVs change from innate immunity to adaptive immunity related signatures with the time after transplantation. Furthermore, the case analysis identified that EVs contain RNA molecules that are closely related to the migration of leukocytes and adaptive immune system during acute rejection episodes. In conclusion, the profiles of EV RNA may reflect the immune responses that are taking place in the recipient’s body. Therefore, it is speculated that EVs may play an essential role in the development of AR by transferring functional mRNA molecules to the allograft, immune cells, and endothelial cells. On that account, EV transcriptome profiling could be used as a diagnostic tool for AR in the future, as well as a therapeutic tool by engineering EVs to target specific genes that may be involved in the development of AR. Keywords: extracellular vesicles, lung transplantation, transplantation immunology, RNA sequencing, acute lung allograft rejection, biomarkers
  • Teikari, Jonna E.; Hou, Shengwei; Wahlsten, Matti; Hess, Wolfgang R.; Sivonen, Kaarina (2018)
    Salinity is an important abiotic factor controlling the distribution and abundance of Nodularia spumigena, the dominating diazotrophic and toxic phototroph, in the brackish water cyanobacterial blooms of the Baltic Sea. To expand the available genomic information for brackish water cyanobacteria, we sequenced the isolate Nodularia spurn/germ UHCC 0039 using an Illumina-SMRT hybrid sequencing approach, revealing a chromosome of 5,294,286 base pairs (bp) and a single plasmid of 92,326 bp. Comparative genomics in Nostocales showed pronounced genetic similarity among Nodularia spumigena strains evidencing their short evolutionary history. The studied Baltic Sea strains share similar sets of CRISPR-Cas cassettes and a higher number of insertion sequence (IS) elements compared to Nodularia spumigena CENA596 isolated from a shrimp production pond in Brazil. Nodularia spumigena UHCC 0039 proliferated similarly at three tested salinities, whereas the lack of salt inhibited its growth and triggered transcriptome remodeling, including the up-regulation of five sigma factors and the down-regulation of two other sigma factors, one of which is specific for strain UHCC 0039. Down-regulated genes additionally included a large genetic region for the synthesis of two yet unidentified natural products. Our results indicate a remarkable plasticity of the Nodularia salinity acclimation, and thus salinity strongly impacts the intensity and distribution of cyanobacterial blooms in the Baltic Sea.
  • Kumar, Ashwini; Adhikari, Sadiksha; Kankainen, Matti; Heckman, Caroline A. (2021)
    Simple Summary The wide variety of next-generation sequencing technologies requires thorough evaluation and understanding of their advantages and shortcomings of these different approaches prior to their implementation in a precision medicine setting. Here, we compared the performance of two DNA sequencing methods, whole-exome and linked-read exome sequencing, to detect large structural variants (SVs) and short variants in eight multiple myeloma (MM) patient cases. For three patient cases, matched tumor-normal samples were sequenced with both methods to compare somatic SVs and short variants. The methods' clinical relevance was also evaluated, and their sensitivity and specificity to detect MM-specific cytogenetic alterations and other short variants were measured. Thus, this study systematically demonstrates and evaluates the performance of whole-exome and linked-read exome sequencing technologies for detecting genetic alterations to aid in selecting the optimal method for clinical application. Linked-read sequencing was developed to aid the detection of large structural variants (SVs) from short-read sequencing efforts. We performed a systematic evaluation to determine if linked-read exome sequencing provides more comprehensive and clinically relevant information than whole-exome sequencing (WES) when applied to the same set of multiple myeloma patient samples. We report that linked-read sequencing detected a higher number of SVs (n = 18,455) than WES (n = 4065). However, linked-read predictions were dominated by inversions (92.4%), leading to poor detection of other types of SVs. In contrast, WES detected 56.3% deletions, 32.6% insertions, 6.7% translocations, 3.3% duplications and 1.2% inversions. Surprisingly, the quantitative performance assessment suggested a higher performance for WES (AUC = 0.791) compared to linked-read sequencing (AUC = 0.766) for detecting clinically validated cytogenetic alterations. We also found that linked-read sequencing detected more short variants (n = 704) compared to WES (n = 109). WES detected somatic mutations in all MM-related genes while linked-read sequencing failed to detect certain mutations. The comparison of somatic mutations detected using linked-read, WES and RNA-seq revealed that WES and RNA-seq detected more mutations than linked-read sequencing. These data indicate that WES outperforms and is more efficient than linked-read sequencing for detecting clinically relevant SVs and MM-specific short variants.
  • Kuosmanen, Anna; Norri, Tuukka; Mäkinen, Veli (2018)
    Transcript prediction can be modeled as a graph problem where exons are modeled as nodes and reads spanning two or more exons are modeled as exon chains. Pacific Biosciences third-generation sequencing technology produces significantly longer reads than earlier second-generation sequencing technologies, which gives valuable information about longer exon chains in a graph. However, with the high error rates of third-generation sequencing, aligning long reads correctly around the splice sites is a challenging task. Incorrect alignments lead to spurious nodes and arcs in the graph, which in turn lead to incorrect transcript predictions. We survey several approaches to find the exon chains corresponding to long reads in a splicing graph, and experimentally study the performance of these methods using simulated data to allow for sensitivity/precision analysis. Our experiments show that short reads from second-generation sequencing can be used to significantly improve exon chain correctness either by error-correcting the long reads before splicing graph creation, or by using them to create a splicing graph on which the long-read alignments are then projected. We also study the memory and time consumption of various modules, and show that accurate exon chains lead to significantly increased transcript prediction accuracy. Availability: The simulated data and in-house scripts used for this article are available at http://www.cs.helsinki.fi/group/gsa/exon-chains/exon-chains-bib.tar.bz2.
  • Helle, Emmi; Ampuja, Minna; Antola, Laura; Kivelä, Riikka (2020)
    The vascular system is essential for the development and function of all organs and tissues in our body. The molecular signature and phenotype of endothelial cells (EC) are greatly affected by blood flow-induced shear stress, which is a vital component of vascular development and homeostasis. Recent advances in differentiation of ECs from human induced pluripotent stem cells (hiPSC) have enabled development of in vitro experimental models of the vasculature containing cells from healthy individuals or from patients harboring genetic variants or diseases of interest. Here we have used hiPSC-derived ECs and bulk- and single-cell RNA sequencing to study the effect of flow on the transcriptomic landscape of hiPSC-ECs and their heterogeneity. We demonstrate that hiPS-ECs are plastic and they adapt to flow by expressing known flow-induced genes. Single-cell RNA sequencing showed that flow induced a more homogenous and homeostatically more stable EC population compared to static cultures, as genes related to cell polarization, barrier formation and glucose and fatty acid transport were induced. The hiPS-ECs increased both arterial and venous markers when exposed to flow. Interestingly, while in general there was a greater increase in the venous markers, one cluster with more arterial-like hiPS-ECs was detected. Single-cell RNA sequencing revealed that not all hiPS-ECs are similar even after sorting, but exposing them to flow increases their homogeneity. Since hiPS-ECs resemble immature ECs and demonstrate high plasticity in response to flow, they provide an excellent model to study vascular development.
  • Andreevskaya, Margarita; Jääskelainen, Elina; Johansson, Per; Ylinen, Anne; Paulin, Lars; Björkroth, Johanna; Auvinen, Petri (2018)
    Psychrotrophic lactic acid bacteria (LAB) are the prevailing spoilage organisms in packaged cold-stored meat products. Species composition and metabolic activities of such LAB spoilage communities are determined by the nature of the meat product, storage conditions, and interspecies interactions. Our knowledge of system level responses of LAB during such interactions is very limited. To expand it, we studied interactions between three common psychrotrophic spoilage LAB (Leuconostoc gelidum, Lactococcus piscium, and Lactobacillus oligofermentans) by comparing their time course transcriptome profiles obtained during their growth in individual, pairwise, and triple cultures. The study revealed how these LAB employed different strategies to cope with the consequences of interspecies competition. The fastest-growing bacterium, Le. gelidum, attempted to enhance its nutrient-scavenging and growth capabilities in the presence of other LAB through upregulation of carbohydrate catabolic pathways, pyruvate fermentation enzymes, and ribosomal proteins, whereas the slower-growing Lc. piscium and Lb. oligofermentans downregulated these functions. These findings may explain the competitive success and predominance of Le. gelidum in a variety of spoiled foods. Peculiarly, interspecies interactions induced overexpression of prophage genes and restriction modification systems (mechanisms of DNA exchange and protection against it) in Lc. piscium and Lb. oligofermentans but not in Le. gelidum. Cocultivation induced also overexpression of the numerous putative adhesins in Lb. oligofermentans. These adhesins might contribute to the survival of this slowly growing bacterium in actively growing meat spoilage communities. IMPORTANCE Despite the apparent relevance of LAB for biotechnology and human health, interactions between members of LAB communities are not well known. Knowledge of such interactions is crucial for understanding how these communities function and, consequently, whether there is any possibility to develop new strategies to interfere with their growth and to postpone spoilage of packaged and refrigerated foods. With the help of controlled experiments, detailed regulation events can be observed. This study gives an insight into the system level interactions and the different competition-induced survival strategies related to enhanced uptake and catabolism of carbon sources, overexpression of adhesins and putative bacteriocins, and the induction of exchange of genetic material. Even though this experiment dealt with only three LAB strains in vitro, these findings agreed well with the relative abundance patterns typically reported for these species in natural food microbial communities.
  • Johansson, Tiira; Yohannes, Dawit A.; Koskela, Satu; Partanen, Jukka; Saavalainen, Paivi (2021)
    The HLA gene complex is the most important single genetic factor in susceptibility to most diseases with autoimmune or autoinflammatory origin and in transplantation matching. Most studies have focused on the vast allelic variation in these genes; only a few studies have explored differences in the expression levels of HLA alleles. In this study, we quantified mRNA expression levels of HLA class I and II genes from peripheral blood samples of 50 healthy individuals. The gene- and allele-specific mRNA expression was assessed using unique molecular identifiers, which enabled PCR bias removal and calculation of the number of original mRNA transcripts. We identified differences in mRNA expression between different HLA genes and alleles. Our results suggest that HLA alleles are differentially expressed and these differences in expression levels are quantifiable using RNA sequencing technology. Our method provides novel insights into HLA research, and it can be applied to quantify expression differences of HLA alleles in various tissues and to evaluate the role of this type of variation in transplantation matching and susceptibility to autoimmune diseases.
  • Hämäläinen, Moona (Helsingin yliopisto, 2019)
    Suomessa tulehduksellisten suolistosairauksien esiintyvyys on kasvussa. Yksi toissijaisista lääkkeistä sairauteen on biologinen lääke infliksimabi, joka on TNF-alfa-estäjä. TNF-alfa on tärkeä sytokiini tulehduksellisissa sairauksissa. Infliksimabista vain noin 1/3 saa hyvän hoitovasteen. 1/3 ei saa vastetta ollenkaan, ja 1/3 menettää saadun vasteen, eikä vastetta osata ennustaa. Tämän tutkimuksen tarkoituksena oli etsiä biomarkkereita geeniekspressioprofiileista RNA-sekvensoinnilla ja ymmärtää erilaisten lääkevasteiden eroja. Tutkielmassa tutkittiin 15 potilaan veren valkosolujen geeniekspressioita ennen (0vk) ja jälkeen (12vk) infliksimabihoidon, sekä verrattiin vasteensaajien ja ei-vasteensaajien geeniekspressioiden profiileja. RNA eristettiin näytteistä, sekvensoitiin ja analysoitiin Chipster-ohjelmalla. Ekspressiotasot erosivat merkitsevästi aikapisteiden välillä 17 geenin osalta, joista tärkein on DUSP2-geeni. DUSP2 laskee epäsuorasti interleukiini-17 pitoisuutta, jolloin tulehdusreaktio vaimenee. Verrattaessa vasteensaajien ja osittaisen vasteensaajien geeniekspressioprofiileja ennen ja jälkeen hoidon, ei selvää trendiä näkynyt geenien ilmentymisessä. Tutkimuksessa ei ollut mukana 12 viikon näytteitä ei-vasteensaajilla, joten siihen ei voitu verrata. Tässä tutkimuksessa ei löytynyt biomarkkeria infliksimabin hoitovasteen ennustamiselle, mutta saatiin tärkeää tietoa DUSP2-geenin aktiivisuudesta. Kaikilla infliksimabihoidon saaneilla DUSP2- geenin ekspressio nousi veressä.
  • Savarese, Marco; Välipakka, Salla; Johari, Mridul; Hackman, Peter; Udd, Bjarne (2020)
    Human genes have a variable length. Those having a coding sequence of extraordinary length and a high number of exons were almost impossible to sequence using the traditional Sanger-based gene-by-gene approach. High-throughput sequencing has partly overcome the size-related technical issues, enabling a straightforward, rapid and relatively inexpensive analysis of large genes. Several large genes (e.g. TTN, NEB, RYR1, DMD) are recognized as disease-causing in patients with skeletal muscle diseases. However, because of their sheer size, the clinical interpretation of variants in these genes is probably the most challenging aspect of the high-throughput genetic investigation in the field of skeletal muscle diseases. The main aim of this review is to discuss the technical and interpretative issues related to the diagnostic investigation of large genes and to reflect upon the current state of the art and the future advancements in the field. © 2020 - IOS Press and the authors. All rights reserved.
  • Pehkonen, Henna; Lento, Mira; von Nandelstadh, Pernilla; Filippou, Artemis; Grenman, Reidar; Lehti, Kaisa; Monni, Outi (2018)
    Background: PPFIA1 is located at the 11q13 region commonly amplified in cancer. The protein liprin-alpha 1 encoded by PPFIA1 contributes to the adhesive and invasive structures of cytoskeletal elements and is located at the invadosomes in cancer cells. However, the precise mechanism of liprin-alpha 1 function in cancer progression has remained elusive. Methods: Invasion regulating activity of liprin-alpha 1 was examined by analyzing the functions of squamous cell carcinoma of head and neck (HNSCC) cell lines in three-dimensional collagen I after RNAi mediated gene knockdown. Transcriptome profiling and Gene Set Enrichment Analysis from HNSCC and breast cancer cells were used to identify expression changes relevant to specific cellular localizations, biological processes and signaling pathways after PPFIA1 knockdown. The significance of the results was assessed by relevant statistical methods (Wald and Benjamini-Hochberg). Localization of proteins associated to liprin-alpha 1 was studied by immunofluorescence in 2D and 3D conditions. The association of PPFIA1 amplification to HNSCC patient survival was explored using The Cancer Genome Atlas data. Results: In this study, we show that liprin-alpha 1 regulates biological processes related to membrane microdomains in breast carcinoma, as well as protein trafficking, cell-cell and cell-substrate contacts in HNSCC cell lines cultured in three-dimensional matrix. Importantly, we show that in all these cancer cells liprin-alpha 1 knockdown leads to the upregulation of transmembrane protein CD82, which is a suppressor of metastasis in several solid tumors. Conclusions: Our results provide novel information regarding the function of liprin-alpha 1 in biological processes essential in cancer progression. The results reveal liprin-alpha 1 as a novel regulator of CD82, linking liprin-alpha 1 to the cancer cell invasion and metastasis pathways.
  • Pehkonen, Henna; Lento, Mira; von Nandelstadh, Pernilla; Filippou, Artemis; Grénman, Reidar; Lehti, Kaisa; Monni, Outi (BioMed Central, 2018)
    Abstract Background PPFIA1 is located at the 11q13 region commonly amplified in cancer. The protein liprin-α1 encoded by PPF1A1 contributes to the adhesive and invasive structures of cytoskeletal elements and is located at the invadosomes in cancer cells. However, the precise mechanism of liprin-α1 function in cancer progression has remained elusive. Methods Invasion regulating activity of liprin-α1 was examined by analyzing the functions of squamous cell carcinoma of head and neck (HNSCC) cell lines in three-dimensional collagen I after RNAi mediated gene knockdown. Transcriptome profiling and Gene Set Enrichment Analysis from HNSCC and breast cancer cells were used to identify expression changes relevant to specific cellular localizations, biological processes and signaling pathways after PPFIA1 knockdown. The significance of the results was assessed by relevant statistical methods (Wald and Benjamini-Hochberg). Localization of proteins associated to liprin-α1 was studied by immunofluorescence in 2D and 3D conditions. The association of PPFIA1 amplification to HNSCC patient survival was explored using The Cancer Genome Atlas data. Results In this study, we show that liprin-α1 regulates biological processes related to membrane microdomains in breast carcinoma, as well as protein trafficking, cell-cell and cell-substrate contacts in HNSCC cell lines cultured in three-dimensional matrix. Importantly, we show that in all these cancer cells liprin-α1 knockdown leads to the upregulation of transmembrane protein CD82, which is a suppressor of metastasis in several solid tumors. Conclusions Our results provide novel information regarding the function of liprin-α1 in biological processes essential in cancer progression. The results reveal liprin-α1 as a novel regulator of CD82, linking liprin-α1 to the cancer cell invasion and metastasis pathways.
  • Santos-Cortez, R.L.P.; Bhutta, M.F.; Earl, J.P.; Hafrén, Lena; Jennings, M.; Mell, J.C.; Pichichero, M.E.; Ryan, A.F.; Tateossian, Hilda; Ehrlich, G.D. (2020)
    Objective: To review the most recent advances in human and bacterial genomics as applied to pathogenesis and clinical management of otitis media. Data sources: PubMed articles published since the last meeting in June 2015 up to June 2019. Review methods: A panel of experts in human and bacterial genomics of otitis media was formed. Each panel member reviewed the literature in their respective fields and wrote draft reviews. The reviews were shared with all panel members, and a merged draft was created. The panel met at the 20th International Symposium on Recent Advances in Otitis Media in June 2019, discussed the review and refined the content. A final draft was made, circulated, and approved by the panel members. Conclusion: Trans-disciplinary approaches applying pan-omic technologies to identify human susceptibility to otitis media and to understand microbial population dynamics, patho-adaptation and virulence mechanisms are crucial to the development of novel, personalized therapeutics and prevention strategies for otitis media. Implications for practice: In the future otitis media prevention strategies may be augmented by mucosal immunization, combination vaccines targeting multiple pathogens, and modulation of the middle ear microbiome. Both treatment and vaccination may be tailored to an individual's otitis media phenotype as defined by molecular profiles obtained by using rapidly developing techniques in microbial and host genomics. © 2020 Elsevier B.V.
  • Holmer, Jacob; Aho, Velma; Eriksdotter, Maria; Paulin, Lars; Pietiäinen, Milla; Auvinen, Petri; Schultzberg, Marianne; Pussinen, Pirkko; Buhlin, Kåre (2021)
    Aim: The aim of this study was to compare the subgingival microbiota of people with Alzheimer ' s disease (AD), mild cognitive impairment (MCI), subjective cognitive decline (SCD) and cognitively healthy individuals. Materials and methods: The study population was recruited from 2013 to 2017 and comprised 132 cases recently diagnosed with AD (n = 46), MCI (n = 40) or SCD (n = 46), and 63 cognitively healthy controls. Subgingival samples were collected, and the microbiotas were characterized by 16S rRNA gene sequencing. Results: The relative abundance of the ten most common genera did not differ between the cases and control groups. However, the microbial richness and evenness were higher in cases than in controls and differed across the four groups. The variables with the greatest influence on the microbial community composition were related to periodontal disease followed by body mass index, study group affiliation and smoking. Ten taxa exhibited significant differences between case participants and controls. Two Operational Taxonomic Units were particularly abundant in AD compared to controls: Slackia exigua, which was also associated with deep periodontal pockets, and a Lachnospiraceae [G-7] bacterium. Conclusion: It is concluded that in individuals with cognitive impairment or AD, the subgingival microbiota exhibits shifts typical of periodontal disease.
  • Johansson, Tiira; Koskela, Satu; Yohannes, Dawit A.; Partanen, Jukka; Saavalainen, Paivi (2021)
    Identification of human leukocyte antigen (HLA) alleles from next-generation sequencing (NGS) data is challenging because of the high polymorphism and mosaic nature of HLA genes. Owing to the complex nature of HLA genes and consequent challenges in allele assignment, Oxford Nanopore Technologies' (ONT) single-molecule sequencing technology has been of great interest due to its fitness for sequencing long reads. In addition to the read length, ONT's advantages are its portability and possibility for a rapid real-time sequencing, which enables a simultaneous data analysis. Here, we describe a targeted RNA-based method for HLA typing using ONT sequencing and SeqNext-HLA SeqPilot software (JSI Medical Systems GmbH). Twelve classical HLA genes were enriched from cDNA of 50 individuals, barcoded, pooled, and sequenced in 10 MinION R9.4 SpotON flow cell runs producing over 30,000 reads per sample. Using barcoded 2D reads, SeqPilot assigned HLA alleles to two-field typing resolution or higher with the average read depth of 1750x. Sequence analysis resulted in 99-100% accuracy at low-resolution level (one-field) and in 74-100% accuracy at high-resolution level (two-field) with the expected alleles. There are still some limitations with ONT RNA sequencing, such as noisy reads, homopolymer errors, and the lack of robust algorithms, which interfere with confident allele assignment. These issues need to be inspected carefully in the future to improve the allele call rates. Nevertheless, here we show that sequencing of multiplexed cDNA amplicon libraries on ONT MinION can produce accurate high-resolution typing results of 12 classical HLA loci. For HLA research, ONT RNA sequencing is a promising method due to its capability to sequence full-length HLA transcripts. In addition to HLA genotyping, the technique could also be applied for simultaneous expression analysis.