Next generation sequencing identifies novel CACNA1A gene mutations in Episodic Ataxia type 2

Episodic Ataxia type 2 (EA2) is a rare autosomal dominantly inherited neurological disorder characterized by recurrent disabling imbalance, vertigo and episodes of ataxia lasting minutes to hours. EA2 is caused most often by loss of function mutations of the calcium channel gene CACNA1A. In addition to EA2, mutations in CACNA1A are responsible for two other allelic disorders: familial hemiplegic migraine type1 (FHM1) and spinocerebellar ataxia type 6 (SCA6). Herein, we have utilised Next Generation Sequencing (NGS) to screen the coding sequence, exon-intron boundaries and UTRs of five genes where mutation is known to produce symptoms related to EA2, including CACNA1A. We performed this screening in a group of 31 unrelated patients with EA2 symptoms. Both novel and known mutations were detected through NGS technology, and confirmed through Sanger sequencing. Genetic testing showed in total 15 mutation bearing patients (48%), of which 9 were novel mutations (6 missense and 3 small frameshift deletion mutations) and six known mutations (4 missense and 2 nonsense).These results demonstrate the efficiency of our NGS-panel for detecting known and novel mutations for EA2 in the CACNA1A gene, also identifying a novel missense mutation in ATP1A2 which is not a normal target for EA2 screening.

Identification and Characterization of a Novel Deoxyhypusine Synthase in Leishmania donovani

Deoxyhypusine synthase, an NAD(+)-dependent enzyme, catalyzes the first step in the post-translational synthesis of an unusual amino acid, hypusine (N-epsilon-(4-amino-2-hydroxybutyl)lysine), in the eukaryotic initiation factor 5A precursor protein. Two putative deoxyhypusine synthase (DHS) sequences have been identified in the Leishmania donovani genome, which are present on chromosomes 20: DHSL20 (DHS-like gene from chromosome 20) and DHS34 (DHS from chromosome 34). Although both sequences exhibit an overall conservation of key residues, DHSL20 protein lacks a critical lysine residue, and the recombinant protein showed no DHS activity in vitro. However, DHS34 contains the critical lysine residue, and the recombinant DHS34 effectively catalyzed deoxyhypusine synthesis. Furthermore, in vivo labeling confirmed that hypusination of eukaryotic initiation factor 5A occurs in intact Leishmania parasites. Interestingly, the DHS34 is much longer, with 601 amino acids, compared with the human DHS enzyme (369 amino acids) and contains several unique insertions. To study the physiological role of DHS34 in Leishmania, gene deletion mutations were attempted via targeted gene replacement. However, chromosomal null mutants of DHS34 could only be obtained in the presence of a DHS34-containing episome. The present data provide evidence that DHS34 is essential for L. donovani and that structural differences in the human and leishmanial DHS enzyme may be exploited for designing selective inhibitors against the parasite.

Division of the Salmonella-Containing Vacuole and Depletion of Acidic Lysosomes in Salmonella-Infected Host Cells Are Novel Strategies of Salmonella enterica To Avoid Lysosomes

Salmonella has evolved several strategies to counteract intracellular microbicidal agents like reactive oxygen and nitrogen species. However, it is not yet clear how Salmonella escapes lysosomal degradation. Some studies have demonstrated that Salmonella can inhibit phagolysosomal fusion, whereas other reports have shown that the Salmonella-containing vacuole (SCV) fuses/interacts with lysosomes. Here, we have addressed this issue from a different perspective by investigating if the infected host cell has a sufficient quantity of lysosomes to target Salmonella. Our results suggest that SCVs divide along with Salmonella, resulting in a single bacterium per SCV. As a consequence, the SCV load per cell increases with the division of Salmonella inside the host cell. This demands more investment from the host cell to counteract Salmonella. Interestingly, we observed that Salmonella infection decreases the number of acidic lysosomes inside the host cell both in vitro and in vivo. These events potentially result in a condition in which an infected cell is left with insufficient acidic lysosomes to target the increasing number of SCVs, which favors the survival and proliferation of Salmonella inside the host cell.

A novel DNA intercalator, butylamino-pyrimido[4',5':4,5]selenolo(2,3-b)quinoline, induces cell cycle arrest and apoptosis in leukemic cells

DNA intercalators are one of the most commonly used chemotherapeutic agents. Novel intercalating compounds of pyrimido[4',5':4,5]selenolo(2,3-b)quinoline series having a butylamino or piperazino group at fourth position (BPSQ and PPSQ, respectively) are studied. Our results showed that BPSQ induced cytotoxicity whereas PPSQ was cytostatic. The cytotoxicity induced by BPSQ was concentration- and time-dependent. Cell cycle analysis and tritiated thymidine assay revealed that BPSQ affects the cell cycle progression by arresting at S phase. The absence of p-histone H3 and reduction in the levels of PCNA in the cells treated with BPSQ further confirmed the cell cycle arrest. Further, annexin V staining, DNA fragmentation, nuclear condensation and changes in the expression levels of BCL2/BAD confirmed the activation of apoptosis. Activation of caspase 8 and lack of cleavage of caspase 9, caspase 3 and PARP suggest the possibility of BPSQ triggering extrinsic pathway for induction of apoptosis, which is discussed. Hence, we have identified a novel compound which would have clinical relevance in cancer chemotherapeutics.

Novel matrix metalloproteinases in intestinal inflammation and in cancer of the gastrointestinal tract

Matrix metalloproteinases (MMPs) comprise a family of 23 zinc-dependent human endopeptidases that can degrade virtually all components of the extracellular matrix (ECM). They are classified into eight subgroups according to their structure and into six subgroups based on their substrate-specificity. MMPs have been implicated in inflammation, tissue destruction, cell migration, arthritis, vascular remodeling, angiogenesis, and tumor growth and invasion. MMPs are inhibited by their natural inhibitors, tissue inhibitors of metalloproteinases (TIMPs). Different MMPs function in the same tasks depending on the tissue or cancer subtype. I investigated the role of recently discovered MMPs, especially MMPs-19 and -26, in intestinal inflammation, in intestinal and cutaneous wound healing, and in intestinal cancer. Several MMPs and TIMPs were studied to determine their exact location at tissue level and to obtain information on possible functions of MMPs in such tissues and diseases as the healthy intestine, inflammatory bowel disease (IBD), neonatal necrotizing enterocolitis (NEC), pyoderma gangrenosum (PG), and colorectal as well as pancreatic cancers. In latent celiac disease (CD), I attempted to identify markers to predict later onset of CD in children and adolescents. The main methods used were immunohistochemistry, in situ hybridization, and Taqman RT-PCR. My results show that MMP-26 is important for re-epithelialization in intestinal and cutaneous wound healing. In colon and pancreatic cancers, MMP-26 seems to be a marker of invasive potential, although it is not itself expressed at the invasive front. MMP-21 is upregulated in pancreatic cancer and may be associated with tumor differentiation. MMPs-19 and -28 are associated with normal tissue turnover in the intestine, but they disappear in tumor progression as if they were protective markers . MMP-12 is an essential protease in intestinal inflammation and tissue destruction, as seen here in NEC and in previous CD studies. In patients with type 1 diabetes (T1D), MMPs-1, -3, and -12 were upregulated in the intestinal mucosa. Furthermore, MMP-7 was strongly elevated in NEC. In a model of aberrant wound repair, PG, MMPs-8, -9, and 10 and TNFα may promote ECM destruction, while absence of MMP-1 and MMP-26 from keratinocytes retards re-epithelialization. Based on my results, I suggest MMP-26 to be considered a putative marker for poor prognosis in pancreatic and colon cancer. However, since it functions differently in various tissues and tumor subtypes, this use cannot be generalized. Furthermore, MMP-26 is a beneficial marker for wound healing if expressed by migrating epithelial cells. MMP-12 expression in latent CD patients warrants research in a larger patient population to confirm its role as a specific marker for CD in pathologically indistinct cases. MMP-7 should be considered one of the most crucial proteases in NEC-associated tissue destruction; hence, specific inhibitors of this MMP are worth investigating. In PG, TNFα inhibitors are potential therapeutic agents, as shown already in clinical trials. In conclusion, studies of several MMPs in specific diseases and in healthy tissues are needed to elucidate their roles at the tissue level. MMPs and TIMPs are not exclusively destructive or reparative in tissues. They seem to function differently in different tissues. To identify selective MMP inhibitors, we must thoroughly understand the MMP profile (degradome) and their functions in various organs not to interfere with normal reparative functions during wound repair or beneficial host-response effects during cancer initiation and growth.

Natively unfolded nucleic acid binding P8 domain of SeMV polyprotein 2a affects the novel ATPase activity of the preceding P10 domain

Open reading frame (ORF) 2a of Sesbania mosaic virus (SeMV) codes for polyprotein 2a (Membrane anchor-protease-VPg-P10-P8). The C-terminal domain of SeMV polyprotein 2a was cloned, expressed and purified in order to functionally characterize it. The protein of size 8 kDa (P8) domain, like viral protein genome linked (VPg), was found to be natively unfolded and could bind to nucleic acids.Interestingly, P10-P8 but not P8 showed a novel Mg2+ dependent ATPase activity that was inhibited in the presence of poly A. In the absence of P8, the ATPase activity of the protein of size 10 kDa (P10) domain was reduced suggesting that the natively unfolded P8 domain influenced the P10 ATPase.

Who really ate the fruit? A novel approach to camera trapping for quantifying frugivory by ruminants

Tropical forest ruminants disperse several plants; yet, their effectiveness as seed dispersers is not systematically quantified. Information on frequency and extent of frugivory by ruminants is lacking. Techniques such as tree watches or fruit traps adapted from avian frugivore studies are not suitable to study terrestrial frugivores, and conventional camera traps provide little quantitative information. We used a novel time-delay camera-trap technique to assess the effectiveness of ruminants as seed dispersers for Phyllanthus emblica at Mudumalai, southern India. After being triggered by animal movement, cameras were programmed to take pictures every 2 min for the next 6 min, yielding a sequence of four pictures. Actual frugivores were differentiated from mere visitors, who did not consume fruit, by comparing the number of fruit remaining across the time-delay photograph sequence. During a 2-year study using this technique, we found that six terrestrial mammals consumed fallen P. emblica fruit. Additionally, seven mammals and one bird species visited fruiting trees but did not consume fallen fruit. Two ruminants, the Indian chevrotain Moschiola indica and chital Axis axis, were P. emblica's most frequent frugivores and they accounted for over 95% of fruit removal, while murid rodents accounted for less than 1%. Plants like P. emblica that are dispersed mainly by large mammalian frugivores are likely to have limited ability to migrate across fragmented landscapes in response to rapidly changing climates. We hope that more quantitative information on ruminant frugivory will become available with a wider application of our time-delay camera-trap technique.

Formation of a novel surface structure encoded by Salmonella Pathogenicity Island 2.

The type III secretion system (T3SS) encoded by Salmonella Pathogenicity Island 2 (SPI2) is essential for virulence and intracellular proliferation of Salmonella enterica. We have previously identified SPI2-encoded proteins that are secreted and function as a translocon for the injection of effector proteins. Here, we describe the formation of a novel SPI2-dependent appendage structure in vitro as well as on the surface of bacteria that reside inside a vacuole of infected host cells. In contrast to the T3SS of other pathogens, the translocon encoded by SPI2 is only present singly or in few copies at one pole of the bacterial cell. Under in vitro conditions, appendages are composed of a filamentous needle-like structure with a diameter of 10 nm that was sheathed with secreted protein. The formation of the appendage in vitro is dependent on acidic media conditions. We analyzed SPI2-encoded appendages in infected cells and observed that acidic vacuolar pH was not required for induction of SPI2 gene expression, but was essential for the assembly of these structures and their function as translocon for delivery of effector proteins.

X-Pro peptides: Synthesis and solution conformation of benzyloxycarbonyl-(Aib-Pro)4methyl ester. Evidence for a novel helical structure

The synthesis of the octapeptide, benzyloxycarbonyl-(-aminoisobutyryl-L-prolyl)4-methyl ester [Z-(Aib-Pro)4-OMe] and an analysis of its solution conformation is reported. The octapeptide is shown to possess three strong intramolecular hydrogen bonds on the basis of studies of the solvent and temperature dependence of NH chemical shifts and rates of hydrogen-deuterium exchange. 13C studies are consistent with a structure involving only trans Aib-Pro bonds, while ir experiments support a hydrogen-bonded conformation. The Aib 3, 5, and 7 NH groups are shown to participate in hydrogen bonding. A 310 helical conformation compatible with the spectroscopic data is suggested. The proposed conformation consists of three type III -turns with Aib and Pro at the corners and stabilized by 4 1 intramolecular hydrogen bonds.

Functional Imagery Training to reduce snacking: Testing a novel motivational intervention based on Elaborated Intrusion theory

Functional Imagery Training (FIT) is a new theory-based, manualized intervention that trains positive goal imagery. Multisensory episodic imagery of proximal personal goals is elicited and practised, to sustain motivation and compete with less functional cravings. This study tested the impact of a single session of FIT plus a booster phone call on snacking. In a stepped-wedge design, 45 participants who wanted to lose weight or reduce snacking were randomly assigned to receive a session of FIT immediately or after a 2-week delay. High-sugar and high-fat snacks were recorded using timeline follow back for the previous 3 days, at baseline, 2 and 4 weeks. At 2 weeks, snacking was lower in the immediate group than in the delayed group, and the reduction after FIT was replicated in the delayed group between 2 and 4 weeks. Frequencies of motivational thoughts about snack reduction rose following FIT for both groups, and this change correlated with reductions in snacking and weight loss. By showing that FIT can support change in eating behaviours, these findings show its potential as a motivational intervention for weight management.

Studies on diamond-like carbon and novel diamond-like carbon polymer hybrid coatings deposited with filtered pulsed arc discharge method

The main obstacle for the application of high quality diamond-like carbon (DLC) coatings has been the lack of adhesion to the substrate as the coating thickness is increased. The aim of this study was to improve the filtered pulsed arc discharge (FPAD) method. With this method it is possible to achieve high DLC coating thicknesses necessary for practical applications. The energy of the carbon ions was measured with an optoelectronic time-of-flight method. An in situ cathode polishing system used for stabilizing the process yield and the carbon ion energies is presented. Simultaneously the quality of the coatings can be controlled. To optimise the quality of the deposition process a simple, fast and inexpensive method using silicon wafers as test substrates was developed. This method was used for evaluating the suitability of a simplified arc-discharge set-up for the deposition of the adhesion layer of DLC coatings. A whole new group of materials discovered by our research group, the diamond-like carbon polymer hybrid (DLC-p-h) coatings, is also presented. The parent polymers used in these novel coatings were polydimethylsiloxane (PDMS) and polytetrafluoroethylene (PTFE). The energy of the plasma ions was found to increase when the anode-cathode distance and the arc voltage were increased. A constant deposition rate for continuous coating runs was obtained with an in situ cathode polishing system. The novel DLC-p-h coatings were found to be water and oil repellent and harder than any polymers. The lowest sliding angle ever measured from a solid surface, 0.15 ± 0.03°, was measured on a DLC-PDMS-h coating. In the FPAD system carbon ions can be accelerated to high energies (≈ 1 keV) necessary for the optimal adhesion (the substrate is broken in the adhesion and quality test) of ultra thick (up to 200 µm) DLC coatings by increasing the anode-cathode distance and using high voltages (up to 4 kV). An excellent adhesion can also be obtained with the simplified arc-discharge device. To maintain high process yield (5µm/h over a surface area of 150 cm2) and to stabilize the carbon ion energies and the high quality (sp3 fraction up to 85%) of the resulting coating, an in situ cathode polishing system must be used. DLC-PDMS-h coating is the superior candidate coating material for anti-soiling applications where also hardness is required.

Development of Novel Assays for Measuring Different Molecular Forms of Prostate Specific Antigen

Prostate cancer (PCa) is the most commonly diagnosed non-skin cancer and second leading cause of cancer-related death of men in developed countries. Measurement of prostate specific antigen (PSA) is a very sensitive method for diagnosing and monitoring of prostate cancer (PCa), but the specificity needs improvement. Measurements of different molecular forms of PSA have been shown to improve differentiation between PCa and benign prostatic diseases. However, accurate measurement of some isoforms has not been achieved in previous assays. The aim of the present study was to develop new assays that reliably measure enzymatically active PSA, PSA-α1-chymotryposin (PSA-ACT) and PSA-α1-protease inhibitor (PSA-API), and to evaluate their diagnostic value. Double-label immunofluorometric assays using a novel monoclonal antibody (MAb) and another antibody to either free PSA (fPSA) or total PSA (tPSA) were developed and used to measure PSA-ACT and fPSA or tPSA at the same time. These assays provide enough sensitivity for measurement of PSA-ACT in sera with low PSA levels. The results obtained confirmed that proportion of PSA-ACT to tPSA (%PSA-ACT) was as useful as proportion of fPSA to tPSA (%fPSA) for discrimination between PCa and benign prostatic hyperplasia (BPH). We developed an immunoassay for detection of PSA-API based on proximity ligation, which improved assay sensitivity 10-fold compared with conventional assays. Our results confirmed previous findings that the PSA-API level is somewhat lower in men with than without PCa, and the combination of %fPSA and proportion of PSA-API to tPSA (%PSA-API) provides diagnostic improvement compared with either method alone. Assays based on this principle should be applicable to other immunoassays in which the nonspecific background is a problem. An immunopeptidometric sandwich assay (IPMA) was developed to measure the enzymatically active PSA. This assay showed high specificity, but sensitivity was not good enough for measurement of PSA concentrations in the gray zone, 2-10 µg/L, in which tPSA does not efficiently differentiate between PCa and BPH. We further developed a solid-phase proximity ligation immunoassay, which provided a 10-fold improvement in sensitivity. This proof of concept study shows that peptides reacting with proteins are potentially useful for sensitive and specific measurement of protein variants for which specific MAbs cannot be obtained.

Identification of novel target genes in different subtypes of cutaneous T-cell lymphoma

Ihon T-solulymfoomat (cutaneous T-cell lymphoma, CTCL) ovat ryhmä imukudossyöpiä, joiden esiintyvyys on nousussa erityisesti länsimaissa. Taudin syntymekanismit ovat suurelta osin tuntemattomat, diagnostiikka on vaikeaa ja siksi usein viivästynyttä eikä parantavaa hoitoa ole. CTCL ilmenee iho-oirein, vaikka syöpäsolut eivät ole iholla normaalisti esiintyviä soluja, vaan elimistön puolustusjärjestelmän soluja, jotka ovat tuntemattomasta syystä vaeltaneet iholle. Syöpäsolut ovat kypsiä T-auttajasoluja (Th-soluja) ja ilmentävät tyypin 2 immuunivasteelle ominaisia sytokiineja. Kromosomaalinen epästabiilius on tautiryhmän keskeinen piirre. CTCL-potilailla on lisääntynyt riski sairastua myös muihin syöpiin, erityisesti keuhkosyöpään ja non-Hodgkin –lymfoomiin. Väitöskirjatutkimuksen tavoitteena oli havaita CTCL:n syntymekanismeja selvittäviä kromosomi- ja geenimuutoksia. Erityisesti tavoitteena oli identifioida molekyylejä, jotka soveltuisivat diagnostisiksi merkkiaineiksi tai täsmähoidon kohteeksi. Työssä on tutkittu kahta tautiryhmän yleisintä muotoa, mycosis fungoidesta (MF) ja Sezaryn syndroomaa (SS) sekä harvinaisempaa vaikeasti diagnosoitavaa subkutaanista pannikuliitin kaltaista T-solulymfoomaa (SPTL). Lisäksi on tutkittu CTCL:ään liittyvää keuhkosyöpää ja verrattu sitä tavalliseen (primaariin) keuhkosyöpään. Tutkimusmenetelminä on käytetty esimerkiksi molekyylisytogeneettisiä metodeja ja mikrosiruja. Väitöskirjatyössä havaittiin ensimmäinen CTCL:lle ominainen toistuva geenitason muutos: puutos- tai katkoskohta NAV3-geenissä. Tämän geenipoikkeavuuden havaittiin esiintyvän useissa taudin alaryhmissä (MF, SS, SPTL). NAV3-geenipuutoksen osoittaminen FISH-tekniikalla on sovellettavissa kliiniseen diagnostiikkaan. Tutkimukset geenipuutoksen aiheuttamista toiminnallisista seurauksista ovat käynnissä. Työssä saatiin myös uutta tietoa taudin syntymekanismeista havaitsemalla useiden Th1-tyypin immuunivasteelle ominaisten geenien alentunut ilmeneminen CTCL-potilailla. Tämän lisäksi potilasnäytteissä havaittiin eräiden solun pinta-antigeenien lisääntynyt ilmeneminen, mikä luo pohjan uusien vasta-ainepohjaisten täsmähoitojen kehittämiselle. Väitöskirjatutkimuksessa todettiin myös CTCL:ään liittyvän keuhkosyövän eroavan kromosomi- ja geenimuutosten suhteen verrokkikeuhkosyövästä, mikä jatkossa antaa aiheen tutkia syöpäkantasolujen merkitystä CTCL:n ja sen liitännäiskasvainten kehittymisen taustalla.

In-situ one-step hydrothermal synthesis of a lead germanate-graphene composite as a novel anode material for lithium-ion batteries

Lead germanate-graphene nanosheets (PbGeO3-GNS) composites have been prepared by an efficient one-step, in-situ hydrothermal method and were used as anode materials for Li-ion batteries (LIBs). The PbGeO3 nanowires, around 100–200 nm in diameter, are highly encapsulated in a graphene matrix. The lithiation and de-lithiation reaction mechanisms of the PbGeO3 anode during the charge-discharge processes have been investigated by X-ray diffraction and electrochemical characterization. Compared with pure PbGeO3 anode, dramatic improvements in the electrochemical performance of the composite anodes have been obtained. In the voltage window of 0.01–1.50 V, the composite anode with 20 wt.% GNS delivers a discharge capacity of 607 mAh g−1 at 100 mA g−1 after 50 cycles. Even at a high current density of 1600 mA g−1, a capacity of 406 mAh g−1 can be achieved. Therefore, the PbGeO3-GNS composite can be considered as a potential anode material for lithium ion batteries.

Novel synthesis of superparamagnetic Ni-Co-B nanoparticles and their effect on superconductor properties of MgB2

A new procedure for the preparation of amorphous Ni-Co-B nanoparticles is reported, with a detailed investigation of their morphology by X-ray diffraction and transmission electron microscopy, as well as their magnetic properties. Many factors, such as chemical composition, anisotropy, size and shape of the particles, were controlled through chemical synthesis, resulting in the control of morphological and magnetic properties of the nanoparticles. Controlling pH values with ethylenediamine and using sodium dodecyl sulfate surfactant lowered the size of the nanoparticles to below 10 nm. Such a small structure and chemical disorder in nanocrystalline materials lead to magnetic properties that are different from those in their bulk-sized counterparts. The obtained nanoparticles can be used for different purposes, from pharmaceutical applications to implementations in different materials technology. The focus of this research is the synthesis of Ni-Co-B nanoparticles in a new way and studying the reaction of Ni-Co-B nanoparticles with Mg and B precursors and their effect on MgB2 properties. New nanostructures are formed in the reaction of Ni-Co-B nanoparticles with Mg: Mg2Ni, Co2Mg and possibly Mg2Co.