On the putative role of Pelota in stem cell differentiation

In dieser Arbeit analysierten wir die Rolle von Pelota in dem conditionalen Knock out Mausmodel . Wir haben licht auf seine Funktion während der Entwicklung geworfen und berichten über defekte die aus dem Pelota Abbau resultieren.Wir haben erfolgreich eine Pelo?/- CreERT ES-Zelllinie erzeugt. Diese Zelllinie hatte keine Anlage zu einer Proliferation Funktionsstörung, aber die Zellen hatten einen Defekt in ihren differenzierungs- Kapazitäten. Sie zeigten eine durchgehende Oct4 Expression und miR-290, clusterspezifisch für Pluripontente Zellen, schien unter differenzierungs Bedingungen herraufreguliert. Die Ectoderm Formation war nicht betroffen, aber eine Analyse von frühen und späten Markern, spezifisch für Mesoderm und Entoderm enthüllte das in den mutanten Zellen Beide Keimschichten nicht ausgebildet werden. Um diese Hypothese zu bestätigen, untersuchten wir die Differenzierungs-Kapazität von adulten Stammzellen, z.B hämatopoetischen Stammzellen.Die Untersuchung der Spermatogenese ergab das alle Entwicklungs Stadien der Keimzellen fehlen. Für umfangreichere Studien erzeugten wir eine Testis spezifische Pelo?/- Stra8-Cre Maus, welche uns den Effekt der Pelo Deletion bei der Spematogenese aufzeigen sollte. Die Analyse der Testes zeigte einen Arrest in der Spermatogenese Entwicklung während des Beginns der meiotischen Teilung (Tag 15). In 10 Tage alten Testes fanden wir eine Abnahme in der Expression von Stra8, Dazl and Ddx4, dieses sind Marker für prämeiotische Stadien von Keimzellen. Trotzdem konnten wir weder auf der Ebene der undifferenzierten ( Plzf und Sox3 ) noch der differenzierten ( c-Kit ) Spermatogonien, einen Pelo deletions Effekt feststellen. Dies legt nahe das Pelota Auswirkungen auf den Beginn der Meiose hat, welche der Beginn der Keimzell Differenzierung ist. Unerwartet fanden wir auch Mosaik Mäuse mit teilweise deletierten Allel (PeloF/?/-Stra-Cre), in verschiedenen somatischen Geweben, inklusive Niere, Leber, Herz, Milz, Lunge, dies Ergebnis deutet Stra8 Expression in der frühen Embryogenese an.Die Studien der gereiften B und T Zellen, die aus Milz und Thymus der Mutanten Mäuse isoliert wurden, offenbarten eine Reduktion der Zell Nummer. Zusätzlich entdeckten wir einen starken Anstieg von doppelt negativen Zellen (CD4- CD8-),diese sind Entwicklungs- Vorstufen von T Zellen. Gleichzeitig bemerkten wir eine dramatische Abnahme auf allen Ebenen der späten Entwicklungsphase der T Zellen. Alle diese Daten bestätigen das Versagen der Differenzierungs-Fähigkeit der Mutanten Zellen.Adham et al. Untersuchte die Pelota Funktion in der Pelota Knock Out Maus. Der Phänotyp der homozygoten Mutanten (Pelo-/-) war letal während der frühen Phase der Embryonal Entwicklung. Um zu bestätigen das dieser Phänotyp aus der Pelota Inaktivierung resultiert, kreuzten wir Pelota+/- Mäuse mit Transgenen Pelo T Mäusen, welches den letalen Phänotyp rettete. Dies demonstriert deutlich den direkten Zusammenhang zwischen der Sterblichkeit und den Pelota Abbau.Die Transgenen Mäuse die Pelo in Testes spezifischer art und weise überexprimierten, zeigten keinen Phänotyp. Die Mäuse waren fertil und die histologischen Analysen kamen zu dem Ergebnis, das die Spermatogenese nicht gestört ist

Single or double-injection technique in axillary block: the success of motor and sensor blockade

Background and Purpose: Axillary brachial plexus block is the method of choice for surgical procedures of upper arm except shoulder region. Distribution of local anaesthetic toward neurovascular space may be a reason for failed block. We investigated the axillary block effectiveness by singeand double-injection technique. Materials and Methods: Ninety patients (21–81 old; ASA I-IV) scheduled for upper arm surgery were divided in three equal groups during prospective, double-blind study. Nerve position was located with neurostimulator (Stimuplex® HNS 11)(0.5 mA, 2Hz and 0.1 ms). In Group S (single-shot), mixture of 30 mL (15 mL 0.5% bupivacaine and 15 mL 2% lidocaine) was injected only above axillary artery (25 mL around median and 5 mL around musculocutaneus nerve). In Group U and R (double-shot), the same mixture of local anaesthetic was applied above (10 mL around median and 5 mL around musculocutaneus nerve) and below axillary artery (15 mL around radial or ulnar nerve). Motor and sensor block were determined (Bromage scale, Pinprick method). Statistic analysis was done (SSP11.0). Results and Conclusions: Effective block analgesia and anaesthesia was achieved in shorter time in Group R (18+/4 and 26+/–3 min)(Group U: 34+/–4 and 41+/–3 min, Group S: 35+/–4 and 45+/–2 min) (P=0.0000) (Table 2). Block effectiveness was significantly higher after radial nerve stimulation (92%)(Group U 88% and S 76%) (P=0.630). Faster motor block was achieved in Group R (18+/–4)(Group U 26+/–3 and S 35+/–4 min) (P=0.000). Double-shot technique with primar radial nerve stimulation, allows better motor and sensor axillary block in comparison with single-shot technique

Comparation of early continuous epidural and intravenous opioid analgesia on haemodynamic changes after several pelvic fractures

Background and Purpose: Continuous epidural analgesia improves excellent pain control in trauma patients with multiple pelvic fractures. Rezidual haemodynamic instability followed by retroperitoneal hemorrhagie in the first 48 hours often post-pones its application with need for parenteral use of high dose of opioids. The aim was to compere the influence of early continuous epidural and intravenous opioid analgesia on haemodynamic changes in these patients. Materials and Methods: After Ethic Committee approval, fifty trauma patients with isolated multiple pelvic fractures were divided in two equal groups and included in prospective, randomized study. In bought groups initial analgesia was started with sufentanil 10 μg h–1 in the first 24h. After that, in Group EP continuous epidural analgesia (levibupivacain O.125%, 5–7 mL h–1) and in Group O continuous infusion of opioid (sufentanil 5–10 μg h–1) was started. The analgesics dose was titrated following the VAS score under 3. PICCO monitoring was established. MAP, CI, HR, SVRI, ITBVI and ELWI was measured during four days. Statistic analysis was done by SPSS 11.0. Results: Study groups were statistic comparable. In the first 24 hours during continuous opioid anaesthesia, bought groups had high need for fluid replacement (Group EP=3.2 ± 0.3, Group O=3.0 ± 0.5 L/24h) (P=0.0928). Second day, SVRI was lower in O Group (1300–1520; EP Group=1700–1810)(P=0.0243) and recovered with 500–750mLof crystalloids infusion. ITBVI was statistical more stable inGroup EP (950 ± 50; Group O (1100 ± 30)(P=0.0002). Only 10% of patients with low CI (<3.0) in Group EP (Group O=32%) needed catecholamin support. Conclusion: Early continuous epidural analgesia with 0.125% levibupivacain is safe as continuous opioid analgesia in patients with multiple pelvic fractures but without opioids complications and better haemodynamic stability

Pelota interacts with HAX1, EIF3G and SRPX and the resulting protein complexes are associated with the actin cytoskeleton

<p>Abstract</p> <p>Background</p> <p>Pelota (PELO) is an evolutionary conserved protein, which has been reported to be involved in the regulation of cell proliferation and stem cell self-renewal. Recent studies revealed the essential role of PELO in the No-Go mRNA decay, by which mRNA with translational stall are endonucleotically cleaved and degraded. Further, PELO-deficient mice die early during gastrulation due to defects in cell proliferation and/or differentiation.</p> <p>Results</p> <p>We show here that PELO is associated with actin microfilaments of mammalian cells. Overexpression of human PELO in Hep2G cells had prominent effect on cell growth, cytoskeleton organization and cell spreading. To find proteins interacting with PELO, full-length human PELO cDNA was used as a bait in a yeast two-hybrid screening assay. Partial sequences of HAX1, EIF3G and SRPX protein were identified as PELO-interacting partners from the screening. The interactions between PELO and HAX1, EIF3G and SRPX were confirmed <it>in vitro </it>by GST pull-down assays and <it>in vivo </it>by co-immunoprecipitation. Furthermore, the PELO interaction domain was mapped to residues 268-385 containing the c-terminal and acidic tail domain. By bimolecular fluorescence complementation assay (BiFC), we found that protein complexes resulting from the interactions between PELO and either HAX1, EIF3G or SRPX were mainly localized to cytoskeletal filaments.</p> <p>Conclusion</p> <p>We could show that PELO is subcellularly localized at the actin cytoskeleton, interacts with HAX1, EIF3G and SRPX proteins and that this interaction occurs at the cytoskeleton. Binding of PELO to cytoskeleton-associated proteins may facilitate PELO to detect and degrade aberrant mRNAs, at which the ribosome is stalled during translation.</p

Expression and Activity of TRPA1 and TRPV1 in the Intervertebral Disc: Association with Inflammation and Matrix Remodeling

Transient receptor potential (TRP) channels have emerged as potential sensors and transducers of inflammatory pain. The aims of this study were to investigate (1) the expression of TRP channels in intervertebral disc (IVD) cells in normal and inflammatory conditions and (2) the function of Transient receptor potential ankyrin 1 (TRPA1) and Transient receptor potential vanilloid 1 (TRPV1) in IVD inflammation and matrix homeostasis. RT-qPCR was used to analyze human fetal, healthy, and degenerated IVD tissues for the gene expression of TRPA1 and TRPV1. The primary IVD cell cultures were stimulated with either interleukin-1 beta (IL-1β) or tumor necrosis factor alpha (TNF-α) alone or in combination with TRPA1/V1 agonist allyl isothiocyanate (AITC, 3 and 10 µM), followed by analysis of calcium flux and the expression of inflammation mediators (RT-qPCR/ELISA) and matrix constituents (RT-qPCR). The matrix structure and composition in caudal motion segments from TRPA1 and TRPV1 wild-type (WT) and knock-out (KO) mice was visualized by FAST staining. Gene expression of other TRP channels (A1, C1, C3, C6, V1, V2, V4, V6, M2, M7, M8) was also tested in cytokine-treated cells. TRPA1 was expressed in fetal IVD cells, 20% of degenerated IVDs, but not in healthy mature IVDs. TRPA1 expression was not detectable in untreated cells and it increased upon cytokine treatment, while TRPV1 was expressed and concomitantly reduced. In inflamed IVD cells, 10 µM AITC activated calcium flux, induced gene expression of IL-8, and reduced disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5) and collagen 1A1, possibly via upregulated TRPA1. TRPA1 KO in mice was associated with signs of degeneration in the nucleus pulposus and the vertebral growth plate, whereas TRPV1 KO did not show profound changes. Cytokine treatment also affected the gene expression of TRPV2 (increase), TRPV4 (increase), and TRPC6 (decrease). TRPA1 might be expressed in developing IVD, downregulated during its maturation, and upregulated again in degenerative disc disease, participating in matrix homeostasis. However, follow-up studies with larger sample sizes are needed to fully elucidate the role of TRPA1 and other TRP channels in degenerative disc disease

Single or double-injection technique in axillary block: the success of motor and sensor blockade

Background and Purpose: Axillary brachial plexus block is the method of choice for surgical procedures of upper arm except shoulder region. Distribution of local anaesthetic toward neurovascular space may be a reason for failed block. We investigated the axillary block effectiveness by singeand double-injection technique. Materials and Methods: Ninety patients (21–81 old; ASA I-IV) scheduled for upper arm surgery were divided in three equal groups during prospective, double-blind study. Nerve position was located with neurostimulator (Stimuplex® HNS 11)(0.5 mA, 2Hz and 0.1 ms). In Group S (single-shot), mixture of 30 mL (15 mL 0.5% bupivacaine and 15 mL 2% lidocaine) was injected only above axillary artery (25 mL around median and 5 mL around musculocutaneus nerve). In Group U and R (double-shot), the same mixture of local anaesthetic was applied above (10 mL around median and 5 mL around musculocutaneus nerve) and below axillary artery (15 mL around radial or ulnar nerve). Motor and sensor block were determined (Bromage scale, Pinprick method). Statistic analysis was done (SSP11.0). Results and Conclusions: Effective block analgesia and anaesthesia was achieved in shorter time in Group R (18+/4 and 26+/–3 min)(Group U: 34+/–4 and 41+/–3 min, Group S: 35+/–4 and 45+/–2 min) (P=0.0000) (Table 2). Block effectiveness was significantly higher after radial nerve stimulation (92%)(Group U 88% and S 76%) (P=0.630). Faster motor block was achieved in Group R (18+/–4)(Group U 26+/–3 and S 35+/–4 min) (P=0.000). Double-shot technique with primar radial nerve stimulation, allows better motor and sensor axillary block in comparison with single-shot technique

Прогностички фактори кај детската акутна лимфобластна леукемија- преглед на литература

The treatment outcome of acute lymphoblastic leukemia (ALL)has remarkably improved over the recent decades, leading to a 5-year overall survival rate up to 90%. This impressive achievement is mainly due to the use of effective multi-agent chemotherapy regimens and the precise stratification of patients into risk groups based on well defined prognostic factors including the clinical features that are present at diagnosis, biologic and genetic features of leukemia cells, and early response to treatment. Patients classified as low risk are treated with less intensive therapy, whereas more aggressive regimens are reserved for those with high-risk features. Currently, minimal residual disease (MRD) is the most important and independent predictor of treatment outcome. This review will describe the clinical, biological, and response-based features and current evidence supporting their clinical application in childhood ALL. &nbsp;Во последните неколку декади е остварен значаен напредок во терапискиот исход кај детската акутна лимфобластна леукемија (АЛЛ), што резултира со денешно 5-годишно вкупно преживување кое достигнува до 90%. Овој импресивен успех се должи на примената на ефикасни, комбинирани хемотераписки режими и прецизна стратификација на пациентите во ризични групи според добро дефинирани прогностички фактори кои ги вклучуваат клиничките карактеристики присутни при дијагнозата, биолошките и генетските карактеристики на леукемиските клетки и раниот одговор на терапија. Пациентите кои се класифицираат во прогностичка група со низок ризик се третираат со помалку интензивна терапија, додека за тие со високо-ризични карактеристики се применуваат поагресивни режими. Минималната резидуална болест (МРБ) во моментов претставува најважен и независен предиктор на терапискиот исход. Во овој ревијален труд се опишани клиничките, биолошките карактеристики и раниот одговор на терапија како и актуелните сознанија кои ја поддржуваат нивната клиничка примена кај детската АЛЛ