Alternative splicing of the human gene SYBL1 modulates protein domain architecture of longin VAMP7/TI-VAMP, showing both non-SNARE and synaptobrevin-like isoforms

<p>Abstract</p> <p>Background</p> <p>The control of intracellular vesicle trafficking is an ideal target to weigh the role of alternative splicing in shaping genomes to make cells. Alternative splicing has been reported for several Soluble <it>N</it>-ethylmaleimide-sensitive factor Attachment protein REceptors of the vesicle (v-SNAREs) or of the target membrane (t-SNARES), which are crucial to intracellular membrane fusion and protein and lipid traffic in Eukaryotes. However, splicing has not yet been investigated in Longins, i.e. the most widespread v-SNAREs. Longins are essential in Eukaryotes and prototyped by VAMP7, Sec22b and Ykt6, sharing a conserved N-terminal Longin domain which regulates membrane fusion and subcellular targeting. Human VAMP7/TI-VAMP, encoded by gene SYBL1, is involved in multiple cell pathways, including control of neurite outgrowth.</p> <p>Results</p> <p>Alternative splicing of SYBL1 by exon skipping events results in the production of a number of VAMP7 isoforms. In-frame or frameshift coding sequence modifications modulate domain architecture of VAMP7 isoforms, which can lack whole domains or domain fragments and show variant or extra domains. Intriguingly, two main types of VAMP7 isoforms either share the inhibitory Longin domain and lack the fusion-promoting SNARE motif, or vice versa. Expression analysis in different tissues and cell lines, quantitative real time RT-PCR and confocal microscopy analysis of fluorescent protein-tagged isoforms demonstrate that VAMP7 variants have different tissue specificities and subcellular localizations. Moreover, design and use of isoform-specific antibodies provided preliminary evidence for the existence of splice variants at the protein level.</p> <p>Conclusions</p> <p>Previous evidence on VAMP7 suggests inhibitory functions for the Longin domain and fusion/growth promoting activity for the Δ-longin molecule. Thus, non-SNARE isoforms with Longin domain and non-longin SNARE isoforms might have somehow opposite regulatory functions. When considering splice variants as "natural mutants", evidence on modulation of subcellular localization by variation in domain combination can shed further light on targeting determinants. Although further work will be needed to characterize identified variants, our data might open the route to unravel novel molecular partners and mechanisms, accounting for the multiplicity of functions carried out by the different members of the Longin proteins family.</p

Longins and the longin domain: pivotal elements in subcellular trafficking and neuronal differentiation

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are the most investigated trafficking proteins due to their role in directing the fusion complex. Among these, long VAMPs, or longins are characterized by N-terminal longin domain (LD), controlling both SNARE complex formation and subcellular localization (SCL) and are prototyped by VAMP7, Sec22b and Ykt6. The LD adopts a stable, closed conformation, but the contribution of this state and each different domain to sorting is still unclear. Human VAMP7, encoded by gene SYBL1, is involved in multiple cell pathways, including control of neurite outgrowth. Furthermore, alternative splicing (AS) of SYBL1 results in the production of two isoform subfamilies that retain an intriguingly domain architecture. Non-SNARE longin variants share the inhibitory LD, whereas non-longin SNARE variants share the SNARE motif. Since previous evidence suggests inhibitory functions for the LD construct and growth promoting activity for the Δ-longin construct, these subfamilies are likely to play opposite functions. Therefore, mechanisms mediating neurotogenesis are not clear, in particular the contribution of extracellular stimuli and different SNAREs. This work focused on the characterization of VAMP7 LD and its isoforms in SCL and neuronal development. Expression analysis in different tissues and cell lines, real time RT-PCR and confocal microscopy analyses demonstrated that VAMP7 variants have different tissue specificities and SCL; furthermore, the LD-only isoform VAMP7i displays also a nuclear localization. Considering their variant domain combinations, these physiological splice variants were used as tools for studying targeting determinants in SCL. Moreover, recombinant fragments of the VAMP7a cytoplasmic region confirmed that individual domains are unable to determine sorting by alone, and open/closed conformational switch is not relevant to SCL in the absence of transmembrane region. Gain-of-function experiments on both neuroblastoma cells and primary neurons revealed that VAMP7 AS is able to regulate neurite outgrowth by balanced production of stimulatory (VAMP7dh) and inhibitory (VAMP7i) isoforms. These effects are also subjected to the substrate (Poly-D-Lysine or Laminin) in which neurons are cultured and to the co-expression of other VAMP7 isoforms or SNAREs (VAMP2), suggesting a fine regulatory mechanism mediated by VAMP7 AS. Additional investigation will be helpful in order to manipulate neuritogenesis in cell therapy and clarify the role of tissue-specific variants in some neurological diseases. Further characterization of VAMP7 LD and isoforms can unravel novel molecular partners and mechanisms, helpful in some biotechnological applications.Le proteine SNARE (Soluble N-ethylmaleimide-sensitive factor attachment protein receptors) sono le più studiate nell’ambito del traffico subcellulare, dato il loro ruolo nella formazione del complesso di fusione delle membrane. In questa famiglia, le VAMP lunghe o longine sono caratterizzate da un dominio N-terminale denominato longin (LD), che ha una funzione sia nella formazione del complesso SNARE, sia nella localizzazione subcellulare delle proteine; le longine trovano inoltre un modello in VAMP7, Sec22b e Ykt6. Il LD adotta una conformazione chiusa che risulta stabile, ma non è ancora chiaro il contributo che tale conformazione e ogni differente dominio proteico portano alla determinazione della localizzazione stessa di tali proteine. VAMP7 umana, codificata dal gene SYBL1 è coinvolta in molteplici pathway subcellulari, compreso il controllo della crescita dei neuriti. Lo splicing alternativo nel locus di SYBL1 produce inoltre due famiglie di isoforme che mantengono un’interessante architettura di domini. Le longine non-SNARE condividono il dominio inibitorio LD, mentre le longine non-LD lo SNARE motif. Date le evidenze preliminari che conferiscono tale funzione inibitoria al costrutto artificiale LD e invece un’attività di promozione della crescita al costrutto non-LD, sembra plausibile che le due sottofamiglie rispecchino questi ruoli contrapposti. Tuttavia, i meccanismi coinvolti nella neuritogenesi non sono ancora completamente chiariti, soprattutto per quanto concerne il contributo degli stimoli extracellulari e delle diverse proteine SNARE. Questo lavoro di tesi si è incentrato sulla caratterizzazione del LD di VAMP7 e delle isoforme di splicing di questa proteina nell’ambito del loro ruolo sia nella localizzazione subcellulare, che nel differenziamento neuronale. Analisi di espressione in diversi tessuti e linee cellulari, dati quantitativi di real time RT-PCR e analisi di microscopia confocale hanno dimostrato come le varianti di VAMP7 presentino differenti tessuto-specificità e localizzazioni subcellulari; l’isoforma VAMP7i mostra inoltre una localizzazione anche nucleare. Considerando la loro diversa combinazione di domini, queste varianti di splicing fisiologiche sono state utilizzzate come strumenti per lo studio dei determinanti di localizzazione. Per di più, frammenti ricombinanti della regione citosolica di VAMP7a hanno confermato che i songoli domini non sono in grado da soli di determinare la localizzaione della proteina e che il cambiamento conformazionale aperto/chiuso non è rilevante per la localizzazione subcellulare, in assenza della regione trasmembrana. Esperimenti di gain-of-function su cellule di neuroblastoma e neuroni primari hanno mostrato l’esistenza di una regolazione della crescita dei neuriti mediata dallo splicing alternativo di VAMP7, con la produzione di isoforme sia inibitorie (VAMP7i) che stimolatorie (VAMP7dh). Tali effetti dipendono anche dal substrato (Poli-D-Lisina o Laminina) in cui i neuroni sono cresciuti e dalla co-espressione con altre isoforme di VAMP7 o con altre SNARE (VAMP2), indicando la presenza di un meccanismo di regolazione fine da parte dello splicing alternativo di VAMP7. Ulteriori investigazioni potranno portare sia alla manipolazione della neuritogenesi per scopi di terapeutici, sia al chiarimento del ruolo che le specifiche varianti di splicing possono avere in alcune malattie neurologiche. La futura caratterizzaione del LD di VAMP7 e delle sue isoforme, ad esempio il ruolo di VAMP7i nel nucleo possono definire nuovi interattori e meccanismi molecolari, utili in alcune applicazioni biotecnologiche

Design of a stirred multiwell bioreactor for expansion of CD34(+) umbilical cord blood cells in hypoxic conditions

Besides having a metabolic role, oxygen is recognized as an important signaling stimulus for stem cells. In hematopoiesis, hypoxia seems to favor stem cell self-renewal. In fact, long-term repopulating hematopoietic stem cells reside in bone marrow at concentrations as low as 1% oxygen. However, O(2) concentration is difficult to control in vitro. Thermodynamically, we found significant differences between O(2) solubility in different media, and in presence of serum. Furthermore, we verified that medium equilibration with a hypoxic atmosphere requires several hours. Thus, in a static culture, the effective O(2) concentration in the cell immediate microenvironment is difficult to control and subject to concentration gradients. Stirred systems improve homogeneity within the culture volume. In this work, we developed a stirred bioreactor to investigate hypoxia effect on the expression of stem cell markers in CD34(+) cells from umbilical cord blood. The stirring system was designed on top of a standard six-well plate to favor continuity with conventional static conditions and transfer of culture protocols. The bioreactor volume (10 mL/well) is suitable for cell expansion and multiparametric flow cytometry analyses. First, it was tested at 21% O(2) for biocompatibility and other possible effects on the cells compared to static conditions. Then, it was used to study c-kit expression of CD34(+) cells at 5% O(2) , using 21%-O(2) cultures as a control. In hypoxia we found that CD34(+) cells maintained a higher expression of c-kit. Further investigation is needed to explore the dynamics of interaction between oxygen- and c-kit-dependent pathways at the molecular level

Tratamiento percutáneo con prótesis endovascular de coartación de aorta abdominal en un adulto

Coarctation of the abdominal aorta is an uncommon, non-inherited vascular condition that affects men and women alike. It has been recently named as "middle aortic syndrome", and the clinical findings are similar to those of typical aortic coarctation. For diagnosis, one must make use of magnetic resonance imaging or arteriography, and therapeutic options include percutaneous balloon catheter dilatation, surgical treatment and, finally, as a more novel option, the implantation of stents. In this paper we present the case of a 45-year-old female, ex-smoker with a history of rheumatoid arthritis and hypertension who presented claudication of lower limbs during gait. There was a marked bilateral decrease of the femoral and Doppler pulses, and showed a damping factor in both femoral and popliteal arteries. The CT angiography found a significant stenosis of the distal third of the infrarenal abdominal aorta with marked hypoplasia of the right iliac. Aortography confirmed the diagnosis (gradient of 80 mmHg). Using two arterial sheaths, two-balloon catheters MATCH-35, 5.0x80 mm were introduced through femoral arteries, simultaneously inflated and subsequently a MEDTRONIC "Bridge Assurant" stent of 10x30 mm was implanted in the stenotic segment; with no complications. The residual gradient was 10 mmHg. The patient improved and was discharged form the hospital 24 hours after the procedureLa coartación de la aorta abdominal es una afección vascular no hereditaria poco frecuente, que afecta a hombres y mujeres por igual. Recientemente ha sido nombrada como �Síndrome aórtico medio�, y los hallazgos clínicos son similares a los de la CoAo típica. Para el diagnóstico, se debe recurrir a la resonancia magnética o a la arteriografía, y las opciones terapéuticas incluyen la dilatación percutánea con catéter-globo, el tratamiento quirúrgico y, por último, como opción más novedosa, la implantación de prótesis endovasculares. En este artículo presentamos el caso de una mujer de 45 años de edad, exfumadora, con antecedentes de artritis reumatoidea e hipertensión arterial que presentaba claudicación de miembros inferiores durante la marcha. Existía una disminución bilateral marcada de los pulsos femorales y el Doppler, y mostró un componente amortiguado en ambas arterias femorales y poplíteas. La AngioTAC encontró una estenosis significativa del tercio distal de la aorta abdominal infrarrenal, con hipoplasia marcada de la ilíaca derecha. La aortografía corroboró el diagnóstico (gradiente de 80 mmHg). A través de dos introductores arteriales por las arterias femorales se avanzaron dos catéteres-globo MATCH-35 de 5.0x80 mm que se inflaron simultáneamente y posteriormente, se implantó un stent MEDTRONIC �Bridge Assurant� de 10 x 30 mm en el segmento estenótico, sin complicaciones. El gradiente residual fue de 10 mmHg. La paciente evolucionó favorablemente y fue egresada a las 24 horas del procedimient

TRATAMIENTO PERCUTÁNEO CON PRÓTESIS ENDOVASCULAR DE COARTACIÓN DE AORTA ABDOMINAL EN UN ADULTO / Percutaneous treatment with endovascular prosthesis of abdominal aortic coarctation in an adult

Resumen: La coartación de la aorta abdominal es una afección vascular no hereditaria poco frecuente, que afecta a hombres y mujeres por igual. Recientemente ha sido nombrada como “Síndrome aórtico medio”, y los hallazgos clínicos son similares a los de la CoAo típica. Para el diagnóstico, se debe recurrir a la resonancia magnética o a la arteriografía, y las opciones terapéuticas incluyen la dilatación percutánea con catéter-globo, el tratamiento quirúrgico y, por último, como opción más novedosa, la implantación de prótesis endovasculares. En este artículo presentamos el caso de una mujer de 45 años de edad, exfumadora, con antecedentes de artritis reumatoidea e hipertensión arterial que presentaba claudicación de miembros inferiores durante la marcha. Existía una disminución bilateral marcada de los pulsos femorales y el Doppler, y mostró un componente amortiguado en ambas arterias femorales y poplíteas. La AngioTAC encontró una estenosis significativa del tercio distal de la aorta abdominal infrarrenal, con hipoplasia marcada de la ilíaca derecha. La aortografía corroboró el diagnóstico (gradiente de 80 mmHg). A través de dos introductores arteriales por las arterias femorales se avanzaron dos catéteres-globo MATCH-35 de 5.0x80 mm que se inflaron simultáneamente y posteriormente, se implantó un stent MEDTRONIC “Bridge Assurant” de 10 x 30 mm en el segmento estenótico, sin complicaciones. El gradiente residual fue de 10 mmHg. La paciente evolucionó favorablemente y fue egresada a las 24 horas del procedimiento. / Abstract: Coarctation of the abdominal aorta is an uncommon, non-inherited vascular condition that affects men and women alike. It has been recently named as "middle aortic syndrome", and the clinical findings are similar to those of typical aortic coarctation. For diagnosis, one must make use of magnetic resonance imaging or arteriography, and therapeutic options include percutaneous balloon catheter dilatation, surgical treatment and, finally, as a more novel option, the implantation of stents. In this paper we present the case of a 45-year-old female, ex-smoker with a history of rheumatoid arthritis and hypertension who presented claudication of lower limbs during gait. There was a marked bilateral decrease of the femoral and Doppler pulses, and showed a damping factor in both femoral and popliteal arteries. The CT angiography found a significant stenosis of the distal third of the infrarenal abdominal aorta with marked hypoplasia of the right iliac. Aortography confirmed the diagnosis (gradient of 80 mmHg). Using two arterial sheaths, two-balloon catheters MATCH-35, 5.0x80 mm were introduced through femoral arteries, simultaneously inflated and subsequently a MEDTRONIC "Bridge Assurant" stent of 10x30 mm was implanted in the stenotic segment; with no complications. The residual gradient was 10 mmHg. The patient improved and was discharged form the hospital 24 hours after the procedure

Reduction of cardiac imaging tests during the COVID-19 pandemic: The case of Italy. Findings from the IAEA Non-invasive Cardiology Protocol Survey on COVID-19 (INCAPS COVID)

Background: In early 2020, COVID-19 massively hit Italy, earlier and harder than any other European country. This caused a series of strict containment measures, aimed at blocking the spread of the pandemic. Healthcare delivery was also affected when resources were diverted towards care of COVID-19 patients, including intensive care wards. Aim of the study: The aim is assessing the impact of COVID-19 on cardiac imaging in Italy, compare to the Rest of Europe (RoE) and the World (RoW). Methods: A global survey was conducted in May–June 2020 worldwide, through a questionnaire distributed online. The survey covered three periods: March and April 2020, and March 2019. Data from 52 Italian centres, a subset of the 909 participating centres from 108 countries, were analyzed. Results: In Italy, volumes decreased by 67% in March 2020, compared to March 2019, as opposed to a significantly lower decrease (p &lt; 0.001) in RoE and RoW (41% and 40%, respectively). A further decrease from March 2020 to April 2020 summed up to 76% for the North, 77% for the Centre and 86% for the South. When compared to the RoE and RoW, this further decrease from March 2020 to April 2020 in Italy was significantly less (p = 0.005), most likely reflecting the earlier effects of the containment measures in Italy, taken earlier than anywhere else in the West. Conclusions: The COVID-19 pandemic massively hit Italy and caused a disruption of healthcare services, including cardiac imaging studies. This raises concern about the medium- and long-term consequences for the high number of patients who were denied timely diagnoses and the subsequent lifesaving therapies and procedures

Impact of COVID-19 on Diagnostic Cardiac Procedural Volume in Oceania: The IAEA Non-Invasive Cardiology Protocol Survey on COVID-19 (INCAPS COVID)

Objectives: The INCAPS COVID Oceania study aimed to assess the impact caused by the COVID-19 pandemic on cardiac procedure volume provided in the Oceania region. Methods: A retrospective survey was performed comparing procedure volumes within March 2019 (pre-COVID-19) with April 2020 (during first wave of COVID-19 pandemic). Sixty-three (63) health care facilities within Oceania that perform cardiac diagnostic procedures were surveyed, including a mixture of metropolitan and regional, hospital and outpatient, public and private sites, and 846 facilities outside of Oceania. The percentage change in procedure volume was measured between March 2019 and April 2020, compared by test type and by facility. Results: In Oceania, the total cardiac diagnostic procedure volume was reduced by 52.2% from March 2019 to April 2020, compared to a reduction of 75.9% seen in the rest of the world (p&lt;0.001). Within Oceania sites, this reduction varied significantly between procedure types, but not between types of health care facility. All procedure types (other than stress cardiac magnetic resonance [CMR] and positron emission tomography [PET]) saw significant reductions in volume over this time period (p&lt;0.001). In Oceania, transthoracic echocardiography (TTE) decreased by 51.6%, transoesophageal echocardiography (TOE) by 74.0%, and stress tests by 65% overall, which was more pronounced for stress electrocardiograph (ECG) (81.8%) and stress echocardiography (76.7%) compared to stress single-photon emission computerised tomography (SPECT) (44.3%). Invasive coronary angiography decreased by 36.7% in Oceania. Conclusion: A significant reduction in cardiac diagnostic procedure volume was seen across all facility types in Oceania and was likely a function of recommendations from cardiac societies and directives from government to minimise spread of COVID-19 amongst patients and staff. Longer term evaluation is important to assess for negative patient outcomes which may relate to deferral of usual models of care within cardiology