A non-radioactive, improved PAR-CLIP and small RNA cDNA library preparation protocol.

Crosslinking and immunoprecipitation (CLIP) methods are powerful techniques to interrogate direct protein-RNA interactions and dissect posttranscriptional gene regulatory networks. One widely used CLIP variant is photoactivatable ribonucleoside enhanced CLIP (PAR-CLIP) that involves in vivo labeling of nascent RNAs with the photoreactive nucleosides 4-thiouridine (4SU) or 6-thioguanosine (6SG), which can efficiently crosslink to interacting proteins using UVA and UVB light. Crosslinking of 4SU or 6SG to interacting amino acids changes their base-pairing properties and results in characteristic mutations in cDNA libraries prepared for high-throughput sequencing, which can be computationally exploited to remove abundant background from non-crosslinked sequences and help pinpoint RNA binding protein binding sites at nucleotide resolution on a transcriptome-wide scale. Here we present a streamlined protocol for fluorescence-based PAR-CLIP (fPAR-CLIP) that eliminates the need to use radioactivity. It is based on direct ligation of a fluorescently labeled adapter to the 3'end of crosslinked RNA on immobilized ribonucleoproteins, followed by isolation of the adapter-ligated RNA and efficient conversion into cDNA without the previously needed size fractionation on denaturing polyacrylamide gels. These improvements cut the experimentation by half to 2 days and increases sensitivity by 10-100-fold.

Covalent Functionalization of Flexible Polyvinyl Chloride Tubing.

Polyvinyl chloride (PVC) tubing is a vital part of many industries, including the medical and food sectors. The ability to chemically modify PVC has previously been reported mainly on powdered or rigid forms of the plastic. Here, we describe the chemical modification of flexible PVC tubing with azide (and the attempted modification with cyanide) facilitated by phase transfer catalysts and characterization of the resulting materials. These modifications provide convenient handles for click chemistry linkages via azide-akyne cycloaddition, which allows for diverse surface functionalization of PVC for a variety of applications.

Continued Ultrasound Surveillance Required after Hand Ischemia Associated with Trauma in Children.

BACKGROUND: Hand ischemia following trauma in children is rare, and the natural history after upper extremity arterial bypass in children is unknown. We hypothesize children with brachial artery repair are at long-term risk of developing aneurysmal degeneration or thrombosis, thus necessitating annual duplex ultrasonography and physical examination. METHODS: A retrospective review of children who had brachial artery repair (bypass or vein patch) for hand ischemia secondary to trauma at a level I trauma pediatric hospital was performed. Telephone interviews were conducted to assess the presence of arm/hand symptoms (pain, weakness, fatigue, sensory function, limb length discrepancy). RESULTS: Between 2003 and 2016, 16 children (12 males), mean age 8 years (3-13 years) underwent brachial artery repair (12 bypass with vein, 4 vein patch). Mechanism of injury included 11 supracondylar fractures and 5 lacerations. All patients were seen at 2 weeks with a duplex ultrasound. Thirteen patients were lost to follow-up. The 3 patients with follow-up had patent bypasses, but one patient 6 years out from the repair had aneurysmal degeneration of the vein graft. Seven patients were never seen again. Phone interviews were conducted for the remaining 6 patients and 2 complained of arm fatigue and intermittent hand pain. Only one patient reported that the pediatrician checked pulses in the affected extremity. CONCLUSIONS: Eighty percentage of children had no further follow-up after the postoperative visit. Asymptomatic aneurysmal degeneration of the vein graft was noted 6 years following repair in one patient, and 2 patients had unevaluated hand complaints. These patients are at risk for late complications and are unlikely to return for routine follow-up. The importance of graft surveillance must be more clearly emphasized at time of initial surgery.

Keratin 19 binds and regulates cytoplasmic HNRNPK mRNA targets in triple-negative breast cancer.

BACKGROUND: Heterogeneous nuclear ribonucleoprotein K (HNRNPK) regulates pre-mRNA processing and long non-coding RNA localization in the nucleus. It was previously shown that shuttling of HNRNPK to the cytoplasm promotes cell proliferation and cancer metastasis. However, the mechanism of HNRNPK cytoplasmic localization, its cytoplasmic RNA ligands, and impact on post-transcriptional gene regulation remain uncharacterized. RESULTS: Here we show that the intermediate filament protein Keratin 19 (K19) directly interacts with HNRNPK and sequesters it in the cytoplasm. Correspondingly, in K19 knockout breast cancer cells, HNRNPK does not localize in the cytoplasm, resulting in reduced cell proliferation. We comprehensively mapped HNRNPK binding sites on mRNAs and showed that, in the cytoplasm, K19-mediated HNRNPK-retention increases the abundance of target mRNAs bound to the 3' untranslated region (3'UTR) at the expected cytidine-rich (C-rich) sequence elements. Furthermore, these mRNAs protected by HNRNPK in the cytoplasm are typically involved in cancer progression and include the p53 signaling pathway that is dysregulated upon HNRNPK knockdown (HNRNPK KD) or K19 knockout (KRT19 KO). CONCLUSIONS: This study identifies how a cytoskeletal protein can directly regulate gene expression by controlling the subcellular localization of RNA-binding proteins to support pathways involved in cancer progression.

EEG en hemodiálisis crónica (A) / EEG in chronic hemodialysis

Se estudian los trazados EEG de 14 pacientes en hemodiálisis crónica. Los registros se obtuvieron en período interdialítico y ninguno de los pacientes presentaba evidencias de compromiso neurológico central. Se encontró un elevado número de trazados alterados (12/14), destacando las actividades paroxísticas (10/14) e irritativas (7/14) por su frecuencia y magnitud, en comparación con comunicaciones previas de la literatura, donde dichos hallazgos son infrecuentes. Debe considerar-se la posibilidad que estos pacientes con EEG alterados con predominancia de actividdad paroxística e irritativa se encuentren en un período previo o inicial de la Encefalopatía o Demencia Dialítica (solamente con manifestaciones electroencefalográficas), dada la elevada incidencia de esta patología en el centro de diálisis donde se realizó este estudio. Esta situación podrá ser corroborada con el seguimiento clínico y EEG de este grupo de pacientes. Se sugiere el control electroencefalográfico periódico para el diagnóstico pre-clínico de la Encefalopatía Dialítica, con miras a un manejo terapéutico oportuno y efectivo