The transcription of the main gene associated with Treacher-Collins syndrome (TCOF1) is regulated by G-quadruplexes and cellular nucleic acid binding protein (CNBP).

Treacle ribosome biogenesis factor 1 (TCOF1) is responsible for about 80% of mandibular dysostosis (MD) cases. We have formerly identified a correlation between TCOF1 and CNBP (CCHC-type zinc finger nucleic acid binding protein) expression in human mesenchymal cells. Given the established role of CNBP in gene regulation during rostral development, we explored the potential for CNBP to modulate TCOF1 transcription. Computational analysis for CNBP binding sites (CNBP-BSs) in the TCOF1 promoter revealed several putative binding sites, two of which (Hs791 and Hs2160) overlap with putative G-quadruplex (G4) sequences (PQSs). We validated the folding of these PQSs measuring circular dichroism and fluorescence of appropriate synthetic oligonucleotides. In vitro studies confirmed binding of purified CNBP to the target PQSs (both folded as G4 and unfolded) with Kd values in the nM range. ChIP assays conducted in HeLa cells chromatin detected the CNBP binding to TCOF1 promoter. Transient transfections of HEK293 cells revealed that Hs2160 cloned upstream SV40 promoter increased transcription of downstream firefly luciferase reporter gene. We also detected a CNBP-BS and PQS (Dr2393) in the zebrafish TCOF1 orthologue promoter (nolc1). Disrupting this G4 in zebrafish embryos by microinjecting DNA antisense oligonucleotides complementary to Dr2393 reduced the transcription of nolc1 and recapitulated the craniofacial anomalies characteristic of Treacher Collins Syndrome. Both cnbp overexpression and Morpholino-mediated knockdown in zebrafish induced nolc1 transcription. These results suggest that CNBP modulates the transcriptional expression of TCOF1 through a mechanism involving G-quadruplex folding/unfolding, and that this regulation is active in vertebrates as distantly related as bony fish and humans. These findings may have implications for understanding and treating MD.

G-Quadruplexes Regulate miRNA Biogenesis in Live Zebrafish Embryos.

RNA guanine quadruplexes (G4s) regulate RNA functions, metabolism, and processing. G4s formed within precursors of microRNAs (pre-miRNAs) may impair pre-miRNAs maturation by Dicer, thus repressing mature miRNA biogenesis. As miRNAs are essential for proper embryonic development, we studied the role of G4s on miRNA biogenesis in vivo during zebrafish embryogenesis. We performed a computational analysis on zebrafish pre-miRNAs to find putative G4 forming sequences (PQSs). The precursor of the miRNA 150 (pre-miR-150) was found to contain an evolutionarily conserved PQS formed by three G-tetrads and able to fold in vitro as G4. MiR-150 controls the expression of myb, which shows a well-defined knock-down phenotype in zebrafish developing embryos. We microinjected zebrafish embryos with in vitro transcribed pre-miR-150 synthesized using either GTP (G-pre-miR-150) or 7-Deaza-GTP, a GTP analogue unable to form G4s (7DG-pre-miR-150). Compared to embryos injected with G-pre-miR-150, embryos injected with 7DG-pre-miR-150 showed higher levels of miRNA 150 (miR-150) and lower levels of myb mRNA and stronger phenotypes associated with myb knock-down. The incubation of pre-miR-150 prior to the injection with the G4 stabilizing ligand pyridostatin (PDS) reverted gene expression variations and rescued the phenotypes related to myb knock-down. Overall, results suggest that the G4 formed in pre-miR-150 functions in vivo as a conserved regulatory structure competing with the stem-loop structure necessary for miRNA biogenesis.

Reporting regression in primary cutaneous melanoma. Part 1: history, histological criteria and pathogenesis.

Regression is an important histopathological parameter reported for the diagnosis of primary cutaneous melanoma. Histological regression is defined by The College of American Pathologists as the replacement of tumour cells by lymphocytic inflammation, with attenuation of the epidermis, and nonlaminated dermal fibrosis with inflammatory cells, melanophagocytosis and telangiectasia. Histological regression may be reported as absent versus present and, if present, as complete, partial or segmental. The stages of histological regression are early, intermediate and late, depending on the extent of histological inflammation and fibrosis. Regression occurs when the host's immune system attacks primary melanocytic tumour cells via tumour-infiltrating lymphocytes, resulting in fibrosis. The immunological mechanisms driving complete, partial and segmental regression may vary. In this first part of this two-part review, we review the history, histological criteria and pathogenesis of regression in primary cutaneous melanoma, while in Part 2 we will review the effect of histological regression on prognosis, evaluation and management.

Reporting regression in primary cutaneous melanoma. Part 2: prognosis, evaluation and management.

The effect of histological regression on patient prognosis for primary cutaneous melanoma is controversial. Some authors hypothesize that regression indicates a robust systemic immune response and may decrease risk of metastasis. Others argue that histological regression calls into question a T0 diagnosis because there may have been an invasive component of the melanoma that is no longer visible but is still active. The literature to date does not suggest that histological regression is associated with increased risk of positive sentinel lymph node status, metastasis or increased risk of mortality. Thus, the presence of histological regression should not change patient staging, evaluation or management. The criteria used for reporting regression have varied dramatically across studies, and standardized reporting is needed to foster evidence-based practices in the future.

CNBP controls transcription by unfolding DNA G-quadruplex structures.

Guanine-rich DNA strands can fold into non-canonical four-stranded secondary structures named G-quadruplexes (G4). Experimental evidences suggest that G4-DNA surrounding transcription start sites act as cis-regulatory elements by either stimulating or inhibiting gene transcription. Therefore, proteins able to target and regulate specific G4 formation/unfolding are crucial for G4-mediated transcriptional control. Here we present data revealing that CNBP acts in vitro as a G4-unfolding protein over a tetramolecular G4 formed by the TG4T oligonucleotide, as well as over the G4 folded in the promoters of several oncogenes. CNBP depletion in cellulo led to a reduction in the transcription of endogenous KRAS, suggesting a regulatory role of CNBP in relieving the transcriptional abrogation due to G4 formation. CNBP activity was also assayed over the evolutionary conserved G4 enhancing the transcription of NOGGIN (NOG) developmental gene. CNBP unfolded in vitro NOG G4 and experiments performed in cellulo and in vivo in developing zebrafish showed a repressive role of CNBP on the transcription of this gene by G4 unwinding. Our results shed light on the mechanisms underlying CNBP way of action, as well as reinforce the notion about the existence and function of G4s in whole living organisms.

Zebrafish Got Spicy: V Course and Symposium of the Latin American Zebrafish Network.

The V Latin American Zebrafish Network Course and Symposium was held from May 4 to 12, 2018 at the Institute of Biotechnology of the National Autonomous University of Mexico. A group of 19 students from eight different countries were selected and trained during a week in a variety of topics and techniques using zebrafish as a model, followed by a 2-day symposium. In this article, we want to point out not only the outstanding activities carried out during the course but also highlight the great experience, the knowledge, and the fantastic relationships we gained from those days.

G-quadruplexes as novel cis-elements controlling transcription during embryonic development.

G-quadruplexes are dynamic structures folded in G-rich single-stranded DNA regions. These structures have been recognized as a potential nucleic acid based mechanism for regulating multiple cellular processes such as replication, transcription and genomic maintenance. So far, their transcriptional role in vivo during vertebrate embryonic development has not yet been addressed. Here, we performed an in silico search to find conserved putative G-quadruplex sequences (PQSs) within proximal promoter regions of human, mouse and zebrafish developmental genes. Among the PQSs able to fold in vitro as G-quadruplex, those present in nog3, col2a1 and fzd5 promoters were selected for further studies. In cellulo studies revealed that the selected G-quadruplexes affected the transcription of luciferase controlled by the SV40 nonrelated promoter. G-quadruplex disruption in vivo by microinjection in zebrafish embryos of either small ligands or DNA oligonucleotides complementary to the selected PQSs resulted in lower transcription of the targeted genes. Moreover, zebrafish embryos and larvae phenotypes caused by the presence of complementary oligonucleotides fully resembled those ones reported for nog3, col2a1 and fzd5 morphants. To our knowledge, this is the first work revealing in vivo the role of conserved G-quadruplexes in the embryonic development, one of the most regulated processes of the vertebrates biology.

Asociación entre maloclusiones y posición de la cabeza y cuello / Association between Malocclusion and Position of the Head and Neck

Con la finalidad de contrastar la hipótesis nula "no hay asociación entre maloclusiones y alteraciones posturales de cabeza y cuello" se realizó un estudio descriptivo transversal en pacientes que consultaron por tratamiento de ortodoncia. La muestra consistió en 116 pacientes con maloclusión atendidos en el Programa de Especialización en Ortodoncia y Ortopedia Dento Máxilo Facial 2007-2009 de la Facultad de Odontología de la Universidad de Chile. Se analizaron las fichas de cada uno de ellos y se buscó asociación entre los valores del Análisis Cráneo Cervical de Rocabado, el cefalograma, estudio de modelos y la evaluación funcional de la lengua. Se encontró asociaciones estadísticas débiles entre la rotación anti horaria del cráneo (ángulo cráneo vertebral y distancia intervertebral C0-C1 disminuidas), con clase III esqueletal que además tenían rotación posterior mandibular y que eran dólico faciales. Altos valores del triángulo hioideo, se asociaron débilmente, con Clase II esqueletal, junto con alteraciones de la posición de la lengua en reposo y retrusión dentaria. Se concluye que en pacientes con maloclusión existe una asociación estadísticamente débil con alteraciones de la postura craneocervical.

In order to test the null hypothesis "there is no association between malocclusion and postural alterations in head and neck" a non-experimental analytical study was carried out in patients with clinical orthodontic treatment. The sample consisted of 116 patients with malocclusion treated at the Specialization Program in Orthodontics and Dento Maxillo Facial Orthopedics from 2007 to 2009, Faculty of Dentistry, Universidad de Chile. We analyzed the records of each patient, for search partnership between the values of Cervical Skull Rocabado Analysis, the cephalogram and study models. We found weak statistical associations between anti-rotation time of the cranium (skull, vertebral angle and distance diminished intervertebral C0-C1), with skeletal class III also had mandibular posterior rotation and were dolicho facial. High values of the hyoid triangle, were weakly associated with skeletal Class II, along with changes in tongue position at rest and dental retrusion. We conclude that in patients with malocclusion there is a statistically weak association with altered craniocervical posture.

Apoptotic and antiapoptotic mechanisms after traumatic brain injury.

Caspase and inhibitor of apoptosis (IAP) expression was examined in rats subjected to moderate traumatic brain injury (TBI) using a parasagittal fluid-percussion brain insult (1.7 to 2.2 atm). Within 1 hour after injury, caspase-8 and -9, two initiators of apoptosis, were predominantly expressed in superficial cortical areas adjacent to the impact site and in the thalamus. Caspase-3, an effector caspase, was evident at 6 hours throughout the traumatized cerebral cortex and hippocampus. Moreover, the authors observed that XIAP, cIAP-1, and cIAP-2, members of the IAP family, were constitutively expressed in the brain. Colocalization of XIAP-immunolabled cells with cell-specific markers indicated that XIAP is expressed within neurons and a subpopulation of oligodendrocytes. Immunoblots of brain extracts revealed that the processed forms of caspase-8, -9, and -3 are present as early as 1 hour after trauma. The appearance of activated caspases corresponded with the detection of cleavage of XIAP into fragments after injury and a concomitant increase in the levels of cIAP-1 and cIAP-2 in the traumatized hemispheres. The current data are consistent with the hypotheses that caspases in both the extrinsic and intrinsic apoptotic pathways are activated after moderate TBI and that IAPs may have a protective role within the brain with alterations in levels and cleavage of IAPs that contribute to cell death in this setting.

Apoptosis after traumatic human spinal cord injury.

OBJECT: Apoptosis is a form of programmed cell death seen in a variety of developmental and disease states, including traumatic injuries. The main objective of this study was to determine whether apoptosis is observed after human spinal cord injury (SCI). The spatial and temporal expression of apoptotic cells as well as the nature of the cells involved in programmed cell death were also investigated. METHODS: The authors examined the spinal cords of 15 patients who died between 3 hours and 2 months after a traumatic SCI. Apoptotic cells were found at the edges of the lesion epicenter and in the adjacent white matter, particularly in the ascending tracts, by using histological (cresyl violet, hematoxylin and eosin) and nuclear staining (Hoechst 33342). The presence of apoptotic cells was supported by staining with the terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick-end labeling technique and confirmed by immunostaining for the processed form of caspase-3 (CPP-32), a member of the interleukin-1beta-converting enzyme/Caenorhabditis elegans D 3 (ICE/CED-3) family of proteases that plays an essential role in programmed cell death. Apoptosis in this series of human SCIs was a prominent pathological finding in 14 of the 15 spinal cords examined when compared with five uninjured control spinal cords. To determine the type of cells undergoing apoptosis, the authors immunostained specimens with a variety of antibodies, including glial fibrillary acidic protein, 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase), and CD45/68. Oligodendrocytes stained with CNPase and a number of apoptotic nuclei colocalized with positive staining for this antibody. CONCLUSIONS: These results support the hypothesis that apoptosis occurs in human SCIs and is accompanied by the activation of caspase-3 of the cysteine protease family. This mechanism of cell death contributes to the secondary injury processes seen after human SCI and may have important clinical implications for the further development of protease inhibitors to prevent programmed cell death.

Intramedullary holocord lipoma in an athlete: case report.

OBJECTIVE AND IMPORTANCE: Holocord intradural lipoma extending from the cervical to the lumbar spine is an exceedingly rare condition. An extensive review of the literature revealed that only eight other cases have been reported, and none occurred during the contemporary magnetic resonance imaging era. CLINICAL PRESENTATION: A case of holocord intradural lipoma that was partially resected with the aid of carbon dioxide laser and intraoperative ultrasound is reported. TECHNIQUE: The intimate relationship of the lipoma to the nerve roots and the absence of a distinct plane between tumor and spinal cord precluded a complete resection of this tumor. At 7 months postoperatively, the patient's dysesthetic pain had resolved almost completely. Proprioception in the lower extremities had improved significantly to the point that the patient was able to walk without a cane and his preoperative Romberg's sign had disappeared. CONCLUSION: Extensive intradural intramedullary spinal lipomas can present in adulthood with symptoms of myelopathy as well as nonradicular pain. We encourage early surgical debulking of the tumor to prevent further progression of symptoms and to offer the possibility of neurological improvement.

Follow-up computerized tomography (CT) scans in moderate and severe head injuries: correlation with Glasgow Coma Scores (GCS), and complication rate.

BACKGROUND: The wide availability of computerized tomography (CT) scan has popularized its use in initial and follow-up evaluations of head trauma patients. Follow-up CT scans of clinically stable patients, however, may not provide additional information, but could potentially subject the patients to secondary injuries. The authors investigated the correlation between CT scans and Glasgow coma score (GCS), and complication rate during follow-up CT scans in an attempt to dissuade clinicians from obtaining unnecessary follow-up CT scans. METHODS: The medical records of 180 patients with blunt head trauma were retrospectively reviewed. Only patients (117) with moderate and severe head injuries were included in the study. A total of 319 follow-up brain CT's for 94 patients were obtained. RESULTS: The change in CT scans was compared to the GCS the day of the scan. These two variables were found to be positively correlated by Chi-square analysis (chi 2 = 81.2; alpha < 0.001). CPP trend was found also be correlated with CT scan evolution by the Chi-square analysis (alpha < 0.001). When patients had unchanged or improved GCS, 73.1% had improved or the same CT appearance. When patients had a worse GCS, the CT was worse in 77.9%. A 16.9% (54/319) complication rate was documented during the follow-up scans (hemodynamic instability, increased intracranial pressure, desaturation, and agitation). This rate was higher in severe head trauma (GCS 3-8) patients than in moderate head injury (GCS 9-12) patients. Hemodynamic instability was the most common complication, which comprises 42.6% (23/54) of all complications. CONCLUSION: Because of the correlation between the CT scan appearance and the clinical status, as well as the detrimental effect of mobilizing critically ill patients, the authors urge the use of follow-up CT scans only in patients with clinical deterioration unexplained by ICP changes alone.

The expression of regenerative growth factors in chronic liver injury and repair.

Acute hepatic injury initiates known cellular and molecular events for regeneration. In contrast, the molecular mechanisms of repair following chronic liver injuries have not been defined. Transforming growth factor alpha (TGF alpha) and hepatocyte growth factor (HGF) are hepatocyte mitogens whose in vivo expression in liver is central to the regulation of regeneration. To study the role of TGF alpha and HGF in liver injury and repair, we used a model of reversible biliary obstruction without a bilioenteric anastomosis. In rats, the common bile duct was obstructed either by a vessel loop suspended from the abdominal wall (LOOP) or by ligation and division (DLD). After 7 days of obstruction, animals were autopsied or were decompressed by subcutaneous release of the loop and then autopsied at 1, 2, 4, 7, or 10 days of postdecompression. Serum bilirubin (mg/dl) increased to 14.8 +/- 2.9 (DLD) and 10.3 +/- 3.0 (LOOP) (+/- SEM, NS, ANOVA) at 7 days of obstruction. Liver sections demonstrated equal ductal hyperplasia and collagen deposition after LOOP and DLD. Biliary decompression reversed bile duct proliferation and normalized bilirubin. Analysis of injured and repairing liver mRNA by ribonuclease protection assay showed that TGF alpha mRNA levels were not significantly altered by injury or during repair. HGF mRNA was elevated following obstruction and showed increased expression 1 day after decompression, peaking at 2 days of repair. This evidence of modulation of HGF during liver repair following chronic cholestatic injury suggests that HGF may have a role in cellular proliferation during repair or act as a compensatory growth factor during injury.

Polychlorinated biphenyls in Mexican cereals and their packings.

The content of polychlorinated biphenyls in Mexican cereals and in their packaging materials was determined. All the samples had polychlorinated biphenyls. In all the food samples and in 73% of the packings, the concentrations of polychlorinated biphenyls exceeded the tolerance limits established by the U.S. Food and Drug Administration. A correlation was found between the values for polychlorinated biphenyls in the cereals and those in their packings, therefore, it is concluded that the main source of polychlorinated biphenyls in Mexican cereals is the transference of those present in the recycled paperboard used for the packings.