Development and internal validation of a clinical score to predict neonatal hypoglycaemia in women with gestational diabetes.

INTRODUCTION: Gestational diabetes (GD) is a risk factor for neonatal hypoglycaemia (NH), but other factors can increase this risk. OBJECTIVES: To create a score to predict NH in women with GD. METHODS: Retrospective study of women with GD with a live singleton birth between 2012 and 2017 from the Portuguese GD registry. Pregnancies with and without NH were compared. A logistic regression was used to study NH predictors. Variables independently associated with NH were used to score derivation. The model's internal validation was performed by a bootstrapping. The association between the score and NH was assessed by logistic regression. RESULTS: We studied 10216 pregnancies, 410 (4.0%) with NH. The model's AUC was 0.628 (95%CI: 0.599-0.657). Optimism-corrected c-index: 0.626. Points were assigned to variables associated with NH in proportion to the model's lowest regression coefficient: insulin-treatment 1, preeclampsia 3, preterm delivery 2, male sex 1, and small-for-gestational-age 2, or large-for-gestational-age 3. NH prevalence by score category 0-1, 2, 3, 4, and ≥5 was 2.3%, 3.0%, 4.5%, 6.0%, 7.4%, and 11.5%, respectively. Per point, the OR for NH was 1.35 (95% CI: 1.27-1.42). A score of 2, 3, 4, 5 or ≥6 (versus ≤1) had a OR for NH of 1.67 (1.29-2.15), 2.24 (1.65-3.04), 2.83 (2.02-3.98), 3.08 (1.83-5.16), and 6.84 (4.34-10.77), respectively. CONCLUSION: Per each score point, women with GD had 35% higher risk of NH. Those with ≥6 points had 6.8-fold higher risk of NH compared to a score ≤1. Our score may be useful for identifying women at a higher risk of NH.

Association between foetal sex and adverse neonatal outcomes in women with gestational diabetes.

AIMS: Foetal male sex is associated with adverse perinatal outcomes. However, studies evaluating the impact of foetal sex on perinatal outcomes in women with gestational diabetes (GDM) are scarce. We studied whether male new-born sex is associated with neonatal outcomes, in women with GDM. METHODS: This is a retrospective study based on the national Portuguese register of GDM. All women with live-born singleton pregnancies between 2012 and 2017 were eligible for study inclusion. Primary endpoints under analysis were neonatal hypoglycaemia, neonatal macrosomia, respiratory distress syndrome (RDS) and neonatal intensive care unit (NICU) admission. We excluded women with missing data on the primary endpoint. Pregnancy data and neonatal outcomes between female and male new-borns were compared. Multivariate logistic regression models were built. RESULTS: We studied 10,768 new-borns in mothers with GDM, 5635 (52.3%) male, 438 (4.1%) had neonatal hypoglycaemia, 406 (3.8%) were macrosomic, 671 (6.2%) had RDS, and 671 (6.2%) needed NICU admission. Male new-borns were more frequently small or large for gestational age. No differences were observed on maternal age, body mass index, glycated haemoglobin, anti-hyperglycaemic treatment, pregnancy complications or gestational age at delivery. In the multivariate regression analysis, male sex was independently associated with neonatal hypoglycaemia [OR 1.26 (IC 95%: 1.04-1.54), p = 0.02], neonatal macrosomia [1.94 (1.56-2.41), p < 0.001], NICU admission [1.29 (1.07-1.56), p = 0.009], and RDS [1.35 (1.05-1.73, p = 0.02]. CONCLUSIONS: Male new-borns have an independent 26% higher risk of neonatal hypoglycaemia, 29% higher risk of NICU admission, 35% higher risk of RDS, and almost twofold higher risk of macrosomia, compared to female new-borns.

Implementation of Evidence-Based Practice in Peripheral Intravenous Catheter Care.

BACKGROUND: Inserting a peripheral intravenous (PIV) catheter is a common health care procedure; however, risks include phlebitis, extravasation, and accidental dislodgement. Using evidence-based practices (EBPs) can reduce these risks. PURPOSE: The purpose of this study was to implement an evidence-based PIV catheter care bundle and a decision-making algorithm. METHODS: A quasi-experimental study design was used. A care bundle and an evidence-based decision-making algorithm were implemented on a medical unit. Outcomes included length of PIV catheter dwell time, phlebitis and other complications, and health professionals' adherence to the interventions. RESULTS: A total of 364 PIV catheters were assessed. PIV catheter dwell time decreased from 3.6 to 2.9 days ( P < .001), and phlebitis rates decreased from 14.8% to 4.9% ( P < .05). Health professionals' adherence increased from 84.3% to 91.8%. CONCLUSIONS: Implementing EBPs can improve care provided to patients with PIV catheters.

Maternal height as a predictor of glucose intolerance in the postpartum and its relationship with maternal pre-gestational weight.

PURPOSE: Short stature predicts higher risk of developing type 2 diabetes. We studied the association between height and glucose intolerance in women with gestational diabetes mellitus (GDM) and whether this association differed according to body mass index (BMI). METHODS: Retrospective study of the Portuguese GDM registry. EXCLUSION CRITERIA: missing data on postpartum oral glucose tolerance test (OGTT) or BMI. ENDPOINT: postpartum glucose intolerance (diabetes mellitus or prediabetes on the 6-8 weeks postpartum OGTT). Women were divided by mean height and compared. A multivariate logistic regression was used, and the analysis was stratified by BMI (cut-off: 30 kg/m2) and interaction was tested. RESULTS: We included 7402 women; mean height was 161.9 ± 6.2 cm. Taller women had lower BMI and lower rates of glucose intolerance (6.8 vs. 8.8%, p = 0.002). Women with BMI < 30 kg/m2 were taller than those with obesity. Height associated with glucose intolerance. The multivariate adjusted OR of glucose intolerance was 0.98 (95% CI 0.96-0.99), p = 0.001, per 1 cm increase in height. This association was only observed in women with BMI < 30 kg/m2: OR 0.97 (95% CI 0.95-0.99), < 0.001. There was no such association in women with BMI ≥ 30 kg/m2: OR 0.99 (95% CI 0.97-1.02), p = 0.65. P for interaction between BMI and height was 0.09. CONCLUSIONS: In non-obese pre-gestational women, height is inversely associated with postpartum glucose intolerance. Per 1 cm increase in height, women present a 3% decrease in the risk of developing diabetes mellitus or prediabetes.

First trimester fasting glucose and glycated haemoglobin cut-offs associated with abnormal glucose homeostasis in the post-partum reclassification in women with hyperglycaemia in pregnancy.

Hyperglycaemia first detected during pregnancy is either gestational diabetes mellitus (GDM) or previous undiagnosed diabetes. We aimed to study if there were a first trimester fasting glycaemia (FTG) and a glycated haemoglobin (HbA1c) cut-off values associated with type 2 diabetes mellitus (T2DM) or abnormal glucose homeostasis (AGH) at the post-partum oral glucose tolerance test (OGTT) reclassification. We retrospectively studied a group of pregnant women from the Portuguese National Registry of GDM. Receiver-operating characteristic (ROC) curves were used to determine the best FTG and HbA1c cut-offs to predict T2DM and AGH. We studied 4068 women. The area under the ROC curves (AUC) for the association with T2DM was 0.85 (0.80-0.90) for FTG and 0.85 (0.80-0.91) for HbA1c. The best FTG cut-off for association with T2DM was 99 mg/dL: sensitivity 77.4%, specificity 74.3%, positive predictive value (PPV) 4.8%, and negative predictive value (NPV) 99.5%. The best HbA1c cut-off for association with T2DM was 5.4%: sensitivity 79.0%, specificity 80.1%, PPV 5.7%, and NPV 99.6%. The AUC for the association of FTG and HbA1c with AGH were 0.73 (0.70-0.76) and 0.71 (0.67-0.74), respectively. The best FTG cut-off for predicting AGH was 99 mg/dL: sensitivity 59.4%, specificity 76.2%, PPV 17.0%, and NPV 95.8%. The best HbA1c cut-off was 5.4%: sensitivity 48.7%, specificity 81.5%, PPV 17.8%, and NPV 95.1%. We suggest an FTG of 99 mg/dL and an HbA1c of 5.4% as the best cut-offs below which T2DM is unlikely to be present. Almost all patients with FTG < 99 mg/dL and HbA1c < 5.4% did not reclassify as T2DM. These early pregnancy cut-offs might alert the physician for the possibility of a previous undiagnosed diabetes and alert them to the importance of testing for it after delivery.

Incremental Learning for Dermatological Imaging Modality Classification.

With the increasing adoption of teledermatology, there is a need to improve the automatic organization of medical records, being dermatological image modality a key filter in this process. Although there has been considerable effort in the classification of medical imaging modalities, this has not been in the field of dermatology. Moreover, as various devices are used in teledermatological consultations, image acquisition conditions may differ. In this work, two models (VGG-16 and MobileNetV2) were used to classify dermatological images from the Portuguese National Health System according to their modality. Afterwards, four incremental learning strategies were applied to these models, namely naive, elastic weight consolidation, averaged gradient episodic memory, and experience replay, enabling their adaptation to new conditions while preserving previously acquired knowledge. The evaluation considered catastrophic forgetting, accuracy, and computational cost. The MobileNetV2 trained with the experience replay strategy, with 500 images in memory, achieved a global accuracy of 86.04% with only 0.0344 of forgetting, which is 6.98% less than the second-best strategy. Regarding efficiency, this strategy took 56 s per epoch longer than the baseline and required, on average, 4554 megabytes of RAM during training. Promising results were achieved, proving the effectiveness of the proposed approach.

Metformin combined with insulin in women with gestational diabetes mellitus: a propensity score-matched study.

AIM: Metformin use in gestational diabetes (GDM) is a common practice. Although its use in combination with insulin might be advantageous, it was never formally tested. We studied whether combined treatment was associated with better obstetric or neonatal outcomes compared to insulin alone. METHODS: This is a retrospective study, using the Portuguese National Registry of GDM (2012-2017), of women treated with insulin ± metformin. Primary endpoints were obstetric and neonatal complications. Secondary endpoints were gestational weight gain (GWG) and insulin dose. A propensity score-matched analysis was performed to balance the distribution of age, BMI, insulin treatment duration, HbA1c, first trimester diagnosis of GDM and previous GDM or macrosomia. Women treated with metformin plus insulin and insulin only were then compared. RESULTS: A total of 4034 women were treated with insulin or insulin plus metformin (10.2%). After propensity score matching, we studied two groups of 386 patients. Obstetric and neonatal complications were similar. Women treated with metformin plus insulin had 201 (52.1%) obstetric complications versus 184 (47.7%) in insulin-only group, p = 0.22; and 112 (29.0%) neonatal complications versus 96 (24.9%), p = 0.19. Patients treated with metformin plus insulin had similar GWG, excessive weight gain and insulin dose compared to the insulin-only group. CONCLUSIONS: Women with GDM treated with insulin plus metformin had similar obstetric and neonatal complications, weight gained and insulin dose compared to those only treated with insulin.

Integrating Domain Knowledge into Deep Learning for Skin Lesion Risk Prioritization to Assist Teledermatology Referral.

Teledermatology has developed rapidly in recent years and is nowadays an essential tool for early diagnosis. In this work, we aim to improve existing Teledermatology processes for skin lesion diagnosis by developing a deep learning approach for risk prioritization with a dataset of retrospective data from referral requests of the Portuguese National Health System. Given the high complexity of this task, we propose a new prioritization pipeline guided and inspired by domain knowledge. We explored automatic lesion segmentation and tested different learning schemes, namely hierarchical classification and curriculum learning approaches, optionally including additional patient metadata. The final priority level prediction can then be obtained by combining predicted diagnosis and a baseline priority level accounting for explicit expert knowledge. In both the differential diagnosis and prioritization branches, lesion segmentation with 30% tolerance for contextual information was shown to improve classification when compared with a flat baseline model trained on original images; furthermore, the addition of patient information was not beneficial for most experiments. Curriculum learning delivered better results than a flat or hierarchical approach. The combination of diagnosis information and a knowledge map, created in collaboration with dermatologists, together with the priority achieved interesting results (best macro F1 of 43.93% for a validated test set), paving the way for new data-centric and knowledge-driven approaches.

MicroRNA-145 Regulates Neural Stem Cell Differentiation Through the Sox2-Lin28/let-7 Signaling Pathway.

MicroRNAs (miRNAs or miRs) regulate several biological functions, including cell fate determination and differentiation. Although miR-145 has already been described to regulate glioma development, its precise role in neurogenesis has never been addressed. miR-145 represses sex-determining region Y-box 2 (Sox2), a core transcription factor of embryonic stem cells (ESCs), to inhibit pluripotency and self-renewal in human ESCs. In addition, the Sox2-Lin28/let-7 signaling pathway regulates proliferation and neurogenesis of neural precursors. In this study, we aimed to investigate the precise role of miR-145 in neural stem cell (NSC) fate decision, and the possible involvement of the Sox2-Lin28/let-7 signaling pathway in miR-145 regulatory network. Our results show for the first time that miR-145 expression significantly increased after induction of mouse NSC differentiation, remaining elevated throughout this process. Forced miR-145 downregulation decreased neuronal markers, namely ßIII-tubulin, NeuN, and MAP2. Interestingly, throughout NSC differentiation, protein levels of Sox2 and Lin28, a well-known suppressor of let-7 biogenesis, decreased. Of note, neuronal differentiation also resulted in let-7a and let-7b upregulation. Transfection of NSCs with anti-miR-145, in turn, increased both Sox2 and Lin28 protein levels, while decreasing both let-7a and let-7b. More importantly, Sox2 and Lin28 silencing partially rescued the impairment of neuronal differentiation induced by miR-145 downregulation. In conclusion, our results demonstrate a novel role for miR-145 during NSC differentiation, where miR-145 modulation of Sox2-Lin28/let-7 network is crucial for neurogenesis progression. Stem Cells 2016;34:1386-1395.

MicroRNA-34a Modulates Neural Stem Cell Differentiation by Regulating Expression of Synaptic and Autophagic Proteins.

We have previously demonstrated the involvement of specific apoptosis-associated microRNAs (miRNAs), including miR-34a, in mouse neural stem cell (NSC) differentiation. In addition, a growing body of evidence points to a critical role for autophagy during neuronal differentiation, as a response-survival mechanism to limit oxidative stress and regulate synaptogenesis associated with this process. The aim of this study was to further investigate the precise role of miR-34a during NSC differentiation. Our results showed that miR-34a expression was markedly downregulated during neurogenesis. Neuronal differentiation and cell morphology, synapse function, and electrophysiological maturation were significantly impaired in miR-34a-overexpressing NSCs. In addition, synaptotagmin 1 (Syt1) and autophagy-related 9a (Atg9a) significantly increased during neurogenesis. Pharmacological inhibition of autophagy impaired both neuronal differentiation and cell morphology. Notably, we showed that Syt1 and Atg9a are miR-34a targets in neural differentiation context, markedly decreasing after miR-34a overexpression. Syt1 overexpression and rapamycin-induced autophagy partially rescued the impairment of neuronal differentiation by miR-34a. In conclusion, our results demonstrate a novel role for miR-34a regulation of NSC differentiation, where miR-34a downregulation and subsequent increase of Syt1 and Atg9a appear to be crucial for neurogenesis progression.

Tauroursodeoxycholic acid increases neural stem cell pool and neuronal conversion by regulating mitochondria-cell cycle retrograde signaling.

The low survival and differentiation rates of stem cells after either transplantation or neural injury have been a major concern of stem cell-based therapy. Thus, further understanding long-term survival and differentiation of stem cells may uncover new targets for discovery and development of novel therapeutic approaches. We have previously described the impact of mitochondrial apoptosis-related events in modulating neural stem cell (NSC) fate. In addition, the endogenous bile acid, tauroursodeoxycholic acid (TUDCA) was shown to be neuroprotective in several animal models of neurodegenerative disorders by acting as an anti-apoptotic and anti-oxidant molecule at the mitochondrial level. Here, we hypothesize that TUDCA might also play a role on NSC fate decision. We found that TUDCA prevents mitochondrial apoptotic events typical of early-stage mouse NSC differentiation, preserves mitochondrial integrity and function, while enhancing self-renewal potential and accelerating cell cycle exit of NSCs. Interestingly, TUDCA prevention of mitochondrial alterations interfered with NSC differentiation potential by favoring neuronal rather than astroglial conversion. Finally, inhibition of mitochondrial reactive oxygen species (mtROS) scavenger and adenosine triphosphate (ATP) synthase revealed that the effect of TUDCA is dependent on mtROS and ATP regulation levels. Collectively, these data underline the importance of mitochondrial stress control of NSC fate decision and support a new role for TUDCA in this process.

Mitochondrial translocation of p53 modulates neuronal fate by preventing differentiation-induced mitochondrial stress.

AIMS: Apoptosis regulatory proteins, such as p53, play a pivotal role in neural differentiation, through mechanisms independent of cell death. In addition, p53 has been identified as an important regulator of mitochondrial survival response, maintaining mitochondrial DNA (mtDNA) integrity and oxidative protection. The aim of this study was to determine the role of mitochondrial p53 in organelle damage and neural differentiation. RESULTS: Our results show that mitochondrial apoptotic events such as reactive oxygen species production, mitochondrial membrane permeabilization, and cytochrome c release are typical of early-stage mouse neural stem cell differentiation, which occurs 3-18 h after induction of differentiation, with no evidence of cell death. In addition, decreased mtDNA content, lipidated LC3 (LC3-II), colocalization of mitochondria and LC3-II puncta, and mitochondria-associated Parkin are consistent with activation of mitophagy. Importantly, at early stages of neural differentiation, p53 was actively translocated to mitochondria and attenuated mitochondrial oxidative stress, cytochrome c release, and mitophagy. Forced mitochondrial translocation of p53 increased neurogenic potential and neurite outgrowth. INNOVATION AND CONCLUSION: In conclusion, our results reveal a novel role for mitochondrial p53, which modulates mitochondrial damage and apoptosis-related events in the context of neural differentiation, thus enhancing neuronal fate.

Death receptors and mitochondria: two prime triggers of neural apoptosis and differentiation.

BACKGROUND: Stem cell therapy is a strategy far from being satisfactory and applied in the clinic. Poor survival and differentiation levels of stem cells after transplantation or neural injury have been major problems. Recently, it has been recognized that cell death-relevant proteins, notably those that operate in the core of the executioner apoptosis machinery are functionally involved in differentiation of a wide range of cell types, including neural cells. SCOPE OF REVIEW: This article will review recent studies on the mechanisms underlying the non-apoptotic function of mitochondrial and death receptor signaling pathways during neural differentiation. In addition, we will discuss how these major apoptosis-regulatory pathways control the decision between differentiation, self-renewal and cell death in neural stem cells and how levels of activity are restrained to prevent cell loss as final outcome. MAJOR CONCLUSIONS: Emerging evidence suggests that, much like p53, caspases and Bcl-2 family members, the two prime triggers of cell death pathways, death receptors and mitochondria, may influence proliferation and differentiation potential of stem cells, neuronal plasticity, and astrocytic versus neuronal stem cell fate decision. GENERAL SIGNIFICANCE: A better understanding of the molecular mechanisms underlying key checkpoints responsible for neural differentiation as an alternative to cell death will surely contribute to improve neuro-replacement strategies.

TAp63γ demethylation regulates protein stability and cellular distribution during neural stem cell differentiation.

p63 is a close relative of the p53 tumor suppressor and transcription factor that modulates cell fate. The full-length isoform of p63, containing a transactivation (TA) domain (TAp63) is an essential proapoptotic protein in neural development. The role of p63 in epithelial development is also well established; however, its precise function during neural differentiation remains largely controversial. Recently, it has been demonstrated that several conserved elements of apoptosis are also integral components of cellular differentiation; p53 directly interacts with key regulators of neurogenesis. The aim of this study was to evaluate the role of p63 during mouse neural stem cell (NSC) differentiation and test whether the histone H3 lysine 27-specific demethylase JMJD3 interacts with p63 to redirect NSCs to neurogenesis. Our results showed that JMJD3 and TAp63γ are coordinately regulated to establish neural-specific gene expression programs in NSCs undergoing differentiation. JMJD3 overexpression increased TAp63γ levels in a demethylase activity-dependent manner. Importantly, overexpression of TAp63γ increased ß-III tubulin whereas downregulation of TAp63γ by specific p63 siRNA decreased ß-III tubulin. Immunoprecipitation assays demonstrated direct interaction between TAp63γ and JMJD3, and modulation of TAp63γ methylation status by JMJD3-demethylase activity. Importantly, the demethylase activity of JMJD3 influenced TAp63γ protein stabilization and cellular distribution, as well as TAp63γ-regulated neurogenesis. These findings clarify the role of p63 in adult neural progenitor cells and reveal TAp63γ as a direct target for JMJD3-mediated neuronal commitment.

Unspliced precursors of NMD-sensitive ß-globin transcripts exhibit decreased steady-state levels in erythroid cells.

Nonsense-mediated mRNA decay (NMD) is a quality control mechanism that detects and rapidly degrades mRNAs carrying premature translation-termination codons (PTCs). Mammalian NMD depends on both splicing and translation, and requires recognition of the premature stop codon by the cytoplasmic ribosomes. Surprisingly, some published data have suggested that nonsense codons may also affect the nuclear metabolism of the nonsense-mutated transcripts. To determine if nonsense codons could influence nuclear events, we have directly assessed the steady-state levels of the unspliced transcripts of wild-type and PTC-containing human ß-globin genes stably transfected in mouse erythroleukemia (MEL) cells, after erythroid differentiation induction, or in HeLa cells. Our analyses by ribonuclease protection assays and reverse transcription-coupled quantitative PCR show that ß-globin pre-mRNAs carrying NMD-competent PTCs, but not those containing a NMD-resistant PTC, exhibit a significant decrease in their steady-state levels relatively to the wild-type or to a missense-mutated ß-globin pre-mRNA. On the contrary, in HeLa cells, human ß-globin pre-mRNAs carrying NMD-competent PTCs accumulate at normal levels. Functional analyses of these pre-mRNAs in MEL cells demonstrate that their low steady-state levels do not reflect significantly lower pre-mRNA stabilities when compared to the normal control. Furthermore, our results also provide evidence that the relative splicing efficiencies of intron 1 and 2 are unaffected. This set of data highlights potential nuclear pathways that might be promoter- and/or cell line-specific, which recognize the NMD-sensitive transcripts as abnormal. These specialized nuclear pathway(s) may be superimposed on the general NMD mechanism.

Distinct regulatory functions of calpain 1 and 2 during neural stem cell self-renewal and differentiation.

Calpains are calcium regulated cysteine proteases that have been described in a wide range of cellular processes, including apoptosis, migration and cell cycle regulation. In addition, calpains have been implicated in differentiation, but their impact on neural differentiation requires further investigation. Here, we addressed the role of calpain 1 and calpain 2 in neural stem cell (NSC) self-renewal and differentiation. We found that calpain inhibition using either the chemical inhibitor calpeptin or the endogenous calpain inhibitor calpastatin favored differentiation of NSCs. This effect was associated with significant changes in cell cycle-related proteins and may be regulated by calcium. Interestingly, calpain 1 and calpain 2 were found to play distinct roles in NSC fate decision. Calpain 1 expression levels were higher in self-renewing NSC and decreased with differentiation, while calpain 2 increased throughout differentiation. In addition, calpain 1 silencing resulted in increased levels of both neuronal and glial markers, ß-III Tubulin and glial fibrillary acidic protein (GFAP). Calpain 2 silencing elicited decreased levels of GFAP. These results support a role for calpain 1 in repressing differentiation, thus maintaining a proliferative NSC pool, and suggest that calpain 2 is involved in glial differentiation.

p53 interaction with JMJD3 results in its nuclear distribution during mouse neural stem cell differentiation.

Conserved elements of apoptosis are also integral components of cellular differentiation. In this regard, p53 is involved in neurogenesis, being required for neurite outgrowth in primary neurons and for axonal regeneration in mice. Interestingly, demethylases regulate p53 activity and its interaction with co-activators by acting on non-histone proteins. In addition, the histone H3 lysine 27-specific demethylase JMJD3 induces ARF expression, thereby stabilizing p53 in mouse embryonic fibroblasts. We hypothesized that p53 interacts with key regulators of neurogenesis to redirect stem cells to differentiation, as an alternative to cell death. Specifically, we investigated the potential cross-talk between p53 and JMJD3 during mouse neural stem cell (NSC) differentiation. Our results demonstrated that JMJD3 mRNA and protein levels were increased early in mouse NSC differentiation, when JMJD3 activity was readily detected. Importantly, modulation of JMJD3 in NSCs resulted in changes of total p53 protein, coincident with increased ARF mRNA and protein expression. ChIP analysis revealed that JMJD3 was present at the promoter and exon 1 regions of ARF during neural differentiation, although without changes in H3K27me3. Immunoprecipitation assays demonstrated a direct interaction between p53 and JMJD3, independent of the C-terminal region of JMJD3, and modulation of p53 methylation by JMJD3-demethylase activity. Finally, transfection of mutant JMJD3 showed that the demethylase activity of JMJD3 was crucial in regulating p53 cellular distribution and function. In conclusion, JMJD3 induces p53 stabilization in mouse NSCs through ARF-dependent mechanisms, directly interacts with p53 and, importantly, causes nuclear accumulation of p53. This suggests that JMJD3 and p53 act in a common pathway during neurogenesis.

Mutational spectrum of delta-globin gene in the Portuguese population.

The phenotype of increased Hb A2 typical of beta-thalassaemia (beta-thal) carriers can be reduced to normal or borderline values because of the co-inheritance of a delta-globin gene (HBD, MIM #142000) mutation, which may lead to misinterpretation of diagnostic results. To know the spectrum of delta-globin mutations in the Portuguese population we performed a mutational analysis of the delta-globin gene in a group of 51 Portuguese beta-thal carriers presenting microcytosis, hypochromia and a normal/borderline Hb A2 level and in another group of 15 individuals suspected to have delta-globin structural abnormalities. The heterozygosity for the beta(+)IVS-I-6T-->C (HBB:c. 92+6T>C) mutation was the main cause for the mentioned atypical beta-thal carrier phenotype. Furthermore, eight individuals were double heterozygous for one common beta-thal mutation and the delta(+)Cd27G-->T mutation (Hb A2-Yialousa; HBD:c.82G>T). One of them also presented a novel delta-globin gene promoter mutation,-80G-->A (HBD:c.-130G>A), responsible for about 25% decrease of the promoter activity in transient expression assays. One the other hand, in the other group of 15 individuals suspected to have delta-globin structural abnormalities observed by biochemical methods, some known Hb A2 variants were identified - Hb A2' (HBD:c.49G>C), Hb A2-Babinga (HBD:c.410G>A), and Hb A2-Wrens (HBD:c.295G>A), and the novel Hb A2-Fogo [delta64(E8)(Gly-->Ser); (HBD:c.193G>A)]. This novel Hb A2 variant was observed segregating in linkage with Hb E (HBB:c.79G>A) in a three generation family. In conclusion, six different delta-globin mutations were found, being two of them new molecular defects. All delta-alleles identified were found linked to the expected beta-globin cluster haplotype. All mutations caused a low Hb A2 level and through this could lead to misdiagnosis when inherited together with a beta-thal allele.