Floral Color, Anthocyanin Synthesis Gene Expression and Control in Cape Erica Species.

Introduction: The Cape Floristic Region (CFR) is a biodiversity hotspot, recognized globally for its unusually high levels of endemism. The origins of this biodiversity are a long-standing topic of research. The largest "Cape clade," Erica, radiated dramatically in the CFR, its ca. 690 species arising within 10-15 Ma. Notable between- and within-species flower color variation in Erica may have contributed to the origins of species diversity through its impact on pollinator efficiency and specificity. Methods: We investigate the expression and function of the genes of the anthocyanin biosynthesis pathway that controls floral color in 12 Erica species groups using RT-qPCR and UPLC-MS/MS. Results: Shifts from ancestral pink- or red- to white- and/or yellow flowers were associated with independent losses of single pathway gene expression, abrogation of the entire pathway due to loss of the expression of a transcription factor or loss of function mutations in pathway genes. Discussion: Striking floral color shifts are prevalent amongst the numerous species of Cape Erica. These results show independent origins of a palette of mutations leading to such shifts, revealing the diverse genetic basis for potentially rapid evolution of a speciation-relevant trait.

An approach to determining anthocyanin synthesis enzyme gene expression in an evolutionary context: an example from Erica plukenetii.

BACKGROUND AND AIMS: Floral colour in angiosperms can be controlled by variations in the expression of the genes of the anthocyanin pathway. Floral colour shifts influence pollinator specificity. Multiple shifts in floral colour occurred in the diversification of the genus Erica (Ericaceae), from plesiomorphic pink to, for example, red or white flowers. Variation in anthocyanin gene expression and its effects on floral colour in the red-, pink- and white-flowered Erica plukenetii species complex was investigated. METHODS: Next generation sequencing, reverse transcriptase PCR and real-time reverse transcriptase quantitative PCR were used to quantify anthocyanin gene expression. KEY RESULTS: Non-homologous mutations causing loss of expression of single genes were found, indicating that the cause was likely to be mutations in transcription factor binding sites upstream of the 5'-untranslated region of the genes, and this was confirmed by sequencing. CONCLUSIONS: Independent evolution and subsequent loss of expression of anthocyanin genes may have influenced diversification in the E. plukenetii species complex. The approach developed here should find more general application in studies on the role of floral colour shifts in diversification.

MR Imaging Scoring System for White Matter Injury after Deep Medullary Vein Thrombosis and Infarction in Neonates.

BACKGROUND AND PURPOSE: Advanced imaging techniques have allowed earlier and more accurate detection of cerebral deep medullary vein thrombosis and infarction. Our objective was to develop an MR imaging scoring system to evaluate the severity of white matter injury in neonates with deep medullary vein thrombosis and infarction. MATERIALS AND METHODS: This was a retrospective study of infants born ≥32 weeks' gestation (2000-2016) diagnosed with deep medullary vein thrombosis and infarction on neuroimaging in the first 30 days of life. A 102-point deep medullary vein white matter injury global severity score was developed. MR images were scored by 2 pediatric radiologists. Subject clinical data and regional and global severity scores were recorded. RESULTS: Fifty-one patients (mean gestational age, 37.3 ± 2.2 weeks; mean birth weight, 3182 ± 720 g) were included with a mean age at diagnosis via MR imaging of postnatal day 10.1 ± 6.1. Global severity scores ranged from 1 to 53, with a median score of 11 (interquartile range, 5-25). Lesions were more common in the frontal and parietal regions and less common in the occipital and temporal regions. Fifty-five percent of the group had neonatal seizures. No difference in perinatal risk factors (gestational age, birthweight, 5-minute Apgar score, chorioamnionitis, delivery room resuscitation, ventilator, or inotrope requirement) was observed among severity score quartiles. CONCLUSIONS: An MR imaging scoring system provides a comprehensive and objective classification of WM injury after deep medullary vein thrombosis and infarction in late preterm and term neonates. The global severity score is independent of gestational age and other antenatal risk factors, consistent with presentation in previously healthy-appearing neonates.

Parenting style impacts cognitive and behavioural outcomes of former preterm infants: A systematic review.

We sought to evaluate published evidence in aggregate regarding the impact of parenting style on the cognitive and behavioural outcomes of former preterm infants. We searched 5 databases using germane MeSH terms. Parenting style was defined as any descriptor of parenting using ≥2 dimensions on published parenting axes. We evaluated studies for quality of evidence and strength of recommendations using standardized tools and categorized summative recommendations by parenting axis and child outcome. Twenty-seven articles met our inclusion criteria. Parental responsivity is the only parenting axis strongly associated with both improved child cognition and behaviour. Parental demandingness is associated only with improved child cognition, and parental warmth and rejection are associated only with child behaviour. Parental coercion is not associated with subsequent child outcomes. Parental responsivity may be essential in optimizing neurodevelopment in former preterm infants. More targeted studies are needed to inform this relationship and identify opportunities for intervention.

The biodiversity hotspot as evolutionary hot-bed: spectacular radiation of Erica in the Cape Floristic Region.

BACKGROUND: The disproportionate species richness of the world's biodiversity hotspots could be explained by low extinction (the evolutionary "museum") and/or high speciation (the "hot-bed") models. We test these models using the largest of the species rich plant groups that characterise the botanically diverse Cape Floristic Region (CFR): the genus Erica L. We generate a novel phylogenetic hypothesis informed by nuclear and plastid DNA sequences of c. 60 % of the c. 800 Erica species (of which 690 are endemic to the CFR), and use this to estimate clade ages (using RELTIME; BEAST), net diversification rates (GEIGER), and shifts in rates of diversification in different areas (BAMM; MuSSE). RESULTS: The diversity of Erica species in the CFR is the result of a single radiation within the last c. 15 million years. Compared to ancestral lineages in the Palearctic, the rate of speciation accelerated across Africa and Madagascar, with a further burst of speciation within the CFR that also exceeds the net diversification rates of other Cape clades. CONCLUSIONS: Erica exemplifies the "hotbed" model of assemblage through recent speciation, implying that with the advent of the modern Cape a multitude of new niches opened and were successively occupied through local species diversification.

Increasing F2-isoprostanes in the first month after birth predicts poor respiratory and neurodevelopmental outcomes in very preterm infants.

OBJECTIVE: This study examined the association between increased early oxidative stress, measured by F2-isoprostanes (IsoPs), and respiratory morbidity at term equivalent age and neurological impairment at 12 months of corrected age (CA). STUDY DESIGN: Plasma samples were collected from 136 premature infants on days 14 and 28 after birth. All participants were infants born at ⩽28 weeks of gestational age enrolled into the Prematurity and Respiratory Outcomes Program (PROP) study. Respiratory morbidity was determined at 40 weeks of postmenstrual age (PMA) by the Respiratory Severity Index (RSI), a composite measure of oxygen and pressure support. Neurodevelopmental assessment was performed using the Developmental Assessment of Young Children (DAYC) at 12 months of CA. Multivariable logistic regression models estimated associations between IsoP change, RSI and DAYC scores. Mediation analysis was performed to determine the relationship between IsoPs and later outcomes. RESULTS: Developmental data were available for 121 patients (90% of enrolled) at 12 months. For each 50-unit increase in IsoPs, regression modeling predicted decreases in cognitive, communication and motor scores of -1.9, -1.2 and -2.4 points, respectively (P<0.001). IsoP increase was also associated with increased RSI at 40 weeks of PMA (odds ratio=1.23; P=0.01). RSI mediated 25% of the IsoP effect on DAYC motor scores (P=0.02) and had no significant impact on cognitive or communication scores. CONCLUSIONS: In the first month after birth, increases in plasma IsoPs identify preterm infants at risk for respiratory morbidity at term equivalent age and worse developmental outcomes at 12 months of CA. Poor neurodevelopment is largely independent of respiratory morbidity.

Adverse neurodevelopmental outcomes after exposure to phenobarbital and levetiracetam for the treatment of neonatal seizures.

OBJECTIVE: Compare neurodevelopment after levetiracetam (LEV) and phenobarbital (PB) for neonatal seizures. STUDY DESIGN: Retrospective study of infants who received antiepileptic drugs (AEDs) for neonatal seizures. Effect of cumulative exposure to LEV and PB on outcomes of death, cerebral palsy (CP) and Bayley Scales of Infant Development (BSID) scores were evaluated at 24 months corrected age. Analyses were adjusted for number of electrographic seizures and gestational age. RESULT: In 280 infants with comparable seizure etiology and cranial imaging results, increased exposure to PB was associated with worse BSID cognitive and motor scores (8.1- and 9-point decrease per 100 mg kg(-1); P=0.01). The effect was less with LEV (2.2- and 2.6-point decrease per 300 mg kg(-1) LEV (P=0.01)). CP probability increased by 2.3-fold per 100 mg kg(-1) PB and was not associated with increasing LEV. CONCLUSION: Increased exposure to PB is associated with worse neurodevelopmental outcomes than LEV. Prospective studies of outcomes of neonatal exposure to AEDs are essential.

Necrotizing enterocolitis in infants with periventricular hemorrhagic infarction: associations and outcomes.

BACKGROUND: Necrotizing enterocolitis (NEC) and periventricular hemorrhagic infarction (PVHI) are complications of prematurity associated with poor neurodevelopmental outcomes. OBJECTIVES: We characterized temporal and causal associations between NEC and type of PVHI as well as associations with outcomes. METHODS: This was a multicenter retrospective study of infants with birth weight <1,500 g and diagnosis of PVHI by a pediatric radiologist at 3 neonatal intensive care units in North Carolina, USA, between January 1998 and December 2004. NEC was confirmed using radiological and surgical pathology findings. Infants were assessed by 3 years using the Bayley Scales of Infant Development, second edition. RESULTS: 35 of 112 (31%) of study patients developed NEC. NEC was diagnosed subsequent to PVHI by a median of 16.6 days (95% CI [9.5, 24.9], p < 0.0001). Indomethacin use and the presence of bilateral PVHI were associated with an increased risk of subsequent NEC (OR 2.8, 95% CI [1.1, 7.2] and OR 2.4, 95% CI [1.1, 5.7], respectively). Having bilateral versus unilateral PVHI was associated with a 2.34-fold increased risk of death (95% CI [1.27, 4.33], p = 0.007). NEC was not associated with worse motor outcomes in this population. Overall, the probability of a mental development index >70 was greatest for infants with unilateral PVHI versus bilateral PVHI, although the presence of NEC was associated with worse cognitive outcomes in both groups. CONCLUSIONS: Premature infants with PVHI often subsequently develop NEC, especially if they have bilateral PVHI and are exposed to indomethacin. While NEC results in worse neurodevelopmental outcomes, PVHI severity appears more important to the outcome of these infants.

[Evaluation of a dysphonic population. Interest of a therapeutic team composed by the phoniatrician and the speech therapist]. / Evaluation d'une population de dysphoniques suivis en équipe phoniatrie/orthophonie.

PURPOSE: To evaluate a population of dysphonic treated in rehabilitation by comparing the VHI score and GRB scale. MATERIALS AND METHODS: 300 questionnaires were completed, only 42 cases were matched before and after 15 rehabilitation sessions; that is 84 questionnaires. Patients were divided into two groups: group 1 (impaired mobility of the vocal cords), group 2 (benign mucosal lesions). All patients completed a VHI questionnaire, a questionnaire evaluating subjective voice abuse (SSVS), a GRB score. The two tests were correlated to the diagnosis of voice pathology but also used for follow up after voice therapy. The tests used for statistical studies were: comparison by pathology by unpaired series tests (theoretical deviation=0); mean tests, Wilcoxon type. RESULTS: Patients were more handicapped by impaired mobility of the vocal cord than by a nodule or a cyst. The patients' vocal handicap (VHI) was significantly lower after 15 therapy sessions, in all of its components. The perceptual evaluation GRB is also significantly better for these patients after 15 therapy sessions. We could not demonstrate a favorable evolution, that is a diminution of the SSVS before and after 15 sessions. CONCLUSION: The efficacy of speech therapy for certain vocal cord pathologies has been demonstrated both in respect of the Vocal Handicap felt by the patient as well as the Hirano scale.

The potential role for prolactin-inducible protein (PIP) as a marker of human breast cancer micrometastasis.

The prolactin-inducible protein (PIP/GCPD15) is believed to originate from a limited set of tissues, including breast and salivary glands, and has been applied as a clinical marker for the diagnosis of metastatic tumours of unknown origin. We have investigated the potential role of PIP mRNA as a marker of human breast cancer metastasis. Using reverse transcription polymerase chain reaction and Southern or dot blot analysis, PIP mRNA was detected in 4/6 breast cell lines, independent of oestrogen receptor (ER) status. In breast primary tumours (n = 97), analysed from histologically characterized sections, PIP mRNA was detected in most cases. Higher PIP mRNA levels correlated with ER+ (P = 0.0004), progesterone receptor positive (PR+) (P = 0.0167), low-grade (P = 0.0195) tumours, and also PIP protein levels assessed by immunohistochemistry (n = 19, P = 0.0319). PIP mRNA expression was also detectable in 11/16 (69%) of axillary node metastases. PIP mRNA expression, however, was also detected in normal breast duct epithelium, skin, salivary gland and peripheral blood leucocyte samples from normal individuals. We conclude that PIP mRNA is frequently expressed in both primary human breast tumours and nodal metastases. However, the presence of PIP expression in skin creates a potential source of contamination in venepuncture samples that should be considered in its application as a marker for breast tumour micrometastases.

Primary T-cell and activated macrophage response associated with tumor protection using peptide/poly-N-acetyl glucosamine vaccination.

The mode of peptide-based cancer vaccine administration critically affects the ability to achieve a clinically relevant tumor-specific response. We have previously shown (Cole et al., Clin. Cancer Res., 3: 867-873, 1997) that a specific formulation of the polysaccharide poly-N-acetyl glucosamine (p-GlcNAc, designated as F2 gel) is an effective vehicle for sustained cytokine and peptide delivery in vitro. The purpose of this study was to evaluate the efficacy of F2 gel/peptide vaccination in the murine EG.7-OVA tumor model and to elucidate potential mechanisms involved in the observed cell-mediated response. C57BL/6 mice were given injections of 200 microl in the base of tail/footpad using either F2 gel alone or 200 microg of: SIINFEKL minimal peptide (OVA) in PBS, OVA peptide/endoplasmic reticulum insertion signal sequence fusion (ESOVA) in PBS, OVA in F2 gel, or ESOVA in F2 gel. Splenocytes were tested 10 days later for a secondary response using a Cr51 assay as well as a primary CTL response using the lactate dehydrogenase cytotoxicity assay. Splenocytes from immunized mice were harvested at specific time points and assayed for cell surface and intracellular markers. On day 10 postvaccination, animals were challenged with EG.7-OVA murine thymoma cells. Tumor size and appearance were recorded. Vaccination with F2 gel/peptide (either OVA or ESOVA) resulted in a primary T-cell response (up to 25% tumor cell-specific lysis) and no tumor growth in 69% of the mice. By 48 h, the proportion of splenic T cells had increased 4-fold compared with B cells. Presence of an increased Th1 CD4 helper population was demonstrated by IFN-gamma production. CD4 cells were activated at 24 and 48 h as shown by IL-2 receptor alpha chain expression (from 2% basal expression to 15.4% at 48 h). Activated splenic macrophages increased from 3 to 8% within 10 h, and their level of B7-2 expression doubled. Depletion of macrophages before vaccine injection abolished any tumor-specific primary CTL response. F2 gel/peptide tumor vaccine can prime the immune system in an antigen-specific manner by generating a measurable primary T-cell response with minimal peptide; this process involves macrophage presence and activation as well as induction of Th1 CD4 cells. This is the first demonstration of a primary CTL response generated with minimal peptide vaccination using a noninfectious delivery system. These results justify additional studies to better define the mechanisms involved in F2 gel/peptide vaccination in preparation for clinical trials.

Detection of occult breast cancer micrometastases in axillary lymph nodes using a multimarker reverse transcriptase-polymerase chain reaction panel.

BACKGROUND: Axillary lymph node status in breast cancer patients remains the single most important predictor of outcomes. Current methods of histopathologic analysis may be inadequate because 30% of node-negative patients recur. The purpose of this study was to test the hypothesis that a multigene reverse transcriptase-polymerase chain reaction (RT-PCR) panel provides a more sensitive method to detect axillary lymph node metastases than routine pathologic examination. STUDY DESIGN: Sixty-one consecutive breast cancer patients were evaluated, with nine normal control patients. Nodes > 1 cm were bisected for histopathologic and RT-PCR analysis. Nodal tissue was homogenized, and total RNA was converted into cDNA with reverse transcriptase. Reverse transcriptase-polymerase chain reaction analysis was performed with primers specific for keratin-19, c-myc, prolactin inducible protein (PIP), and beta-actin using ethidium bromide gel electrophoresis. Reverse transcriptase-polymerase chain reaction positive/ pathology negative axillary lymph nodes were reevaluated using step sectioning and immunohistochemical staining. RESULTS: Thirty-seven patients had pathologically negative axillary lymph nodes, of which 15 (40%) were positive by RT-PCR analysis. Two RT-PCR negative results (one probably from tissue processing error and the other secondary to sampling error) among the 24 histologically positive specimens were detected (8%). The number of patients in each pathologic stage was 26 patients in stage I; 18, stage IIA; 7, stage IIB; 7, stage IIIA; 3, stage IIIB; and 0 patients in stage IV. By RT-PCR staging, 8 of 26 patients went from stage I to IIA (30%), and 7 of 18 from stage IIA to IIB (39%). Of the RT-PCR positive individuals who were stage I by pathologic analysis, 100% were found to be c-myc positive, 0% keratin-19 positive, and 0% PIP positive; for stage IIIB patients these markers were 50%, 100%, and 100% respectively. Additionally, an increasing number of positive markers per specimen appeared to correlate with larger primary tumor size (p < 0.01) and decreased predicted 5-year survival (r = 0.950, p < 0.002). CONCLUSIONS: Multimarker RT-PCR analysis appears to be a readily available and highly sensitive method for the detection of axillary lymph node micrometastases. Longterm followup of RT-PCR positive patients will be required to determine its clinical relevance. If validated as a predictor of disease recurrence, this method would provide a powerful complement to routine histopathologic analysis of axillary lymph nodes.