Lakshmi Srinivasan

Novel FOXF1 Mutations in Sporadic and Familial Cases of Alveolar Capillary Dysplasia with Misaligned Pulmonary Veins Imply a Role for its DNA Binding Domain

Alveolar capillary dysplasia with misalignment of pulmonary veins (ACD/MPV) is a rare and lethal developmental disorder of the lung defined by a constellation of characteristic histopathological features. Nonpulmonary anomalies involving organs of gastrointestinal, cardiovascular, and genitourinary systems have been identified in approximately 80% of patients with ACD/MPV. We have collected DNA and pathological samples from more than 90 infants with ACD/MPV and their family members. Since the publication of our initial report of four point mutations and 10 deletions, we have identified an additional 38 novel nonsynonymous mutations of FOXF1 (nine nonsense, seven frameshift, one inframe deletion, 20 missense, and one no stop). This report represents an up to date list of all known FOXF1 mutations to the best of our knowledge. Majority of the cases are sporadic. We report four familial cases of which three show maternal inheritance, consistent with paternal imprinting of the gene. Twenty five mutations (60%) are located within the putative DNA‐binding domain, indicating its plausible role in FOXF1 function. Five mutations map to the second exon. We identified two additional genic and eight genomic deletions upstream to FOXF1. These results corroborate and extend our previous observations and further establish involvement of FOXF1 in ACD/MPV and lung organogenesis.

New technologies for the rapid diagnosis of neonatal sepsis.

Purpose of review To present recent literature on novel diagnostic tests in neonatal sepsis. Recent findings Our review of technologies for the rapid diagnosis of neonatal sepsis includes new adaptations of time-honored tests as well as advances on the forefront of medicine. A recent study demonstrates that age-specific likelihood values for the complete blood count may determine risk of infection. Systematic reviews of procalcitonin, mannose-binding lectin and molecular amplification techniques provide summary data from accumulated literature on these tests. Proteomics-based and genomics-based exploratory researches suggest new combinations of markers as important signals of sepsis, whereas damage-associated molecular patterns, a class of inflammatory mediators now viewed as key players in the inflammatory cascade, may be useful predictors of disease progression and severity. Heart rate variability monitoring has also been suggested as a way to reduce mortality in very low birth weight neonates. Finally, molecular techniques are rapidly advancing in sophistication and may soon be useful as adjunctive bacterial identification tests. Summary Several novel tests show promise in the early detection of sepsis. Highlights include new combinations of biomarkers unearthed by proteomics-based research and identification of sepsis based on gene expression profiling. Future research should focus on validation of these findings and further refinement of molecular techniques.

Protein Acetylation Regulates Both PU.1 Transactivation and Igκ 3′ Enhancer Activity

Igκ gene expression and chromatin structure change during B cell development. At the pre-B cell stage, the locus is relatively hypoacetylated on histone H3, whereas it is hyperacetylated at the plasma cell stage. We find in this study that the histone deacetylase inhibitor, trichostatin A (TSA) stimulated 3′ enhancer activity through the PU.1 binding site. TSA also stimulated PU.1 transactivation potential. PU.1 activity was increased by the coactivator acetyltransferase protein, p300, and p300 physically interacted with PU.1 residues 7–30. PU.1 served as a substrate for p300 and was acetylated on lysine residues 170, 171, 206, and 208. Mutation of PU.1 lysines 170 and 171 did not affect PU.1 DNA binding, but did lower the ability of PU.1 to activate transcription in association with p300. Lysine 170 was acetylated in pre-B cells and plasmacytoma cells, but TSA treatment did not stimulate PU.1 acetylation at this residue arguing that a second mechanism can stimulate 3′ enhancer activity. Using chromatin immunoprecipitation assays we found that TSA caused preferential acetylation of histone H3 at the 3′ enhancer. The relevance of these studies for PU.1 function in transcription and hemopoietic development is discussed.

Transient requirements of YY1 expression for PcG transcriptional repression and phenotypic rescue

A hallmark of PcG transcriptional repression is stability of the repressed state, although the mechanism of this stability is unclear. The mammalian transcription factor YY1 can function as a PcG protein in Drosophila resulting in transcriptional repression and correction of phenotypic defects caused by mutation of its homologue, Pleiohomeotic (PHO). To discern the temporal requirements of YY1 expression for these functions, and to study its mechanism of stable repression in vivo, we used a Drosophila larval wing imaginal disc transcriptional repression system. We found that YY1 was needed transiently at day 3 or later of embryonic development to stably repress a reporter transgene at day 8 in wing imaginal discs. Stable transcriptional repression did not correlate with maintenance of YY1 or Polycomb DNA binding, but did correlate with persistence of histone H3 methylation on lysine 27. We also found that YY1 expressed during the first 7 days of development was sufficient for rescue of fly development (a 14 day process) in pho mutant flies. Therefore, YY1 was transiently required for correction of fly defects and was dispensable past the pharate adult stage. Possible mechanisms of repression by YY1 are discussed.

Cerebrospinal Fluid Reference Ranges in Term and Preterm Infants in the Neonatal Intensive Care Unit

OBJECTIVE To determine reference ranges of cerebrospinal fluid (CSF) laboratory findings in term and preterm infants in the neonatal intensive care unit. STUDY DESIGN Data were collected prospectively as part of a multisite study of infants aged <6 months undergoing lumbar puncture for evaluation of suspected sepsis. Infants with a red blood cell count >500 cells/μL or a known cause of CSF pleocytosis were excluded from the analysis. RESULTS A total of 318 infants met the inclusion criteria. Of these, 148 infants (47%) were preterm, and 229 (72%) received antibiotics before undergoing lumbar puncture. The upper reference limit of the CSF white blood cell (WBC) count was 12 cells/μL in preterm infants and 14 cells/μL in term infants. CSF protein levels were significantly higher in preterm infants (upper reference limit, 209 mg/dL vs 159 mg/dL in term infants; P < .001), and declined with advancing postnatal age in both groups (preterm, P = .008; term, P < .001). CSF glucose levels did not differ in term and preterm infants. Antibiotic exposure did not significantly affect CSF WBC, protein, or glucose values. CONCLUSIONS CSF WBC counts are not significantly different in preterm and term infants. CSF protein levels are higher and decline more slowly with postnatal age in preterm infants compared with term infants. This study provides CSF reference ranges for hospitalized preterm and term infants, particularly in the first month of life.

Lumbar Puncture in the Neonate: Challenges in Decision Making and Interpretation

Multiple studies have provided normative ranges for cerebrospinal fluid (CSF) parameters in term and preterm infants and described changes with advancing postnatal age, as well as in special circumstances, such as traumatic lumbar puncture (LP), previous antibiotic administration, seizures, and concomitant infections at other sites. Although guidelines exist for the interpretation of CSF parameters in neonates, there appears to be no single combination of parameters that conclusively excludes meningitis. It remains important for clinicians to perform LPs early in the course of illness, ideally before the administration of antibiotic therapy. This review presents currently available literature on the indications for LP as well as guidelines for the interpretation of CSF parameters in neonates.

Traumatic lumbar punctures in infants hospitalized in the neonatal intensive care unit.

Traumatic lumbar punctures occur frequently in the neonatal intensive care unit, making the interpretation of cerebrospinal fluid values difficult. We report correction factors for cerebrospinal fluid protein and white blood cells in the face of red blood cell contamination. These correction factors should facilitate the diagnosis of bacterial meningitis in highrisk hospitalized infants.

Risk Factors Associated With Laboratory-confirmed Bloodstream Infections in a Tertiary Neonatal Intensive Care Unit

Background: Bloodstream infections (BSI) remain a leading cause of morbidity and mortality among infants admitted to neonatal intensive care units (NICUs). At the time of evaluation for suspected BSI, presenting signs may be nonspecific. We sought to determine the clinical signs and risk factors associated with laboratory-confirmed BSI among infants evaluated for late-onset sepsis in a tertiary NICU. Methods: This prospective cohort study included infants >3 days of age admitted to a level 4 NICU from July 2006 to October 2009 for whom a blood culture was drawn for suspected sepsis. Clinicians documented presenting signs at the time of culture. Laboratory-confirmed BSI was defined as per the National Healthcare Safety Network. Multivariate analyses were performed using a logistic regression random effects model. Results: Six-hundred and eighty eligible episodes of suspected BSI were recorded in 409 infants. Enteral contrast within the preceding 48 hours was the most significant risk factor for laboratory-confirmed BSI [Odds Ratio: 9.58 (95% confidence interval: 2.03–45.19)] followed by presence of a central venous catheter. Apnea and hypotension were the most strongly associated presenting signs. Conclusion: Among infants evaluated in a tertiary NICU, recent exposure to enteral contrast was associated with increased odds of developing BSI. Apnea and hypotension were the most strongly associated clinical signs of infection.

Elucidating the role of genomics in neonatal sepsis

Sepsis is a major cause of neonatal morbidity and mortality, especially in vulnerable preterm populations. Immature immune defenses, and environmental and maternal factors contribute to this risk, with as many as a third of very preterm infants experiencing sepsis during their stay in the neonatal intensive care unit (NICU). Epidemiologic and twin studies have suggested that there is a genetic contribution to sepsis predilection. Several investigators have conducted candidate gene association studies on variants of specific interest and potential functional significance in neonatal sepsis. In this review, we describe details of studies that have evaluated genetic susceptibility in neonatal sepsis, and summarize findings from a review of candidate gene association studies.

Glutaminase is essential for the growth of triple-negative breast cancer cells with a deregulated glutamine metabolism pathway and its suppression synergizes with mTOR inhibition

Tumor cells display fundamental changes in metabolism and nutrient uptake in order to utilize additional nutrient sources to meet their enhanced bioenergetic requirements. Glutamine (Gln) is one such nutrient that is rapidly taken up by tumor cells to fulfill this increased metabolic demand. A vital step in the catabolism of glutamine is its conversion to glutamate by the mitochondrial enzyme glutaminase (GLS). This study has identified GLS a potential therapeutic target in breast cancer, specifically in the basal subtype that exhibits a deregulated glutaminolysis pathway. Using inducible shRNA mediated gene knockdown, we discovered that loss of GLS function in triple-negative breast cancer (TNBC) cell lines with a deregulated glutaminolysis pathway led to profound tumor growth inhibition in vitro and in vivo. GLS knockdown had no effect on growth and metabolite levels in non-TNBC cell lines. We rescued the anti-tumor effect of GLS knockdown using shRNA resistant cDNAs encoding both GLS isoforms and by addition of an α-ketoglutarate (αKG) analog thus confirming the critical role of GLS in TNBC. Pharmacological inhibition of GLS with the small molecule inhibitor CB-839 reduced cell growth and led to a decrease in mammalian target of rapamycin (mTOR) activity and an increase in the stress response pathway driven by activating transcription factor 4 (ATF4). Finally, we found that GLS inhibition synergizes with mTOR inhibition, which introduces the possibility of a novel therapeutic strategy for TNBC. Our study revealed that GLS is essential for the survival of TNBC with a deregulated glutaminolysis pathway. The synergistic activity of GLS and mTOR inhibitors in TNBC cell lines suggests therapeutic potential of this combination for the treatment of vulnerable subpopulations of TNBC.