Pathophysiologic mechanisms involved in neonatal hypoxic ischemic encephalopathy (HIE) are associated with complex changes of blood flow and metabolism. Therapeutic hypothermia (TH) is effective in reducing the extent of brain injury, but it remains uncertain how TH affects cerebral blood flow (CBF) and metabolism. Ten neonates undergoing TH for HIE and seventeen healthy controls were recruited from the NICU and the well baby nursery, respectively. A combination of frequency domain near infrared spectroscopy (FDNIRS) and diffuse correlation spectroscopy (DCS) systems was used to non-invasively measure cerebral hemodynamic and metabolic variables at the bedside. Results showed that cerebral oxygen metabolism (CMRO 2i ) and CBF indices (CBF i ) in neonates with HIE during TH were significantly lower than post-TH and age-matched control values. Also, cerebral blood volume (CBV) and hemoglobin oxygen saturation (SO 2 ) were significantly higher in neonates with HIE during TH compared with age-matched control neonates. Post-TH CBV was significantly decreased compared with values during TH whereas SO 2 remained unchanged after the therapy. Thus, FDNIRS-DCS can provide information complimentary to SO 2 and can assess individual cerebral metabolic responses to TH. Combined FDNIRS-DCS parameters improve the understanding of the underlying physiology and have the potential to serve as bedside biomarkers of treatment response and optimization. Keywords: cerebral blood circulation; cerebral hemoglobin oxygen saturation; cerebral metabolic rate of oxygen consumption; near infrared spectroscopy; neonatal hypoxic ischemic encephalopathy; therapeutic hypothermia INTRODUCTION Neonatal hypoxic ischemic encephalopathy (HIE) occurs in B2 to 3 per 1,000 live births in the USA, with devastating consequences including neurodevelopmental disabilities such as cerebral palsy, life-long cognitive impairment, and epilepsy.1 In HIE, there is a period of reduced blood flow (ischemia) and oxygen delivery (hypoxia) followed by reperfusion with transient energy recovery, and then a secondary energy failure.2 Treatment is aimed at preventing the cascade of events that begin with reperfusion and lead to secondary energy failure and inevitable cell death; this is the 'window of opportunity.' 3 Theories for injury mechanisms in this window suggest a central role for excitotoxicity and oxidative stress with the injury evolving over hours to weeks.2 Therapeutic hypothermia (TH) is the treatment of choice for HIE, as it is the only treatment with proven efficacy and acts through a variety of mechanisms, including those associated with decreased neuronal energy metabolism. 4 Although early studies showed that TH decreased rates of both mortality and disability, recently published long-term outcomes have demonstrated significance only for decreased mortality rates.5 In addition, a recent

Despite recent advances in instrumentation and analytical strategies for identification and quantitation of protein phosphorylation, a highly specific enrichment protocol is still a challenge in large-scale studies. Here, we report a simple pH/acid control method that addresses the poor specificity seriously criticized in IMAC. Detailed evaluation of the capture and release mechanism in IMAC revealed that pH, buffer and salt yield a complex interplay in enrichment of phosphopeptides, yet they play individual roles in recovery and specificity. A revised one-step IMAC method with low sample loss and high specificity can be rationally designed by controlling salt, pH and the structure and concentration of organic acid. Without methyl esterification, the one-step IMAC enrichment with single LC-MS/MS identified 386 phosphoproteins in 550 mug of non-small-cell lung cancer cell lysate with 96% specificity. Additional fractionation by SDS-PAGE from 4 mg of cell lysate revealed the comprehensive proteome map, identifying 2747 phosphorylation sites from 2360 nondegenerate phosphopeptides and 1219 phosphoproteins with a false discovery rate of 0.63%. To our knowledge, this pH/acid-controlled IMAC procedure provides higher specificity than any other one-step IMAC purification procedure. Furthermore, the simple and reproducible IMAC protocol can be adapted to other solid supports, fully automated or manual, for large-scale identification of the vastly under-explored phosphoproteome.

Early brain development, from the embryonic period to infancy, is characterized by rapid structural and functional changes. These changes can be studied using structural and physiological neuroimaging methods. In order to optimally acquire and accurately interpret this data, concepts from adult neuroimaging cannot be directly transferred. Instead, one must have a basic understanding of fetal and neonatal structural and physiological brain development, and the important modulators of this process. Here, we first review the major developmental milestones of transient cerebral structures and structural connectivity (axonal connectivity) followed by a summary of the contributions from ex vivo and in vivo MRI. Next, we discuss the basic biology of neuronal circuitry development (synaptic connectivity, i.e. ensemble of direct chemical and electrical connections between neurons), physiology of neurovascular coupling, baseline metabolic needs of the fetus and the infant, and functional connectivity (defined as statistical dependence of low-frequency spontaneous fluctuations seen with functional magnetic resonance imaging (fMRI)). The complementary roles of magnetic resonance imaging (MRI), electroencephalography (EEG), magnetoencephalography (MEG), and near-infrared spectroscopy (NIRS) are discussed. We include a section on modulators of brain development where we focus on the placenta and emerging placental MRI approaches. In each section we discuss key technical limitations of the imaging modalities and some of the limitations arising due to the biology of the system. Although neuroimaging approaches have contributed significantly to our understanding of early brain development, there is much yet to be done and a dire need for technical innovations and scientific discoveries to realize the future potential of early fetal and infant interventions to avert long term disease.

Cerebrospinal fluid (CSF) provides vital support for the brain. Abnormal CSF accumulation, such as hydrocephalus, can negatively affect perinatal neurodevelopment. The mechanisms regulating CSF clearance during the postnatal critical period are unclear. Here, we show that CSF K+, accompanied by water, is cleared through the choroid plexus (ChP) during mouse early postnatal development. We report that, at this developmental stage, the ChP showed increased ATP production and increased expression of ATP-dependent K+ transporters, particularly the Na+, K+, Cl−, and water cotransporter NKCC1. Overexpression of NKCC1 in the ChP resulted in increased CSF K+ clearance, increased cerebral compliance, and reduced circulating CSF in the brain without changes in intracranial pressure in mice. Moreover, ChP-specific NKCC1 overexpression in an obstructive hydrocephalus mouse model resulted in reduced ventriculomegaly. Collectively, our results implicate NKCC1 in regulating CSF K+ clearance through the ChP in the critical period during postnatal neurodevelopment in mice.

Methodologies to enrich heterogeneous types of phosphopeptides are critical for comprehensive mapping of the under-explored phosphoproteome. Taking advantage of the distinct binding affinities of Ga(3+) and Fe(3+) for phosphopeptides, we designed a metal-directed immobilized metal ion affinity chromatography for the sequential enrichment of phosphopeptides. In Raji B cells, the sequential Ga(3+)-Fe(3+)-immobilized metal affinity chromatography (IMAC) strategy displayed a 1.5-3.5-fold superior phosphoproteomic coverage compared to single IMAC (Fe(3+), Ti(4+), Ga(3+), and Al(3+)). In addition, up to 92% of the 6283 phosphopeptides were uniquely enriched in either the first Ga(3+)-IMAC (41%) or second Fe(3+)-IMAC (51%). The complementary properties of Ga(3+) and Fe(3+) were further demonstrated through the exclusive enrichment of almost all of 1214 multiply phosphorylated peptides (99.4%) in the Ga(3+)-IMAC, whereas only 10% of 5069 monophosphorylated phosphopeptides were commonly enriched in both fractions. The application of sequential Ga(3+)-Fe(3+)-IMAC to human lung cancer tissue allowed the identification of 2560 unique phosphopeptides with only 8% overlap. In addition to the above-mentioned mono- and multiply phosphorylated peptides, this fractionation ability was also demonstrated on the basic and acidic phosphopeptides: acidophilic phosphorylation sites were predominately enriched in the first Ga(3+)-IMAC (72%), while Pro-directed (85%) and basophilic (79%) phosphorylation sites were enriched in the second Fe(3+)-IMAC. This strategy provided complementary mapping of different kinase substrates in multiple cellular pathways related to cancer invasion and metastasis of lung cancer. Given the fractionation ability and ease of tip preparation of this Ga(3+)-Fe(3+)-IMAC, we propose that this strategy allows more comprehensive characterization of the phosphoproteome both in vitro and in vivo.

S-nitrosylation, a post-translational modification of cysteine residues induced by nitric oxide, mediates many physiological functions. Due to the labile nature of S-nitrosylation, detection by mass spectrometry (MS) is challenging. Here, we developed an S-alkylating labeling strategy using the irreversible biotinylation on S-nitrosocysteines for site-specific identification of the S-nitrosoproteome by LC-MS/MS. Using COS-7 cells without endogenous nitric oxide synthase, we demonstrated that the S-alkylating labeling strategy substantially improved the blocking efficiency of free cysteines, minimized the false-positive identification caused by disulfide interchange, and increased the digestion efficiency for improved peptide identification using MS analyses. Using this strategy, we identified total 586 unique S-nitrosylation sites corresponding to 384 proteins in S-nitroso-N-acetylpenicillamine (SNAP)/l-cysteine-treated mouse MS-1 endothelial cells, including 234 previously unreported S-nitrosylated proteins. When the topologies of 84 identified transmembrane proteins were further analyzed, their S-nitrosylation sites were found to mostly face the cytoplasmic side, implying that S-nitrosylation occurs in the cytoplasm. In addition to the previously known acid/basic motifs, the ten deduced consensus motifs suggested that combination of local hydrophobicity and acid/base motifs in the tertiary structure contribute to the specificity of S-nitrosylation. Moreover, the S-nitrosylated cysteines showed preference on beta-strand, having lower relative surface accessibility at the S-nitrosocysteines.

Membrane proteins are crucial targets for cancer biomarker discovery and drug development. However, in addition to the inherent challenges of hydrophobicity and low abundance, complete membrane proteome coverage of clinical specimen is usually hindered by the requirement of large amount of starting materials. Toward comprehensive membrane proteomic profiling for small amounts of samples (10 μg), we developed high-pH reverse phase (Hp-RP) combined with stop-and-go extraction tip (StageTip) technique, as a fast (∼15 min.), sensitive, reproducible, high-resolution and multiplexed fractionation method suitable for accurate quantification of the membrane proteome. This approach provided almost 2-fold enhanced detection of peptides encompassing transmembrane helix (TMH) domain, as compared with strong anion exchange (SAX) and strong cation exchange (SCX) StageTip techniques. Almost 5000 proteins (∼60% membrane proteins) can be identified in only 10 μg of membrane protein digests, showing the superior sensitivity of the Hp-RP StageTip approach. The method allowed up to 9- and 6-fold increase in the identification of unique hydrophobic and hydrophilic peptides, respectively. The Hp-RP StageTip method enabled in-depth membrane proteome profiling of 11 lung cancer cell lines harboring different EGFR mutation status, which resulted in the identification of 3983 annotated membrane proteins. This provides the largest collection of reference peptide spectral data for lung cancer membrane subproteome. Finally, relative quantification of membrane proteins between Gefitinib-resistant and -sensitive lung cancer cell lines revealed several up-regulated membrane proteins with key roles in lung cancer progression.

Understanding the evolution of regional and hemispheric asymmetries in the early stages of life is essential to the advancement of developmental neuroscience. By using 2 noninvasive optical methods, frequency-domain near-infrared spectroscopy and diffuse correlation spectroscopy, we measured cerebral hemoglobin oxygenation (SO(2)), blood volume (CBV), an index of cerebral blood flow (CBF(i)), and the metabolic rate of oxygen (CMRO(2i)) in the frontal, temporal, and parietal regions of 70 premature and term newborns. In concordance with results obtained using more invasive imaging modalities, we verified both hemodynamic (CBV, CBF(i), and SO(2)) and metabolic (CMRO(2i)) parameters were greater in the temporal and parietal regions than in the frontal region and that these differences increased with age. In addition, we found that most parameters were significantly greater in the right hemisphere than in the left. Finally, in comparing age-matched males and females, we found that males had higher CBF(i) in most cortical regions, higher CMRO(2i) in the frontal region, and more prominent right-left CBF(i) asymmetry. These results reveal, for the first time, that we can detect regional and hemispheric asymmetries in newborns using noninvasive optical techniques. Such a bedside screening tool may facilitate early detection of abnormalities and delays in maturation of specific cortical areas.