The Maize PI/GLO Ortholog Zmm16/sterile tassel silky ear1 Interacts with the Zygomorphy and Sex Determination Pathways in Flower Development.

In monocots and eudicots, B class function specifies second and third whorl floral organ identity as described in the classic ABCE model. Grass B class APETALA3/DEFICIENS orthologs have been functionally characterized; here, we describe the positional cloning and characterization of a maize (Zea mays) PISTILLATA/GLOBOSA ortholog Zea mays mads16 (Zmm16)/sterile tassel silky ear1 (sts1). We show that, similar to many eudicots, all the maize B class proteins bind DNA as obligate heterodimers and positively regulate their own expression. However, sts1 mutants have novel phenotypes that provide insight into two derived aspects of maize flower development: carpel abortion and floral asymmetry. Specifically, we show that carpel abortion acts downstream of organ identity and requires the growth-promoting factor grassy tillers1 and that the maize B class genes are expressed asymmetrically, likely in response to zygomorphy of grass floral primordia. Further investigation reveals that floral phyllotactic patterning is also zygomorphic, suggesting significant mechanistic differences with the well-characterized models of floral polarity. These unexpected results show that despite extensive study of B class gene functions in diverse flowering plants, novel insights can be gained from careful investigation of homeotic mutants outside the core eudicot model species.

Discovery and structure-based design of 4,6-diaminonicotinamides as potent and selective IRAK4 inhibitors.

The identification of small molecule inhibitors of IRAK4 for the treatment of autoimmune diseases has been an area of intense research. We discovered novel 4,6-diaminonicotinamides which potently inhibit IRAK4. Optimization efforts were aided by X-ray crystal structures of inhibitors bound to IRAK4. Structure activity relationship (SAR) studies led to the identification of compound 29 which exhibited sub-micromolar potency in a LTA stimulated cellular assay.

What Definition Is Used to Describe Second Impact Syndrome in Sports? A Systematic and Critical Review.

Concern about what has been termed, "second impact syndrome" (SIS) is a major factor determining return-to-play decisions after concussion. However, definitions of SIS vary. We used Scopus to conduct a systematic review and categorize the definitions used to describe SIS. Of the 91 sources identified, 79 (87%) clearly specified that SIS involved either cerebral edema or death after a concussion when a prior concussion had not resolved. Twelve articles (13%) could be interpreted as merely the events of two consecutive concussions. Among the articles that listed mortality rates, nearly all (33/35, 94%) said the rate of death was "high" (e.g., 50% to 100%). Our review found that most articles define SIS as a syndrome requiring catastrophic brain injury after consecutive concussive episodes. Given that it is unclear how common it is to have a second concussion while not fully recovered from a first concussion, the actual mortality rate of SIS is unknown.

TCP transcription factor, BRANCH ANGLE DEFECTIVE 1 (BAD1), is required for normal tassel branch angle formation in maize.

In grass inflorescences, a structure called the "pulvinus" is found between the inflorescence main stem and lateral branches. The size of the pulvinus affects the angle of the lateral branches that emerge from the main axis and therefore has a large impact on inflorescence architecture. Through EMS mutagenesis we have identified three complementation groups of recessive mutants in maize having defects in pulvinus formation. All mutants showed extremely acute tassel branch angles accompanied by a significant reduction in the size of the pulvinus compared with normal plants. Two of the complementation groups correspond to mutations in the previously identified genes, RAMOSA2 (RA2) and LIGULELESS1 (LG1). Mutants corresponding to a third group were cloned using mapped-based approaches and found to encode a new member of the plant-specific TCP (TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL NUCLEAR ANTIGEN FACTOR) family of DNA-binding proteins, BRANCH ANGLE DEFECTIVE 1 (BAD1). BAD1 is expressed in the developing pulvinus as well as in other developing tissues, including the tassels and juvenile leaves. Both molecular and genetics studies show that RA2 is upstream of BAD1, whereas LG1 may function in a separate pathway. Our findings demonstrate that BAD1 is a TCP class II gene that functions to promote cell proliferation in a lateral organ, the pulvinus, and influences inflorescence architecture by impacting the angle of lateral branch emergence.

BARREN STALK FASTIGIATE1 is an AT-hook protein required for the formation of maize ears.

Ears are the seed-bearing inflorescences of maize (Zea mays) plants and represent a crucial component of maize yield. The first step in the formation of ears is the initiation of axillary meristems in the axils of developing leaves. In the classic maize mutant barren stalk fastigiate1 (baf1), first discovered in the 1950s, ears either do not form or, if they do, are partially fused to the main stalk. We positionally cloned Baf1 and found that it encodes a transcriptional regulator containing an AT-hook DNA binding motif. Single coorthologs of Baf1 are found in syntenic regions of brachypodium (Brachypodium distachyon), rice (Oryza sativa), and sorghum (Sorghum bicolor), suggesting that the gene is likely present in all cereal species. Protein-protein interaction assays suggest that BAF1 is capable of forming homodimers and heterodimers with other members of the AT-hook family. Another transcriptional regulator required for ear initiation is the basic helix-loop-helix protein BARREN STALK1 (BA1). Genetic and expression analyses suggest that Baf1 is required to reach a threshold level of Ba1 expression for the initiation of maize ears. We propose that Baf1 functions in the demarcation of a boundary region essential for the specification of a stem cell niche.

The control of axillary meristem fate in the maize ramosa pathway.

Plant axillary meristems are composed of highly organized, self-renewing stem cells that produce indeterminate branches or terminate in differentiated structures, such as the flowers. These opposite fates, dictated by both genetic and environmental factors, determine interspecific differences in the architecture of plants. The Cys(2)-His(2) zinc-finger transcription factor RAMOSA1 (RA1) regulates the fate of most axillary meristems during the early development of maize inflorescences, the tassel and the ear, and has been implicated in the evolution of grass architecture. Mutations in RA1 or any other known members of the ramosa pathway, RAMOSA2 and RAMOSA3, generate highly branched inflorescences. Here, we report a genetic screen for the enhancement of maize inflorescence branching and the discovery of a new regulator of meristem fate: the RAMOSA1 ENHANCER LOCUS2 (REL2) gene. rel2 mutants dramatically increase the formation of long branches in ears of both ra1 and ra2 mutants. REL2 encodes a transcriptional co-repressor similar to the TOPLESS protein of Arabidopsis, which is known to maintain apical-basal polarity during embryogenesis. REL2 is capable of rescuing the embryonic defects of the Arabidopsis topless-1 mutant, suggesting that REL2 also functions as a transcriptional co-repressor throughout development. We show by genetic and molecular analyses that REL2 physically interacts with RA1, indicating that the REL2/RA1 transcriptional repressor complex antagonizes the formation of indeterminate branches during maize inflorescence development. Our results reveal a novel mechanism for the control of meristem fate and the architecture of plants.

A conserved mechanism of bract suppression in the grass family.

Suppression of inflorescence leaf, or bract, growth has evolved multiple times in diverse angiosperm lineages, including the Poaceae and Brassicaceae. Studies of Arabidopsis thaliana mutants have revealed several genes involved in bract suppression, but it is not known if these genes play a similar role in other plants with suppressed bracts. We identified maize (Zea mays) tassel sheath (tsh) mutants, characterized by the loss of bract suppression, that comprise five loci (tsh1-tsh5). We used map-based cloning to identify Tsh1 and found that it encodes a GATA zinc-finger protein, a close homolog of HANABA TARANU (HAN) of Arabidopsis. The bract suppression function of Tsh1 is conserved throughout the grass family, as we demonstrate that the rice (Oryza sativa) NECK LEAF1 (NL1) and barley (Hordeum vulgare) THIRD OUTER GLUME (TRD) genes are orthologous with Tsh1. Interestingly, NL1/Tsh1/TRD expression and function are not conserved with HAN. The existence of paralogous NL1/Tsh1/TRD-like genes in the grasses indicates that the NL1/Tsh1/TRD lineage was created by recent duplications that may have facilitated its neofunctionalization. A comparison with the Arabidopsis genes regulating bract suppression further supports the hypothesis that the convergent evolution of bract suppression in the Poaceae involved recruitment of a distinct genetic pathway.

Neurophysiologic intraoperative monitoring in children with Down syndrome.

OBJECTIVE: Neurophysiological monitoring during complex spine procedures may reduce risk of injury by providing feedback to the operating surgeon. This tool is a well-established and important surgical adjunct in adults, but clinical data in children are not well described. Moreover, to the best of our knowledge, neurophysiologic intraoperative monitoring data have not been reported in children with neurodevelopmental disorders, such as Down syndrome, who commonly present with craniocervical instability requiring internal fixation. The purpose of this study is to determine the reliability and safety of neurophysiologic intraoperative monitoring in a group of children with Down syndrome undergoing neurosurgical spine procedures. METHODS: A total of six consecutive spinal procedures in six children with Down syndrome (three boys and three girls; mean age 10 years, range 4-16 years) were analyzed between January 1, 2008 and June 31, 2011. Somatosensory evoked potentials were stimulated at the ulnar nerve and tibial nerve for upper and lower extremities, respectively, and recorded at Erb's point and the scalp. Motor evoked potentials were elicited by transcranial electrical stimulation and recorded at the extensor carpi ulnaris muscle and tibialis anterior muscle for upper and lower extremities, respectively. A standardized anesthesia protocol for monitoring consisted of a titrated propofol drip combined with bolus dosing of fentanyl or sufentanil. RESULTS: Somatosensory and motor evoked potentials were documented at the beginning and end of the procedure in all six patients. Changes during the surgery were recorded. Five patients maintained somatosensory potentials throughout surgery. One patient demonstrated a >10% increase in latency or >50% decrease in amplitude suggesting spinal cord dysfunction. A mean baseline stimulation threshold for motor evoked potentials of 485 + 85 V (range 387-600 V) was used. Four patients maintained motor evoked potentials throughout surgery. One patient had loss of left lower somatosensory evoked potentials (SSEPs) and motor evoked potentials (MEPs) after rod placement; upon removal of the rod, SSEPs returned but not MEPs. Another patient did not have consistent MEPs on one side and had absent MEPs on the contralateral side throughout the case. Loss of MEPs in these two patients did not correlate with postoperative neurological status. There were no complications directly related to neurophysiologic intraoperative monitoring technique. CONCLUSIONS: Neurophysiologic intraoperative monitoring during neurosurgical procedures in children with Down syndrome may be reliably and safely implemented. Changes in neurophysiologic parameters during surgery must be carefully interpreted, and discussed with the neurosurgeon, neurophysiologist, and neuroanesthesiologist, and may not correlate with postoperative clinical changes. These changes may be related to abnormal physiology rather than an insult at the time of surgery. Nonetheless, the authors advocate routine neurophysiologic intraoperative monitoring in this special group of children undergoing neurosurgical spine procedures.

Identification of a phenylacylsulfonamide series of dual Bcl-2/Bcl-xL antagonists.

A series of phenylacylsulfonamides has been prepared as antagonists of Bcl-2/Bcl-xL. In addition to potent binding affinities for both Bcl-2 and Bcl-xL, these compounds were shown to induce classical markers of apoptosis in isolated mitochondria. Overall weak cellular potency was improved by the incorporation of polar functionality resulting in compounds with moderate antiproliferative activity.

Design and synthesis of new tetrahydroquinolines derivatives as CETP inhibitors.

This letter describes the discovery and SAR optimization of tetrazoyl tetrahydroquinoline derivatives as potent CETP inhibitors. Compound 6m exhibited robust HDL-c increase in hCETP/hApoA1 double transgenic model and favorable pharmacokinetic properties.

Pyrazole and pyrimidine phenylacylsulfonamides as dual Bcl-2/Bcl-xL antagonists.

5-Butyl-1,4-diphenyl pyrazole and 2-amino-5-chloro pyrimidine acylsulfonamides were developed as potent dual antagonists of Bcl-2 and Bcl-xL. Compounds were optimized for binding to the I88, L92, I95, and F99 pockets normally occupied by pro-apoptotic protein Bim. An X-ray crystal structure confirmed the proposed binding mode. Observation of cytochrome c release from isolated mitochondria in MV-411 cells provides further evidence of target inhibition. Compounds demonstrated submicromolar antiproliferative activity in Bcl-2/Bcl-xL dependent cell lines.

Design, synthesis and structure-activity-relationship of 1,5-tetrahydronaphthyridines as CETP inhibitors.

This Letter describes the discovery and SAR optimization of 1,5-tetrahydronaphthyridines, a new class of potent CETP inhibitors. The effort led to the identification of 21b and 21d with in vitro human plasma CETP inhibitory activity in the nanomolar range (IC(50)=23 and 22nM, respectively). Both 21b and 21d exhibited robust HDL-c increase in hCETP/hApoA1 dual heterozygous mice model.

Evacetrapib is a novel, potent, and selective inhibitor of cholesteryl ester transfer protein that elevates HDL cholesterol without inducing aldosterone or increasing blood pressure.

Cholesteryl ester transfer protein (CETP) catalyses the exchange of cholesteryl ester and triglyceride between HDL and apoB containing lipoprotein particles. The role of CETP in modulating plasma HDL cholesterol levels in humans is well established and there have been significant efforts to develop CETP inhibitors to increase HDL cholesterol for the treatment of coronary artery disease. These efforts, however, have been hampered by the fact that most CETP inhibitors either have low potency or have undesirable side effects. In this study, we describe a novel benzazepine compound evacetrapib (LY2484595), which is a potent and selective inhibitor of CETP both in vitro and in vivo. Evacetrapib inhibited human recombinant CETP protein (5.5 nM IC(50)) and CETP activity in human plasma (36 nM IC(50)) in vitro. In double transgenic mice expressing human CETP and apoAI, evacetrapib exhibited an ex vivo CETP inhibition ED(50) of less than 5 mg/kg at 8 h post oral dose and significantly elevated HDL cholesterol. Importantly, no blood pressure elevation was observed in rats dosed with evacetrapib at high exposure multiples compared with the positive control, torcetrapib. In addition, in a human adrenal cortical carcinoma cell line (H295R cells), evacetrapib did not induce aldosterone or cortisol biosynthesis whereas torcetrapib dramatically induced aldosterone and cortisol biosynthesis. Our data indicate that evacetrapib is a potent and selective CETP inhibitor without torcetrapib-like off-target liabilities. Evacetrapib is currently in phase II clinical development.

sparse inflorescence1 encodes a monocot-specific YUCCA-like gene required for vegetative and reproductive development in maize.

The plant growth hormone auxin plays a critical role in the initiation of lateral organs and meristems. Here, we identify and characterize a mutant, sparse inflorescence1 (spi1), which has defects in the initiation of axillary meristems and lateral organs during vegetative and inflorescence development in maize. Positional cloning shows that spi1 encodes a flavin monooxygenase similar to the YUCCA (YUC) genes of Arabidopsis, which are involved in local auxin biosynthesis in various plant tissues. In Arabidopsis, loss of function of single members of the YUC family has no obvious effect, but in maize the mutation of a single yuc locus causes severe developmental defects. Phylogenetic analysis of the different members of the YUC family in moss, monocot, and eudicot species shows that there have been independent expansions of the family in monocots and eudicots. spi1 belongs to a monocot-specific clade, within which the role of individual YUC genes has diversified. These observations, together with expression and functional data, suggest that spi1 has evolved a dominant role in auxin biosynthesis that is essential for normal maize inflorescence development. Analysis of the interaction between spi1 and genes regulating auxin transport indicate that auxin transport and biosynthesis function synergistically to regulate the formation of axillary meristems and lateral organs in maize.

The role of barren stalk1 in the architecture of maize.

The architecture of higher plants is established through the activity of lateral meristems--small groups of stem cells formed during vegetative and reproductive development. Lateral meristems generate branches and inflorescence structures, which define the overall form of a plant, and are largely responsible for the evolution of different plant architectures. Here, we report the isolation of the barren stalk1 gene, which encodes a non-canonical basic helix-loop-helix protein required for the initiation of all aerial lateral meristems in maize. barren stalk1 represents one of the earliest genes involved in the patterning of maize inflorescences, and, together with the teosinte branched1 gene, it regulates vegetative lateral meristem development. The architecture of maize has been a major target of selection for early agriculturalists and modern farmers, because it influences harvesting, breeding strategies and mechanization. By sampling nucleotide diversity in the barren stalk1 region, we show that two haplotypes entered the maize gene pool from its wild progenitor, teosinte, and that only one was incorporated throughout modern inbreds, suggesting that barren stalk1 was selected for agronomic purposes.

Secreted proprotein convertase subtilisin/kexin type 9 reduces both hepatic and extrahepatic low-density lipoprotein receptors in vivo.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a serine protease that is known to reduce hepatic low-density lipoprotein receptor (LDLR) levels and increase plasma LDL cholesterol. It is not clear, however, whether secreted PCSK9 degrades extrahepatic LDLRs. We present evidence that recombinant PCSK9, either injected intravenously into or expressed in the liver of C57BL/6 mice, significantly reduced LDLR levels in multiple extrahepatic tissues. During the initial characterization, we found that injected human recombinant PCSK9 at 30 microg/mouse had a half-life of 15 min in serum in mice. Hepatic LDLR levels were reduced within 30min and the degradation of hepatic LDLR reached the maximum 2h after the initial protein injection. Endocytosis of PCSK9 in liver occurred within 5min of protein injection and internalized PCSK9 was only barely detectable within 1h. When extrahepatic LDLRs were examined by Western blotting analysis, we found significant reductions of LDLRs in multiple extrahepatic tissues including lung, adipose and kidney along with the more dramatic reduction of LDLRs in liver. These studies were further extended using adenoviral expression of human PCSK9 in C57BL/6 mice to demonstrate that PCSK9 produced in liver impacted extrahepatic tissue LDLR levels as well. Taken together, our studies indicate that secreted PCSK9 can potentially impact extrahepatic tissue cholesterol homeostasis by regulating extrahepatic tissue LDLR levels.

Discovery of orally active pyrrolopyridine- and aminopyridine-based Met kinase inhibitors.

A series of acylurea analogs derived from pyrrolopyridine and aminopyridine scaffolds were identified as potent inhibitors of Met kinase activity. The SAR at various positions of the two kinase scaffolds was investigated. These studies led to the discovery of compounds 3b and 20b, which demonstrated favorable pharmacokinetic properties in mice and significant antitumor activity in a human gastric carcinoma xenograft model.

Peroxisome proliferator-activated receptor alpha agonists regulate cholesterol ester transfer protein.

Peroxisome proliferator-activated receptor alpha (PPARalpha) belongs to the nuclear receptor superfamily that regulates multiple target genes involved in lipid metabolism. Cholesterol ester transfer protein (CETP) is a secreted glycoprotein that modifies high-density lipoprotein (HDL) particles. In humans, plasma CETP activity is inversely correlated with HDL cholesterol levels. We report here that PPARalpha agonists increase CETP mRNA, protein and accordingly its activity. In a human CETP transgenic animal model harboring the natural flanking regions (Jiang et al. in J Clin Investigat 90:1290-1295, 1992), both fenofibrate and a specific synthetic PPARalpha agonist LY970 elevated human CETP mRNA in liver, serum protein and CETP activity. In hamsters, the endogenous liver CETP mRNA level and the serum CETP activity were dose-dependently upregulated by fenofibrate. In addition Wy14643, a PPARalpha agonist, also significantly elevated CETP mRNA and activity. In a carcinoma cell line of hepatic origin, HepG2 cells, overexpression of PPARalpha resulted in increased CETP mRNA and agonist treatment further elevated CETP mRNA levels. We conclude that PPARalpha agonists upregulate CETP expression and activity and may play an important role in PPARalpha (agonist mediated HDL cholesterol homeostasis in humans.

Dimeric Macrocyclic Antagonists of Inhibitor of Apoptosis Proteins for the Treatment of Cancer.

A series of dimeric macrocyclic compounds were prepared and evaluated as antagonists for inhibitor of apoptosis proteins. The most potent analogue 11, which binds to XIAP and c-IAP proteins with high affinity and induces caspase-3 activation and ultimately cell apoptosis, inhibits growth of human melanoma and colorectal cell lines at low nanomolar concentrations. Furthermore, compound 11 demonstrated significant antitumor activity in the A875 human melanoma xenograft model at doses as low as 2 mg/kg on a q3d schedule.

Discovery of N-(2-chloro-6-methyl- phenyl)-2-(6-(4-(2-hydroxyethyl)- piperazin-1-yl)-2-methylpyrimidin-4- ylamino)thiazole-5-carboxamide (BMS-354825), a dual Src/Abl kinase inhibitor with potent antitumor activity in preclinical assays.

A series of substituted 2-(aminopyridyl)- and 2-(aminopyrimidinyl)thiazole-5-carboxamides was identified as potent Src/Abl kinase inhibitors with excellent antiproliferative activity against hematological and solid tumor cell lines. Compound 13 was orally active in a K562 xenograft model of chronic myelogenous leukemia (CML), demonstrating complete tumor regressions and low toxicity at multiple dose levels. On the basis of its robust in vivo activity and favorable pharmacokinetic profile, 13 was selected for additional characterization for oncology indications.