Hemodynamic and Metabolic Correlates of Perinatal White Matter Injury Severity

Background and Purpose

Although the spectrum of perinatal white matter injury (WMI) in preterm infants is shifting from cystic encephalomalacia to milder forms of WMI, the factors that contribute to this changing spectrum are unclear. We hypothesized that the variability in WMI quantified by immunohistochemical markers of inflammation could be correlated with the severity of impaired blood oxygen, glucose and lactate.

Methods

We employed a preterm fetal sheep model of in utero moderate hypoxemia and global severe but not complete cerebral ischemia that reproduces the spectrum of human WMI. Since there is small but measurable residual brain blood flow during occlusion, we sought to determine if the metabolic state of the residual arterial blood was associated with severity of WMI. Near the conclusion of hypoxia-ischemia, we recorded cephalic arterial blood pressure, blood oxygen, glucose and lactate levels. To define the spectrum of WMI, an ordinal WMI rating scale was compared against an unbiased quantitative image analysis protocol that provided continuous histo-pathological outcome measures for astrogliosis and microgliosis derived from the entire white matter.

Results

A spectrum of WMI was observed that ranged from diffuse non-necrotic lesions to more severe injury that comprised discrete foci of microscopic or macroscopic necrosis. Residual arterial pressure, oxygen content and blood glucose displayed a significant inverse association with WMI and lactate concentrations were directly related. Elevated glucose levels were the most significantly associated with less severe WMI.

Conclusions

Our results suggest that under conditions of hypoxemia and severe cephalic hypotension, WMI severity measured using unbiased immunohistochemical measurements correlated with several physiologic parameters, including glucose, which may be a useful marker of fetal response to hypoxia or provide protection against energy failure and more severe WMI.     

Structural and gene expression analyses of uptake hydrogenases and other proteins involved in nitrogenase protection in Frankia

The actinorhizal bacterium Frankia expresses nitrogenase and can therefore convert molecular nitrogen into ammonia and the by-product hydrogen. However, nitrogenase is inhibited by oxygen. Consequently, Frankia and its actinorhizal hosts have developed various mechanisms for excluding oxygen from their nitrogen-containing compartments. These include the expression of oxygen-scavenging uptake hydrogenases, the formation of hopanoid-rich vesicles, enclosed by multi-layered hopanoid structures, the lignification of hyphal cell walls, and the production of haemoglobins in the symbiotic nodule. In this work, we analysed the expression and structure of the so-called uptake hydrogenase (Hup), which catalyses the in vivo dissociation of hydrogen to recycle the energy locked up in this ‘waste’ product. Two uptake hydrogenase syntons have been identified in Frankia: synton 1 is expressed under free-living conditions while synton 2 is expressed during symbiosis. We used qPCR to determine synton 1 hup gene expression in two Frankia strains under aerobic and anaerobic conditions. We also predicted the 3D structures of the Hup protein subunits based on multiple sequence alignments and remote homology modelling. Finally, we performed BLAST searches of genome and protein databases to identify genes that may contribute to the protection of nitrogenase against oxygen in the two Frankia strains. Our results show that in Frankia strain ACN14a, the expression patterns of the large (HupL1) and small (HupS1) uptake hydrogenase subunits depend on the abundance of oxygen in the external environment. Structural models of the membrane-bound hydrogenase subunits of ACN14a showed that both subunits resemble the structures of known [NiFe] hydrogenases (Volbeda et al. 1995), but contain fewer cysteine residues than the uptake hydrogenase of the Frankia DC12 and Eu1c strains. Moreover, we show that all of the investigated Frankia strains have two squalene hopane cyclase genes (shc1 and shc2). The only exceptions were CcI3 and the symbiont of Datisca glomerata, which possess shc1 but not shc2. Four truncated haemoglobin genes were identified in Frankia ACN14a and Eu1f, three in CcI3, two in EANpec1 and one in the Datisca glomerata symbiont (Dg).     

Cancer cell survival following DNA damage-mediated premature senescence is regulated by mammalian target of rapamycin (mTOR)-dependent Inhibition of sirtuin 1

DNA-damaging agents can induce premature senescence in cancer cells, which contributes to the static effects of cancer. However, senescent cancer cells may re-enter the cell cycle and lead to tumor relapse. Understanding the mechanisms that control the viability of senescent cells may be helpful in eliminating these cells before they can regrow. Treating human squamous cell carcinoma (SCC) cells with the anti-cancer compounds, resveratrol and doxorubicin, triggered p53-independent premature senescence by invoking oxidative stress-mediated DNA damage. This process involved the mTOR-dependent phosphorylation of SIRT1 at serine 47, resulting in the inhibition of the deacetylase activity of SIRT1. SIRT1 phosphorylation caused concomitant increases in p65/RelA NF-κB acetylation and the expression of an anti-apoptotic Bfl-1/A1. SIRT1 physically interacts with the mTOR-Raptor complex, and a single amino acid substitution in the TOS (TOR signaling) motif in the SIRT1 prevented Ser-47 phosphorylation and Bfl-1/A1 induction. The pharmacologic and genetic inhibition of mTOR, unphosphorylatable S47A, or F474A TOS mutants restored SIRT1 deacetylase activity, blocked Bfl-1/A1 induction, and sensitized prematurely senescent SCC cells for apoptosis. We further show that the treatment of UVB-induced SCCs with doxorubicin transiently stabilized tumor growth but was followed by tumor regrowth upon drug removal in p53(+/-)/SKH-1 mice. The subsequent treatment of stabilized SCCs with rapamycin decreased tumor size and induced caspase-3 activation. These results demonstrate that the inhibition of SIRT1 by mTOR fosters survival of DNA damage-induced prematurely senescent SCC cells via Bfl-1/A1 in the absence of functional p53.     

Overexpression of a Defensin Enhances Resistance to a Fruit-Specific Anthracnose Fungus in Pepper

Functional characterization of a defensin, J1-1, was conducted to evaluate its biotechnological potentiality in transgenic pepper plants against the causal agent of anthracnose disease, Colletotrichum gloeosporioides. To determine antifungal activity, J1-1 recombinant protein was generated and tested for the activity against C. gloeosporioides, resulting in 50% inhibition of fungal growth at a protein concentration of 0.1 mg·mL⁻¹. To develop transgenic pepper plants resistant to anthracnose disease, J1-1 cDNA under the control of 35S promoter was introduced into pepper via Agrobacterium-mediated genetic transformation method. Southern and Northern blot analyses confirmed that a single copy of the transgene in selected transgenic plants was normally expressed and also stably transmitted to subsequent generations. The insertion of T-DNA was further analyzed in three independent homozygous lines using inverse PCR, and confirmed the integration of transgene in non-coding region of genomic DNA. Immunoblot results showed that the level of J1-1 proteins, which was not normally accumulated in unripe fruits, accumulated high in transgenic plants but appeared to differ among transgenic lines. Moreover, the expression of jasmonic acid-biosynthetic genes and pathogenesis-related genes were up-regulated in the transgenic lines, which is co-related with the resistance of J1-1 transgenic plants to anthracnose disease. Consequently, the constitutive expression of J1-1 in transgenic pepper plants provided strong resistance to the anthracnose fungus that was associated with highly reduced lesion formation and fungal colonization. These results implied the significance of the antifungal protein, J1-1, as a useful agronomic trait to control fungal disease.     

Hexanedioic acid from Hermetia illucens larvae (Diptera: Stratiomyidae) protects mice against Klebsiella pneumoniae infection

The antibacterial effects of larval extract from Hermetia illucens, commonly known as the black soldier fly, have been demonstrated in vitro. In this study, gas chromatography–mass spectrometry analysis identified the active compound within this larval extract as hexanedioic acid. The antibacterial effects of hexanedioic acid were investigated in mice infected with Klebsiella pneumoniae. After administration of hexanedioic acid, infected mice showed decreased lung bacterial loads and lower rates of body weight loss compared to those in the infection‐only control group. Based on lung bacterial loads, oral hexanedioic acid treatment showed better protection than intraperitoneal treatment. Histopathology confirmed that daily administration of hexanedioic acid for 10 days showed zero toxicity to the kidneys or livers of mice. Therefore, hexanedioic acid may be a novel antibacterial agent.     

Structure Driven Design of Novel Human Ether-A-Go-Go-Related-Gene Channel (hERG1) Activators

One of the main culprits in modern drug discovery is apparent cardiotoxicity of many lead-candidates via inadvertent pharmacologic blockade of K⁺, Ca²⁺ and Na⁺ currents. Many drugs inadvertently block hERG1 leading to an acquired form of the Long QT syndrome and potentially lethal polymorphic ventricular tachycardia. An emerging strategy is to rely on interventions with a drug that may proactively activate hERG1 channels reducing cardiovascular risks. Small molecules-activators have a great potential for co-therapies where the risk of hERG-related QT prolongation is significant and rehabilitation of the drug is impractical. Although a number of hERG1 activators have been identified in the last decade, their binding sites, functional moieties responsible for channel activation and thus mechanism of action, have yet to be established. Here, we present a proof-of-principle study that combines de-novo drug design, molecular modeling, chemical synthesis with whole cell electrophysiology and Action Potential (AP) recordings in fetal mouse ventricular myocytes to establish basic chemical principles required for efficient activator of hERG1 channel. In order to minimize the likelihood that these molecules would also block the hERG1 channel they were computationally engineered to minimize interactions with known intra-cavitary drug binding sites. The combination of experimental and theoretical studies led to identification of functional elements (functional groups, flexibility) underlying efficiency of hERG1 activators targeting binding pocket located in the S4–S5 linker, as well as identified potential side-effects in this promising line of drugs, which was associated with multi-channel targeting of the developed drugs.     

Methanol elicits the biosynthesis of 4-coumaroylserotonin and feruloylserotonin in rice seedlings

Rice (Oryza sativa cv. Dongjin) plants responded to treatment with methanol by inducing the synthesis of secondary metabolites such as serotonin derivatives, which include feruloylserotonin and 4-coumaroylserotonin. This response was not only a dose dependence on methanol showing a maximum effect with 1% methanol concentration, but also methanol specific. No other solvents such as ethanol, atetaldehyde, isopropanol, formaldehyde and formic acid showed the induced synthesis of serotonin derivatives as methanol did. The methanol induced synthesis of serotonin derivatives was completely blocked by the addition of abscisic acid (ABA), and significantly inhibited by the additions of zeatin and indoleacetic acid (IAA). However, gibberellic acid (GA) had little effect on the action of methanol. Finally, the induced synthesis of serotonin derivatives upon methanol treatment was closely associated with the transient increase in the activity of key enzyme of serotonin N-hydroxycinnamoyl transferase (SHT) which catalyzes the condensation of serotonin and phenolic-CoA into serotonin derivatives.Key words: elicitor, methanol, 4-coumaroylserotonin, feruloylserotonin, serotonin N-hydroxycinnamoyl transferase, rice seedlingsElicitor broadly refers to molecules and stimuli that either induce or control gene expression and metabolism.¹ To date, a series of elicitors have been reported and include various cell wall constituents of plant and microbe origins, avirulence gene products from microbes, and a lots of chemical and physical stimuli such as CuSO₄, CuCl₂, ozone and UV light.² Among chemical elicitors, CuSO₄ is well known to elicit the accumulation of sesquiterpene lubimin in fruit cavities of Datura stramonium.³ Aluminum chloride (AlCl₃) induces resveratrol synthesis in grapevine leaves.⁴ It is also reported that Arabidopsis induces camalexin synthesis in response to α-aminobutyric acid.⁵ Recently, it was found that rice leaves upon senescence produced methanol which then triggered the synthesis of tryptophan and serotonin, suggestive of a key role of methanol as an endogenous elicitor for both primary and secondary metabolites.⁶ Here, we further examined the role of methanol in rice leaves as an elicitor on the biosynthesis of serotonin derivatives such as 4-coumaroylserotonin (CS) and feruloylserotonin (FS) which show antifungal activity as well as antioxidant activity.^7,8     

Novel major quantitative trait loci regulating the content of isoflavone in soybean seeds

Despite their medicinal, pharmaceutical, and nutritional importance of isoflavones, the genetic basis controlling the amounts of isoflavones in soybean seeds is still not well understood. The main obstacle is the great variability in the content of isoflavone in seeds harvested from different environments. In this study, quantitative trait loci (QTL) for the content of different isoflavones including daidzein, genistein, and glycitein were investigated in a population of recombinant inbred lines derived from the cross of “Hwangkeum” (Glycine max) by “IT182932” (Glycine soja). Seeds analyzed were harvested in three different experimental environments. QTL analyses for isoflavone content were conducted by composite interval mapping across a genomewide genetic map. Two major QTL were mapped to soybean chromosomes 5 and 8, which were designated QDZGT1 and QDZGT2, respectively. Both loci have not been previously reported in other isoflavone sources. The results from this study will be useful in cloning genes that can control the contents of isoflavones in soybean and for the development of soybean lines containing a high or low isoflavone content.     

TRPV1 in GABAergic interneurons mediates neuropathic mechanical allodynia and disinhibition of the nociceptive circuitry in the spinal cord

Neuropathic pain and allodynia may arise from sensitization of central circuits. We report a mechanism of disinhibition-based central sensitization resulting from long-term depression (LTD) of GABAergic interneurons as a consequence of TRPV1 activation in the spinal cord. Intrathecal administration of TRPV1 agonists led to mechanical allodynia that was not dependent on peripheral TRPV1 neurons. TRPV1 was functionally expressed in GABAergic spinal interneurons and activation of spinal TRPV1 resulted in LTD of excitatory inputs and a reduction of inhibitory signaling to spinothalamic tract (STT) projection neurons. Mechanical hypersensitivity after peripheral nerve injury was attenuated in TRPV1(-/-) mice but not in mice lacking TRPV1-expressing peripheral neurons. Mechanical pain was reversed by a spinally applied TRPV1 antagonist while avoiding the hyperthermic side effect of systemic treatment. Our results demonstrate that spinal TRPV1 plays a critical role as a synaptic regulator and suggest the utility of central nervous system-specific TRPV1 antagonists for treating neuropathic pain.