High osmolarity, bound water, and hydrostatic pressure contribute to notochord mechanics and its morphogenesis into the nucleus pulposus (NP) compartment of the intervertebral disc. Indeed, the osmoadaptive transcription factor, nuclear factor of activated T-cells 5 (NFAT5 aka TonEBP), is robustly expressed by resident cells of the notochord and NP. Nevertheless, the molecular mechanisms that drive notochord osmoregulation and the functions of NFAT5 in disc embryogenesis remain largely unexplored. In this study, we show that deletion of NFAT5 in mice results in delayed vertebral column development and a reduced NP aspect ratio in the caudal spine. This phenotype is associated with lower levels of the T-box transcription factor, Brachyury, delayed expression of notochord phenotypic markers, and decreased collagen II deposition in the perinotochordal sheath and condensing mesenchyme. In addition, NFAT5 mutants showed a stage-dependent dysregulation of sonic hedgehog (Shh) signaling with non-classical expression of Gli1. Generation of mice with notochord-specific deletion of IFT88 (ShhcreERT2;Ift88f/f) supported this mode of Gli1 regulation. Using isolated primary NP cells and bioinformatics approaches, we further show that Ptch1 and Smo expression is controlled by NFAT5 in a cell autonomous manner. Altogether, our results demonstrate that NFAT5 contributes to notochord and disc embryogenesis through its regulation of hallmark notochord phenotypic markers, extracellular matrix, and Shh signaling.

Cerebrovascular remodeling is the leading factor for stroke and characterized by increased extracellular matrix deposition, migration and proliferation of vascular smooth muscle cells, and inhibition of their apoptosis. TMEM16A is an important component of Ca2+-activated Cl- channels. Previously, we showed that downregulation of TMEM16A in the basilar artery was negatively correlated with cerebrovascular remodeling during hypertension. However, it is unclear whether TMEM16A participates in angiotensin II (Ang II)-induced vascular remodeling in mice that have TMEM16A gene modification. In this study, we generated a transgenic mouse that overexpresses TMEM16A specifically in vascular smooth muscle cells. We observed that vascular remodeling in the basilar artery during Ang II-induced hypertension was significantly suppressed upon vascular smooth muscle-specific overexpression of TMEM16A relative to control mice. Specifically, we observed a large reduction in the deposition of fibronectin and collagen I. The expression of matrix metalloproteinases (MMP-2, MMP-9, and MMP-14), and tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) were upregulated in the basilar artery during Ang II-induced hypertension, but this was suppressed upon overexpression of TMEM16A in blood vessels. Furthermore, TMEM16A overexpression alleviated the overactivity of the canonical TGF-β1/Smad3, and non-canonical TGF-β1/ERK and JNK pathways in the basilar artery during Ang II-induced hypertension. These in vivo results were similar to the results derived in vitro with basilar artery smooth muscle cells stimulated by Ang II. Moreover, we observed that the inhibitory effect of TMEM16A on MMPs was mediated by decreasing the activation of WNK1, which is a Cl--sensitive serine/threonine kinase. In conclusion, this study demonstrates that TMEM16A protects against cerebrovascular remodeling during hypertension by suppressing extracellular matrix deposition. We also showed that TMEM16A exerts this effect by reducing the expression of MMPs via inhibiting WNK1, and decreasing the subsequent activities of TGF-β1/Smad3, ERK, and JNK. Accordingly, our results suggest that TMEM16A may serve as a novel therapeutic target for vascular remodeling.

Cancer is a major cause of death in both developed and developing countries. Polyphenols, abundantly found in plants, possess many anticarcinogenic properties, including inhibition of cancer cell proliferation, tumor growth, angiogenesis, metastasis and inflammation, as well as pro-apoptotic effects. Our study aimed to investigate the effects of a complex of (+)-catechin with 2 lysines (Cat:Lys) on cancer and non-cancer cells. For this, the in vitro effects of Cat:Lys on the viability, growth, proliferation, apoptosis, nutrient uptake and migration of breast, pancreatic and colorectal cancer and non-cancer cell lines was evaluated. We found that Cat:Lys exerted antiproliferative and cytotoxic effects in all breast, pancreatic and colorectal cell lines tested, but with a much less marked amplitude in non-cancer cell lines. It nevertheless interfered with nutrient (3H-deoxy-D-glucose and 3H-lactate) uptake and with lactate production in both cancer and non-cancer cell lines. Cat:Lys was found to possess selective antimigratory effects in breast, pancreatic and colorectal cancer cell lines compared to non-cancer cell lines. Cat:Lys also exerted pro-apoptotic effects in all the cancer cell lines that we tested, but not in non-cancer breast and pancreatic cell lines. The antimigratory, but not the pro-apoptotic, effects of Cat:Lys were found to be mediated by JAK2/STAT3 and Wnt pathway inhibition. In conclusion, Cat:Lys is a strong candidate for the development of new, effective anticancer agents against cancer.

JTZ-951 (enarodustat) is an oral hypoxia-inducible factor (HIF) prolyl hydroxylase inhibitor. JTZ-951 has inhibitory activities on human HIF-prolyl hydroxylase 1-3, but not on various receptors or enzymes. In Hep3B cells, JTZ-951 increased HIF-1α and HIF-2α protein levels, erythropoietin (EPO) mRNA levels, and EPO production. In normal rats, after a single oral dose of JTZ-951, the hepatic and renal EPO mRNA levels and plasma EPO concentrations were also increased. In 5/6-nephrectomized rats, repeated oral doses of JTZ-951 once daily or intermittent dosing showed the erythropoiesis stimulating effect. The administration of JTZ-951 at a high dose increased plasma vascular endothelial growth factor (VEGF) levels; however, retinal VEGF mRNA levels and the retinal vascular permeability were not changed. Finally, we evaluated the effect of JTZ-951 in a colorectal cancer cell-inoculated mouse model. Although JTZ-951 at a high dose increased the plasma VEGF, it had no effect on tumor growth. In summary, JTZ-951 induces erythropoiesis without affecting VEGF function. Therefore, it is expected that JTZ-951 will be a new oral candidate that increases and maintains hemoglobin concentrations in renal anemia patients.

A recent report of autosomal-recessive primary isolated dystonia (DYT2 dystonia) identified mutations in HPCA, a gene encoding a neuronal calcium sensor protein, hippocalcin (HPCA), as the cause of this disease. However, how mutant HPCA leads to neuronal dysfunction remains unknown. Using a multidisciplinary approach, we demonstrated the failure of dystonic N75K HPCA mutant to decode short bursts of action potentials and theta rhythms in hippocampal neurons by its Ca2+-dependent translocation to the plasma membrane. This translocation suppresses neuronal activity via slow afterhyperpolarization (sAHP) and we found that the N75K mutant could not control sAHP during physiologically relevant neuronal activation. Simulations based on the obtained experimental results directly demonstrated an increased excitability in neurons expressing N75K mutant instead of wild type (WT) HPCA. In conclusion, our study identifies sAHP as a downstream cellular target perturbed by N75K mutation in DYT2 dystonia, demonstrates its impact on neuronal excitability, and suggests a potential therapeutic strategy to efficiently treat DYT2.

Optimal decision-making requires humans to predict the value and probability of prospective (rewarding) outcomes. The aim of the present study was to evaluate and dissociate the cortical mechanisms activated by information on an upcoming potentially rewarded target stimulus with varying probabilities. Electro-cortical activity was recorded during a cued Go/NoGo experiment, during which cue letters signaled upcoming target letters to which participants had to respond. The probability of target letter appearance after the cue letter and the amount of money that could be won for correct and fast responses were orthogonally manipulated across four task blocks. As expected, reward availability affected a prefrontally distributed reward-related positivity, and a centrally distributed P300-like event-related potential (ERP). Moreover, a late prefrontally distributed ERP was affected by probability information. These results show that information on value and probability, respectively, activates separate mechanisms in the cortex. These results contribute to a further understanding of the neural underpinnings of normal and abnormal reward processing.

The developmentally indispensable Notch pathway exhibits a high grade of pleiotropism in its biological output. Emerging evidence supports the notion of post-translational modifications (PTMs) as a modus operandi controlling dynamic fine-tuning of Notch activity. Although, the intricacy of Notch post-translational regulation, as well as how these modifications lead to multiples of divergent Notch phenotypes is still largely unknown, numerous studies show a correlation between the site of modification and the output. These include glycosylation of the extracellular domain of Notch modulating ligand binding, and phosphorylation of the PEST domain controlling half-life of the intracellular domain of Notch. Furthermore, several reports show that multiple PTMs can act in concert, or compete for the same sites to drive opposite outputs. However, further investigation of the complex PTM crosstalk is required for a complete understanding of the PTM-mediated Notch switchboard. In this review, we aim to provide a consistent and up-to-date summary of the currently known PTMs acting on the Notch signaling pathway, their functions in different contexts, as well as explore their implications in physiology and disease. Furthermore, we give an overview of the present state of PTM research methodology, and allude to a future with PTM-targeted Notch therapeutics.

Understanding and predicting the intentions of others through limb movements are vital to social interaction. The processing of biological motion is unique from the processing of motion of inanimate objects. Presently, there is controversy over whether visual consciousness of biological motion is regulated by visual attention. In addition, the neural mechanisms involved in biological motion-related visual awareness are not known. In the current study, the relationship between visual awareness (aware vs unaware), represented by a point-light walker and biological-motion-based attention, manipulated by a difference in congruence (congruent, incongruent) between the direction of a pre-cue and that of biological motion was explored. The neural mechanisms involved in processing the stimuli were explored through electroencephalography. Both early (50-150 ms, 100-200 ms, and 174-226 ms after target presentation) and late (350-550 ms after target presentation) awareness-related neural processings were observed during a biological motion-based congruency task. Early processing was localized to occipital-parietal regions, such as the left postcentral gyrus, the left middle occipital gyrus, and the right precentral gyrus. In the 174-226-ms window, the activity in the occipital region was gradually replaced by activity in the parietal and frontal regions. Late processing was localized to frontal-parietal regions, such as the right dorsal superior frontal gyrus, the left medial superior frontal gyrus, and the occipito-temporal regions. Congruency-related processing occurred in the 246-260-ms window and was localized to the right superior occipital gyrus. In summary, due to its complexity, biological motion awareness has a unique neural basis.

Food allergy is a growing health problem worldwide because of its increasing prevalence, life-threatening potential, and shortage of effective preventive treatments. In an outbreak of wheat allergy in Japan, thousands of patients had allergic reactions to wheat after using soap containing hydrolyzed wheat protein (HWP).

Specific interactions between the helical membrane-spanning domains of transmembrane proteins play central roles in the proper folding and oligomerization of these proteins. However, the relationship between the hydrophobic amino acid sequences of transmembrane domains and their functional interactions is in most cases unknown. Here, we use ultra-simple artificial proteins to systematically study the sequence basis for transmembrane domain interactions. We show that most short homopolymeric polyleucine transmembrane proteins containing single amino acid substitutions can activate the platelet-derived growth factor β receptor or the erythropoietin receptor in cultured mouse cells, resulting in cell transformation or proliferation. These proteins displayed complex patterns of activity that were markedly affected by seemingly minor sequence differences in the ultra-simple protein itself or in the transmembrane domain of the target receptor, and the effects of these sequence differences are not additive. In addition, specific leucine residues along the length of these proteins are required for activity, and the positions of these required leucines differ based on the identity and position of the central substituted amino acid. Our results suggest that these ultra-simple proteins use a variety of molecular mechanisms to activate the same target and that diversification of transmembrane domain sequences over the course of evolution minimized off-target interactions.

Vagus nerve stimulation (VNS) is being explored as a potential therapeutic for Parkinson's disease (PD). VNS is less invasive than other surgical treatments and has beneficial effects on behavior and brain pathology. It has been suggested that VNS exerts these effects by increasing brain-derived neurotrophic factor (BDNF) to enhance pro-survival mechanisms of its receptor, tropomyosin receptor kinase-B (TrkB). We have previously shown that striatal BDNF is increased after VNS in a lesion model of PD. By chronically administering ANA-12, a TrkB-specific antagonist, we aimed to determine TrkB's role in beneficial VNS effects for a PD model. In this study, we administered a noradrenergic neurotoxin, DSP-4, intraperitoneally and one week later administered a bilateral intrastriatal dopaminergic neurotoxin, 6-OHDA. At this time, the left vagus nerve was cuffed for stimulation. Eleven days later, rats received VNS twice per day for ten days, with daily locomotor assessment. Daily ANA-12 injections were given one hour prior to the afternoon stimulation and concurrent locomotor session. Following the final VNS session, rats were euthanized, and left striatum, bilateral substantia nigra and locus coeruleus were sectioned for immunohistochemical detection of neurons, α-synuclein, astrocytes, and microglia. While ANA-12 did not avert behavioral improvements of VNS, and only partially prevented VNS-induced attenuation of neuronal loss in the locus coeruleus, it did stop neuronal and anti-inflammatory effects of VNS in the nigrostriatal system, indicating a role for TrkB in mediating VNS efficacy. However, our data also suggest that BDNF-TrkB is not the sole mechanism of action for VNS in PD.

Glossina palpalis palpalis remains the major vector of sleeping sickness in Côte d'Ivoire. The disease is still active at low endemic levels in Bonon and Sinfra foci in the western-central part of the country. In this study, we investigated the impact of a control campaign on G. p. palpalis population structure in Bonon and Sinfra foci in order to adapt control strategies. Genetic variation at microsatellite loci was used to examine the population structure of different G. p. palpalis cohorts before and after control campaigns. Isolation by distance was observed in our sampling sites. Before control, effective population size was high (239 individuals) with dispersal at rather short distance (731 m per generation). We found some evidence that some of the flies captured after treatment come from surrounding sites, which increased the genetic variance. One Locus, GPCAG, displayed a 1000% increase of subdivision measure after control while other loci only exhibited a substantial increase in variance of subdivision. Our data suggested a possible trap avoidance behaviour in G. p. palpalis. It is important to take into account and better understand the possible reinvasion from neighboring sites and trap avoidance for the sake of sustainability of control campaigns effects.

Pogostemon cablin is an important commercial source of patchouli oil, whose main active ingredient is patchouli alcohol. This sesquiterpene is a product of the mevalonate pathway, in which 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR) is the rate-limiting enzyme. In this study, P. cablin HMGCR cDNA, comprising 2209 nucleotides encoding 425 amino acid residues was isolated, and bioinformatics analysis was used to analyze the protein sequence. Based on this analysis, a C-terminal truncated variant was engineered for recombinant expression in E. coli. The 38 kDa recombinant protein was identified by SDS-PAGE, and assayed for mevalonolactone production. According to the PcHMGCR1 gene sequence alignment with other species, the HMGCR protein had obvious resemblance with other plants HMG coenzyme A reductase and had homology with other species including plants, fungi, archaebacteria and animals. The prokaryotic expression vector was constructed by restriction enzyme digestion to be transformed into E. coli to express the recombinant protein, and 38 kDa recombinant protein was identified by the SDS-PAGE. Enzymatic activity was detected using GC-MS and, as a result, mevalonolactone was detected in the in vitro reaction mixture. Differential expression analysis showed that PcHMGCR1 expressed the highest amount in roots. The research results are of great significance for further research on the molecular biosynthesis mechanism of Patchouli alcohol in P. cablin.

Deuterium incorporation in solid-state hydrogen deuterium exchange with mass spectrometry (ssHDX-MS) has been correlated with protein aggregation on storage in sugar-based solid matrices. Here, the effects of sucrose, arginine and histidine buffer on the rate of aggregation of a lyophilized monoclonal antibody (mAb) were assessed using design of experiments (DoE) and response surface methodology. Lyophilized formulations were characterized using ssHDX-MS and Fourier transform infrared spectroscopy (ssFTIR) to assess potential correlation with stability in solid state. The samples were subjected to storage stability at 5 °C and stressed stability at 40 °C/75% RH for 6 months, and the aggregation rate was measured using size exclusion chromatography (SEC). Different levels of arginine had no significant effect on deuterium uptake in ssHDX-MS, although stability studies showed that aggregation rate decreased with increasing arginine concentration. Similarly, when histidine buffer was replaced with phosphate buffer at the same pH and molarity, ssHDX-MS showed no differences in deuterium uptake, but storage stability studies showed a significant increase in aggregation rate. The results suggest that proteins can be stabilized in amorphous solids by ionic interactions which ssHDX-MS does not detect, an important indication of the limitations of the method.

Induced pluripotent stem cell (iPSC) line were generated from erythroblasts of a Brazilian patient with familiar form of amyotrophic lateral sclerosis (ALS). NGS analysis demonstrated that patient carried a mutation in SOD1 gene, as well as a deletion in FUS gene. CytoTune™-iPS 2.0 Sendai Reprogramming Kit (containing the reprogramming factors OCT3/4, KLF4, SOX2 and cMYC) was used to generate the cell lines. The iPSCs express pluripotency markers, have normal karyotype and differentiated spontaneously in the three germ layers. The expression of Sendai virus was lost in all iPSC lines after 15 passages.

Phenyl- and bioisosteric ferrocenyl-derived aminoquinoline-benzimidazole hybrid compounds were synthesised and evaluated for their in vitro antiplasmodial activity against the chloroquine-sensitive NF54 and multi-drug resistant K1 strains of the human malaria parasite, Plasmodium falciparum. All compounds were active against the two strains, generally showing enhanced activity in the K1 strain, with resistance indices less than 1. Cytotoxicity studies using Chinese hamster ovarian cells revealed that the hybrids were relatively non-cytotoxic and demonstrated selective killing of the parasite. Based on favourable in vitro antiplasmodial and cytotoxicity data, the most active phenyl (4c) and ferrocenyl (5b) hybrids were tested in vivo against the rodent Plasmodium berghei mouse model. Both compounds caused a reduction in parasitemia relative to the control, with 5c displaying superior activity (92% reduction in parasitemia at 4 × 50 mg/kg oral doses). The most active phenyl and ferrocenyl derivatives showed inhibition of β-haematin formation in a NP-40 detergent-mediated assay, indicating a possible contributing mechanism of antiplasmodial action. The most active ferrocenyl hybrid did not display appreciable reactive oxygen species (ROS) generation in a ROS-induced DNA cleavage gel electrophoresis study. The compounds were also screened for their in vitro activity against Mycobacterium tuberculosis. The hybrids containing a more hydrophobic substituent had enhanced activity (<32.7 μM) compared to those with a less hydrophobic substituent (>62.5 μM).

A series of pyrazole-thiophene derivatives exhibiting good Akt inhibitory activities were obtained on the basis of conformational restriction strategy, leading to the discovery of compound 1d and 1o which showed excellent in vitro antitumor effect against a variety of hematologic cancer cells and their potential of inducing apoptosis, blocking the cell cycles at S phase and significantly inhibiting the phosphorylation of downstream biomarkers of Akt kinase of cancer cells. Amongst, compound 1o also exhibited good PK profiles and inhibited about 40% tumor growth in MM1S xenograft model. Compound 1o might be a potential candidate for further development.

The interconversion of sequences that constitute the genome and the proteome is becoming increasingly important due to the generation of large amounts of DNA sequence data. Following mapping of DNA segments to the genome, one fundamentally important task is to find the amino acid sequences which are coded within a list of genomic sections. Conversely, given a series of protein segments, an important task is to find the genomic loci which code for a list of protein regions. To perform these tasks on a region by region basis is extremely laborious when a large number of regions are being studied. We have therefore implemented an R package geno2proteo which performs the two mapping tasks and subsequent sequence retrieval in a batch fashion. In order to make the tool more accessible to users, we have created a web interface of the R package which allows the users to perform the mapping tasks by going to the web page http://sharrocksresources.manchester.ac.uk/tofigaps and using the web service.

Adenoid cystic carcinoma (ACC) is a slow growing salivary gland malignancy that is molecularly characterized by t(6:9)(q22-23;p23-24) translocations which predominantly result in MYB-NFIB gene fusions in nearly half of tumours. Detection of MYB-NFIB transcripts is typically performed with fresh ACC tissue using conventional RT-PCR fragment analysis or FISH techniques, which are prone to failure when only archival formalin fixed paraffin embedded (FFPE) tissue is available. The purpose of this pilot study was to evaluate the utility of NanoString probe technology for the detection of MYB-NFIB transcripts in archival ACC tissue.

To understand the physiological functions of polyamine oxidases (PAOs) in plants, we analyzed the effects of exogenous polyamines during seed germination and early seedling development, using Arabidopsis thaliana lines independently harboring T-DNA insertions in each PAO gene. Spermine caused seedling growth inhibition but did not affect the germination in all lines including wild-type Col-0. However, an AtPAO2 knockout mutant, -pao2, could not germinate under excess spermidine (Spd) conditions. The root growth rates of post-germination -pao2 seedlings were also strongly inhibited by the Spd treatment compared with the wild-type plants. AtPAO2 has a conserved peroxisome-targeting signal sequence at its C-terminus. We prepared two types of AtPAO2 expression plants in a -pao2 background. In -pao2/PAO2 plants a 5.8-kbp genomic fragment containing the complete coding sequence of AtPAO2 was introduced, while in -pao2/PAO2ΔC plants the same fragment lacking the peroxisome-targeting signal was introduced. The Spd-sensitive phenotypes observed in -pao2 were completely recovered in both of the transgenic complementation lines. Thus, AtPAO2 appears to be involved in excess Spd catabolism during seed germination and early seedling development irrespective of subcellular localization.