Simian Research Articles

Characterization of an RNase with two catalytic centers. Human RNase6 catalytic and phosphate-binding site arrangement favors the endonuclease cleavage of polymeric substrates

BackgroundHuman RNase6 is a small cationic antimicrobial protein that belongs to the vertebrate RNaseA superfamily. All members share a common catalytic mechanism, which involves a conserved catalytic triad, constituted by two histidines and a lysine (His15/His122/Lys38 in RNase6 corresponding to His12/His119/Lys41 in RNaseA). Recently, our first crystal structure of human RNase6 identified an additional His pair (His36/His39) and suggested the presence of a secondary active site. MethodsIn this work we have explored RNase6 and RNaseA subsite architecture by X-ray crystallography, site-directed mutagenesis and kinetic characterization. ResultsThe analysis of two novel crystal structures of RNase6 in complex with phosphate anions at atomic resolution locates a total of nine binding sites and reveals the contribution of Lys87 to phosphate-binding at the secondary active center. Contribution of the second catalytic triad residues to the enzyme activity is confirmed by mutagenesis. RNase6 catalytic site architecture has been compared with an RNaseA engineered variant where a phosphate-binding subsite is converted into a secondary catalytic center (RNaseA-K7H/R10H). ConclusionsWe have identified the residues that participate in RNase6 second catalytic triad (His36/His39/Lys87) and secondary phosphate-binding sites. To note, residues His39 and Lys87 are unique within higher primates. The RNaseA/RNase6 side-by-side comparison correlates the presence of a dual active site in RNase6 with a favored endonuclease-type cleavage pattern. General significanceAn RNase dual catalytic and extended binding site arrangement facilitates the cleavage of polymeric substrates. This is the first report of the presence of two catalytic centers in a single monomer within the RNaseA superfamily.

A Single Pheromone Receptor Gene Conserved across 400 My of Vertebrate Evolution.

Pheromones are crucial for eliciting social and sexual behaviors in diverse animal species. The vomeronasal receptor type-1 (V1R) genes, encoding members of a pheromone receptor family, are highly variable in number and repertoire among mammals due to extensive gene gain and loss. Here, we report a novel pheromone receptor gene belonging to the V1R family, named ancient V1R (ancV1R), which is shared among most Osteichthyes (bony vertebrates) from the basal lineage of ray-finned fishes to mammals. Phylogenetic and syntenic analyses of ancV1R using 115 vertebrate genomes revealed that it represents an orthologous gene conserved for >400 My of vertebrate evolution. Interestingly, the loss of ancV1R in some tetrapods is coincident with the degeneration of the vomeronasal organ in higher primates, cetaceans, and some reptiles including birds and crocodilians. In addition, ancV1R is expressed in most mature vomeronasal sensory neurons in contrast with canonical V1Rs, which are sparsely expressed in a manner that is consistent with the "one neuron-one receptor" rule. Our results imply that a previously undescribed V1R gene inherited from an ancient Silurian ancestor may have played an important functional role in the evolution of vertebrate vomeronasal organ.

Salivary peptide histatin 1 mediated cell adhesion: a possible role in mesenchymal-epithelial transition and in pathologies.

Histatins are histidine-rich peptides present in the saliva of humans and higher primates and have been implicated in the protection of the oral cavity. Histatin 1 is one of the most abundant histatins and recent reports show that it has a stimulating effect on cellular adherence, thereby suggesting a role in maintaining the quality of the epithelial barrier and stimulating mesenchymal-to-epithelial transition. Here we summarize these findings and discuss them in the context of previous reports. The recent findings also provide new insights in the physiological functions of histatin 1, which are discussed here. Furthermore, we put forward a possible role of histatin 1 in various pathologies and its potential function in clinical applications.

Characterization of the ICCE Repeat in Mammals Reveals an Evolutionary Relationship with the DXZ4 Macrosatellite through Conserved CTCF Binding Motifs.

Appreciation is growing for how chromosomes are organized in three-dimensional space at interphase. Microscopic and high throughput sequence-based studies have established that the mammalian inactive X chromosome (Xi) adopts an alternate conformation relative to the active X chromosome. The Xi is organized into several multi-megabase chromatin loops called superloops. At the base of these loops are superloop anchors, and in humans three of these anchors are composed of large tandem repeat DNA that include DXZ4, Functional Intergenic Repeating RNA Element, and Inactive-X CTCF-binding Contact Element (ICCE). Each repeat contains a high density of binding sites for the architectural organization protein CCCTC-binding factor (CTCF) which exclusively associates with the Xi allele in normal cells. Removal of DXZ4 from the Xi compromises proper folding of the chromosome. In this study, we report the characterization of the ICCE tandem repeat, for which very little is known. ICCE is embedded within an intron of the Nobody (NBDY) gene locus at Xp11.21. We find that primary DNA sequence conservation of ICCE is only retained in higher primates, but that ICCE orthologs exist beyond the primate lineage. Like DXZ4, what is conserved is organization of the underlying DNA into a large tandem repeat, physical location within the NBDY locus and conservation of short DNA sequences corresponding to specific CTCF and Yin Yang 1 binding motifs that correlate with female-specific DNA hypomethylation. Unlike DXZ4, ICCE is not common to all eutherian mammals. Analysis of certain ICCE CTCF motifs reveal striking similarity with the DXZ4 motif and support an evolutionary relationship between DXZ4 and ICCE.

LRRTM4-C538Y novel gene mutation is associated with hereditary macular degeneration with novel dysfunction of ON-type bipolar cells

The macula is a unique structure in higher primates, where cone and rod photoreceptors show highest density in the fovea and the surrounding area, respectively. The hereditary macular dystrophies represent a heterozygous group of rare disorders characterized by central visual loss and atrophy of the macula and surrounding retina. Here we report an atypical absence of ON-type bipolar cell response in a Japanese patient with autosomal dominant macular dystrophy (adMD). To identify a causal genetic mutation for the adMD, we performed whole-exome sequencing (WES) on four affected and four-non affected members of the family for three generations, and identified a novel p.C538Y mutation in a post-synaptic gene, LRRTM4. WES analysis revealed seven rare genetic variations in patients. We further referred to our in-house WES data from 1360 families with inherited retinal diseases, and found that only p.C538Y mutation in LRRTM4 was associated with adMD-affected patients. Combinatorial filtration using public database of single-nucleotide polymorphism frequency and genotype-phenotype annotated database identified novel mutation in atypical adMD.

Justice, Reciprocity and the Internalisation of Punishment in Victims of Crime

This paper is published as part of special issue on the theme of ‘justice without retribution’. Any attempt to consider how justice may be achieved without retribution has to begin with a consideration of what we mean by justice. The most powerful pleas for justice usually come from those who feel that they have been harmed by the wrongful acts of others. This paper will explore this intuition about justice and will argue that it arises from the central importance of reciprocity, in the form of equity, balance and fairness, in human relationships. This is expressed in our image of justice, one of whose core symbols is a set of scales. I will use a clinical case to illustrate what can happen when criminal harm is not followed by any form of restitution. In this case, the punitive impulse was internalised in the victim and turned against herself in the form of deliberate self harm and, ultimately, death by suicide. I will argue that human relationships largely consist of reciprocal acts of good or harm and that we constantly strive for a balanced reciprocity in our relationships with others. This theme has been played out throughout human history in personal relationships, social customs, legal systems, religion and culture. It is also seen in higher primates. When someone is a victim of wrongful harm, there is a need to take measures to restore the balance which has been lost. I will attempt to draw a distinction between retributivism as the term is usually understood and the application of the principle of reciprocity in criminal justice. This distinction is one that has found expression in what has become known as ‘restorative justice’ as opposed to conventional justice. There are two cardinal features of restorative justice in this context. The first is that it brings offender and victim into a relationship with the aim of repairing the harm that has been caused. The second is that it gives the offender the opportunity to give something back to his victim. I will conclude that our responses to criminal wrong-doing should be based on restorative principles and that punishment can sometimes be part of the process by which moral harm is made good.

Adrenocortical development: Lessons from mouse models

The adrenocortical gland undergoes structural and functional remodelling in the fetal and postnatal periods. After birth, the fetal zone of the gland undergoes rapid involution in favor of the definitive cortex, which reaches maturity with the emergence of the zona reticularis(zR) at the adrenarche. The mechanisms underlying the adrenarche, the process leading to pre-puberty elevation of plasma androgens in higher primates, remain unknown, largely due to lack of any experimental model. By following up fetal and definitive cortex cell lines in mice, we showed that activation of protein kinase A (PKA) signaling mainly impacts the adult cortex by stimulating centripetal regeneration, with differentiation and then conversion of the zona fasciculata into a functional zR. Animals developed Cushing syndrome associated with primary hyperaldosteronism, suggesting possible coexistence of these hypersecretions in certain patients. Remarkably, all of these traits were sex-dependent: testicular androgens promoted WNT signaling antagonism on PKA, slowing cortical renewal and delaying onset of Cushing syndrome and the establishment of the zR in male mice, this being corrected by orchidectomy. In conclusion, zR derives from centripetal conversion of the zona fasciculata under cellular renewal induced by PKA signaling, determining the size of the adult cortex. Finally, we demonstrated that this PKA-dependent mobilization of cortical progenitors is sexually dimorphic and could, if confirmed in humans, account for female preponderance in adrenocortical pathologies.

Dominant functional role of the novel phosphorylation site S811 in the human renal NaCl cotransporter.

The NaCl cotransporter (NCC) is essential for electrolyte homeostasis and control of blood pressure. The human SLC12A3 gene, which encodes NCC, gives rise to 3 isoforms, of which only the shortest isoform [NaCl cotransporter isoform 3 (NCC3)] has been studied extensively. All NCC isoforms share key phosphorylation sites at T55 and T60 that are essential mediators of NCC function. Recently, a novel phosphorylation site at S811 was identified in isoforms 1 and 2 [NaCl cotransporter splice variant (NCCSV)], which are only present in humans and higher primates. The aim of the current study, therefore, is to investigate the role of S811 phosphorylation in the regulation of NCC by a combination of biochemical and fluorescent microscopy analyses. We demonstrate that hypotonic low-chloride buffer increases S811 phosphorylation, whereas phosphorylation-deficient S811A mutant hinders phosphorylation at T55 and T60 in NCCSV and NCC3. NCCSV S811A impairs NCC3 activity in a dominant-negative fashion, although it does not affect plasma membrane abundance. This effect may be explained by the heterodimerization of NCCSV with NCC3. Taken together, our study highlights the dominant-negative effect of NCCSV on T55 and T60 phosphorylation and NCC activity. Here, we reveal a new function of NCCSV in humans that broadens the understanding on NCC regulation in blood pressure control.-Tutakhel, O. A. Z., Bianchi, F., Smits, D. A., Bindels, R. J. M., Hoenderop, J. G. J., van der Wijst, J. Dominant functional role of the novel phosphorylation site S811 in the human renal NaCl cotransporter.

The Special Case of Human Astrocytes

In this first issue of Neuroglia, it is highly appropriate that Professor Jorge A. Colombo at the Unit of Applied Neurobiology (UNA, CEMIC-CONICET) in Buenos Aires, Argentina, writes a perspective of idiosyncrasies of astrocytes in the human brain. Much of his work has been focused on the special case of interlaminar astrocytes, so-named because of their long straight processes that traverse the layers of the human cerebral cortex. Notably, interlaminar astrocytes are primate-specific and their evolutionary development is directly related to that of the columnar organization of the cerebral cortex in higher primates. The human brain also contains varicose projection astrocytes or polarized astrocytes which are absent in lower animals. In addition, classical protoplasmic astrocytes dwelling in the brains of humans are ≈15-times larger and immensely more complex than their rodent counterparts. Human astrocytes retain their peculiar morphology even after grafting into rodent brains; that is, they replace the host astrocytes and confer certain cognitive advantages into so-called ‘humanised’ chimeric mice. Recently, a number of innovative studies have highlighted the major differences between human and rodent astrocytes. Nonetheless, these differences are not widely recognized, and we hope that Jorge Colombo’s Perspective and our associated Commentary will help stimulate appreciation of human astrocytes by neuroscientists and glial cell biologists alike.

Simian, Amphibian, and Able: Reevaluating Browning’s Caliban

Simian, Amphibian, and Able: Reevaluating Browning’s Caliban

Analysis of chromatin accessibility in decidualizing human endometrial stromal cells.

Spontaneous decidualization of the endometrium in response to progesterone signaling is confined to menstruating species, including humans and other higher primates. During this process, endometrial stromal cells (EnSCs) differentiate into specialized decidual cells that control embryo implantation. We subjected undifferentiated and decidualizing human EnSCs to an assay for transposase accessible chromatin with sequencing (ATAC-seq) to map the underlying chromatin changes. A total of 185,084 open DNA loci were mapped accurately in EnSCs. Altered chromatin accessibility upon decidualization was strongly associated with differential gene expression. Analysis of 1533 opening and closing chromatin regions revealed over-representation of DNA binding motifs for known decidual transcription factors (TFs) and identified putative new regulators. ATAC-seq footprint analysis provided evidence of TF binding at specific motifs. One of the largest footprints involved the most enriched motif—basic leucine zipper—as part of a triple motif that also comprised the estrogen receptor and Pax domain binding sites. Without exception, triple motifs were located within Alu elements, which suggests a role for this primate-specific transposable element (TE) in the evolution of decidual genes. Although other TEs were generally under-represented in open chromatin of undifferentiated EnSCs, several classes contributed to the regulatory DNA landscape that underpins decidual gene expression.—Vrljicak, P., Lucas, E. S., Lansdowne, L., Lucciola, R., Muter, J., Dyer, N. P., Brosens, J. J., Ott, S. Analysis of chromatin accessibility in decidualizing human endometrial stromal cells.

Microarray for gene expression analysis of defined subtypes of motoneurons

Background: Some cells in the motor-related cortices directly control muscles via spinal motoneurons (MNs) by monosynaptic connection in higher primates. It is generally accepted that this direct connection in primates is essential for dexterous hand movements.

Assessing the Circulating Placental-Specific Anti-angiogenic Protein sFLT-1 e15a in Preeclampsia.

Preeclampsia is a common obstetric complication globally responsible for a significant burden of maternal and perinatal morbidity and mortality. The anti-angiogenic protein, sFLT-1, plays a central role in its pathophysiology. sFLT-1 is released from a range of tissues into the circulation, where it antagonizes the activity of vascular endothelial growth factor and placental growth factor leading to endothelial dysfunction. The resulting widespread endothelial dysfunction produces the clinical features of preeclampsia including hypertension and proteinuria. Multiple splice variants of sFLT-1 have been identified, with one, known as sFLT-1 e15a, present only in humans and higher-order primates. This sFLT-1 variant is also the main form of sFLT-1 produced by the placenta. Recent work has shown that sFLT-1 e15a is significantly elevated in the placenta and circulation of women with preeclampsia. It is also biologically active, capable of causing endothelial dysfunction and end-organ dysfunction seen in preeclampsia. Indeed, overexpression of sFLT-1 e15a in mice recapitulates the preeclamptic phenotype in pregnancy. No commercial assay currently exists to analyze sFLT-1 e15a protein levels. Here, a new ELISA method to determine circulating sFLT-1 variant levels is described.

Control of species-dependent cortico-motoneuronal connections underlying manual dexterity.

Superior manual dexterity in higher primates emerged together with the appearance of cortico-motoneuronal (CM) connections during the evolution of the mammalian corticospinal (CS) system. Previously thought to be specific to higher primates, we identified transient CM connections in early postnatal mice, which are eventually eliminated by Sema6D-PlexA1 signaling. PlexA1 mutant mice maintain CM connections into adulthood and exhibit superior manual dexterity as compared with that of controls. Last, differing PlexA1 expression in layer 5 of the motor cortex, which is strong in wild-type mice but weak in humans, may be explained by FEZF2-mediated cis-regulatory elements that are found only in higher primates. Thus, species-dependent regulation of PlexA1 expression may have been crucial in the evolution of mammalian CS systems that improved fine motor control in higher primates.

A rare missense variant in RCL1 segregates with depression in extended families

Depression is the most prevalent psychiatric disorder with a complex and elusive etiology that is moderately heritable. Identification of genes would greatly facilitate the elucidation of the biological mechanisms underlying depression, however, its complex etiology has proved to be a major bottleneck in the identification of its genetic risk factors, especially in genome-wide association-like studies. In this study, we exploit the properties of a genetic isolate and its family-based structure to explore whether relatively rare exonic variants influence the burden of depressive symptoms in families. Using a multistep approach involving linkage and haplotype analyses followed by exome sequencing in the Erasmus Rucphen Family (ERF) study, we identified a rare (minor allele frequency (MAF)=1%) missense c.1114C>T mutation (rs115482041) in the RCL1 gene segregating with depression across multiple generations. Rs115482041 showed significant association with depressive symptoms (N=2393, βT-allele=2.33, P-value=1 × 10−4) and explained 2.9% of the estimated genetic variance of depressive symptoms (22%) in ERF. Despite being twice as rare (MAF<0.5%), c.1114C>T showed similar effect and significant association with depressive symptoms in samples from the independent population-based Rotterdam study (N=1604, βT-allele=3.60, P-value=3 × 10−2). A comparison of RCL1 expression in human and mouse brain revealed a striking co-localization of RCL1 with the layer 1 interlaminar subclass of astrocytes found exclusively in higher-order primates. Our findings identify RCL1 as a novel candidate gene for depression and offer insights into mechanisms through which RCL1 may be relevant for depression.

The relationship between the Fermi–Dirac distribution and statistical distributions in languages

In this article, we study, from the mathematical point of view, the analogies between language and multi-particle systems in thermodynamics. We attempt to introduce an appropriate mathematical apparatus and the technical tools of statistical physics to descriptions of language. In particular, we apply the notions of number of degrees of freedom, Bose condensate, phase transition and others to linguistics objects. On the basis of a statistical analysis of dictionaries and statistical distributions in languages, we conjecture that the transition from the semiotic communication system of the higher primates to human language can be described as a phase transition of the first kind. We show that the number of words appearing with frequency 1 in a corpus of texts is equal to the number of ones in the corresponding Fermi–Dirac distribution, while the high frequency of stop-words corresponds to the large number of particles in the Bose condensate, when the number of degrees of freedom is less than two, provided there is a gap in the spectrum. The presented considerations are illustrated by examples from the Russian language. Some of the illustrative examples are untranslatable into English, and so they were replaced in translation by similar examples from the English language.

Evolution of Brain Active Gene Promoters in Human Lineage Towards the Increased Plasticity of Gene Regulation.

Adaptability to a variety of environmental conditions is a prominent feature of Homo sapiens. We hypothesize that this feature can be explained by evolutionary changes in gene promoters active in the brain prefrontal cortex leading to a more flexible gene regulation network. The genotype-dependent range of gene expression can be broader in humans than in other higher primates. Thus, we searched for specific signatures of evolutionary changes in promoter architectures of multiple hominid genes, including the genes active in human cortical neurons that may indicate an increase of variability of gene expression rather than just changes in the level of expression, such as downregulation or upregulation of the genes. We performed a whole-genome search for genetic-based alterations that may impact gene regulation "flexibility" in a process of hominids evolution, such as (i) CpG dinucleotide content, (ii) predicted nucleosome-DNA dissociation constant, and (iii) predicted affinities for TATA-binding protein (TBP) in gene promoters. We tested all putative promoter regions across the human genome and especially gene promoters in active chromatin state in neurons of prefrontal cortex, the brain region critical for abstract thinking and social and behavioral adaptation. Our data imply that the origin of modern man has been associated with an increase of flexibility of promoter-driven gene regulation in brain. In contrast, after splitting from the ancestral lineages of H. sapiens, the evolution of ape species is characterized by reduced flexibility of gene promoter functioning, underlying reduced variability of the gene expression.

Human Salivary Protein Histatin 5 Has Potent Bactericidal Activity against ESKAPE Pathogens.

ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanni, Pseudomonas aeruginosa, and Enterobacter species) pathogens have characteristic multiple-drug resistance and cause an increasing number of nosocomial infections worldwide. Peptide-based therapeutics to treat ESKAPE infections might be an alternative to conventional antibiotics. Histatin 5 (Hst 5) is a salivary cationic histidine-rich peptide produced only in humans and higher primates. It has high antifungal activity against Candida albicans through an energy-dependent, non-lytic process; but its bactericidal effects are less known. We found Hst 5 has bactericidal activity against S. aureus (60–70% killing) and A. baumannii (85–90% killing) in 10 and 100 mM sodium phosphate buffer (NaPB), while killing of >99% of P. aeruginosa, 60–80% E. cloacae and 20–60% of E. faecium was found in 10 mM NaPB. Hst 5 killed 60% of biofilm cells of P. aeruginosa, but had reduced activity against biofilms of S. aureus and A. baumannii. Hst 5 killed 20% of K. pneumonia biofilm cells but not planktonic cells. Binding and uptake studies using FITC-labeled Hst 5 showed E. faecium and E. cloacae killing required Hst 5 internalization and was energy dependent, while bactericidal activity was rapid against P. aeruginosa and A. baumannii suggesting membrane disruption. Hst 5-mediated killing of S. aureus was both non-lytic and energy independent. Additionally, we found that spermidine conjugated Hst 5 (Hst5-Spd) had improved killing activity against E. faecium, E. cloacae, and A. baumannii. Hst 5 or its derivative has antibacterial activity against five out of six ESKAPE pathogens and may be an alternative treatment for these infections.

Has evolution ‘prepared’ us to deal with death? Paleoanthropological aspects of the enigma of &lt;i&gt;Homo naledi’s&lt;/i&gt; disposal of their dead

The Homo naledi discovery introduced questions that had not been previously posed regarding fossil finds. This is because, apart from their fascinating physiology, they seemingly deliberately disposed of their dead in a ritualised way. Although this theory may still be disproved in future, the present article provisionally accepts it. This evokes religious questions because it suggests the possibility of causal thinking, wilful and cooperative behaviour, and the possibility that this behaviour entails traces of proto-religious ideas. This poses the challenge to develop a hominin hermeneutics that endeavours to reconstruct the possible motivation behind this action. The relatively larger brain with its enlarged Broca’s area suggests the possibility of a sophisticated communication system and an enhanced way of dealing with emotion. We know that almost all life forms have some form of awareness and that more sophisticated degrees of consciousness may be present in the higher primates. Various ‘clues’ are investigated to try and understand the H. naledi phenomenon: lessons from chimpanzee studies, the implications of tool making for hominin development, the possibility of a proto-language and the role symbol formation may have played. The H. naledi case also indicates on a theological level that religion is natural. Some attention is given to this thesis. Biological and environmental factors come into play to illuminate biological factors like emotion and higher cognition without which religion would not be possible. Sophisticated cognition is coloured by affect (basic emotions are typical of all mammals) and this makes some form of reflection on the fate of loved ones who have died a strong possibility.

Histatin 1 Enhances Cell Adhesion to Titanium in an Implant Integration Model

Cellular adhesion is essential for successful integration of dental implants. Rapid soft tissue integration is important to create a seal around the implant and prevent infections, which commonly cause implant failure and can result in bone loss. In addition, soft tissue management is important to obtain good dental aesthetics. We previously demonstrated that the salivary peptide histatin 1 (Hst1) causes a more than 2-fold increase in the ability of human adherent cells to attach and spread on a glass surface. Cells treated with Hst1 attached more rapidly and firmly to the substrate and to each other. In the current study, we examine the potential application of Hst1 for promotion of dental implant integration. Our results show that Hst1 enhances the attachment and spreading of soft tissue cell types (oral epithelial cells and fibroblasts) to titanium (Ti) and hydroxyapatite (HAP), biomaterials that have found wide applications as implant material in dentistry and orthopedics. For improved visualization of cell adhesion to Ti, we developed a novel technique that uses sputtering to deposit a thin, transparent layer of Ti onto glass slides. This approach allows detailed, high-resolution analysis of cell adherence to Ti in real time. Furthermore, our results suggest that Hst1 has no negative effects on cell survival. Given its natural occurrence in the oral cavity, Hst1 could be an attractive agent for clinical application. Importantly, even though Hst1 is specific for saliva of humans and higher primates, it stimulated the attachment and spreading of canine cells, paving the way for preclinical studies in canine models.