pIChemiSt ─ Free Tool for the Calculation of Isoelectric Points of Modified Peptides.

The isoelectric point (pI) is a fundamental physicochemical property of peptides and proteins. It is widely used to steer design away from low solubility and aggregation and guide peptide separation and purification. Experimental measurements of pI can be replaced by calculations knowing the ionizable groups of peptides and their corresponding pKa values. Different pKa sets are published in the literature for natural amino acids, however, they are insufficient to describe synthetically modified peptides, complex peptides of natural origin, and peptides conjugated with structures of other modalities. Noncanonical modifications (nCAAs) are ignored in the conventional sequence-based pI calculations, therefore producing large errors in their pI predictions. In this work, we describe a pI calculation method that uses the chemical structure as an input, automatically identifies ionizable groups of nCAAs and other fragments, and performs pKa predictions for them. The method is validated on a curated set of experimental measures on 29 modified and 119093 natural peptides, providing an improvement of R2 from 0.74 to 0.95 and 0.96 against the conventional sequence-based approach for modified peptides for the two studied pKa prediction tools, ACDlabs and pKaMatcher, correspondingly. The method is available in the form of an open source Python library at https://github.com/AstraZeneca/peptide-tools, which can be integrated into other proprietary and free software packages. We anticipate that the pI calculation tool may facilitate optimization and purification activities across various application domains of peptides, including the development of biopharmaceuticals.

Unraveling the Allosteric Cross-Talk between the Coactivator Peptide and the Ligand-Binding Site in the Glucocorticoid Receptor.

The glucocorticoid receptor (GR) is a nuclear receptor that controls critical biological processes by regulating the transcription of specific genes. There is a known allosteric cross-talk between the ligand and coregulator binding sites within the GR ligand-binding domain that is crucial for the control of the functional response. However, the molecular mechanisms underlying such an allosteric control remain elusive. Here, molecular dynamics (MD) simulations, bioinformatic analysis, and biophysical measurements are integrated to capture the structural and dynamic features of the allosteric cross-talk within the GR. We identified a network of evolutionarily conserved residues that enables the allosteric signal transduction, in agreement with experimental data. MD simulations clarify how such a network is dynamically interconnected and offer a mechanistic explanation of how different peptides affect the intensity of the allosteric signal. This study provides useful insights to elucidate the GR allosteric regulation, ultimately providing a foundation for designing novel drugs.

Reduced T cell-dependent humoral immune response in microsomal prostaglandin E synthase-1 null mice is mediated by nonhematopoietic cells.

Microsomal PGE synthase-1 (mPGES-1) is an inducible enzyme that specifically catalyzes the conversion of PGH2 to PGE2. We showed that mPGES-1 null mice had a significantly reduced incidence and severity of collagen-induced arthritis compared with wild-type (WT) mice associated with a marked reduction in Abs to type II collagen. In this study, we further elucidated the role of mPGES-1 in the humoral immune response. Basal levels of serum IgM and IgG were significantly reduced in mPGES-1 null mice. Compared with WT mice, mPGES-1 null mice exhibited a significant reduction of hapten-specific serum Abs in response to immunization with the T cell-dependent (TD) Ag DNP-keyhole limpet hemocyanin. Immunization with the T cell-independent type 1 Ag trinitrophenyl-LPS or the T cell-independent type 2 Ag DNP-Ficoll revealed minimal differences between strains. Germinal center formation in the spleen of mPGES-1 null and WT mice were similar after immunization with DNP-keyhole limpet hemocyanin. To determine whether the effect of mPGES-1 and PGE2 was localized to hematopoietic or nonhematopoietic cells, we generated bone marrow chimeras. We demonstrated that mPGES-1 deficiency in nonhematopoietic cells was the critical factor for reduced TD Ab production. We conclude that mPGES-1 and PGE2-dependent phenotypic changes of nonhematopoietic/mesenchymal stromal cells play a key role in TD humoral immune responses in vivo. These findings may have relevance to the pathogenesis of rheumatoid arthritis and other autoimmune inflammatory diseases associated with autoantibody formation.

Niemann-pick type C2 deficiency in human fibroblasts confers robust and selective activation of prostaglandin E2 biosynthesis.

Activated fibroblasts, also known as myofibroblasts, are mediators of several major human pathologies including proliferative fibrotic disorders, invasive tumor growth, rheumatoid arthritis, and atherosclerosis. We previously identified Niemann-Pick type C2 (NPC2) protein as a negative regulator of fibroblast activation (Csepeggi, C., Jiang, M., Kojima, F., Crofford, L. J., and Frolov, A. (2011) J. Biol. Chem. 286, 2078-2087). Here we report that NPC2-deficiency leads to a dramatic up-regulation of the arachidonic acid (AA) metabolic pathway in human fibroblasts. The major enzymes in this pathway, cPLA2 type IVA, COX-2, and mPGES-1, were dramatically up-regulated at both the transcriptional and translational levels. The specific phenotypic changes resulted in a >10-fold increase in the production and secretion of a key modulator of inflammation and immunity, prostaglandin E2. More importantly, AA metabolome profiling by liquid chromatography/tandem mass-spectrometry revealed the very specific nature of prostaglandin E2 up-regulation as the other analyzed AA metabolites derived from the COX-2, cytochrome P450, 5/15-lipoxygenase, and non-enzymatic oxidative pathways were mostly down-regulated. Blocking activity of cPLA2 efficiently suppressed expression of inflammatory cytokines, IL-1ß and IL-6, thereby identifying cPLA2 as an important regulator of the inflammatory program in NPC2-null cells. Altogether, these studies highlight NPC2 as a specific regulator of AA metabolism and inflammation that suggests potential for NPC2 protein or its related signaling in the treatment of inflammatory diseases characterized by the presence of activated fibroblasts.

Dung-beetles (Coleoptera, Scarabaeidae, Aphodiinae, Scarabaeinae) feeding on faeces of steppe marmots Marmotabobak (Rodentia, Sciuridae) in Middle Volga territory.

Background: In open terrestrial biomes of Holarctic realm, ground squirrels are recognised as keystone species inhabiting steppes. They shape the plant species composition and diversity and support a fauna of species associated with their burrows. Ground squirrels and associated dung-beetles are important elements of the steppe food webs, yet the trophic associations between species are still poorly studied. New information: The area in the northern outskirts of Obshchy Syrt plateau, on the border of Samara and Orenburg Provinces of Russia was surveyed and scarab beetles (Scarabaeidae) feeding on steppe marmot (Marmotabobak (Müller, 1776)) faeces were collected from six localities. Twenty eight species of two subfamilies - Aphodiinae and Scarabaeinae, - were identified with the majority of species belonging the genus Aphodius Hellwig, 1798. Seven species are recorded as consumers of marmot faeces for the first time. Only two nidicolous specialist species were found which suggests that the studied population of steppe marmots is as result of the recent secondary colonisation and not all the associated scarab beetle faunas were re-established.

Two Cases of Sporadic Amyotrophic Lateral Sclerosis With Contrasting Clinical Phenotypes: Genetic Insights.

Amyotrophic lateral sclerosis (ALS) is a fatal neuromuscular disease that affects individuals of diverse racial and ethnic backgrounds. There is currently no cure for ALS, and the number of efficient disease-modifying drugs for ALS is limited to a few, despite the large number of clinical trials conducted in recent years. The latter could be attributed to the significant heterogeneity of ALS clinical phenotypes even in their familial forms. To address this issue, we conducted postmortem genetic screening of two female patients with sporadic ALS (sALS) and contrasting clinical phenotypes. The results demonstrated that despite their contrasting clinical phenotypes, both patients had rare pathologic/deleterious mutations in five genes: ACSM5, BBS12, HLA-DQB1, MUC20, and OBSCN, with mutations in three of those genes being identical: BBS12, HLA-DQB1, and MUC20. Additional groups of mutated genes linked to ALS, other neurologic disorders, and ALS-related pathologies were also identified. These data are consistent with a hypothesis that an individual could be primed for ALS via mutations in a specific set of genes not directly linked to ALS. The disease could be initiated by a concerted action of several mutated genes linked to ALS and the disease's clinical phenotype will evolve further through accessory gene mutations associated with other neurological disorders and ALS-related pathologies.

Phylogenetic analysis of the Neotropical scarab beetle tribe Aegidiini (Coleoptera, Scarabaeidae, Orphninae) with description of new taxa.

In the Neotropics, orphnine scarab beetles are represented by the endemic tribe Aegidiini Paulian, 1984 with five genera and over 50 species. Phylogenetic analysis based on morphological characters of all supraspecific taxa of Orphninae showed that Aegidiini is comprised of two lineages. New subtribes, Aegidiina subtr. nov. (Aegidium Westwood, 1845, Paraegidium Vulcano et al., 1966, Aegidiellus Paulian, 1984, and Onorius Frolov & Vaz-de-Mello, 2015, and Aegidininasubtr. nov. (Aegidinus Arrow, 1904) are proposed to better reflect this phylogeny. Two new species of Aegidinus are described: A.alexanderisp. nov., from the Yungas in Peru and A.elbaesp. nov. from the Caqueta moist forests ecoregion in Colombia. A diagnostic key to Aegidinus species is given.

A Rare Case of Penoscrotal Webbing and Extensive Hernias: An Anatomical Report With Genetic Insights.

During a routine anatomical dissection of an 81-year-old male cadaver received through the Gift Body Program of Saint Louis University School of Medicine (SLU SOM), a massive bulging in the abdominal area was observed that was consistent with numerous hernia repairs noted in the donor's self-reported medical history. Gross anatomical dissection of the cadaveric body revealed extensive herniation of portions of the small intestine and peritoneal sac along the costal margin and extending to the left aspect of the abdomen. Additionally, an uncircumcised phallus was buried within the suprapubic fat pad and demonstrated simple, grade III penoscrotal webbing (PSW), creating an impression of micropenis presence. To gain additional insights into the current case, analysis of the coding regions (exomes) of DNA procured from the body for putative genetic variants was performed using next-generation sequencing (NGS) technology. This analysis revealed 110 rare (minor allele frequency (MAF) ≤ 0.01), pathologic/deleterious genetic mutations. The most relevant variants to this case were the ones associated with male sexual development, BMP1 and BMP4; connective tissue development, COL3A1 and COL5A3; cilia morphogenesis and function, DNAH5 and MAPK15; as well as hormonal homeostasis, ESR1. Direct involvement of BMP1 both in male sexual development and hernia genesis makes it a strong candidate for linking the two pathologies, PSW and multiple hernias, observed in the present case. Yet the presence of a group of mutated genes linked to myopathies (ITGA7, NRAP, POLM, SCN5A, XIRP2) and muscular dystrophy (ITGA7) raises a question about the involvement of these muscular pathologies in hernia genesis and unsuccessful hernia repairs associated with the current case.

Amplicon metagenomics of dung beetles (Coleoptera, Scarabaeidae, Scarabaeinae) as a proxy for lemur (Primates, Lemuroidea) studies in Madagascar.

Dung beetles (Scarabaeidae, Scarabaeinae) are among the most cost-effective and informative biodiversity indicator groups, conveying rich information about the status of habitats and faunas of an area. Yet their use for monitoring the mammal species, that are the main providers of the food for the dung beetles, has only recently been recognized. In the present work, we studied the diet of four endemic Madagascan dung beetles (Helictopleurusfissicollis (Fairmaire), H.giganteus (Harold), Nanosagaboides (Boucomont), and Epilissussplendidus Fairmaire) using high-throughput sequencing and amplicon metagenomics. For all beetle species, the ⅔-¾ of reads belonged to humans, suggesting that human feces are the main source of food for the beetles in the examined areas. The second most abundant were the reads of the cattle (Bostaurus Linnaeus). We also found lower but significant number of reads of six lemur species belonging to three genera. Our sampling localities agree well with the known ranges of these lemur species. The amplicon metagenomics method proved a promising tool for the lemur inventories in Madagascar.

Combining structural and coevolution information to unveil allosteric sites.

Understanding allosteric regulation in biomolecules is of great interest to pharmaceutical research and computational methods emerged during the last decades to characterize allosteric coupling. However, the prediction of allosteric sites in a protein structure remains a challenging task. Here, we integrate local binding site information, coevolutionary information, and information on dynamic allostery into a structure-based three-parameter model to identify potentially hidden allosteric sites in ensembles of protein structures with orthosteric ligands. When tested on five allosteric proteins (LFA-1, p38-α, GR, MAT2A, and BCKDK), the model successfully ranked all known allosteric pockets in the top three positions. Finally, we identified a novel druggable site in MAT2A confirmed by X-ray crystallography and SPR and a hitherto unknown druggable allosteric site in BCKDK validated by biochemical and X-ray crystallography analyses. Our model can be applied in drug discovery to identify allosteric pockets.

Somatic cell plasticity and Niemann-Pick type C2 protein: fibroblast activation.

A growing body of evidence points toward activated fibroblasts, also known as myofibroblasts, as one of the leading mediators in several major human pathologies including proliferative fibrotic disorders, invasive tumor growth, rheumatoid arthritis, and atherosclerosis. Niemann-Pick Type C2 (NPC2) protein has been recently identified as a product of the second gene in NPC disease. It encodes ubiquitous, highly conserved, secretory protein with the poorly defined function. Here we show that NPC2 deficiency in human fibroblasts confers their activation. The activation phenomenon was not limited to fibroblasts as it was also observed in aortic smooth muscle cells upon silencing NPC2 gene by siRNA. More importantly, activated synovial fibroblasts isolated from patients with rheumatoid arthritis were also identified as NPC2-deficient at both the NPC2 mRNA and protein levels. The molecular mechanism responsible for activation of NPC2-null cells was shown to be a sustained phosphorylation of ERK 1/2 mitogen-activated protein kinase (MAPK), which fulfills both the sufficient and necessary fibroblast activation criteria. All of these findings highlight a novel mechanism where NPC2 by negatively regulating ERK 1/2 MAPK phosphorylation may efficiently suppress development of maladaptive tissue remodeling and inflammation.

New taxa of Athyreacaridae (Acari: Heterostigmata) from Neotropical and Afrotropical realms.

A new genus and three new species of Athyreacaridae (Acari: Heterostigmata) are described associated with beetles of subfamily Bolboceratinae (Coleoptera: Geotrupidae) in Neotropical and Afrotropical realms: Neoathyreacarus pygmephoroides gen., sp. nov. collected on Bolborhinum tubericeps in Chile; Athyreacarus staturosus sp. nov. collected on Zefevazia cantisani  in Argentina; and Athyreacarus camerikae sp. nov. collected on Namibiobolbus helgae in South Africa. An updated key to genera and species of Athyreacaridae is provided. Athyreacarus staturosus sp. nov. is the largest ever described non-physogastric female among Heterostigmata.

Sex-Related Pain Behavioral Differences following Unilateral NGF Injections in a Rat Model of Low Back Pain.

Low back pain (LBP) is a globally prevalent and costly societal problem with multifactorial etiologies and incompletely understood pathophysiological mechanisms. To address such shortcomings regarding the role of neurotrophins in the underlying mechanisms of pain, an LBP model was developed in rats involving two unilateral intramuscular injections of nerve growth factor (NGF) into deep trunk muscles. To date, behavioral investigations of this NGF-LBP model have been limited, especially as it pertains to female pain behaviors. This study compared mechanical sensitivity to noxious (hyperalgesia) and non-noxious (hypersensitivity) stimuli in control and NGF-injected male and female rats through pain resolution. Although the baseline testing revealed no differences between males and females, NGF-injected females demonstrated prolonged ipsilateral deep trunk mechanical hyperalgesia that resolved seven days later than males. Moreover, females showed bilateral trunk mechanical sensitivity to noxious and non-noxious stimuli compared to only ipsilateral behaviors in males. Sex differences were also observed in the severity of behavioral responses, with females displaying greater mean differences from baseline at several timepoints. Overall, these NGF-LBP behavioral findings mirror some of the sex differences reported in the clinical presentation of LBP and accentuate the translatability of this NGF-LBP model. Future studies using this LBP-NGF model could help to elucidate the neurobiological mechanisms responsible for the development, severity, and/or resolution of muscular LBP as well as to provide insights into the processes governing the transition from acute to chronic LBP.

Somatosensory behavioral alterations in a NGF-induced persistent low back pain model.

Low back pain (LBP) is a major global burden in part due to the underlying pathophysiological mechanisms being poorly understood. A LBP rat model involving two injections of nerve growth factor (NGF, an endogenous pain-related neurotrophin) into trunk musculature was recently developed. Additional behavioral work in this NGF-LBP rat model is required to better characterize local and remote somatosensory alterations related to NGF-induced peripheral and central sensitization. This work characterizes the time-dependent development of hypersensitivity to trunk and hindpaw cutaneous mechanical stimulation and deep muscle mechanical hyperalgesia in adult male Sprague-Dawley rats (n = 6/group). Behavioral assays were performed at baseline (Day 0, D0), D2, D5 (pre- and 4 h post-2nd NGF or control injection), D7, D10, and D14 in NGF and control groups. Trunk and hindpaw cutaneous mechanical hypersensitivity were tested using von Frey filaments. Deep trunk mechanical hyperalgesia was determined using a small animal algometer. NGF rats demonstrated increased cutaneous sensitivity to ipsilateral trunk mechanical stimuli at D7, D10, and D14. NGF rats also demonstrated ipsilateral deep mechanical hyperalgesia on D2, D5 + 4 h, D7, D10, and D14. Cutaneous hypersensitivity was delayed compared to deep hyperalgesia in NGF rats. No additional sensory changes were noted. Together, these results indicate that male mechanical somatosensory changes develop primarily locally in the ipsilateral trunk following unilateral NGF injections. These findings contrast with a previous report in female rats using this NGF-LBP model showing more widespread (bilateral) hyperalgesia and remote mechanical hypersensitivity. Future studies will examine potential sex-related pain behavioral differences in the NGF model.

Somatic cell plasticity and Niemann-pick type C2 protein: adipocyte differentiation and function.

The phenotypic stability of somatic cells is essential for the maintenance of both structural and functional organ integrity of the adult human body. Deregulated cell plasticity could result in the development of debilitating diseases such as cancer, fibrosis, atherosclerosis, obesity, and type 2 diabetes. We have previously demonstrated that a nonsense mutation in the NPC2 gene, which encodes ubiquitous, highly conserved, secretory protein with unknown function, leads to activation of human skin fibroblasts. The activated fibroblasts, also known as myofibroblasts, have the properties of mesenchymal stem cells and are able to differentiate along the mesodermal and endodermal lineages. Here we show that NPC2-null, but not the normal skin fibroblasts, possess characteristics of adipogenic progenitors as demonstrated by their specific gene expression pattern as well as the ability for efficient differentiation into white adipocytes. The presence of NPC2 in mature white adipocytes was also necessary for their maintenance because silencing NPC2 in differentiated cells by siRNA stimulated PPARG expression, which was followed by a shift toward a more favorable, brown adipocyte-like metabolic state characterized by up-regulated lipolysis and increased insulin sensitivity. It appears that NPC2 controls both the adipogenesis and the metabolic state of mature white adipocytes through a common mechanism that is linked to activation of FGFR2 that could be followed by induction of PPARG expression. Altogether, the current study highlights NPC2 as a novel intracrine/autocrine factor that controls adipocyte differentiation and function as well as potential therapeutic target for the treatment of type 2 diabetes and related metabolic disorders.

Toward a universal model to calculate the solvation thermodynamics of druglike molecules: the importance of new experimental databases.

We demonstrate that a new free energy functional in the integral equation theory of molecular liquids gives accurate calculations of hydration thermodynamics for druglike molecules. The functional provides an improved description of excluded volume effects by incorporating two free coefficients. When the values of these coefficients are obtained from experimental data for simple organic molecules, the hydration free energies of an external test set of druglike molecules can be calculated with an accuracy of about 1 kcal/mol. The 3D RISM/UC method proposed here is easily implemented using existing computational software and allows in silico screening of the solvation thermodynamics of potential pharmaceutical molecules at significantly lower computational expense than explicit solvent simulations.

Salting out in organic solvents: a new route to carbon nanotube bundle engineering.

In this study we investigate salt effects on bundle formation of carbon nanotubes (CNTs) dispersed in an organic solvent, N-methyl-2-pyrrolidone (NMP). Addition of NaI salt leads to self-assembly of CNTs into well-recognizable bundles. It is possible to control the size of the CNT bundles by varying the salt concentration.

A new species of Aegidium Arrow (Coleoptera: Scarabaeidae: Orphninae) from Colombia.

In the Neotropical Region, Orphninae (Coleoptera: Scarabaeidae) are represented by the endemic tribe Aegidiini, which comprises five genera and 49 species (Paulian 1948; Colby 2009; Frolov Vaz-de-Mello 2015; Frolov et al. 2017a, 2017b, 2017c, 2019). Aegidium Westwood is the largest genus of the tribe and it comprises 24 valid species known from the southern Mexico in the north to Central Bolivia in the south (Frolov et al. 2015; Rojkoff Frolov 2017; Frolov Akhmetova 2020).

Identification of larvae of two Aphodius Helwig, 1798 (Coleoptera: Scarabaeidae: Aphodiinae) species using morphology and DNA barcode.

The third instar larvae of Aphodius (Alocoderus) hydrochaeris (Fabricius, 1798) and A. (Bodilus) ictericus (Laicharting, 1781) are described based on scanning electron microscopy and COI sequences. COI barcode sequence for A. (A.) hydrohaeris is provided for the first time. Two haplotypes are discovered in A. (B.) ictericus.

Ion interactions with the carbon nanotube surface in aqueous solutions: understanding the molecular mechanisms.

We study the molecular mechanisms of alkali halide ion interactions with the single-wall carbon nanotube surface in water by means of fully atomistic molecular dynamics simulations. We focus on the basic physical-chemical principles of ion-nanotube interactions in aqueous solutions and discuss them in light of recent experimental findings on selective ion effects on carbon nanotubes.