Insert Domain Research Articles

A Cipadesin Limonoid and a Tirucallane Triterpene from the Fruit of Sandoricum koetjape and their Inhibitory Properties against Receptor Tyrosine Kinases

A new cipadesin limonoid, i.e. 3-epi-cipadonoid C (1), and a new tirucallane triterpene, i.e. hispidol B 3-palmitate (3), have been isolated from the seeds and fruit peels extract of Sandoricum koetjape, respectively. Along with these compounds the known limonoid, cipaferen G (2), and two pentacyclic triterpenes, bryonolic (4) and bryononic (5) acids, were also isolated. The strucrures of the new compounds were elucidated by the analysis of NMR and mass spectral data. Compounds 1 - 5 were evaluated as the inhibitor of receptor tyrosine kinases (EGFR, Epidermal Growth Factor Receptor; HER2, HER4, Human Epidermal growth factor Receptor 2, -4; IGFR, Insulin-like Growth Factor Receptor; InsR, Insulin Receptor; KDR, Kinase insert Domain Receptor; PDGFRα, and PDGFRβ, Platelet-Derived Growth Factor Receptor-α and -β). The results showed only 1 and 3 that have weak activity against InsR.

The Spliceosomal Component Sf3b1is Essential for Hematopoietic Stem Cell Formation through the Regulation of the Jak/Stat Signaling Pathway

Hematopoietic stem cells (HSCs) maintain multi-lineage hematopoiesis for the lifetime of an organism. During embryogenesis, HSCs first emerge from specialized hemogenic endothelial cells within the dorsal aorta through a process termed the endothelial-to-hematopoietic transition. While much is known about the role of transcription factors and transcriptional control in HSC specification, it is ill defined how post-transcriptional RNA processing influences this first HSC fate decision. Using zebrafish loss-of-function mutants for the pre-mRNA spliceosomal component sf3b1 (splicing factor 3b, subunit 1), we demonstrated that impairing splicing hindered HSC production, specifically at the endothelial-to-hematopoietic transition. Loss-of-function sf3b1hi3394 mutants have diminished expression of thehemogenic endothelial and HSC markers runx1 and gata2b, while the pan-endothelial marker kdrl (kinase insert domain receptor like) and aorta-specific markers notch1b and notch3 are normally expressed at 24 hpf (hours post fertilization). These data indicate that although sf3b1 is ubiquitously expressed, hemogenic endothelium and the downstream HSCs are more sensitive to its loss than other endothelial cells. To uncover the mechanism underlying the defect in hemogenic endothelium and HSCs in sf3b1 mutants, we performed RNA-sequencing on purified kdrl:gfp- positiveendothelial cells from sf3b1 mutants and wild-type siblings at 24 hpf. We observed nearly 900 genes were mis-spliced and of those only 144 were differentially expressed. This group was significantly enriched for genes involved in mRNA processing and Jak (Janus kinase)/Stat (Signaling transducer and activator of transcription) signaling. In particular, target genes of the Jak/Stat pathway were significantly enriched in the downregulated gene list and many components of the signaling pathway were mis-spliced including il6st (interleukin 6 signal transducer), the gene encoding the IL6-family common receptor Gp130 (Glycoprotein 130).To determine if mis-splicing of il6st was sufficient to decrease HSC production, we injected antisense morpholinos into wild-type zebrafish to mimic the sf3b1 mutant - specific exon-skipping event in il6st and found significantly diminished HSC levels at 28 hpf. Stat3 is the major transcription factor downstream of Gp130, thus we ubiquitously overexpressed a constitutively-active form of Stat3 and found it could significantly suppress the HSC defects in sf3b1 mutants. Together, these data indicate that Sf3b1-mediated splicing regulation of the Jak/Stat pathway is critical for HSC emergence. Mutations in SF3B1 are prevalent in myelodysplastic syndrome (MDS), a disorder originating in HSC dysfunction. Altered STAT3 signaling is also common in MDS, thus our data suggest that SF3B1 disruptions in HSCs could be contributing to MDS via mis-regulation of JAK/STAT signaling. DisclosuresNo relevant conflicts of interest to declare.

Epigenetic reader BRD4 supports mycobacterial pathogenesis by co-modulating host lipophagy and angiogenesis

Mycobacterium tuberculosis (Mtb)-driven lipid accumulation is intricately associated with the progression of tuberculosis (TB) disease. Although several studies elucidating the mechanisms for lipid droplet (LD) biosynthesis exist, we provide evidence for the significance of their regulated turnover via macroautophagy/autophagy during Mtb infection. We demonstrate that Mtb utilizes EGFR (epidermal growth factor receptor) signaling to induce the expression of the histone acetylation reader, BRD4 (bromodomain containing 4). The EGFR-BRD4 axis suppresses lipid-specific autophagy, and hence favors cellular lipid accumulation. Specifically, we found that pharmacological inhibition or knockdown of Egfr or Brd4 enhances autophagic flux and concomitantly decreases cellular LDs that is otherwise maintained at a significant level in chloroquine-treated or Atg5 knocked down autophagy-compromised host cells. In line with the enhanced lipophagy, we found that loss of EGFR or BRD4 function restricts mycobacterial burden that is rescued by external replenishment with oleic acid. We also report that the EGFR-BRD4 axis exerts additional effects by modulating pro-angiogenic gene expression and consequently aberrant angiogenesis during mycobacterial infection. This is important in the context of systemic Mtb dissemination as well as for the efficient delivery of anti-mycobacterial therapeutics to the Mtb-rich core of TB granuloma. Finally, utilizing an in vivo mouse model of TB, we show that pharmacological inhibition of EGFR and BRD4 compromises LD build-up via enhanced lipophagy and normalizes angiogenesis, thereby restricting Mtb burden and rescuing mice from severe TB-like pathology. These findings shed light on the novel roles of BRD4 during Mtb infection, and its possible implication in potentiating anti-TB responses.

A Large Insertion Domain in the Rho Factor From a Low G + C, Gram-negative Bacterium is Critical for RNA Binding and Transcription Termination Activity

Rho-dependent termination of transcription (RDTT) is a critical regulatory mechanism specific to bacteria. In a subset of species including most Actinobacteria and Bacteroidetes, the Rho factor contains a large, poorly conserved N-terminal insertion domain (NID) of cryptic function. To date, only two NID-bearing Rho factors from high G + C Actinobacteria have been thoroughly characterized. Both can trigger RDTT at promoter-proximal sites or with structurally constrained transcripts that are unsuitable for the archetypal, NID-less Rho factor of Escherichia coli (EcRho). Here, we provide the first biochemical characterization of a NID-bearing Rho factor from a low G + C bacterium. We show that Bacteroides fragilis Rho (BfRho) is a bona fide RNA-dependent NTPase motor able to unwind long RNA:DNA duplexes and to disrupt transcription complexes. The large NID (~40% of total mass) strongly increases BfRho affinity for RNA, is strictly required for RDTT, but does not promote RDTT at promoter-proximal sites or with a structurally constrained transcript. Furthermore, the NID does not preclude modulation of RDTT by transcription factors NusA and NusG or by the Rho inhibitor bicyclomycin. Although the NID contains a prion-like Q/N-rich motif, it does not spontaneously trigger formation of β-amyloids. Thus, despite its unusually large RNA binding domain, BfRho behaves more like the NID-less EcRho than NID-bearing counterparts from high G + C Actinobacteria. Our data highlight the evolutionary plasticity of Rho’s N-terminal region and illustrate how RDTT is adapted to distinct genomic contents.

Mutational analysis of rare insertions and deletions in exon 18 and 19 of HER2 in Chinese patients with different cancer types.

3069 Background: HER2 belongs to the same family with EGFR and is known as an important cancer driver gene. Kinase domain insertions and deletions (KD indels) are frequent driver mutations in both EGFR and HER2. The most common HER2 KD indels are the exon 20 insertions while others are rarely reported. Our study aimed to investigate the role of less common HER2 KD indels in solid tumors. Methods: This study was performed in 63,267 Chinese patients including 53,591 patients with lung cancer, 5,888 patients with colorectal cancer, 3,774 patients with breast cancer and 14 patients with renal pelvis cancer. Tissue or plasma samples from the patients were subjected to capture-based targeted sequencing covering HER2 and other cancer related genes. The sequencing data of each patient were retrospectively collected and analyzed. The HER2 exon 18/19 indels identified in our study were compared with data from COSMIC and MSKCC. In vitro analysis in Ba/F3 cell lines was performed to assess drug response of different HER2 exon 18/19 indels. Results: We identified recurrent HER2 KD indels in exon 18 and 19, with a frequency of 0.04% (25/63,267). The data from COSMIC and MSKCC reported the prevalence of HER2 exon 18/19 indels ranging from 0.04% to 0.23% among breast, cervical, and pancreatic cancers. In our study, HER2 exon 18/19 indels were identified in 20 patients with lung cancer (0.037%), two with colorectal cancer (0.034%), two with breast cancer (0.053%) and one with renal pelvis cancer (7.143%). Only two patients (8%) harbored concurrent actionable driver mutations including EGFR mutation and MET amplification. Meanwhile, high level of normalized allelic frequency of HER2 exon 18/19 indels was presented in most patients (22/25, 88%). In lung cancer, the presence of EGFR driver mutation was less common in patients with HER2 exon 18/19 indels than wild type HER2 (5% vs. 47.4%, p < 0.01). The rates of concurrent driver mutations in lung cancer were comparable between HER2 exon 18/19 indels and the two established HER2 drivers, exon 20 insertions and S310 mutations. The in vitro proliferation assay demonstrated that E698_P699insLL mutation in HER2 exon 18 and L755_E757delinsPQ mutation in HER2 exon 19 both conferred a survival advantage to Ba/F3 cells. Dose-response curves showed inhibitory effects on cell viability of several HER2 tyrosine kinase inhibitors including neratinib, lapatinib, poziotinib and afatinib. In particular, lapatinib, poziotinib and afatinib demonstrated comparable or stronger inhibitory ability toward the two HER2 mutants than wild type HER2 in terms of IC50. Conclusions: Our study revealed a novel class of HER2 KD indels in exon 18/19 that may act as driver mutations in several cancer types. The drug response observed in vitro indicated the potential to use anti- HER2 targeted therapies for HER2 exon 18/19 indels. Further studies on this rare type of HER2 mutation are warranted.

Chimeric Phi29 DNA polymerase with helix-hairpin-helix motifs shows enhanced salt tolerance and replication performance.

SummaryPhi29 DNA polymerase (Phi29 Pol) has been successfully applied in DNA nanoball‐based sequencing, real‐time DNA sequencing from single polymerase molecules and nanopore sequencing employing the sequencing by synthesis (SBS) method. Among these, polymerase‐assisted nanopore sequencing technology analyses nucleotide sequences as a function of changes in electrical current. This ionic, current‐based sequencing technology requires polymerases to perform replication at high salt concentrations, for example 0.3 M KCl. Nonetheless, the salt tolerance of wild‐type Phi29 Pol is relatively low. Here, we fused helix–hairpin–helix (HhH)2 domains E‐L (eight repeats in total) of topoisomerase V (Topo V) from the hyperthermophile Methanopyrus kandleri to the Phi29 Pol COOH terminus, designated Phi29EL DNA polymerase (Phi29EL Pol). Domain fusion increased the overall enzyme replication efficiency by fourfold. Phi29EL Pol catalysed rolling circle replication in a broader range of salt concentrations than did Phi29 Pol, extending the KCl concentration range for activity up to 0.3 M. In addition, the mutation of Glu375 to Ser or Gln increased Phi29EL Pol activity in the presence of KCl. In this work, we produced a salt‐tolerant Phi29 Pol derivative by means of (HhH)2 domain insertion. The multiple advantages of this insertion make it a good substitute for Phi29 Pol, especially for use in nanopore sequencing or other circumstances that require high salt concentrations.

Network pharmacology-based evaluation of natural compounds with paclitaxel for the treatment of metastatic breast cancer

Metastatic breast cancer is a prevalent life-threatening disease. Paclitaxel (PTX) is widely used in metastatic breast cancer therapy, but the side effects limit its chemotherapeutic application. Multidrug strategies have recently been used to maximize potency and decrease the toxicity of a particular drug by reducing its dosage. Therefore, we have evaluated the combined anti-cancerous effect of PTX with tested natural compounds (andrographolide (AND), silibinin (SIL), mimosine (MIM) and trans-anethole (TA)) using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, trypan blue dye exclusion assay, proliferating cell nuclear antigen (PCNA) staining, network pharmacology, molecular docking, molecular dynamics (MD) and in vivo chick chorioallantoic membrane (CAM) angiogenesis assay. We observed a reduction in the IC50 value of PTX with tested natural compounds. Further, the network pharmacology-based analysis of compound-disease-target (C-D-T) network showed that PTX, AND, SIL, MIM and TA targeted 55, 61, 56, 31 and 18 proteins of metastatic breast cancer, respectively. Molecular docking results indicated that AND and SIL inhibited the C-D-T network's core target kinase insert domain receptor (KDR) protein more effectively than others. While MD showed that the binding of AND with KDR was stronger and more stable than others. In trypan blue dye exclusion assay and PCNA staining, AND and SIL along with PTX were found to be more effective than PTX alone. CAM assay results suggested that AND, SIL and TA increase the anti-angiogenic potential of PTX. Thus, natural compounds can be used to improve the anti-cancer potential of PTX.

Structural characterisation of the Chaetomium thermophilum Chl1 helicase.

Chl1 is a member of the XPD family of 5'-3' DNA helicases, which perform a variety of roles in genome maintenance and transmission. They possess a variety of unique structural features, including the presence of a highly variable, partially-ordered insertion in the helicase domain 1. Chl1 has been shown to be required for chromosome segregation in yeast due to its role in the formation of persistent chromosome cohesion during S-phase. Here we present structural and biochemical data to show that Chl1 has the same overall domain organisation as other members of the XPD family, but with some conformational alterations. We also present data suggesting the insert domain in Chl1 regulates its DNA binding.

Entamoeba histolytica ‐Induced Activation of Caspase‐4 Regulates Gasdermin D Cleavage to Mediate IL‐1β Secretion in Macrophages

A hallmark of Entamoeba histolytica (Eh) invasion in the gut is acute intestinal inflammation dominated by the secretion of pro-inflammatory cytokines. Eh in contact with macrophages activates caspase-1 by the recruitment of the NLRP3 inflammasome complex in a Gal-lectin and Eh cysteine proteases 5 (EhCP-A5)-dependent manner, resulting in the maturation and secretion of interleukin (IL)-1β and IL-18. Although it is well-defined that caspase-1 activation is important in mediating the secretion of IL-1β and IL-18, the role caspase-4 play in the overall pro-inflammatory response is less identified. We have recently shown that Eh activates caspase-1/4 in macrophage that cleaved gasdermin D (GSDMD) leading to the insertion of N-terminal effector domain into the cell membrane, driving both pore formation and IL-1β secretion without causing significant cell death. In this study, we interrogated the requirements and activation mechanism for Eh-induced caspase-4 activation in cleaving GSDMD and in mediating bioactive IL-1β release. Similar to caspase-1, caspase-4 activation was dependent on Eh Gal-lectin and EhCP-A5 binding to macrophages that triggered ATP release that acted as a second signal. To determine if Eh-induced activation of caspase-4 interacted with the canonical NLRP3 inflammasome to regulate GSDMD-mediated pro-inflammatory cytokine release, CRISPR-Cas9 gene editing of caspase-1, 4, ASC, and NLRP3 THP-1 cells were utilized. Unlike caspase-1, caspase-4 activation in response to Eh was triggered within 10 min and was independent of ASC, NLRP3, and caspase-1. In the absence of caspase-1, caspase-4 activation was significantly up regulated that enhanced the cleavage of GSDMD to induce robust IL-1β secretion. Eh-induced caspase-4 played a major role in triggering GSDMD pore formation and bioactive IL-1β release as quantified by HEK-Blue™ reporter cell IL-1β assay independent of pyroptosis. This was in marked contrast to the positive control, LPS + Nigericin that induced high expression of caspase-1, enhanced GSDMD cleavage, IL-1β secretion, and massive pyroptosis. These results suggest that Eh induced “hyperactivated macrophages” led to caspase-4 dependent GSDMD cleavage and IL-1β secretion in the absence of pyroptosis important in disease pathogenesis.

MODELING CONSTRUCT OF TECHNICAL TERMINOLOGY VALUING IN THE ROMANIAN LANGUAGE BY ALLOPHONE STUDENTS

The aim of the article is to reflect the theoretical premises in the construction of the specific model of technical terminology exploitation in the Romanian language by allophone students. The theoretical and practical studies devoted to professional languages, mentioned in this paper, have been fundamental elements of the pedagogical model developed for the objective of the research. The models reflected in the Common European Framework of Reference for languages, designed for formative actions, which constitute a privileged opening in supporting the formation of communicative competence in a language other than the mother tongue, served as the basis: Model of development of generalindividual competences; Model of extension and diversification of linguistic communicative competence; Model of performing the determined communicative activity (-ies); Model of optimal functional insertion in a particular domain; Model of enrichment/diversification of strategies in terms of performing tasks. Communicative competence is a category that reflects the authors' point of view equating communicative competence and terminological competence, element in the reference system ofprofessional and personal training purposes. In conclusion, the modeling construct represented in Figure 3 defines the plan to affirm the coordinates of the methodology for valuing technical terminology in higher education (formative and operational), which will be concretized at the level of performance achieved at graduation integrating the technical terminology into a message for various communication contexts.

The Use of RUVBL1‐2 in the Oncogenic Process

RUVBL1 and RUVBL2 are two highly conserved eukaryotic AAA+ (ATPases Associated with diverse cellular Activities) proteins that engage in a diverse array of cellular functions. These functions can range from ribonucleoprotein complex assembly and cellular transformation to chromatin remodeling and DNA damage response. Individually, RUVBL1 and RUVBL2 form homohexamers with weak ATPase activities. However, together they form highly active heterohexameric or dodecameric complexes made up of alternating units of RUVBL1 and RUVBL2. Like all other AAA+ proteins, RUVBL1 and RUVBL2 ATPases contain a Walker A and a Walker B motif. These motifs are required for nucleotide binding and hydrolysis, respectively. RUVBL1 and RUVBL2 are each made up of three distinct domains: an N terminal αβα subdomain of the AAA+ domain, a 170 amino-acid insertion domain, and an all α subdomain of the AAA+ domain. The amino-acid insertion domain is unique to RUVBL1 and RUVBL2 and can mediate DNA or RNA binding. Despite its many essential functions, the complex of RUVBL1 and RUVBL2 (referred to as RUVBL1/2 here) is present at low levels in eukaryotic cells. It is therefore thought that RUVBL1/2 interacts only transiently with its binding partners and acts as a chaperone for assembly of other complexes. The molecular mechanism by which the RUVBL1/2 ATPase engages in cellular activities remains elusive. Interestingly, RUVBL1/2 is overexpressed in numerous human cancers including gastric, bladder, pancreatic, and breast cancers. For example, researchers have found that in pancreatic ductal adenocarcinoma (PDAC), which is among the deadliest cancers, RUVBL1 helps the formation of membrane protrusions by promoting actin polymerization. These protrusions increase the invasiveness and metastatic properties of PDAC cells. Due to their strong link to oncogenesis, design of inhibitors against RUVBL1/2 is a major area of research. Although members of the AAA+ family are challenging drug targets, recent high-resolution structures and new drug screening strategies have enabled advances in design of novel AAA+ inhibitors. For example, the small molecules Sorafenib and CB-6644 were recently reported to inhibit the ATPase activity of the RUVBL1/2 complex. With further optimizations and testing, these drugs may be improved and potentially considered as potent therapeutic inhibitors of RUVBL1/2 activity. The Walton SMART team has designed a 3D model of RUVBL1/2 to investigate the relationship between the molecular structure and function of this ATPase complex.

Engineering the acyltransferase domain of epothilone polyketide synthase to alter the substrate specificity

BackgroundPolyketide synthases (PKSs) include ketone synthase (KS), acyltransferase (AT) and acyl carrier protein (ACP) domains to catalyse the elongation of polyketide chains. Some PKSs also contain ketoreductase (KR), dehydratase (DH) and enoylreductase (ER) domains as modification domains. Insertion, deletion or substitution of the catalytic domains may lead to the production of novel polyketide derivatives or to the accumulation of desired products. Epothilones are 16-membered macrolides that have been used as anticancer drugs. The substrate promiscuity of the module 4 AT domain of the epothilone PKS (EPOAT4) results in production of epothilone mixtures; substitution of this domain may change the ratios of epothilones. In addition, there are two dormant domains in module 9 of the epothilone PKS. Removing these redundant domains to generate a simpler and more efficient assembly line is a desirable goal.ResultsThe substitution of module 4 drastically diminished the activity of epothilone PKS. However, with careful design of the KS-AT linker and the post-AT linker, replacing EPOAT4 with EPOAT2, EPOAT6, EPOAT7 or EPOAT8 (specifically incorporating methylmalonyl-CoA (MMCoA)) significantly increased the ratio of epothilone D (4) to epothilone C (3) (the highest ratio of 4:3 = 4.6:1), whereas the ratio of 4:3 in the parental strain Schlegelella brevitalea 104-1 was 1.4:1. We also obtained three strains by swapping EPOAT4 with EPOAT3, EPOAT5, or EPOAT9, which specifically incorporate malonyl-CoA (MCoA). These strains produced only epothilone C, and the yield was increased by a factor of 1.8 compared to that of parental strain 104-1. Furthermore, mutations of five residues in the AT domain identified Ser310 as the critical factor for MMCoA recognition in EPOAT4. Then, the mutation of His308 to valine or tyrosine combined with the mutation of Phe310 to serine further altered the product ratios. At the same time, we successfully deleted the inactive module 9 DH and ER domains and fused the ΨKR domain with the KR domain through an ~ 25-residue linker to generate a productive and simplified epothilone PKS.ConclusionsThese results suggested that the substitution and deletion of catalytic domains effectively produces desirable compounds and that selection of the linkers between domains is crucial for maintaining intact PKS catalytic activity.

Circular RNA circ-ARFIP2 regulates proliferation, migration and invasion in human vascular smooth muscle cells via miR-338-3p-dependent modulation of KDR

Dysfunction of vascular smooth muscle cells (VSMCs) plays a critical role in the pathogenesis of intracranial aneurysm (IA). Circular RNAs (circRNAs) have been implicated in the pathogenesis of IA by reducing microRNA (miRNA) activity. In this paper, we investigated the precise roles of circRNA ADP ribosylation factor interacting protein 2 (circ-ARFIP2, circ_0021001) in VSMC dysfunction. The levels of circ-ARFIP2, miR-338-3p and kinase insert domain receptor (KDR) were detected by quantitative real-time polymerase chain reaction (qRT-PCR) or western blot. Ribonuclease (RNase) R and subcellular fractionation assays were used to assess the stability and localization of circ-ARFIP2, respectively. Cell viability was detected by Cell Counting Kit-8 (CCK-8) assay, and cell invasion was measured by transwell assay. Cell proliferation was gauged by 5-Ethynyl-2'-Deoxyuridine (EdU) assay. Cell migration was evaluated by transwell and wound-healing assays. Targeted correlations among circ-ARFIP2, miR-338-3p and KDR were validated by dual-luciferase reporter and RNA immunoprecipitation (RIP) assays. Circ-ARFIP2 and KDR were underexpressed and miR-338-3p was overexpressed in the arterial wall tissues of IA patients. Overexpression of circ-ARFIP2 in human umbilical artery smooth muscle cells (HUASMCs) showed a significant promotion in cell proliferation, migration and invasion. Mechanistically, circ-ARFIP2 targeted miR-338-3p, and circ-ARFIP2 regulated cell behaviors by miR-338-3p. KDR was a direct and functional target of miR-338-3p. Moreover, KDR was a downstream effector of circ-ARFIP2 function. Circ-ARFIP2 regulated KDR expression by targeting miR-338-3p. Our present findings demonstrated that the increased level of circ-ARFIP2 enhanced HUASMC proliferation, migration and invasion at least in part by the miR-338-3p/KDR axis.

Engineering Protease-Resistant Peptides to Inhibit Human Parainfluenza Viral Respiratory Infection.

The lower respiratory tract infections affecting children worldwide are in large part caused by the parainfluenza viruses (HPIVs), particularly HPIV3, along with human metapneumovirus and respiratory syncytial virus, enveloped negative-strand RNA viruses. There are no vaccines for these important human pathogens, and existing treatments have limited or no efficacy. Infection by HPIV is initiated by viral glycoprotein-mediated fusion between viral and host cell membranes. A viral fusion protein (F), once activated in proximity to a target cell, undergoes a series of conformational changes that first extend the trimer subunits to allow insertion of the hydrophobic domains into the target cell membrane and then refold the trimer into a stable postfusion state, driving the merger of the viral and host cell membranes. Lipopeptides derived from the C-terminal heptad repeat (HRC) domain of HPIV3 F inhibit infection by interfering with the structural transitions of the trimeric F assembly. Clinical application of this strategy, however, requires improving the in vivo stability of antiviral peptides. We show that the HRC peptide backbone can be modified via partial replacement of α-amino acid residues with β-amino acid residues to generate α/β-peptides that retain antiviral activity but are poor protease substrates. Relative to a conventional α-lipopeptide, our best α/β-lipopeptide exhibits improved persistence in vivo and improved anti-HPIV3 antiviral activity in animals.

Compositional change of gut microbiome and osteocalcin expressing endothelial progenitor cells in patients with coronary artery disease.

Osteogenic endothelial progenitor cells (EPCs) contribute to impaired endothelial repair and promote coronary artery disease (CAD) and vascular calcification. Immature EPCs expressing osteocalcin (OCN) has been linked to unstable CAD; however, phenotypic regulation of OCN-expressing EPCs is not understood. We hypothesized that gut-microbiome derived pro-inflammatory substance, trimethylamine N-oxide (TMAO) might be associated with mobilization of OCN-expressing EPCs. This study aimed to investigate the association between dysbiosis, TMAO, and circulating mature and immature OCN-expressing EPCs levels in patients with and without CAD. We included 202 patients (CAD N = 88; no CAD N = 114) who underwent assessment of EPCs using flow cytometry and gut microbiome composition. Mature and immature EPCs co-staining for OCN were identified using cell surface markers as CD34+/CD133-/kinase insert domain receptor (KDR)+ and CD34-/CD133+/KDR+ cells, respectively. The number of observed operational taxonomy units (OTU), index of microbial richness, was used to identify patients with dysbiosis. The number of immature OCN-expressing EPCs were higher in patients with CAD or dysbiosis than patients without. TMAO levels were not associated with circulating levels of OCN-expressing EPCs. The relative abundance of Ruminococcus gnavus was moderately correlated with circulating levels of immature OCN-expressing EPCs, especially in diabetic patients. Gut dysbiosis was associated with increased levels of TMAO, immature OCN-expressing EPCs, and CAD. The relative abundance of Ruminococcus gnavus was correlated with immature OCN-expressing EPCs, suggesting that the harmful effects of immature OCN-expressing EPCs on CAD and potentially vascular calcification might be mediated by gut microbiome-derived systemic inflammation.

Spatial and Frequency Approaches for Audio File Protection

In order to protect audio files, we propose in this paper a new integration scheme for blind audio file watermarking. The goal is to find a compromise between capacity and imperceptibility in order to hide as much data as possible while minimizing file degradation. This integration scheme is implemented in the three insertion domains: spatial, frequency and multi-resolution domains. For the spatial-domain integration, the mark is inserted directly into the data samples. For the frequency-domain integration, a Discrete Cosine Transform is applied to the audio frames; after the thresholding and quantification step, the watermark is inserted into the Discrete Cosine Transform coefficients to obtain the watermarked file. For the multi-resolution-domain insertion, a single-level Discrete Wavelet Transform is applied using the scaling low-pass filter and wavelet high-pass filter. The watermark integration is then performed using the obtained AC coefficients. The proposed concealment process combines three values to integrate two bits and only one may be modified, which reduces the probability of change unlike other approaches. This implies less modification and therefore less distortion of the host file; this explains the good Signal-to-Noise Ratio obtained of more than 59[Formula: see text]dB for the spatial-domain integration and therefore a reasonable imperceptibility. An evaluation of the watermark’s robustness demonstrates that the proposed schemes generate reasonably robust watermarked samples against various attacks with a high-quality watermark with normalized cross-correlation greater than 0.9 for the three insertion domains.

Crystal structure of P. falciparum Cpn60 bound to ATP reveals an open dynamic conformation before substrate binding

Plasmodium falciparum harbors group 1 and group 2 chaperonin systems to mediate the folding of cellular proteins in different cellular locations. Two distinct group 1 chaperonins operate in the organelles of mitochondria and apicoplasts, while group 2 chaperonins function in the cytosol. No structural information has been reported for any chaperonin from plasmodium. In this study, we describe the crystal structure of a double heptameric ring Plasmodium falciparum mitochondrial chaperonin 60 (Cpn60) bound with ATP, which differs significantly from any known crystal structure of chaperonin 60. The structure likely represents a unique intermediate state during conformational conversion from the closed state to the opened state. Three of the seven apical domains are highly dynamic while the equatorial domains form a stable ring. The structure implies large movements of the apical domain in the solution play a role in nucleotide-dependent regulation of substrate binding and folding. A unique 26–27 residue insertion in the equatorial domain of Plasmodium falciparum mitochondrial chaperonin greatly increases both inter-ring and intra-ring subunit–subunit interactions. The present structure provides new insights into the mechanism of Cpn60 in chaperonin assembly and function.

Aging Regulated Through a Stability Model of Insulin/Insulin Growth Factor Receptor Function.

Mutations of the insulin-like receptor in Drosophila extend lifespan. New research suggests this receptor operates in two modes. The first extends lifespan while slowing reproduction and reducing growth. The second strongly extends lifespan without impairing growth or reproduction; it confers longevity assurance. The mutation that confers longevity assurance resides in the kinase insert domain, which contains a potential SH2 binding site for substrate proteins. We apply a recent model for the function of receptor tyrosine kinases to propose how insulin receptor structure can modulate aging. This concept hypothesizes that strong insulin-like ligands promote phosphorylation of high threshold substrate binding sites to robustly induce reproduction, which impairs survival as a consequence of trade-offs. Lower levels of receptor stimulation provide less kinase dimer stability, which reduces reproduction and extends lifespan by avoiding reproductive costs. Environmental conditions that favor diapause alter the expression of insulin ligands to further repress the stability of the interacting kinase domains, block phosphorylation of low threshold substrates and thus induce a unique molecular program that confers longevity assurance. Mutations of the insulin receptor that block low-phosphorylation site interactions, such as within the kinase insert domain, can extend lifespan while maintaining overall dimer stability. These flies are long-lived while maintaining reproduction and growth. The kinase insert domain of Drosophila provides a novel avenue from which to seek signaling of the insulin/insulin-like growth factor system of humans that modulate aging without impacting reproduction and growth, or incurring insulin resistance pathology.

Intermittent hypoxia: Friend or foe on endothelial repair in mouse model

Aim of the study: Obstructive sleep apnea, which is characterized by intermittent hypoxia (IH), is a common respiratory disease. The aim of the present study was to explore the relationship between hypoxia and endothelial progenitor cell (EPC) function, and explain the role of IH in endothelial repair. Materials and methods: Peripheral blood mononuclear cells (PBMCs) were isolated from a mouse model of IH. The number of CD133+ kinase insert domain receptor (KDR)+, CD133+CD34+, CD34+KDR+ and ALDHlowCD34+KDR+ EPCs was determined by flow cytometry. HIF-1α, stromal-derived factor-1 (SDF-1) α and VEGF were measured by ELISA. The proliferative ability of PBMCs was determined. EPC migration was assessed by Transwell assay and surface proteins by western blot analysis. EPCs were co-cultured with mouse brain endothelial cells and their angiogenic ability was analyzed. Results: The number of CD133+KDR+, CD133+CD34+ and CD34+KDR+ EPCs increased with IH ingravescence. The number of ALDHlowCD34+KDR+ EPCs with mild IH stimulation was higher and gradually decreased in the moderate and severe IH groups. The release of HIF-1α, SDF-1α and VEGF in the serum increased with the increase in the degree of IH. In the mild IH treatment, the migration and angiogenesis of EPCs, as well as the expression of vascular endothelial growth factor receptor 2 and cysteine-X-cysteine receptor 4, were higher than those in the control group, but progressively decreased in the groups with moderate and severe IH. Conclusion: Increased levels of IH accelerated the increase in vasoactive factors in peripheral blood, thereby mobilizing a large number of EPCs. Increasing of IH diminished the mobilization, chemotactic and angiogenetic ability of EPCs.

Sperm chromatin-condensing protamine enhances SMYD5 thermal stability

Studying thermal stability of proteins not only provides insight into protein structure but also is instrumental in identifying previously unknown interaction partners. We develop a machine learning strategy that combines orthogonal partial least squares regression and stability screening of Silver Bullets Bio library to identify biologically active molecules that enhance protein stability. This strategy proves effective in extracting the stability-enhancing molecules for SMYD5, a histone lysine methyltransferase that regulates chromosome integrity. Protamine, a histone substitute in chromatin condensation during spermatogenesis, is identified as the most influential molecule to enhance SMYD5 thermal stability. We find that the C-terminal poly-glutamic acid tract (poly-E) and a 30-residue insertion in MYND domain (M-insertion), which are unique to SMYD5, regulate the structural stability. However, protamine plays a dominant role in SMYD5 stability, and in the presence of protamine, the poly-E tract or M-insertion loses its ability to affect the stability. The stability-enhancing effect of protamine is SMYD5 specific, and for SMYD2, a closely related homolog, protamine exhibits opposite, destabilizing effects. We find that both SMYD5 and SMYD2 interact with protamine, where SMYD5 interaction is independent of the poly-E tract and M-insertion. Protamine not only helps provide insight into the structure-stability relationships of SMYD5, but also suggests a potential functional link of SMYD5 to spermatogenesis. SMYD5 is a ubiquitously expressed gene with the highest expression in testis, especially in the seminiferous ducts that contain germ cells. Thus, our study opens up avenues that could help delineate major mechanisms underlying chromatin dynamics during spermatogenesis.