Rendomab B4, a monoclonal antibody that discriminates the human endothelin B receptor of melanoma cells and inhibits their migration

Metastatic melanoma is an aggressive cancer with a poor prognostic, and the design of new targeted drugs to treat melanoma is a therapeutic challenge. A promising approach is to produce monoclonal antibodies (mAbs) against the endothelin B receptor (ETB), which is known to be overexpressed in melanoma and to contribute to proliferation, migration and vasculogenic mimicry associated with invasiveness of this cancer.

We previously described rendomab-B1, a mAb produced by DNA immunization. It is endowed with remarkable characteristics in term of affinity, specificity and antagonist properties against human ETB expressed by the endothelial cells, but, surprisingly, had poor affinity for ETB expressed by melanoma cells. This characteristic strongly suggested the existence of a tumor-specific ETB form. In the study reported here, we identified a new mAb, rendomab-B4, which, in contrast to rendomab-B1, binds ETB expressed on UACC-257, WM-266-4 and SLM8 melanoma cells. Moreover, after binding to UACC-257 cells, rendomab-B4 is internalized and colocalizes with the endosomal protein EEA-1. Interestingly, rendomab-B4, despite its inability to compete with endothelin binding, is able to inhibit phospholipase C pathway and migration induced by endothelin. By contrast, rendomab-B4 fails to decrease ERK1/2 phosphorylation induced by endothelin, suggesting a biased effect on ETB.

These particular properties make rendomab-B4 an interesting tool to analyze ETB-structure/function and a promising starting point for the development of new immunological tools in the field of melanoma therapeutics.     

Massive parallel screening of phage libraries for the generation of repertoires of human immunomodulatory monoclonal antibodies

Immune checkpoints are emerging as novel targets for cancer therapy, and antibodies against them have shown remarkable clinical efficacy with potential for combination treatments to achieve high therapeutic index. This work aims at providing a novel approach for the generation of several novel human immunomodulatory antibodies capable of binding their targets in their native conformation and useful for therapeutic applications.

We performed a massive parallel screening of phage libraries by using for the first time activated human lymphocytes to generate large collections of single-chain variable fragments (scFvs) against 10 different immune checkpoints: LAG-3, PD-L1, PD-1, TIM3, BTLA, TIGIT, OX40, 4-1BB, CD27 and ICOS. By next-generation sequencing and bioinformatics analysis we ranked individual scFvs in each collection and identified those with the highest level of enrichment.

As a proof of concept of the quality/potency of the binders identified by this approach, human IgGs from three of these collections (i.e., PD-1, PD-L1 and LAG-3) were generated and shown to have comparable or better binding affinity and biological activity than the clinically validated anti-PD-1 mAb nivolumab.

The repertoires generated in this work represent a convenient source of agonistic or antagonistic antibodies against the ‘Checkpoint Immunome’ for preclinical screening and clinical implementation of optimized treatments.     

Modification of cysteine 457 in plakoglobin modulates the proliferation and migration of colorectal cancer cells by altering binding to E-cadherin/catenins

Objectives: In tissue samples from patients with colorectal cancer (CRC), oxidation of C420 and C457 of plakoglobin (Pg) within tumor tissue was identified by proteomic analysis. The aim of this study was to identify the roles of Pg C420 and C457.

Methods: Human CRC tissues, CRC and breast cancer cells, and normal mouse colon were prepared to validate Pg oxidation. MC38 cells were co-transfected with E-cadherin plus wild type (WT) or mutant (C420S or C457S) Pg to evaluate protein interactions and cellular localization, proliferation, and migration.

Results: Pg was more oxidized in stage III CRC tumor tissue than in non-tumor tissue. Similar oxidation of Pg was elicited by H₂O₂ treatment in normal colon and cancer cells. C457S Pg exhibited diminished binding to E-cadherin and α-catenin, and reduced the assembly of E-cadherin–α-/β-catenin complexes. Correspondingly, immunofluorescent analysis of Pg cellular localization suggested impaired binding of C457S Pg to membranes. Cell migration and proliferation were also suppressed in C457S-expressing cells.

Discussion: Pg appears to be redox-sensitive in cancer, and the C457 modification may impair cell migration and proliferation by affecting its interaction with the E-cadherin/catenin axis. Our findings suggest that redox-sensitive cysteines of Pg may be the targets for CRC therapy.     

Litter and its interaction with standing vegetation affect seedling recruitment in Tibetan alpine grasslands

Background: Seedling recruitment is widely recognised to be important in the maintenance of diversity and coexistence of species. It is not clear how local abiotic factors mediated by litter and biotic interactions influence plant species recruitment in alpine grasslands.

Aims: To determine how litter and standing vegetation affected seedling recruitment in plant communities in Tibetan alpine grasslands.

Methods: Seedling recruitment was quantified in response to experimental treatments: (1) removal of standing vegetation and litter; (2) removal of standing vegetation with litter intact; (3) intact standing vegetation and removal of litter and (4) control: intact standing vegetation and litter.

Results: Litter removal increased seedling numbers, while standing vegetation removal had no effect. An interaction between litter and standing vegetation removal marginally increased seedling number. Species richness of seedlings was not related to either litter removal or standing vegetation removal. Species composition of seedling differed significantly between removal of both litter and standing vegetation and control.

Conclusions: Litter and its interaction with standing vegetation has an important role in affecting plant species recruitment in this alpine plant community. In contrast, biotic interactions, such as competition or facilitation from standing vegetation, appear to have only minor effects on recruitment.     

Insects in the Red Middle Permian of Southern France: first Protanisoptera (Odonatoptera) and new Caloneurodea (Panorthoptera), with biostratigraphical implications

The Permian odonatopteran Protanisoptera are discovered for the first time in the playa palaeoenvironments of Gonfaron and Lodève (Southern France). The new genus and species Bansheepteron gonfaronensis is erected and described on the basis of a distal half of a wing from the Guadalupian of Gonfaron. It is compared with all the previously described protanisopterans. Another specimen consisting of a basal half of a wing from the Guadalupian of Lodève, attributed to cf. Bansheepteron gonfaronensis, is also described. Furthermore, three new panorthopteran Caloneurodea are described from the Early to Middle Permian (Cisuralian and Guadalupian) of Southern France, viz. Gallogramma galadrieli gen. et sp. nov. from the le Luc Basin (Gonfaron, Var), and Paleuthygramma cf. acuta Carpenter, 1943 from the Lodève Basin (Hérault). These new fossils increase the palaeodiversity of the Caloneurodea, an interesting clade which now gathers six species in the red Permian of the Southern France, making it one of the most diverse clade in these palaeoenvironments after the Odonatoptera. The present discoveries better support a Guadalupian age for the Gonfaron Formation, even if a Late Cisuralian affinities remains possible.

http://zoobank.org/urn:lsid:zoobank.org:pub:1955790C-EA66-4137-9300-3E1B76C1585F

http://zoobank.org/urn:lsid:zoobank.org:act:0E77F461-6096-4567-8477-AA3D0D1037D3

http://zoobank.org/urn:lsid:zoobank.org:act:1D6DE829-BB0D-4EDA-9707-BFF442581601     

Potential use of transrenal DNA for non-invasive monitoring and prognosis of colorectal cancer

Background: Anti-EGFR mAb are recommended treatment for metastatic colorectal cancer (mCRC). Accurate mutation profiling and disease monitoring are challenging. The current study investigates the potential use of transrenal DNA as a biomarker for disease management.

Methods: Agreement between archival tissue specimens and transrenal DNA extracted from 200 post-treated mCRC patients was determined. Total DNA concentrations were measured and mutations within the KRAS and EGFR genes were profiled for each specimen. To ascertain therapy resistance; patients were serially monitored monthly.

Results: Concordance measurement with matched tissues at baseline was remarkably high (92%) for EGFR and KRAS mutations. Sensitivity and specificity were 98.4% and 89.1% respectively. Newly detectable mutations for a subgroup of patients with initial wildtype characteristics were evident after 4?months of anti-EGFR mAb therapy. Survival analysis using adjusted estimates showed that patients detected by transrenal DNA for key mutations or had higher mutant DNA content had poorer outcome.

Conclusion: Transrenal DNA offers new options to follow clinical treatment in mCRC. It demonstrates the ability to capture newly acquired mutations that has strong associative links to therapy resistance. Patients with these mutations fared poorly for survival outcomes and indicated possible prognostic value for transrenal DNA detection.     

The oldest ‘amphipterygid’ damselfly of tropical affinities in the Paleocene of Menat (Zygoptera: Eucaloptera)

The new damselfly genus and species Valerea multicellulata is described from the Paleocene of Menat (France), a Lagerstatte with many fossil insects, plants and vertebrates with high paleontological value. Aquatic insects are very scarce in this outcrop, this damselfly being the fourth described Odonata. Its closest modern relatives belong to the Amphipterygidae or the Devadattidae, families with very narrow tropical extant distributions. This new fossil allows us to confirm the tropical affinities of the odonatan fauna of the Menat paleolake communities. It also shows that the amphipterygids were clearly more widespread during the Paleogene than today, probably in relation to the worldwide warm and equable climate in the Paleocene.

http://zoobank.org/urn:lsid:zoobank.org:act:3F631097-DE0B-40FA8227-9C12F55DBAB4     

Dynamic variation of histone H3 trimethyl Lys4 (H3K4me3) and heterochromatin protein 1 (HP1) with employment length in nickel smelting workers

Objective: To investigate the dynamic variation in H3K4me3 and HP1 with employment length in nickel smelting workers.

Methods: Blood samples were collected from 140 nickel smelting workers and 140 age-matched office workers to test for H3K4me3, and HP1 levels.

Results: H3K4me3 was statistically significantly different (p?<?0.05) between the two groups and positively correlated with employment length (r_s?=_?0.267). HP1 was not correlated with employment length (p?=?0.066) but was significantly different between the two groups.

Conclusions: Chronic exposure to nickel can induce oxidative damage, and increase H3K4me3 expression and inhibit HP1 expression.     

CXCR4 as a prognostic biomarker in gastrointestinal cancer: a meta-analysis

Background: CXCR4 is a member of the C-X-C chemokine receptor family, which is associated with multiple types of cancer. Although it has been widely reported, the prognostic value of CXCR4 expression in gastrointestinal (GI) cancer remains controversial.

Methods: A meta-analysis was conducted to investigate the relationship between CXCR4 and prognosis of patients with GI cancer. Subgroup analysis was also performed according to tumour subtypes and heterogeneity test.

Results: A total of 24 studies including 3637 cases suggested that overexpression of CXCR4 is significantly associated with overall survival (OS) for patients with GI cancer (HR = 1.71, 95% CI = 1.45–2.03, p?=?0.000). Subgroup analysis also indicated that high CXCR4 expression in oesophagus, gastric and colorectal cancer all predicted a worse prognosis (HR = 1.52, 95% CI = 1.26–1.84, p?=?0.001 for oesophagus cancer; HR = 1.59, 95% CI = 1.10–2.30, p?=?0.015 for gastric cancer; HR = 2.21, 95% CI = 1.56–3.14, p?=?0.000 for colorectal cancer).

Conclusions: CXCR4 may serve as a prognostic indicator in GI cancer patients.     

Increase in the thermostability of Bacillus sp. strain TAR-1 xylanase using a site saturation mutagenesis library

Site saturation mutagenesis library is a recently developed technique, in which any one out of all amino acid residues in a target region is substituted into other 19 amino acid residues. In this study, we used this technique to increase the thermostability of a GH10 xylanase, XynR, from Bacillus sp. strain TAR-1. We hypothesized that the substrate binding region of XynR is flexible, and that the thermostability of XynR will increase if the flexibility of the substrate binding region is decreased without impairing the substrate binding ability. Site saturation mutagenesis libraries of amino acid residues Tyr43–Lys115 and Ala300–Asn325 of XynR were constructed. By screening 480 clones, S92E was selected as the most thermostable one, exhibiting the residual activity of 80% after heat treatment at 80°C for 15 min in the hydrolysis of Remazol Brilliant Blue-xylan. Our results suggest that this strategy is effective for stabilization of GH10 xylanase.

Abbreviations: DNS: 3,5-dinitrosalicylic acid; RBB-xylan: Remazol Brilliant Blue-xylan     

Structure investigation,enrichment analysis and structure-based repurposing of FDA-approved drugs as inhibitors of BET-BRD4

We report herein detailed structural insights into the ligand recognition modes guiding bromodomain selectivity, enrichment analysis and docking-based database screening for the identification of the FDA-approved drugs that have potential to be the human BRD4 inhibitors. Analysis of multiple X-ray structures prevailed that the lysine-recognition sites are highly conserved, and apparently, the dynamic ZA loop guides the specific ligand-recognition. The protein–ligand interaction profiling revealed that both BRD2 and BRD4 shared hydrophobic interaction of bound ligands with PRO-98/PRO-82, PHE-99/PHE-83, LEU-108/LEU-92 and direct H-bonding with ASN-156/ASN-140 (BRD2/BRD4), while on the other hand the water-mediated H-bonding of bound ligands with PRO-82, GLN-85, PRO-86, VAL-87, ASP-88, LEU-92, TYR-97 and MET-132, and aromatic π–π stacking with TRP-81 prevailed as unique interaction in BRD4, and were not observed in BRD2. Subsequently, through ROC curve analysis, the best enrichment was found with PDB-ID 4QZS of BRD4 structures. Finally, through docking-based database screening study, we found that several drugs have better binding affinity than the control candidate lead (+)-JQ1 (Binding affinity?=?-7.9?kcal/mol), a well-known BRD4 inhibitor. Among the top-ranked drugs, azelastine, a selective histamine H1 receptor antagonist, showed the best binding affinity of –9.3?kcal/mol and showed interactions with several key residues of the acetyl lysine binding pocket. Azelastine may serve as a promising template for further medicinal chemistry. These insights may serve as basis for structure-based drug design, drug repurposing and the discovery of novel BRD4 inhibitors.

Communicated by Ramaswamy H. Sarma     

Taxonomical notes on Chinese camaenids with description of three new species (Gastropoda: Pulmonata)

The present study deals with four Chinese camaenid species based on museum collections and newly obtained materials. Pseudiberus liuae Wu, n. sp., diagnosed by two long mucous glands and the smallest shell size in the genus and inhabiting bare rock like other congeneric members, is described from southern Gansu. Aegista (Plectotropis) wardi (Preston, 1912) is conchologically re-described and moved out of Aegista Alber, 1860 to Pseudiberus Ancey, 1887 based on the keeled periphery and the absence of hairs, scales or their scars on the teleoconch, which are present in Aegista but partially absent in Pseudiberus. The first Chinese fluorescent snail Bradybaena qixiaensis Wu & Asami n. sp. is reported from Nanjing, Jiangsu. The species shares many characters with the Japanese fluorescent snail Bradybaena pellucida Kuroda & Habe, 1953 but is distinct in the pattern of microsculpture on the internal surface of the penis. Nesiohelix yeni Wu & Asami n. sp., sympatric with N. moreletiana (Heude, 1882), is distinguished from its congener by possession of a bubble-shaped penial caecum.

Pseudiberus liuae Wu in Wu & Asami, In Press

LSID urn:lsid:zoobank.org:act:3C9299AA-5089-4E43-9B26-85A0D6C23B66

Bradybaena qixiaensis Wu & Asami, In Press

LSID urn:lsid:zoobank.org:act:7991C4D5-5E0B-46DF-8B19-BE26511806CD

Nesiohelix yeni Wu & Asami, In Press

LSID urn:lsid:zoobank.org:act:68CFF173-AACC-4DA8-B347-9ABB5CA569A3     

A density functional theory-based analysis of the structural,topological and electronic properties of gemcitabine drug adsorption on the pyrrolidine functionalized single-walled carbon nanotube

The stability of gemcitabine anticancer drug on the functionalized (8,0) zigzag carbon nanotube as a drug delivery vehicle is studied within the formalism of the density functional theory calculations to understand the role of the pyrrolidine functional group in binding the adsorbed molecule to the drug delivery system as well as improving water solubility. The binding energies, natural bond orbital calculations, and the quantum theory of atoms in molecules results are obtained to provide more evidences related to the intermolecular interaction between gemcitabine drug and the functionalized nanotube. The negative binding energy corresponds to favorable binding of gemcitabine drug to the functionalized nanotube and presence of the active sites available for hydrogen bond formation facilitates better drug binding to the nanotube sidewall. The results presented in this article indicate that pyrrolidine functionalized carbon nanotube seems to be a novel material for drug delivery applications.

Communicated by Heidar Moradnia     

Reliability of two-point discrimination thresholds using a 4-2-1 stepping algorithm

Purpose: A 4-2-1 stepping algorithm reliably captures light touch thresholds but has not been used to assess two-point discrimination (TPD) thresholds. Therefore, the purpose of this investigation was to determine the intra- and inter-rater reliability of a 4-2-1 stepping algorithm at determining TPD thresholds.

Materials and methods: Fifteen healthy, physically active young adults were assessed twice over a 1-week period using digital calipers and a 4-2-1 stepping algorithm. TPD thresholds were assessed by an expert and a novice examiner at each time point. Reliability was assessed on the plantar surface of the foot at the head of the first and base of the fifth metatarsal.

Results: Three intra-rater intraclass correlation coefficient (ICC) values exceeded 0.75 and were interpreted as good. The inter-rater reliability was good with ICC values ranging from 0.76 to 0.93 at both sites during both test sessions.

Conclusions: The 4-2-1 stepping algorithm demonstrates good intra- and inter-tester reliability at determining TPD thresholds on the plantar surface of the foot at the head of the first and base of the fifth metatarsal in young healthy adults.     

Effect of lipid composition on incorporation of trastuzumab-PEG-lipid into nanoliposomes by post-insertion method: physicochemical and cellular characterization

Context: Anti-HER2 immunoliposomes are promising nanotechnology based systems for active targeting of breast tumors, which depends on the amount of incorporated antibody.

Objective/Aim: In this work, we investigated the possible effect of lipid composition on the incorporation of trastuzumab-PEG-PE micelles into nanoliposomes and on their subsequent specific cellular targeting.

Materials and methods: Trastuzumab (anti-HER2 monoclonal antibody) was monothiolated and conjugated to maleimide-PEG-PE micelles. Liposomes of different lipid compositions were prepared by the thin layer hydration. Trastuzumab-PEG-PE micelles were incorporated into the liposomes by the post-insertion method. The percentage of lipid mixing was determined based on fluorescence resonance energy transfer. Cellular binding and uptake of rhodamine-labeled immunoliposomes were studied in SKBR-3 (HER2⁺⁺⁺) and MCF-7 (HER2⁺) cells. Also, antitumor cell activity of the immunoliposomes was compared to free trastuzumab and the liposomes.

Results: The lipid mixing of trastuzumab-PEG-PE micelles depended on the liposome composition. The immunoliposomes containing DPPC, cholesterol and PEG-PE showed prominent lipid mixing. The lipid mixing was consistent with the cell binding results which showed an efficient and specific binding of the immunoliposomes to SKBR-3 cells. Antitumor cell activity of the immunoliposomes in SKBR-3, unlike MCF-7 cells, depended on the content of trastuzumab.

Discussion: Cholesterol and PEG-PE in the liposome composition are prerequisites for a successful lipid mixing due to their ability to facilitate fusion. The higher lipid mixing results in higher antibody incorporation and consequently higher targeted cell binding.

Conclusions: The lipid mixing depends on the liposome composition, which reflects targeted cell binding of the immunoliposomes.     

Molecular dynamics simulations provide insights into the origin of gleevec’s selectivity toward human tyrosine kinases

Protein kinases are critical drug targets against cancer. Since the discovery of Gleevec, a specific inhibitor of Abl kinase, the capability of this drug to distinguish between Abl and other tyrosine kinases, such as Src, has been intensely investigated but the origin of Gleevec’s selectivity to Abl against Src is less studied. Here, we performed molecular dynamics (MD) simulations, dynamical cross-correlation matrices (DCCM), dynamical network analysis, and binding free energy calculations to explore Gleevec’s selectivity based on the crystal structures of Abl, Src, and their common ancestors (ANC-AS) and the two constructed mutation systems (AS→Abl and AS→Src). MD simulations revealed that the conformation of the phosphate-binding loop (P-loop) was altered significantly in the AS→Abl system. DCCM results unraveled that mutations increased anticorrelated motions in the AS→Abl system. Community network analysis suggested that the P-loop established special contacts in the AS→Abl system that are devoid in the AS→Src system. The binding free energy calculations unveiled that the affinity of Gleevec to AS→Abl increased to near the Abl level, whereas its affinity to AS→Src decreased to near the Src level. Analysis of individual residue contributions showed that the differences were located mainly at the P-loop. This study is valuable for understanding the sensitivity of Gleevec to human tyrosine kinases.

Communicated by Ramaswamy H. Sarma     

Tyrosine-phosphorylation of the scaffold protein ADAP and its role in T cell signaling

Introduction: The Adhesion and Degranulation promoting Adaptor Protein (ADAP) is phosphorylated upon T cell activation and acts as a scaffold for the formation of a signaling complex that integrates molecular interactions between T cell or chemokine receptors, the actin cytoskeleton, and integrin-mediated cellular adhesion and migration.

Areas covered: This article reviews current knowledge of the functions of the adapter protein ADAP in T cell signaling with a focus on the role of individual phosphotyrosine (pY) motifs for SH2 domain mediated interactions. The data presented was obtained from literature searches (PubMed) as well as the authors own research on the topic.

Expert commentary: ADAP can be regarded as a paradigmatic example of how tyrosine phosphorylation sites serve as dynamic interaction hubs. Molecular crowding at unstructured and redundant sites (pY595, pY651) is contrasted by more specific interactions enabled by the three-dimensional environment of a particular phosphotyrosine motif (pY571).