D2 Region Research Articles

The Dugald River-type, shear zone hosted, Zn-Pb-Ag mineralisation, Mount Isa Inlier, Australia

The Dugald River Zn-Pb-Ag mine is situated in the Mount Isa Inlier, a globally significant base metal province. Zn-Pb deposits in the Mount Isa Inlier are stratabound with four main genetic models, including SEDEX-style, remobilised SEDEX, epigenetic and Broken Hill-type mineralisation applied to interpret their formation. We propose that the Zn-Pb-Ag mineralisation at Dugald River represents a unique, shear zone hosted deposit type that formed through a series of successive deformation events during the Paleoproterozoic Isan Orogeny that concentrated the mineralisation within the Dugald River Shear Zone during two main mineralising phases. The first phase of mineralisation occurred during regional D2 shortening, which is associated with the formation of large-scale F2 folds and a regionally penetrative S2 fabric. During this phase, progressive tightening of upright F2 folds resulted in several sets of secondary space accommodating quartz-carbonate veins that were progressively rotated into parallelism with the pervasive, steep, W-dipping S2 cleavage. The quartz-carbonate veins were coevally replaced by sulphides, which migrated to extensional sites (boudin necks and fold hinges) in tight folds. Thereby creating a sulphide-rich horizon within a developing high strain zone, which during D4 developed into the Dugald River Shear Zone. The second phase of mineralisation occurred during the regional D4 transpressional deformation event and resulted in significant metal enrichment and the current geometry of the ore bodies. The significant enrichment of the mineralisation during D4 resulted from further fold tightening within the high strain zone, which resulted in the attenuation and dismembering of folds and produced a transposed fabric (S4). The sulphide veins were transposed into parallelism with S4 forming sulphide-rich planar ore textures. Strain partitioning at the contact between the ductile deforming sulphide horizon and the brittle deforming slates resulted in the development of an anastomosing shear zone, known as the Dugald River Shear Zone. A right-handed releasing bend in the shear zone produced a dilational jog and a thick, high-grade ore body. The mobilisation of sulphides within the dilational jog involved fragmentation of sulphides and wall rock, brecciation, rotation and rolling of fragments, and the formation of durchbewegung texture.

Metabarcoding the zooplankton species of the Saudi Arabian Gulf: A study employing mock communities and two gene markers

Ecosystem health can be monitored by analyzing the species diversity and abundance in a region. The process of recognizing species based on morphology takes a lot of time and expertise. Metabarcoding and other modern molecular taxonomic methods can help speed up species identification. However, the choice of gene markers and primers, the lack of reference sequences in public databases, and the choice of tools used in bioinformatic analyses have a substantial impact on the observed diversity. To test the effectiveness of metabarcoding in assessing the species diversity, mock communities of zooplankton species found in the Saudi Arabian Gulf were assembled and subjected to high-throughput sequencing (HTS) using an Illumina MiSeq platform. Short CO1 and 28S nuclear gene fragments, containing the D2 region, of lengths 313 bp and 400 bp, respectively, were used as gene markers. Trimmomatic, Cutadapt, the FLASH read-merge software tool, and the USEARCH pipeline for OTU clustering were used in the bioinformatic analyses. Around 90% of the zooplankton species in the mock communities were detected. We conclude that the combination of CO1 and 28S markers is a quick and effective tool for evaluating zooplankton species diversity.

Cryo-EM analyses of KIT and oncogenic mutants reveal structural oncogenic plasticity and a target for therapeutic intervention.

The receptor tyrosine kinase KIT and its ligand stem cell factor (SCF) are required for the development of hematopoietic stem cells, germ cells, and other cells. A variety of human cancers, such as acute myeloid leukemia, gastrointestinal stromal tumor, and mast cell leukemia, are driven by somatic gain-of-function KIT mutations. Here, we report cryo electron microscopy (cryo-EM) structural analyses of full-length wild-type and two oncogenic KIT mutants, which show that the overall symmetric arrangement of the extracellular domain of ligand-occupied KIT dimers contains asymmetric D5 homotypic contacts juxtaposing the plasma membrane. Mutational analysis of KIT reveals in D5 region an "Achilles heel" for therapeutic intervention. A ligand-sensitized oncogenic KIT mutant exhibits a more comprehensive and stableD5 asymmetric conformation. A constitutively active ligand-independent oncogenic KIT mutant adopts a V-shaped conformation solely held by D5-mediated contacts. Binding of SCF to this mutant fully restores the conformation of wild-type KIT dimers, including the formation of salt bridges responsible for D4 homotypic contacts and other hallmarks of SCF-induced KIT dimerization. These experiments reveal an unexpected structural plasticity of oncogenic KIT mutants and a therapeutic target in D5.

Preference of CAMSAP3 for expanded microtubule lattice contributes to stabilization of the minus end.

CAMSAPs are proteins that show microtubule minus-end-specific localization, decoration, and stabilization. Although the mechanism for minus-end recognition via their C-terminal CKK domain has been well described in recent studies, it is unclear how CAMSAPs stabilize microtubules. Our several binding assays revealed that the D2 region of CAMSAP3 specifically binds to microtubules with the expanded lattice. To investigate the relationship between this preference and the stabilization effect of CAMSAP3, we precisely measured individual microtubule lengths and found that D2 binding expanded the microtubule lattice by ∼3%. Consistent with the notion that the expanded lattice is a common feature of stable microtubules, the presence of D2 slowed the microtubule depolymerization rate to ∼1/20, suggesting that the D2-triggered lattice expansion stabilizes microtubules. Combining these results, we propose that CAMSAP3 stabilizes microtubules by lattice expansion upon D2 binding, which further accelerates the recruitment of other CAMSAP3 molecules. Because only CAMSAP3 has D2 and the highest microtubule-stabilizing effect among mammalian CAMSAPs, our model also explains the molecular basis for the functional diversity of CAMSAP family members.

Phylogenetic relationships, host associations, and three new species of a poorly known group of “tetraphyllidean” tapeworms from elasmobranchs

This paper aims to expand understanding of a poorly known group of cestodes that parasitize an intriguingly diverse suite of elasmobranchs. The group's three currently described members (i.e., Pentaloculum macrocephalum, Pentaloculum hoi, and Zyxibothrium kamienae) parasitize an electric ray, a carpet shark, and a skate, respectively. Pentaloculum grahami n. sp. is described from a second genus of carpet shark, specifically Parascyllium collare, in Australia. Zyxibothrium duffyi n. sp. and Zyxibothrium healyae n. sp. are described from the deep-sea skates Brochiraja asperula and Brochiraja spinifera, respectively off New Zealand. The three new species share distinctive bothridia that bear a small number of large, circular, facial loculi and lateral bands of vitelline follicles that converge posterior to the ovary-features which are found in all other members of these genera. Zyxibothrium healyae n. sp. is unique in possessing three, rather than four or five, facial loculi. Zyxibothrium duffyi n. sp. possesses a combination of five facial loculi and vitelline follicles that stop short of the anterior margin of the proglottid. Pentaloculum grahami n. sp. is the largest member of the group with the greatest number of proglottids. Based on striking similarities in scolex morphology, Pentaloculum and Zyxibothrium have been hypothesized to belong to a distinct subgroup of "tetraphyllideans" provisionally designated as Clade 1. Based on sequence data for the D1-D3 region of the 28S rDNA gene generated for species of Zyxibothrium for the first time, we confirm the reciprocal monophyly of both genera as well as the monophyly of Clade 1 and its status as a distinct lineage among the "Tetraphyllidea". This work also suggests that the presence of five facial loculi is homoplasious given this character state is found in members of both genera. The new species expand the host associations of Clade 1 to include additional skate and carpet shark genera. Moving forward we would expect to find additional members of this group parasitizing other species of parascyliid carpet sharks as well as other species of the rajid genus Malacoraja and the arhynchobatid genus Brochiraja. Here we have doubled the number of described species in the taxon referred to as Clade 1 while simultaneously expanding our understanding of the morphology and anatomy of its members. This additional information will help inform the ultimate revision of the ordinal classification of the cestodes to address the highly polyphyletic nature of the order "Tetraphyllidea" as it is currently configured.

Cutaneous transcriptomic profiling and candidate pigment genes in the wild discus (Symphysodon spp.)

ABSTRACT Discus (Symphysodon spp.) is a popular freshwater ornamental fish with high market value due to its extraordinary skin pigmentation. The molecular mechanisms that govern skin colouration have yet to be identified. Six skin samples from wild discus were collected in the D1 (dorsal intense black bar stripe), D2 (ventral lighter bar stripes and orange carotenoid spots) and D3 (posterior without stripe nor spot) regions of wild discus for transcriptomic profiling and analysis. Differential analysis was conducted in 3 pair-wise comparisons, D1vsD3, D2vsD3 and D2vsD1. The results demonstrated that the melanin synthesis-associated genes (ERBB4, ADCY9, WNT2, 14-3-3 proteins and TSPAN genes) were upregulated in D1 and D2 regions, whereas carotenoids metabolism-associated genes (PLIN2, AHCY, RETSAT, STARD10, TTC39B and RDH1 genes) were uniquely expressed in D2 region. Amino acid metabolism-related pathways were enriched in both the D1 and D2 regions. MAPK pathway enrichment was detected merely in the D1 region, while various transporter activities were enriched in the D2 region. Further investigations speculated the key regulator roles of GNAQ, ADCY9, 14-3-3 and TSPAN genes in black vertical bar formation. This is the first fundamental report on discus skin pigment genes that could be extended into more detailed studies in the future.

Cloning, DNA sequence, and expression of flagellins from high and low virulence strains of Edwardsiella tarda and their macrophage-stimulating activities.

Edwardsiella tarda is a causative pathogen of edwardsiellosis in fish. Our previous studies on high (NUF251) and low (NUF194) virulent strains of E. tarda demonstrated that NUF251 strain induced significantly higher levels of NO and TNF-α from fish and mouse macrophages than NUF194 strain. Subsequent studies suggested that a flagellin-like protein secreted from E. tarda might be a responsible factor for the macrophage-stimulating activities. To evaluate the activities of flagellins of E. tarda, in this study, the flagellin genes of NUF251 and NUF194 strains were isolated by PCR and cloned into pQE-30 and pCold I expression vectors, and then the recombinant flagellins of two strains were overexpressed in E. coli JM109 and pG-Tf/BL21, respectively. The molecular weight of the purified recombinant flagellins of NUF251 and NUF194 strains were estimated to be 45kDa and 37kDa, respectively on SDS-PAGE analysis. Referring the three-dimensional structure of Salmonella flagellin, which has been reported to have 4 domains (D0, D1, D2, and D3), high sequence homology between two flagellins of E. tarda was observed at conservative domain (D0 and D1) regions, whereas the sequences equivalent to D2 and D3 domains were different, and even equivalent to 57 amino acids were deleted in NUF194. Both recombinant flagellins induced NO production, mRNA expression level of inducible NO synthase (iNOS), and intercellular ROS production in mouse macrophage cell line RAW264.7cells. Also, the secretion of TNF-α and its mRNA expression level were increased by treatment of both recombinant flagellins. These results indicate that the recombinant flagellins from different virulent E. tarda strains can stimulate macrophages with nearly equal levels as judged by the parameters tested, even though they are differences in the structure and molecular weight, suggesting that conservative D0 and D1 domains are sufficient structural elements for the recombinant flagellins to induce a certain level of macrophage-stimulation in vitro. Further studies are necessary focusing on the role of D2 and D3 domain regions of the recombinant flagellins as macrophage-stimulating agent as well as their influence on host immune system in vivo.

Morphological, Phylogenetic and Toxinological Characterization of Potentially Harmful Algal Species from the Marine Coastal Waters of Buenos Aires Province (Argentina)

In the framework of a monitoring program of harmful microalgae from the marine coastal waters of the Buenos Aires Province, seven strains were isolated and characterized by morphological and molecular analysis (LSU rDNA partial sequencing, D1–D3 regions). Established strains belonged to Alexandrium catenella, Protoceratium reticulatum and Pseudo-nitzschia multiseries. The toxinological profile of the target strains were determined by UHPLC-FLD equipment for paralytic shellfish toxins (PSTs) and LC-MS/MS for lipophilic (LSTs) and amnesic toxins (ASTs). The toxin profile varied in the four strains of A. catenella, the predominant compounds were gonyautoxins (GTXs) GTX2,3 and GTX1,4 for strains LPCc001 and LPCc004, and N-sulfocarbamoyl toxins (Cs) C1,2 and GTX1,4 for strains LPCc002 and LPCc008. The obtained cellular toxicity values were moderate-to-high (12.38–46.40 pg saxitoxin equiv. cell−1). The toxin profile of P. reticulatum was dominated by yessotoxins (YTXs) (up to 94.40 pg cell−1) accompanied by homo-yessotoxin (Homo-YTX) traces. In P. multiseries, the toxin profiles were dominated by domoic acid (DA) (1.62 pg cell−1 and 1.09 pg cell−1) and secondarily by Isomer A (Iso-A), Epi-domoic acid (Epi-DA), Isomer-E (Iso-E) and Isomer-D (Iso-D). This study provides detailed information about representative HAB species in the area, useful for resource management, risk evaluation and related research on toxic dinoflagellates and diatoms.

Genetic analysis of the tomato russet mite provides evidence of oligophagy and a widespread pestiferous haplotype.

Worldwide, the tomato russet mite (TRM), Aculops lycopersici (Eriophyidae), is a key pest on cultivated tomato in addition to infesting other cultivated and wild Solanaceae; however, basic information on TRM supporting effective control strategies is still lacking, mainly regarding its taxonomic status and genetic diversity and structure. As A. lycopersici is reported on different species and genera of host plants, populations associated with different host plants may constitute specialized cryptic species, as shown for other eriophyids previously considered generalists. The main aims of this study were to (i) confirm the TRM taxonomic unity of populations from different host plants and localities as well as the species' oligophagy, and (ii) to advance the understanding of TRM host relationship and invasion history. For this purpose, we evaluated the genetic variability and structure of populations from different host plants along crucial areas of occurrence, including the area of potential origin, based on DNA sequences of mitochondrial (cytochrome c oxidase subunit I) and nuclear (internal transcribed spacer, D2 28S) genomic regions. Specimens from South America (Brazil) and Europe (France, Italy, Poland, The Netherlands) were collected from tomato and other solanaceous species from the genera Solanum and Physalis. Final TRM datasets were composed of 101, 82 and 50 sequences from the COI (672bp), ITS (553bp) and D2 (605bp) regions, respectively. Distributions and frequencies of haplotypes (COI) and genotypes (D2 and ITS1) were inferred; pairwise genetic distance comparisons, and phylogenetic analysis were performed, including Bayesian Inference (BI) combined analysis. Our results showed that genetic divergences for mitochondrial and nuclear genomic regions from TRM associated with different host plants were lower than those observed in other eriophyid taxa, confirming conspecificity of TRM populations and oligophagy of this eriophyid mite. Four haplotypes (cH) were identified from the COI sequences with cH1 being the most frequent, representing 90% of all sequences occurring in all host plants studied (Brazil, France, The Netherlands); the other haplotypes were present exclusively in Brazilian populations. Six variants (I) were identified from the ITS sequences: I-1 was the most frequent (76.5% of all sequences), spread in all countries and associated with all host plants, except S. nigrum. Just one D2 sequence variant was found in all studied countries. The genetic homogeneity among populations highlights the occurrence of a highly invasive and oligophagous haplotype. These results failed to corroborate the hypothesis that differential symptomatology or damage intensity among tomato varieties and solanaceous host plants could be due to the genetic diversity of the associated mite populations. The genetic evidence, along with the history of spread of cultivated tomato, corroborates the hypothesis of a South American origin of TRM.

Molecular identification of root-lesion nematodes, Pratylenchus spp. in agricultural crops from Costa Rica

Introduction. The root-lesion nematodes, Pratylenchus spp., have a wide host range and reduce the yield of different crops. Information on the diversity of Pratylenchus species is scarce in Costa Rica. Objective. To identify the Pratylenchus species associated with 12 crops based on the D3 region of the 28S rDNA gene. Materials and methods. During 2013 to 2015, root samples were collected in Alajuela, Cartago, Guanacaste, Heredia, and San José in crops of rice (Oryza sativa), black pepper (Piper nigrum), sugarcane (Saccharum officinarum), aster (Aster sp.), coffee (Coffea arabica), banana (Musa paradisiaca), lily (Lilium sp.), gypsophila (Gypsophila sp.), onion (Allium cepa), potato (Solanum tuberosum), strawberry (Fragaria x ananassa), and leather-leaf fern (Rumohra adiantiformis). The D3 region of the 28S rDNA gene from each population was amplified and sequenced. A GenBank Blast Search was performed for each sequence. The phylogenetic relationships were established by Bayesian Inference. Results. Blast Search indicated the presence of P. pseudocoffeae in aster, P. brachyurus in black pepper, P. crenatus in onion and potato, P. hippeastri and P. gutierrezi in sugarcane and coffee, respectively. Pratylenchus bolivianus in leather-leaf fern and potato, P. penetrans in onion, strawberry, gypsophila, and lily, P. zeae in rice and sugarcane, while P. speijeri in banana. The phylogenetic analysis corroborated the Pratylenchus species identity with exceptions of sequences from 1) banana, grouped to P. coffeae complex group, 2) sugar cane, grouped to P. hippeastri complex group 3) onion and potato were related with P. crenatus, in an independent group, and 4) leather-leaf fern and potato were grouped with P. bolivianus with low resolution. Conclusions. Nine genetic groups of Pratylenchus were found, some of those should be verified with other molecular markers to get a conclusive identification.

New Morphological and Molecular Data Reveal an Underestimation of Species Diversity of Mites of the Genus Geckobia (Acariformes: Pterygosomatidae) in India

Mites of the genus Geckobia (Acariformes: Pterygosomatidae) are permanent and highly specialised ectoparasites of geckos (Gekkota). We conducted a local study on Geckobia mites associated with the geckos of the family Gekkonidae found mainly in the territory of the Indian Institute of Science’s campus (Bangalore, India). In total, we examined 208 lizards belonging to two genera: Hemidactylus and Cnemaspis. We assessed the prevalence of the mites and identified the preferred site for their infestation. We extended the standard morphological identification of the mite species by using DNA barcode markers, partial sequences of the mitochondrial cytochrome c oxidase subunit I (COI) gene and nuclear ribosomal gene sequences: 18S rRNA and hypervariable region D2 of nuclear 28S rRNA. We checked the suitability of COI and nuclear (D2 of 28S rRNA) markers for species delimitations and identification purposes of the genus. The distance- and phylogeny-based approaches were applied: (i) to test the presence of a barcoding gap, we used the automated barcoding gap discovery tool (ABGD) and investigated intra- and interspecific genetic distances, and (ii) to reconstruct evolutionary relationships within the species, we performed maximum likelihood (ML) and Bayesian inference with Markov-Chain Monte Carlo (BI) analyses. As a result, we described five new species—Geckobia gigantea sp. n., G. treutleri sp. n., G. unica sp. n. and G. brevicephala sp. n.—from four Hemidactylus species: H. giganteus, H. treutleri, H. parvimaculatus and H. frenatus, respectively, and G. mysoriensis sp. n. from Cnemaspis mysoriensis. Additionally, we found three already described species: Geckobia indica Hirst, 1917 on H. treutleri (new host), Geckobia bataviensis Vitzhum, 1926 on H. parvimaculatus (new host) and H. frenatus (new locality) and Geckobia phillipinensis Lawrence, 1953 on H. frenatus (new locality). The diagnoses of G. indica and G. phillipinensis were improved and supplemented by descriptions of the males and juveniles. Both topologies of the BI and ML phylogenetic trees, as well as genetic distances, supported the species boundaries in the mite population shown by the morphological data. Hemidactylus frenatus was the most infected gecko species (61% prevalence), with the highest number of mite species (three spp.). The scale-mite richness was higher than expected; therefore, further research is required to evaluate the true diversity of Geckobia mites.

New Records of Neobenedenia girellae (Hargis, 1955) (Monogenea: Capsalidae) in Marine Ornamental Fish Imported to Yucatan, Mexico

We detected Neobenedenia girellae infections in 40 species belonging to 12 families of imported marine ornamental fish from a public aquarium in the Mexican state of Yucatan. A total of 348 fish specimens were examined for monogeneans in January 2018 to December 2020. Monogeneans were corroborated morphologically and molecularly with a partial sequence of 28S (region D1–D3) ribosomal DNA and analyzed in a molecular phylogenetic context in combination with other N. girellae sequences available in GenBank. The phylogenetic tree revealed that the specimen found consistently belonged to the N. girellae clade. High infection parameters were detected of N. girellae in most hosts. This identification is relevant to aquarists and aquaculturists in the Gulf of Mexico because N. girellae is considered highly pathogenic in confined fish. This work demonstrates that the importation of ornamental fish coupled with deficient sanitary measures (lack of quarantine areas in distribution centers) contributes to spread of parasites and their establishment within Mexico.

A new species of Cleruchus (Hymenoptera, Mymaridae), an egg parasitoid of the invasive Cis chinensis (Coleoptera, Ciidae) in Switzerland, with new records of other congeners in Europe

A fairyfly (Hymenoptera, Mymaridae) Cleruchus breviclava Triapitsyn & Coray, sp. nov. is described and illustrated. The new species is an egg parasitoid of the invasive Cis chinensis Lawrence (Coleoptera, Ciidae) in Antrodia xantha fungus (Polyporales, Fomitopsidaceae) in Basel, Switzerland; it is also known from low mountains in Germany and Switzerland. Supporting data on the “barcoding” region of the mitochondrial cytochrome c oxidase subunit I gene, as well as separate regions of nuclear ribosomal RNA, the D2 region of 28S and the internal transcribed spacer 2, provide strong evidence of conspecificity of the morphologically variable macropterous and strongly brachypterous individuals of C. breviclava. Macropterous females of the new species are most similar to those of C. detritus Bakkendorf, also known from Switzerland. New records are provided for some other species of Cleruchus Enock in Europe. A key to both sexes of the described European species of the genus is given.

Reappraisal of the hyperdiverse

Morphologically similar species are often overlooked but molecular techniques have been effective in signalling potential hidden diversity, boosting the documentation of unique evolutionary lineages and ecological diversity. Platynereis dumerilii and Platynereis massiliensis are part of a recognised species complex, where differences in the reproductive biology have mainly been highlighted to date. Analyses of DNA sequence data (COI, 16S rDNA and D2 region of the 28S rDNA) of populations of the apparent morphotype of P. dumerilii obtained from a broader sampling area along European marine waters, including the Azores and Webbnesia islands (Madeira and Canaries), provided compelling evidence for the existence of at least 10 divergent evolutionary lineages. Complementing the genetic data, morphological observations of the better represented lineages revealed two major groups with distinctive paragnath patterns. Two new Platynereis species were erected: P. nunezi sp. nov., widespread in the Azores and Webbnesia islands, and P. jourdei sp. nov., restricted to the western Mediterranean. The new combination P. agilis is also proposed for Nereis agilis, previously unaccepted for one of the lineages present both in the Northeast Atlantic and western Mediterranean. Platynereis dumerilii is redescribed based on topotypic material. However, uncertainty in the identity of P. massiliensis due to the original brief description and the absence of type and topotypic material prevents the unequivocal assignment to the lineage assumed in this and previous studies. The remaining five lineages are represented by only a few small specimens with morphological features poorly preserved and were therefore not described in this study. ZooBank: urn:lsid:zoobank.org:pub:50079615-85E5-447E-BDD7-21E81C2A6F4D

Novel Sirpα-Fc Fusion Protein IMM01 Exhibits Dual Anti-Tumor Activities By Targeting CD47/Sirpα Signal Pathway

Background: This study aims to evaluate the anti-tumor mechanism of IMM01, a novel recombinant SIRPα-Fc fusion protein targeting CD47-SIRPα pathway. Methods: IMM01 was a recombinant fusion protein of SIRPα (V2) extracellular segment domain 1 and human IgG1 Fc, with the N glycosylation site N80 mutated to A (Alanine) in D1 region. IMM01 fusion proteins were produced using in house developed CHO-K1 cell expression system. IMM01 blocking activities on Jurkat-CSR (Jurkat-CD47KO-CARSIRPα) cells, antibody-dependent cellular phagocytosis (ADCP), antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) assays were done by flow cytometry. Detection of cytokines of IL-1β, IL-2, IL-4, IL-5, IL-6, GM-CSF, IFN-γ, IL-10 and TNF production by PBMC was done CBA assay. In vivo mechanism of action study was done in different mouse models including HL-60 CB17-SCID, Daudi CB17-SCID, SNU-1 CB17-SCID, Raji CB17-SCID and CT26-hPDL1(Tg) hCD47(Tg) mPDL1(KO) mCD47(KO). In addition, human red blood cells binding and erythrocyte agglutination of IMM01 were also evaluated. Results:In vitro binding assay revealed that IMM01 has a strong binding affinity to CD47 with lowest EC50 at 0.4967 nM. The removal of N-glycosylation modification improves the consistence and purity of the IMM01 protein expression, and the potential mitigation of immunogenicity. Using a chimeric SIRPα receptor protein expressing cell line, IMM01 not only can block the CD47 at the protein level, but also block the signal transduction at the cellular levels. Compared to negative (hIgG1-Fc) controls, IMM01 stimulated secretion of minimal amount of IL-10 and TNF but inhibited IL-8 production by PBMC. IMM01 can induce strong ADCP and moderate ADCC, but not the CDC activities. The strong phagocytosis against tumor cells is dependent on interaction of Fc with activating FcγRs on the presence of macrophages. Mechanism of action studies demonstrated that IMM01 induced strong phagocytosis against tumor cells was attributed to dual activities of blocking "don't eat me" signal and activating "eat me" signal. IMM01 has strong and robust in vivo anti-tumor activities in different mouse tumor models either as monotherapy on hematological malignancies, or in combination therapy with PD-L1 mAb, PD-1 mAb, CD33 mAb, and HER-2 mAb, respectively. The in vivo therapeutic efficacy of IMM01 was done in HL-60 xenograft model showed that 100% of the mice achieved CR after administration of IMM01 at 5mg/kg for 2 weeks. Also, 100% of mice achieved CR after IMM01 combined with the above mentioned mAbs in different mouse models, respectively. This illustrated that IMM01 has a great therapeutic potential in combination with these commonly used immunotherapy or targeted therapy agents to be used in the treatment of solid tumors and hematological malignancies. Finally, IMM01 demonstrated a favorable safety profile with no human RBC binding, nor hemagglutination induction. Conclusion: Our study results demonstrated that, as a novel bi-functional SIRPα-Fc recombinant protein, IMM01 has very strong anti-tumor activities with a good curative effect on mouse xenograft tumor models with dual anti-tumor activities by blocking CD47 "don't eat me" signal and activating the phagocytosis "eat me" signal. Furthermore, IMM01 demonstrated a well synergistic effect when combined with different immunotherapeutic agents. Importantly, our results revealed that IMM01 has a favorable safety profile with no human RBC binding activity and no hemagglutination induction. Phase I clinical study is ongoing, and the clinical trial results will be reported soon. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

First detection of the potato cyst nematode (Globodera rostochiensis) in a major potato production region of China.

The potato cyst nematodes (PCN) Globodera rostochiensis and Globodera pallida are the very important quarantine nematode pests of potato [Stone 1973]. Both species cause serious potato tuber yield losses. These species are subjected to strict quarantine regulations in many countries (EPPO 2017). G. rostochiensis was detected in Sichuan and Yunnan province, China in 2022 (Jiang et al. 2022). A survey for cyst nematodes in potato fields was conducted in Guizhou Province from 2018 to 2020. A total of 200 samples, including roots and soil, were collected from 40 potato fields in Hezhang (N27 06.145, E104 39.153) and Weining (N26 50.541, E104 09.885) counties in Guizhou Province, China. The Cobb decanting and sieving method was used to isolate cysts and J2s from the soil samples (Southey 1986). The potato roots were stained with acid fusion to observe cyst development. Morphological and molecular analyses indicated that 27 (13.5%) of the samples contained G. rostochiensis. The cyst density ranged between 1-85 cysts per 100 cm3 of soil and a mean density was 15 cysts per 100 cm3 soil. The smoothly rounded cysts were in brown and golden color, and the terminal cone was absent and circumfenestrate. The key morphometrics of cysts (n=20) were 695 ± 26 (685-757) μm in length excluding neck and 690 ± 30 (668 to769) μm in width; the number of cuticular ridges between anus and vulval fenestra was 16.3 ±2.1 (14 to 18); fenestral length was 15.1 ± 2.1 μm (13.18 to 19.27); distance from anus to the edge of fenestra was 61.12 ± 8.9 (49.22 to76.27) μm; and Granek's ratio was 4.54 ± 0.8 (3.97-5.26). The key morphometrics of J2 (n = 20): 468.0 ± 20.1 (427 to - 521) μm in body length, 20.58 ± 0.7 (20.2 to 21.8) μm in stylet length, 43.9 ± 5.6 (40.3 to 53.9) μm in tail length, and 23.1 ± 1.8 (21.77 to 25.32) μm in hyaline region length. The cyst and J2 morphologies were consistent with those of G. rostochiensis (Subbotin et al. 2010, EPPO 2017). Genomic DNA was isolated from cysts (n=20). DNA extraction was performed in a volume of 20 μl containing 3 μl 10× PCR buffer, 3 μl Proteinase K (600 μg μl-1), 14 μl distilled water and a single cyst was added and ground in an ice bath as described by Ou et al. (2008). The internal transcribed spacer (ITS) regions were amplified using the universal primers: rDNA1 (5'-TTGATTACGTCCCTGCCCTTT-3') and rDNA2 (5'-TTTCACTCGCCGTTACTAAGG-3') (Fleming 1998 ), and the 28S rDNA-D2/D3 regions were amplified using the primers: D2A (5´-ACAAGTACCGTGAGGGAAAGTTG-3´) and D3B (5´-TCGGAAGGAACCAGCTACTA-3´) (Subbotin et al. 2006). After the brackets at the beginning and end of the sequences were closed-up, the ITS rDNA sequences (GenBank Accession No. MZ042367 and MZ042368) showed 99.66% - 99.92% identity to G. rostochiensis sequences available in GenBank (FJ212166.1, GQ294513, FJ212164.1 and KJ409617.1). Sequences from the 28S region (GenBank Accession No. MZ057597 and MZ057598) were 99.23% - 99.74% similar to those of G. rostochiensis isolate from Slovakia (KJ409625.1), Italy (KJ409631.1) and United Kingdom (KJ409633.1). We used species specific primers ITS5（5'-GGAAGTAAAAGTCGTAACAAGG-3'）and PITSr3 (5'AGCGCAGACATGCCGCAA-3') to amplify the product (Bulman & Marshall 1997; EPPO 2017). A single 434bp fragment was obtained from Hezhang and Weining populations. A host test for the Hezhang and Weining populations were performed by inoculating 1,000 eggs per plant of varieties Qingshu 9, Huize 2 and Hezuo 88 grown in the pots containing 800 cm3 of sterilized soil (soil: sand ratio was 3:1), and four replications were tested in greenhouse under 16 h light, 22°C in the day and 8 h dark in the night. At 90 days post inoculation, 32.6 ± 7, 31.2 ± 8, and 29.5 ± 8 females and cysts were extracted from the infected roots and soils of the varieties Qingshu 9, Huize 2 and Hezuo 88, respectively. No females and cysts were observed on the control plants. The trial indicated that potato cultivars Qingshu 9, Huize 2 and Hezuo 88 are hosts for the Hezhang and Weining populations of Globodera rostochiensis. To the best of our knowledge, this is the first detection of potato cyst nematode Globodera rostochiensis in Guizhou Province, China.

SRSF1 promotes ASMC proliferation in asthma by competitively binding CCND2 with miRNA-135a

BackgroundAsthma is an inflammatory syndrome characterized by airway hyperresponsiveness, bronchial inflammation, and airway remodeling. Abnormal proliferation of airway smooth muscle cells (ASMCs) is the main pathological feature of asthma. This study investigated the function and mechanism of serine arginine-rich splicing factor 1 (SRSF1) in ASMC proliferation in asthma. MethodsSRSF1 expressions in the bronchi of ovalbumin-induced asthmatic mice and IgE-treated mouse ASMCs (mASMCs) were evaluated using quantitative real-time PCR and Western blot. The localization and expression of SRSF1 in the bronchi of asthmatic mice were assessed by immunohistochemistry. Functionally, gain- and loss-of-function assays, flow cytometry, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays were conducted. Mechanistically, RNA degradation assay, RNA immunoprecipitation, RNA pull-down, and dual-luciferase reporter gene assays were carried out. ResultsSRSF1 was highly expressed in the bronchi of ovalbumin-induced asthma mice and IgE-treated mASMCs and was mainly located in the nucleus. Experiments on the function of SRSF1 showed that the silencing of SRSF1 induced the cell cycle of mASMC arrest and restrained mASMC proliferation. Investigations into the mechanism of SRSF1 revealed that SRSF1 and miR-135a are competitively bound to the 3′UTR region of Cyclin D2 (CCND2). SRSF1 overexpression repressed the degradation of CCND2 mRNA, and miR-135a negatively regulated CCND2 expression. Furthermore, SRSF1 knockdown inhibited ASMC proliferation in asthma mouse models by regulating the levels of miR-135a and CCND2. ConclusionSRSF1 knockdown repressed ASMC proliferation in asthma by regulating miR-135a/CCND2 levels.

The Effect of VWF Propeptide on VWF Mutant in D1 Domain

To investigate whether co-transfection of wild-type VWFpp with VWF mutant in D1 region is able to correct VWF defects in biosynthesis and secretion. Four VWF mutant plasmids were single transfected into HEK 293 cells, or co-transfected into HEK 293 cells with the wild type VWFpp plasmids. The VWF in supernatant and lysate of transfected cells were analyzed by ELISA, vertical VWF multimer electrophoresis. The retention of VWF in endoplasmic reticulum of transfected cells were detected by immunofluorescence confocal microscope. In the vertical VWF multimer analysis, with co-expressing VWF mutant and VWFpp, the VWF multimer bands disappeared, and the VWF antigen in both supernatant and lysate of cells decreased, compared with the single expression of VWF mutant. Although the intracellular levels of VWF antigens decreased after co-expression, the retention rate of VWF mutant decreased in endoplasmic reticulum. VWFpp can reduce the retention of VWF in endoplasmic reticulum, assists the transport of VWF between subcellular organelles. However, VWFpp inhibits the biosynthesis and secretion of VWF about the mutant in D1 domain.

Common variant c.-22 + 155C > T of BDNF as a genetic risk factor of opium addiction

Opioid use disorder is largely genetic in nature. The common genetic variants present in population might be modulating the risk by affecting expression level of genes in the brain. Here, we study common variants in promoter region of brain-derived neurotrophic factor (BDNF) and Dopamine receptor D2 (DRD2) in a highly prevalent opium addiction geographical region. We identify highly expressed isoforms using CAGE datasets and its associated promoter. Common variants were extracted from promoters and genotyped in addicts compared to controls. The associated variants obtained through hitchhiking events were removed and eQTL analysis was performed. We identified two mutations (rs7944119:G > T and rs13306221:C > T) in the promoter of BDNF to be significantly associated with the addiction. In the dominant inheritance model, both rs7944119 and rs13306221 increases the risk of addiction. Of these two, rs7944119 was in linkage disequilibrium with rs13306221 and showed association due to hitchhiking. The rs13306221-T was associated with a lower expression level of the short BDNF isoform in the Cerebellar cortex. This finding suggests that rs13306221 (c.-22 + 155C > T) could increase risk of addiction by decreasing the expression level of the short isoform of BDNF, therefore, changes in the expression of the BDNF might not be the effect, but rather a cause of opium addiction disorder. Or, subjects with less expression of BDNF are more prone to addiction and addiction further decreases expression of BDNF short isoform in the brain. Although, it should be explored further in more detail.

Toxares koreanus sp. nov. – a new Toxares species from South Korea (Hymenoptera, Braconidae, Aphidiinae)

The genusToxaresHaliday, 1840 is a small taxon of Aphidiinae, consisting four valid species in the world. OneToxaresspecies is recorded as new to science from South Korea, in this study. Descriptions and illustrations of the new species,T. koreanussp. nov., are provided, together with their mitochondrialcytochrome c oxidase subunit I(COI) and D2 region of the nuclear gene for28S rRNA(28S) sequences. The phylogenetic tree reconstructed using a combination ofCOIand28Srevealed the phylogenetic position of the genusToxareswithin Aphidiinae.