A non-neutralizing antibody broadly protects against influenza virus infection by engaging effector cells.

Hemagglutinin (HA) is the immunodominant protein of the influenza virus. We previously showed that mice injected with a monoglycosylated influenza A HA (HAmg) produced cross-strain-reactive antibodies and were better protected than mice injected with a fully glycosylated HA (HAfg) during lethal dose challenge. We employed a single B-cell screening platform to isolate the cross-protective monoclonal antibody (mAb) 651 from mice immunized with the HAmg of A/Brisbane/59/2007 (H1N1) influenza virus (Bris/07). The mAb 651 recognized the head domain of a broad spectrum of HAs from groups 1 and 2 influenza A viruses and offered prophylactic and therapeutic efficacy against A/California/07/2009 (H1N1) (Cal/09) and Bris/07 infections in mice. The antibody did not possess neutralizing activity; however, antibody-dependent cellular cytotoxicity and antibody-dependent cellular phagocytosis mediated by natural killer cells and alveolar macrophages were important in the protective efficacy of mAb 651. Together, this study highlighted the significance of effector functions for non-neutralizing antibodies to exhibit protection against influenza virus infection.

Long-Read Genome Sequencing of Abscondita cerata (Coleoptera: Lampyridae), the Endemic Firefly of Taiwan.

Abscondita cerata is the most abundant and widely distributed endemic firefly species in Taiwan and is considered a key environmental and ecological indicator organism. In this study, we report the first long-read genome sequencing of Abs. cerata sequenced by Nanopore technology. The draft genome size, 967 Mb, was measured through a hybrid approach that consisted of assembling using 11.25-Gb Nanopore long reads and polishing using 9.47-Gb BGI PE100 short reads. The drafted genome was assembled into 4,855 contigs, with the N50 reaching 325.269 kb length. The assembled genome was predicted to possess 55,206 protein-coding genes, of which 20,862 (37.78%) were functionally annotated with public databases. 47.11% of the genome sequences consisted of repeat elements; among them DNA transposons accounted for the largest proportion (26.79%). A BUSCO (Benchmarking Universal Single Copy Orthologs) evaluation demonstrated that the genome and gene completeness were 84.8% and 79%, respectively. The phylogeny constructed using 1,792 single copy genes was consistent with previous studies. The comparative transcriptome between adult male head and lantern tissues revealed (1) the vision of Abs. cerata is primarily UV-sensitive to environmental twilight, which determines when it begins its nocturnal activity, (2) the major expressed OR56d receptor may be correlated to suitable humidity sensing, and (3) Luc1-type luciferase is responsible for Abs. cerata's luminescent spectrum.

Species-Specific Flash Patterns Track the Nocturnal Behavior of Sympatric Taiwanese Fireflies.

It is highly challenging to evaluate the species' content and behavior changes in wild fireflies, especially for a sympatric population. Here, the flash interval (FI) and flash duration (FD) of flying males from three sympatric species (Abscondita cerata, Luciola kagiana, and Luciola curtithorax) were investigated for their potentials in assessing species composition and nocturnal behaviors during the A. cerata mating season. Both FI and FD were quantified from the continuous flashes of adult fireflies (lasting 5-30 s) via spatiotemporal analyses of video recorded along the Genliao hiking trail in Taipei, Taiwan. Compared to FD patterns and flash colors, FI patterns exhibited the highest species specificity, making them a suitable reference for differentiating firefly species. Through the case study of a massive occurrence of A. cerata (21 April 2018), the species contents (~85% of the flying population) and active periods of a sympatric population comprising A. cerata and L. kagiana were successfully evaluated by FI pattern matching, as well as field specimen collections. Our study suggests that FI patterns may be a reliable species-specific luminous marker for monitoring the behavioral changes in a sympatric firefly population in the field, and has implication values for firefly conservation.

Luminescent characteristics and mitochondrial COI barcodes of nine cohabitated Taiwanese fireflies.

Background: Over 50 Taiwanese firefly species have been discovered, but scientists lack information regarding most of their genetics, bioluminescent features, and cohabitating phenomena. In this study, we focus on morphological species identification and phylogeny reconstructed by COI barcoding, as well as luminescent characteristics of cohabited Taiwanese firefly species to determine the key factors that influenced how distinct bioluminescent species evolved to coexist and proliferate within the same habitat. Methods: In this study, 366 specimens from nine species were collected in northern Taiwan from April to August, 2016-2019. First, the species and sex of the specimens were morphologically and genetically identified. Then, their luminescent spectra and intensities were recorded using a spectrometer and a power meter, respectively. The habitat temperature, relative humidity, and environmental light intensity were also measured. The cytochrome oxidase I (COI) gene sequence was used as a DNA barcode to reveal the phylogenetic relationships of cohabitated species. Results: Nine species-eight adult species (Abscondita chinensis, Abscondita cerata, Aquatica ficta, Luciola curtithorax, Luciola kagiana, Luciola filiformis, Curtos sauteri, and Curtos costipennis) and one larval Pyrocoelia praetexta-were morphologically identified. The nine species could be found in April-August. Six of the eight adult species shared an overlap occurrence period in May. Luminescent spectra analysis revealed that the λ max of studied species ranged from 552-572 nm (yellow-green to orange-yellow). The average luminescent intensity range of these species was about 1.2-14 lux (182.1-2,048 nW/cm2) for males and 0.8-5.8 lux (122.8-850 nW/cm2) for females, and the maximum luminescent intensity of males was 1.01-7.26-fold higher than that of females. Compared with previous studies, this study demonstrates that different λ max, species-specific flash patterns, microhabitat choices, nocturnal activity time, and/or an isolated mating season are key factors that may lead to the species-specific courtship of cohabitated fireflies. Moreover, we estimated that the fireflies start flashing or flying when the environmental light intensity decreased to 6.49-28.1 lux. Thus, based on a rough theoretical calculation, the sensing distance between male and female fireflies might be 1.8-2.7 m apart in the dark. In addition, the mitochondrial COI barcode identified species with high resolution and suggested that most of the studied species have been placed correctly with congeners in previous phylogenies. Several cryptic species were revealed by the COI barcode with 3.27%-12.3% variation. This study renews the idea that fireflies' luminescence color originated from the green color of a Lampyridae ancestor, then red-shifted to yellow-green in Luciolinae, and further changed to orange-yellow color in some derived species.

Structures of two elapid snake venom metalloproteases with distinct activities highlight the disulfide patterns in the D domain of ADAMalysin family proteins.

The structures of snake venom metalloproteases (SVMPs) are proposed to be useful models to understand the structural and functional relationship of ADAM (a disintegrin and metalloprotease) which are membrane-anchored proteins involved in multiple human diseases. We have purified, sequenced and determined the structures of two new P-III SVMPs - atragin and kaouthiagin-like (K-like) from Naja atra. Atragin exhibits a known C-shaped topology, whereas K-like adopts an I-shaped conformation because of the distinct disulfide pattern in the disintegrin-like (D) domain. K-like exhibits an enzymatic specificity toward pro-TNFalpha with less inhibition of cell migration, but atragin shows the opposite effect. The specificity of the enzymatic activity is indicated to be dominated mainly by the local structures of SVMP in the metalloprotease (M) domain, whereas the hyper-variable region (HVR) in the cysteine-rich (C) domain is involved in a cell-migration activity. We demonstrate also a pH-dependent enzymatic activity of atragin that we correlate with the structural dynamics of a Zn(2+)-binding motif and the Met-turn based on the structures determined with a pH-jump method. The structural variations between the C- and I-shapes highlight the disulfide bond patterns in the D domain of the ADAM/adamalysin/reprolysins family proteins.

Lymphatic vessel remodeling and invasion in pancreatic cancer progression.

BACKGROUND: The lymphatic system is involved in metastasis in pancreatic cancer progression. In cancer staging, lymphatic spread has been used to assess the invasiveness of tumor cells. However, from the endothelium's perspective, the analysis downplays the peri-lesional activities of lymphatic vessels. This unintended bias is largely due to the lack of 3-dimensional (3-D) tissue information to depict the lesion microstructure and vasculature in a global and integrated fashion. METHODS: We targeted the pancreas as the model organ to investigate lymphatic vessel remodeling in cancer lesion progression. Transparent pancreases were prepared by tissue clearing to facilitate deep-tissue, tile-scanning microscopy for 3-D lymphatic network imaging. FINDINGS: In human pancreatic ductal adenocarcinoma, we identify the close association between the pancreatic intraepithelial neoplasia (PanIN) lesions and the lymphatic network. In mouse models of PanIN (elastase-CreER;LSL-KrasG12D and elastase-CreER;LSL-KrasG12D;p53+/-), the 3-D image data reveal the peri-lesional lymphangiogenesis, endothelial invagination, formation of the bridge/valve-like luminal tubules, vasodilation, and luminal invasion. In the orthotopic mouse model of pancreatic cancer, we identify the localized, graft-induced lymphangiogenesis and the peri- and intra-tumoral lymphatic vessel invasion. INTERPRETATION: The integrated view of duct lesions and vascular remodeling suggests an active role, rather than a passive target, of lymphatic vessels in the metastasis of pancreatic cancer. Our 3-D image data provide insights into the pancreatic cancer microenvironment and establish the technical and morphological foundation for systematic detection and 3-D analysis of lymphatic vessel invasion. FUND: Taiwan Academia Sinica (AS-107-TP-L15 and AS-105-TP-B15), Ministry of Science and Technology (MOST 106-2321-B-001-048, 106-0210-01-15-02, 106-2321-B-002-034, and 106-2314-B-007-004-MY2), and Taiwan National Health Research Institutes (NHRI EX107-10524EI).

Biochemical Mechanisms and Catabolic Enzymes Involved in Bacterial Estrogen Degradation Pathways.

Estrogens have been classified as group 1 carcinogens by the World Health Organization and represent a significant concern given that they are found in surface waters worldwide, and long-term exposure to estrogen-contaminated water can disrupt sexual development in animals. To date, the estrogen catabolic enzymes and genes remain unknown. Using a tiered functional genomics approach, we identified three estrogen catabolic gene clusters in Sphingomonas sp. strain KC8. We identified several estrone-derived compounds, including 4-hydroxyestrone, a meta-cleavage product, and pyridinestrone acid. The yeast-based estrogen assay suggested that pyridinestrone acid exhibits negligible estrogenic activity. We characterized 17ß-estradiol dehydrogenase and 4-hydroxyestrone 4,5-dioxygenase, responsible for the 17-dehydrogenation and meta-cleavage of the estrogen A ring, respectively. The characteristic pyridinestrone acid was detected in estrone-spiked samples collected from two wastewater treatment plants and two suburban rivers in Taiwan. The results significantly expand our understanding of microbial degradation of aromatic steroids at molecular level.

Predominant structural configuration of natural antibody repertoires enables potent antibody responses against protein antigens.

Humoral immunity against diverse pathogens is rapidly elicited from natural antibody repertoires of limited complexity. But the organizing principles underlying the antibody repertoires that facilitate this immunity are not well-understood. We used HER2 as a model immunogen and reverse-engineered murine antibody response through constructing an artificial antibody library encoded with rudimentary sequence and structural characteristics learned from high throughput sequencing of antibody variable domains. Antibodies selected in vitro from the phage-displayed synthetic antibody library bound to the model immunogen with high affinity and specificities, which reproduced the specificities of natural antibody responses. We conclude that natural antibody structural repertoires are shaped to allow functional antibodies to be encoded efficiently, within the complexity limit of an individual antibody repertoire, to bind to diverse protein antigens with high specificity and affinity. Phage-displayed synthetic antibody libraries, in conjunction with high-throughput sequencing, can thus be designed to replicate natural antibody responses and to generate novel antibodies against diverse antigens.

Uric acid spherulites in the reflector layer of firefly light organ.

BACKGROUND: In firefly light organs, reflector layer is a specialized tissue which is believed to play a key role for increasing the bioluminescence intensity through reflection. However, the nature of this unique tissue remains elusive. In this report, we investigated the role, fine structure and nature of the reflector layer in the light organ of adult Luciola cerata. PRINCIPAL FINDINGS: Our results indicated that the reflector layer is capable of reflecting bioluminescence, and contains abundant uric acid. Electron microscopy (EM) demonstrated that the cytosol of the reflector layer's cells is filled with densely packed spherical granules, which should be the uric acid granules. These granules are highly regular in size (∼700 nm in diameter), and exhibit a radial internal structure. X-ray diffraction (XRD) analyses revealed that an intense single peak pattern with a d-spacing value of 0.320 nm is specifically detected in the light organ, and is highly similar to the diffraction peak pattern and d-spacing value of needle-formed crystals of monosodium urate monohydrate. However, the molar ratio evaluation of uric acid to various cations (K(+), Na(+), Ca(2+) and Mg(2+)) in the light organ deduced that only a few uric acid molecules were in the form of urate salts. Thus, non-salt uric acid should be the source of the diffraction signal detected in the light organ. CONCLUSIONS: In the light organ, the intense single peak diffraction signal might come from a unique needle-like uric acid form, which is different from other known structures of non-salt uric acid form. The finding of a radial structure in the granules of reflector layer implies that the spherical uric acid granules might be formed by the radial arrangement of needle-formed packing matter.

A photocytes-associated fatty acid-binding protein from the light organ of adult Taiwanese firefly, Luciola cerata.

BACKGROUND: Intracellular fatty acid-binding proteins (FABPs) are considered to be an important energy source supplier in lipid metabolism; however, they have never been reported in any bioluminescent tissue before. In this study, we determined the structural and functional characteristics of a novel FABP (lcFABP) from the light organ of adult Taiwanese firefly, Luciola cerata, and showed anatomical association of lcFABP with photocytes. PRINCIPAL FINDINGS: Our results demonstrated the primary structure of lcFABP deduced from the cDNA clone of light organ shares structural homologies with other insect and human FABPs. In vitro binding assay indicated the recombinant lcFABP binds saturated long chain fatty acids (C14-C18) more strongly than other fatty acids and firefly luciferin. In addition, tissue distribution screening assay using a rabbit antiserum specifically against the N-terminal sequence of lcFABP confirmed the light organ-specific expression of lcFABP. In the light organ, the lcFABP constituted about 15% of total soluble proteins, and was detected in both cytosol and nucleus of photocytes. CONCLUSIONS: The specific localization of abundant lcFABP in the light organ suggests that sustained bioluminescent flashes in the light organ might be a high energy demanding process. In photocytes, lcFABP might play a key role in providing long chain fatty acids to peroxisomes for the luciferase-catalyzed long chain acyl-CoA synthetic reaction.

Structural difference between group I and group II cobra cardiotoxins: X-ray, NMR, and CD analysis of the effect of cis-proline conformation on three-fingered toxins.

Natural homologues of cobra cardiotoxins (CTXs) were classified into two structural subclasses of group I and II based on the amino acid sequence and circular dichroism analysis, but the exact differences in their three-dimensional structures and biological significance remain elusive. We show by circular dichroism, NMR spectroscopic, and X-ray crystallographic analyses of a newly purified group I CTX A6 from eastern Taiwan cobra (Naja atra) venoms that its loop I conformation adopts a type VIa turn with a cis peptide bond located between two proline residues of PPxY. A similar "banana-twisted" conformation can be observed in other group I CTXs and also in cyclolinopeptide A and its analogues. By binding to the membrane environment, group I CTX undergoes a conformational change to adopt a more extended hydrophobic domain with beta-sheet twisting closer to the one adopted by group II CTX. This result resolves a discrepancy in the CTX structural difference reported previously between solution as well as crystal state and shows that, in addition to the hydrophobicity, the exact loop I conformation also plays an important role in CTX-membrane interaction. Potential protein targets of group I CTXs after cell internalization are also discussed on the basis of the determined loop I conformation.