A lineage-resolved cartography of microRNA promoter activity in C. elegans empowers multidimensional developmental analysis.

Elucidating the expression of microRNAs in developing single cells is critical for functional discovery. Here, we construct scCAMERA (single-cell cartography of microRNA expression based on reporter assay), utilizing promoter-driven fluorescent reporters in conjunction with imaging and lineage tracing. The cartography delineates the transcriptional activity of 54 conserved microRNAs in lineage-resolved single cells throughout C. elegans embryogenesis. The combinatorial expression of microRNAs partitions cells into fine clusters reflecting their function and anatomy. Notably, the expression of individual microRNAs exhibits high cell specificity and divergence among family members. Guided by cellular expression patterns, we identify developmental functions of specific microRNAs, including miR-1 in pharynx development and physiology, miR-232 in excretory canal morphogenesis by repressing NHR-25/NR5A, and a functional synergy between miR-232 and miR-234 in canal development, demonstrating the broad utility of scCAMERA. Furthermore, integrative analysis reveals that tissue-specific fate determinants activate microRNAs to repress protein production from leaky transcripts associated with alternative, especially neuronal, fates, thereby enhancing the fidelity of developmental fate differentiation. Collectively, our study offers rich opportunities for multidimensional expression-informed analysis of microRNA biology in metazoans.

Spatiotemporal analysis of mRNA-protein relationships enhances transcriptome-based developmental inference.

Elucidating the complex relationships between mRNA and protein expression at high spatiotemporal resolution is critical for unraveling multilevel gene regulation and enhancing mRNA-based developmental analyses. In this study, we conduct a single-cell analysis of mRNA and protein expression of transcription factors throughout C. elegans embryogenesis. Initially, cellular co-presence of mRNA and protein is low, increasing to a medium-high level (73%) upon factoring in delayed protein synthesis and long-term protein persistence. These factors substantially affect mRNA-protein concordance, leading to potential inaccuracies in mRNA-reliant gene detection and specificity characterization. Building on the learned relationship, we infer protein presence from mRNA expression and demonstrate its utility in identifying tissue-specific genes and elucidating relationships between genes and cells. This approach facilitates identifying the role of sptf-1/SP7 in neuronal lineage development. Collectively, this study provides insights into gene expression dynamics during rapid embryogenesis and approaches for improving the efficacy of transcriptome-based developmental analyses.

Metabolic plasticity sustains the robustness of Caenorhabditis elegans embryogenesis.

Embryogenesis is highly dependent on maternally loaded materials, particularly those used for energy production. Different environmental conditions and genetic backgrounds shape embryogenesis. The robustness of embryogenesis in response to extrinsic and intrinsic changes remains incompletely understood. By analyzing the levels of two major nutrients, glycogen and neutral lipids, we discovered stage-dependent usage of these two nutrients along with mitochondrial morphology changes during Caenorhabditis elegans embryogenesis. ATGL, the rate-limiting lipase in cellular lipolysis, is expressed and required in the hypodermis to regulate mitochondrial function and support embryogenesis. The embryonic lethality of atgl-1 mutants can be suppressed by reducing sinh-1/age-1-akt signaling, likely through modulating glucose metabolism to maintain sustainable glucose consumption. The embryonic lethality of atgl-1(xd314) is also affected by parental nutrition. Parental glucose and oleic acid supplements promote glycogen storage in atgl-1(xd314) embryos to compensate for the impaired lipolysis. The rescue by parental vitamin B12 supplement is likely through enhancing mitochondrial function in atgl-1 mutants. These findings reveal that metabolic plasticity contributes to the robustness of C. elegans embryogenesis.

Isolation and Identification of Novel Highly Pathogenic Avian Influenza Virus (H5N8) Subclade 2.3.4.4b from Geese in Northeastern China.

H5N8, a highly pathogenic avian influenza, has become a new zoonotic threat in recent years. As of December 28, 2021, at least 3,206 H5N8 cases had been reported in wild birds and poultry worldwide. In January 2021, a novel virus strain named A/goose/China/1/2021 was isolated during an H5N8 goose influenza outbreak in northeastern China. The PB2, PB1, HA, and M genes of A/goose/China/1/2021 were highly identical to those of H5N8 strains emerging in Kazakhstan and Russia in Central Asia from August to September 2020, while the remaining four genes had the closest homology to those of H5N8 viruses isolated in South Korea in East Asia from November to December 2020. We thus speculate that A/goose/China/1/2021 is likely a reassortant virus that formed in the 2020 to 2021 influenza season and that the migratory birds via the two migration routes of Central Asia and East Asia-Australia may have played an essential role in the genetic reassortment of this virus. The phylogenetic analysis indicated that the HA genes of H5N8 viruses belonging to group II of subclade 2.3.4.4b, including A/goose/China/1/2021, may be derived from strains in Central Asia. Given the complex global spread of H5N8 virus, our study highlights the necessity to strengthen the function of the global surveillance network for H5N8 virus and to accelerate the pace of vaccine development to confront the current challenges posed by H5N8 virus of subclade 2.3.4.4. IMPORTANCE H5N8, a highly pathogenic avian influenza, not only has an impact on public health, but also has a huge negative impact on animal health, food safety, safety, and even on the local and international economy. The migratory wild birds play a vital role in the intercontinental transmission of H5N8 virus. It is urgent that we should strengthen the function of the global surveillance network for H5N8 virus and accelerate the pace of vaccine development to confront the current challenges posed by H5N8 virus of subclade 2.3.4.4.

Defect-buffering cellular plasticity increases robustness of metazoan embryogenesis.

Developmental processes are intrinsically robust so as to preserve a normal-like state in response to genetic and environmental fluctuations. However, the robustness and potential phenotypic plasticity of individual developing cells under genetic perturbations remain to be systematically evaluated. Using large-scale gene perturbation, live imaging, lineage tracing, and single-cell phenomics, we quantified the phenotypic landscape of C. elegans embryogenesis in >2,000 embryos following individual knockdown of over 750 conserved genes. We observed that cellular genetic systems are not sufficiently robust to single-gene perturbations across all cells; rather, gene knockdowns frequently induced cellular defects. Dynamic phenotypic analyses revealed many cellular defects to be transient, with cells exhibiting phenotypic plasticity that serves to alleviate, correct, and accommodate the defects. Moreover, potential developmentally related cell modules may buffer the phenotypic effects of individual cell position changes. Our findings reveal non-negligible contributions of cellular plasticity and multicellularity as compensatory strategies to increase developmental robustness.

A SYBR Green-based real-time RT-PCR assay to differentiate the H1N1 influenza virus lineages.

The H1N1 subtype influenza viruses (H1N1) have been causing persistent epidemics in human, swine and poultry populations since 1918. This subtype has evolved into four relatively stable genetic lineages, including classical swine influenza virus lineage, seasonal human influenza virus lineage, avian influenza virus lineage and Eurasian avian-like swine influenza virus lineage. In this study, four pairs of primers, based on the relatively conserved HA nucleotide regions of each H1N1 genetic lineage, were designed to establish a SYBR Green-based real-time quantitative RT-PCR (qPCR) assay to differentiate between the H1N1 genetic lineages. The results of qPCR assay showed that the lineage-specific primers designed for each H1N1 lineage were intra-lineage-specific, without mismatch of inter-lineage or inter-subtype and there appeared specific amplification curves when the concentrations of H1N1 plasmids were greater than or equal to 1.0 × 101 copies/reaction. Thus, this qPCR assay can specifically differentiate between the four lineages of H1N1 with a good specificity and sensitivity, which would assist in recognizing the infection and epidemic status of different H1N1 genetic lineages.

Assessing the effects of a two-amino acid flexibility in the Hemagglutinin 220-loop receptor-binding domain on the fitness of Influenza A(H9N2) viruses.

The enzootic and zoonotic nature of H9N2 avian influenza viruses poses a persistent threat to the global poultry industry and public health. In particular, the emerging sublineage h9.4.2.5 of H9N2 viruses has drawn great attention. In this study, we determined the effects of the flexibility at residues 226 and 227 in the hemagglutinin on the receptor avidity and immune evasion of H9N2 viruses. The solid-phase direct binding assay showed that residue 226 plays a core role in the receptor preference of H9N2 viruses, while residue 227 affects the preference of the virus for a receptor. Consequently, each of these two successive residues can modulate the receptor avidity of H9N2 viruses and influence their potential of zoonotic infection. The antigenic map based on the cross-hemagglutination inhibition (HI) titers revealed that amino acid substitutions at positions 226 or 227 appear to be involved in antigenic drift, potentially resulting in the emergence of H9N2 immune evasion mutants. Further analysis suggested that increased receptor avidity facilitated by residue 226Q or 227M resulted in a reduction in the HI titer. Among the four naturally-occurring amino acid combinations comprising QQ, MQ, LQ, and LM, the number of viruses with LM accounted for 79.64% of the sublineage h9.4.2.5 and the rescued virus with LM exhibited absolute advantages of in vitro and in vivo replication and transmission. Collectively, these data demonstrate that residues 226 and 227 are under selective pressure and their synergistic regulation of receptor avidity and antigenicity is related to the evolution of circulating H9N2 viruses.

Characterization of Newcastle disease virus obtained from toco toucan.

Given that the current Newcastle disease virus (NDV) infection in wild birds poses the threat to poultry, surveillance of Newcastle disease in captive wild birds was carried out in Jilin, China in 2018. Here, an NDV strain obtained from toco toucan was firstly characterized. The results showed that the F gene of the NDV isolate Toucan/China/3/2018 is classified as genotype II in class II. Sequence analysis of the F0 cleavage site was 113RQGR/L117, which supports the result of the intracerebral pathogenicity index assay indicating classification of the isolate as low-pathogenicity. Experimental infection demonstrated that Toucan/China/3/2018 can effectively replicate and transmit among chickens. To our knowledge, this is the first report on genetically and pathogenically characterizing NDV strain isolated from toucan, which enriches the epidemiological information of NDV in wild birds.

Nanomaterials-based Electrochemical Immunosensors.

With the development of nanomaterials and sensor technology, nanomaterials-based electrochemical immunosensors have been widely employed in various fields. Nanomaterials for electrode modification are emerging one after another in order to improve the performance of electrochemical immunosensors. When compared with traditional detection methods, electrochemical immunosensors have the advantages of simplicity, real-time analysis, high sensitivity, miniaturization, rapid detection time, and low cost. Here, we summarize recent developments in electrochemical immunosensors based on nanomaterials, including carbon nanomaterials, metal nanomaterials, and quantum dots. Additionally, we discuss research challenges and future prospects for this field of study.