Salmonella enterica serovar Kentucky sequence type 198 (ST198) isolates from chicken meat products. Ten S. Kentucky strains obtained from chicken meat products in Xuancheng, China, carried 12 to 17 resistance genes, such as blaCTX-M-55, rmtB, tet(A), floR, and fosA3, combined with mutations within gyrA (S83F and D87N) and parC (S80I), resulting in resistance to numerous antimicrobial agents, including the clinically important antibiotics cephalosporin, ciprofloxacin, tigecycline, and fosfomycin. These S. Kentucky isolates shared a close phylogenetic relationship (21 to 36 single-nucleotide polymorphisms [SNPs]) and showed close genetic relatedness to two human clinical isolates from China. Three S. Kentucky strains were subjected to whole-genome sequencing using Pacific Biosciences (PacBio) single-molecule real-time (SMRT) technology. All antimicrobial resistance genes were located on their chromosomes and clustered in one multiresistance region (MRR) and Salmonella genomic island (SGI) SGI1-K. The MRRs in three S. Kentucky strains were bounded by IS26 at both ends and were inserted downstream of the bcfABCDEFG cluster with 8-bp direct repeats. The MRRs were related to those of IncHI2 plasmids but differed by insertions, deletions, and rearrangements of multiple segments involving resistance genes and plasmid backbones. This finding suggests that the MRR fragment possibly originates from IncHI2 plasmids. Four SGI1-K variants with slight differences were identified in 10 S. Kentucky strains. Mobile elements, particularly IS26, play an essential role in forming distinct MRRs and SGI1-K structures. In conclusion, the emergence of extensively drug-resistant S. Kentucky ST198 strains containing numerous chromosomally located resistance genes is alarming and needs continued surveillance.The purpose of this study was to characterize extensively drug-resistant IMPORTANCESalmonella spp. are important foodborne pathogens, and multidrug-resistant (MDR) Salmonella strains have become a serious threat to clinical therapy. MDR S. Kentucky ST198 strains have been increasingly reported from various sources and have become a global risk. In this study, we described extensively drug-resistant S. Kentucky ST198 strains from chicken meat products from a city in China. Numerous resistance genes are clustered in the chromosomes of S. Kentucky ST198 strains, possibly acquired with the help of mobile elements. This would facilitate the spread of numerous resistance genes as intrinsic chromosomal genes within this global epidemic clone, with the potential to capture more resistance genes. The emergence and dissemination of extensively drug-resistant S. Kentucky ST198 pose a severe clinical and public health threat; therefore, continuous surveillance is warranted. Salmonella is one of the most common foodborne pathogens causing human gastroenteritis and is often associated with the contamination of poultry products  emerged due to the chromosomal integration of Salmonella genomic island (SGI) SGI1-K, probably in the mid-1990s , cephalosporinase (CMY), or carbapenemase  genes  of S. Kentucky ST198 was identified among S. enterica strains from patients, poultry, and meat products in China in 2013 to 2017  genes , 8, 9. Ml spread , 9. Howe to 2016 . MDR S. in China –15. In tSalmonella strains (17.3%) were isolated from 75 chicken meat and internal organ samples. Three serotypes were identified among 13 Salmonella strains. S. Kentucky (n = 10) was the most prevalent serotype, and all of the S. Kentucky strains belonged to ST198. Salmonella enterica serovar Agona ST13 (n = 2) and Salmonella enterica serovar Infantis ST32 (n = 1) were also detected. The high prevalence of S. Kentucky ST198 (13.33% [10/75 strains]) in this study is noteworthy; it was much higher than those in the chicken supply chain  and among patients  in China between 2010 and 2016  in the production chain of broiler chickens in Sichuan Province, China  China 1619.Salmonella isolates were tested for the MICs of 15 antimicrobial agents. They were all susceptible to meropenem and colistin. Additionally, 10 S. Kentucky strains were resistant to ampicillin, cefazolin, cefotaxime, gentamicin, streptomycin, amikacin, tetracycline, tigecycline, chloramphenicol, florfenicol, nalidixic acid, ciprofloxacin, and sulfamethoxazole-trimethoprim, and 8 of them also exhibited resistance to fosfomycin -Ia, aac(3)-IId, aadA17, and rmtB, tetracycline resistance gene tet(A), florfenicol exporter floR, lincomycin resistance gene lnu(F), and rifampin resistance gene arr-2 were identified in all 10 S. Kentucky isolates, whereas the resistance genes aadA7, aac(3)-Iv, qnrS1, sul1, mph(A), and fosA3 were detected in 1 to 9 S. Kentucky strains  genes (S. Kentucky strains in this study had mutations within gyrA (S83F and D87N) and parC (S80I) (qnrS1 or gyrA mutation (S83Y) resulted in nalidixic acid resistance only in S. Infantis and S. Agona strains  , account strains .S. Kentucky and S. Agona strains exhibited resistance to tigecycline (MICs of 2 to 8 mg/L), a last-resort antibiotic to treat serious infections caused by MDR bacteria. Mobile tigecycline resistance determinants tet(X) and tmexCD1-toprJ1 or amino acid changes within AcrAB-TolC, RamA, RamR, MarA, and MarR were not identified in 12 tigecycline-resistant strains in this study. We previously confirmed the association of a tetracycline resistance gene tet(A) variant with tigecycline resistance in S. Kentucky (tet(A) variant in all S. Kentucky and S. Agona isolates in this study may account for their tigecycline resistance. It further highlights the important role of the tet(A) variant in the wide dissemination of tigecycline resistance.Of concern, all Kentucky . The preS. Kentucky ST198 strains in this study, we performed a phylogenetic analysis based on core genome single-nucleotide polymorphisms (cgSNPs). They showed a close genetic relationship (21 to 36 single-nucleotide polymorphisms [SNPs]), differing from two clinical S. Kentucky ST198 strains (GenBank accession no. PRJNA820366) recovered in Nantong, Jiangsu Province, China, in 2021 by only 16 or 19 SNPs , AH19MCS8 (slaughterhouse), and AH19MCS11 (slaughterhouse), were selected based on their sources and susceptible phenotypes and were further sequenced using Pacific Biosciences (PacBio) single-molecule real-time (SMRT) sequencing. The complete genome sequences of three S. Kentucky isolates were obtained . In these S. Kentucky isolates, all antimicrobial resistance genes were located on their chromosomes and were clustered in one mosaic multiresistance region (MRR) and SGI1-K. No resistance genes were identified on five plasmids in AH19MCS8 and AH19MCS11 (see Table S1).In order to clarify the genetic structures of numerous resistance genes in S. Kentucky strains shared similar MRRs. The MRRs were bounded at both ends by IS26 and inserted into the chromosome with an identical location downstream of the bcfABCDEFG cluster encoding fimbrial proteins. The insertion of MRR interrupted a putative DsbA family protein and generated 8-bp direct repeats (DRs) (5′-GTGGTGGC-3′). We further compared the contigs in other S. Kentucky strains and found that fragments of IS26 with identical 8-bp DRs were inserted downstream of the bcfABCDEFG cluster in the remaining seven strains, suggesting that similar insertion of MRRs possibly occurred in all S. Kentucky strains in this study.As shown in 26 (26-aph(3′)-Ia-IS26] and a variety of open reading frames (ORFs) such as the transcriptional regulators hipA/hipB and the conjugal transfer gene traJ, which was highly (>99.9%) similar to two separate segments corresponding to IncHI2 plasmid backbones, such as pSG17-135-HI2 (S. Agona [GenBank accession no. CP048776]) and pMTY18780-1_IncHI2 (Escherichia coli [GenBank accession no. AP023198]), except that one copy of ISEc78 was inserted into a putative hypothetical protein with 8-bp DRs (5′-GAATTTTT-3′) in AH19MCS1. The second part (~69.0 kb) contained numerous resistance genes, such as mph(A), aadA17, lnu(F), aac(3)-IId, blaTEM-1b, rmtB, fosA3, blaCTX-M-55, qnrS1, and floR, associated with insertion sequences (ISs), transposons, and integron .The MRR of AH19MCS1 was the largest (~92.9 kb) and consisted of two parts bounded by IS26 . The firdfrA14/arr-2/ΔcmlA5 cassette array associated with an incomplete class 1 integrase IntI1 truncated by IS26 and the absence of an ~28.4-kb segment involving the ΔtnpA-qnrS1-ISKpn19-ΔIS26-fosA3-IS26-Δaph(4)-Ia-aac(3)-Ia-ΔIS26-ΔISKpn19-qnrS1-ΔISEcp1-blaCTX-M-55-ΔblaTEM-1b-IS26-IS26 structure (fosA3 resistance module (fosA3-orf1-orf2-Δorf3-IS26) or a 4,373-bp macrolide phosphotransferase region [IS26-mph(A)-mrx-mphR(A)-ΔIS6100] was observed in AH19MCS11 or AH19MCS8, respectively, and an approximately 29.5-kb segment (IS26-ΔTn1721-gshB-ΔIS4321-IS26-ISCR2-floR-ΔtetA-IS26-ISEcp1-blaCTX-M-55-ΔblaTEM-1b-IS26-fosA3-IS26-rmtB-blaTEM-1b-IS26) was in the opposite location in the latter one , in S. Kentucky ST198 were likely carried by plasmids except for blaCTX-M-14b  being the most common type. As previously described for numerous S. Kentucky ST198 strains (trmE gene in our S. Kentucky strains (AY463797), one copy of ISEc78 was inserted into backbone gene traG and generated 8-bp DRs in four SGI1-K variants, and an additional insertion of IS640 into S005 flanked by 4-bp DRs was observed in AH19MCS9 and AH19MCS11 (Four SGI1-K variants (types I to IV) with slight differences were identified in 10  strains , 15, SGI strains . CompareH19MCS11 .26 with opposite orientations. The insertion of MRR resulted in the absence of large backbone segments, including S013 (13 bp), S014 to S026, and resG, as well as ΔS044, the downstream 18-bp recombination site duplication, and 524 bp of the 5′ end of yidY, compared with SGI1-K (intI1-ΔaacCA5), 417-bp transposon Tn1721, ΔfipA, core SGI1-K resistance genes strA and strB within Tn5393, and blaTEM-1b within Tn2, and IS26, compared with SGI1-K. An additional deletion of a 2,065-bp segment (ΔaacCA5-aadA7-qacEΔ1-Δsul1) was observed in AH19MCS9 and AH19MCS11.The MRRs of four SGI1-K variants were bounded by two copies of ISh SGI1-K . The MRR26 was inserted into one hypothetical protein downstream of the yidZ gene in AH19MCS8 and AH19MCS11, with 8-bp DRs. Homologous recombination between this IS26 element and the upstream IS26 with opposite orientation could explain the inversion of the 2,426-bp intervening segment (ΔyidY-yidZ-Δhy) observed in the remaining eight S. Kentucky strains  and chicken internal organs (n = 16) were collected from two supermarkets and a slaughterhouse in Xuancheng, Anhui Province, China. The samples were incubated in 100 mL buffered peptone water (BPW) for 18 to 24 h at 37°C with constant shaking at 180 rpm. Then, the enriched 1-mL BPW cultures were transferred to 10 mL Rappaport-Vassiliadis R10 broth (RVR10) and incubated for 24 h at 42°C. The cultures were further inoculated onto XLT4 agar plates and incubated for 24 h at 37°C. One suspicious isolate per plate was purified and confirmed by detection of the stn gene for Salmonella identification using PCR and sequencing . The following antimicrobial agents were tested: ampicillin, cefazolin, cefotaxime, meropenem, gentamicin, streptomycin, amikacin, tetracycline, tigecycline, chloramphenicol, florfenicol, nalidixic acid, ciprofloxacin, colistin, fosfomycin, and sulfamethoxazole-trimethoprim. E. coli ATCC 25922 was used as the quality control strain. The results were interpreted according to EUCAST clinical breakpoints  and ECOFF (https://www.eucast.org/mic_and_zone_distributions_and_ecoffs).The antimicrobial agent MICs of Salmonella isolates were sequenced on the Illumina HiSeq platform. The raw reads were assembled into contigs with SPAdes v.3.8.2. The serotypes were analyzed by the SalmonellaIn Silico Typing Resource (SISTR)  and analysis of chromosomal mutations, resistance genes, and plasmid replicons with the Center for Genomic Epidemiology (CGE) platform (http://www.genomicepidemiology.org). The SGI and MRR structures in AH19MCS1, AH19MCS8, and AH19MCS11 were analyzed by the RAST server (https://www-is.biotoul.fr), and BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Resistance genes and plasmid replicons were identified with >90% sequence homology and coverage. Contigs carrying SGI1-K fragments were extracted and assembled by PCR and sequencing  using SGI1-K variants detected in isolates AH19MCS1, AH19MCS8, and AH19MCS11 as references. The phylogenetic tree of 10 S. Kentucky strains in this study and 2 S. Kentucky strains from patients in China (https://harvest.readthedocs.io/en/latest/content/parsnp.html) and visualized with iTOL . All  (SISTR) . Three ST server , ISfindein China  was consith iTOL .Salmonella isolates have been deposited in GenBank under accession no. PRJNA868865.The whole-genome sequences of all


Photosynthesis Research (2023) 157:133–146 10.1007/s11120-023-01033-xThe original article has been corrected: Figure legends 3 and 5 had been inversed and have now been correctly rearranged.


The patient was diagnosed with IgG4-related pericarditis. Glucocorticoids resolved serological and imaging abnormalities. Prompt treatment improves the disease status.IgG4-related pericarditis has rarely been reported. Here, we report a case of IgG4-related disease that presented with pericardial effusion. A 67-year-old female who presented with palpitations and chest pain was admitted because of a large amount of pericardial effusion that required drainage. The patient underwent pericardial drainage, and the symptoms were gradually alleviated. IgG4 levels were elevated in the serum and pericardial effusions. A biopsy specimen of  IgG4-realated disease (IgG4-RD) is a relatively rare autoimmune disorder associated with elevated serum IgG4 concentration, lymphoplasmacytic infiltration of IgG4-positive plasma cells, and storiform fibrosis in various organs. Here, we present a case of IgG4-RD presenting with pericardial effusion.A 67-year-old female presented to our hospital with palpitations and chest pain. The patient had a history of asthma and diabetes mellitus (DM). Computed tomography (CT) revealed a large amount of pericardial effusion, and the patient was admitted .μL, eosinophil of 790/μL, total protein concentration of 9.3 mg/dL, C-reactive protein (CRP) concentration of 1.53 mg/dL, and N-terminal probrain natriuretic peptide (NT-proBNP) of 223 pg/mL. IgG and IgG4 levels were markedly elevated in the serum  and pericardial fluid . The IgE levels were also elevated (271 IU/mL). Elevations in viral antibody and tumor marker levels and serum autoantibodies related to collagen diseases were not observed. Electrocardiogram revealed a sinus tachycardia. The patient underwent pericardial drainage, and her symptoms were gradually alleviated. Positron emission tomography (PET)/CT revealed 18F-fluorodeoxyglucose (18F-FDG) uptake in the pericardium and submandibular gland  and an IgG4-positive/IgG-positive cell ratio of 40%  one or more organs showing diffuse or localized swelling or a mass or nodule characteristic of IgG4-RD, (2) serum IgG4 levels greater than 135 mg/dL, and (3) positivity for two of the following three criteria: (1) dense lymphocyte and plasma cell infiltration with fibrosis, (2) ratio of IgG4-positive plasma cells/IgG-positive cells greater than 40% and the number of IgG4-positive plasma cells greater than 10 per high-power field, and (3) typical tissue fibrosis, particularly storiform fibrosis, or obliterative phlebitis . Horie eMost patients with IgG4-related pericarditis show a rapid clinical response to steroid therapy , 9 and wThe diagnosis of IgG4-related pericarditis is often difficult based on histological analysis. PET/CT helps identify IgG4-related organs and is useful for evaluating the efficacy of steroid therapy for IgG4-related diseases.


FRK, a gene associated with various types of cancer. In this work, we precisely modeled the disease-associated missense mutation in theC. elegansFRK orthologsrc-2,using CRISPR/Cas9 gene editing, and investigated the physiological role of this mutation and thesrc-2gene. In addition, we generated a conserved variant insrc-1(FYNortholog) to assess the functional redundancy of the conserved variant. The putative pathogenic variantssrc-1orsrc-2caused only subtle phenotypes, suggesting that these mutations alone are not sufficient to explain the facial deformities observed in the Goldenhar Syndrome patient. However, thesrc-2mutant exhibited reduced brood size and moderately enhanced embryonic developmental phenotypes, including epidermal and neuronal patterning defects, in thesrc-1(RNAi)condition, indicating that thesrc-2locus could play a supportive role during developmental processes. Overall, however, these studies showed thatsrc-1/FYNis essential for regulating embryogenesis and morphogenesis, whilesrc-2/FRKis largely dispensable for normal embryonic development, suggestingFYN, notFRK, is the dominant non-receptor protein kinase during embryonic development inC. elegans.Goldenhar Syndrome is a rare congenital disorder characterized by hemifacial microsomia. Although select mutations have been mapped for this disorder, the genetic etiologies in the majority of cases remain unknown. A recent clinical report of a Goldenhar Syndrome patient identified a homozygous missense mutation in FRKgene has been linked to various types of cancers, including breast and brain cancers . In a recent clinical report, a homozygous missense mutation of theFRKgene, NM_002031.3 (FRK): c.484G>A  (https://www.ncbi.nlm.nih.gov/clinvar/variation/VCV001162776.2), was identified in a Goldenhar Syndrome patient presenting with hemifacial microsomia (HFM). HFM is a common birth defect and although several chromosomal abnormalities and causal gene variants have been identified, genetic etiologies in the majority of cases remain unknown. The phenotypic features associated with this disease suggest that defects may arise from abnormities in cranial neural crest cells (CNCC) , which are an ectoderm-derived set of cells that migrate to the pharyngeal arches and facilitate craniofacial development . To investigate the physiological effect of this putative pathogenic variant, we precisely modeled the Val170Ile variant in theC. elegansFRKorthologsrc-2, using CRISPR/Cas9 gene editing. Since a sequence alignment indicated thatsrc-1(FYNortholog) is the paralog ofsrc-2inC. elegans, we also generated the conserved variant Val190Ile insrc-1to characterize the functional redundancy of the variant .Fyn-related Src family tyrosine kinase (FRK) belongs to the family of non-receptor protein kinases, serving as a signaling intermediary during G1 and S phase to negatively regulate cell proliferation . Dysfunction ofsrc-1mutantalone,src-2 mutant alone, andsrc-1;src-2double mutant fail to exhibit obvious morphological defects in hatched animals . However, animals carrying thesrc-2mutation have reduced brood sizes, similar to those of a nullsrc-2mutant,src-2(ok819), which bears a full-length deletion of thesrc-2gene . Thesrc-1variant alone does not cause a significant reduction of brood size compared to wild type . Additionally, thesrc-1src-2double mutant shows a similar brood size when compared to thesrc-2mutant alone, suggesting that thesrc-1variant does not have functional redundancy with thesrc-2variant  with respect to its role regulating brood size.Homozygous animals carryingsrc-2 mutant alone does not result in morphological or embryonic lethality defects , depletion ofsrc-1,by feeding RNAi, in either wildtype orsrc-2is associated with a variety of tissue morphogenesis defects, including abnormal pharyngeal, intestinal, and epidermal morphogenesis ; larval morphogenesis defects and embryonic lethality phenotypes are enhanced whensrc-1(RNAi)is combined withsrc-2 mutant condition . These findings are consistent with prior work suggestingsrc-1is the predominant FRK/SRC during embryonic development inC. elegans; lethality studies have shown essential roles forsrc-1, but notsrc-2during embryogenesissrc-1is widely expressed across all tissues during development, whilesrc-2expression is restricted to developing pharynx and adjacent regions .While theFRKgene expression is disrupted, we employed a previously described automated 4D imaging regime, which enables collection of semi-high throughput embryogenesis data and allows for profiling of embryonic developmental phenotypes following perturbation  (dlg-1Δ7::DLG-1::GFP) and a mCherry-tagged plasma membrane marker in a subset of the developing neurons (Pcnd-1::mCherry::PH). In this strain, or in the same strain background carrying thesrc-2orsrc-1;src-2double mutation, created by CRISPR gene editing, we depletedsrc-1andsrc-2by injection RNAi . Consistent with our previous results, thesrc-2mutant alone and thesrc-1(V190I)src-2double mutant embryos do not exhibit significant defects or embryonic lethality in this strain background , suggesting that the patient isolated mutation is not sufficient to trigger developmental defects inC. elegans. Next, we performedsrc-1(RNAi), by injection, in wild type or mutantsrc-2conditions to examine if these mutations can enhancesrc-1(RNAi)phenotypes. We found thatsrc-1(RNAi)alone or in combination with thesrc-2mutation results in penetrant embryonic lethality, whereas control andsrc-2(RNAi)depletion conditions do not .To further assess the nature of the embryonic defects when, 2019) -N. For src-1(RNAi)and determine if enhancement is observed when thesrc-2missense mutation is combined with this condition, we analyzed the high-resolution 4D embryonic development data; this revealed that most embryos arrest at or near comma stage insrc-1(RNAi)depleted embryos in control or thesrc-2strain (62% and 74% respectively) . We did not observe gross abnormalities in the proliferation of cells, prior to arrest, in the morphogenesis reporter strain in any background. Similarly, the same perturbation conditions were tested using a germ-layer reporter strain, which marks endoderm, ectoderm, and mesoderm cells . We found the nuclear counts for each germ layer at the comma stage to be largely comparable to controls. However, despite largely normal nuclear counts, the arrested embryos undersrc-1(RNAi)conditions frequently exhibit enclosure defects, disrupted germ-layer organization, and irregular neuronal and hypodermal patterning , suggesting that tissue mis-patterning may underpin the observed enclosure defects. While bothsrc-1(RNAi)in control embryos and in thesrc-2strain tended to result in defects during epidermal enclosure,src-1(RNAi)in combination withsrc-2was more likely to result in embryos that undergo a dramatic protrusive rupture event, whereby the enclosing epidermal tissue retracts and releases or partially releases neuronal tissue (33% vs 63% respectively) . A similar uptick in prevalence was observed in irregular neuronal and epidermal patterning insrc-1(RNAi)in thesrc-2strain, relative tosrc-1(RNAi)alone . During development, neuronal precursors are established in four quadrants in control embryos, with precise symmetry along the anterior-posterior axis. However, insrc-1(RNAi)control embryos and in thesrc-1(RNAi),src-2strain condition, abnormal patterning phenotypes were observed in 71% and 95% of the imaged embryos (respectively); both conditions frequently exhibit three asymmetrical loci instead of the expected 4 symmetrical loci . Similarly, epidermal patterning in control embryos is characterized by an organized strip of dorsal hypodermal cells and a single row of seam cells (arrows) that stretch along each of the lateral surfaces during enclosure. In contrast, insrc-1(RNAi)and in thesrc-1(RNAi),src-2condition, a disorganized epidermis is observed, with ill-defined dorsal hypodermis and seam cells . The co-occurrence of neuronal and epidermal asymmetrical patterning defects in these conditions may indicate a general disruption of ectoderm-derived tissues or may suggest a feedback mechanism between neuronal and epidermal tissue morphogenic processes in thesrc-1(RNAi)condition. Altogether, developmental phenotypes, including severe neuronal patterning, epidermal patterning, and protrusive rupture phenotypes were enhanced in thesrc-1(RNAi)src-2combined condition compared tosrc-1(RNAi)controls alone by 24%, 24%, and 30%, respectively  . Thesrc-2allele modestly enhances thesrc-1(RNAi)phenotype suggesting thatSRC-2has a role in supporting the function of its paralogSRC-1during embryonic development. Alternatively, we cannot rule out the possibility that the brood size effects insrc-2may synergize withsrc-1(RNAi)in unexpected ways to manifest in developmental defects, due to altered loading kinetics of the injected RNA into forming embryos.To understand the nature of the embryonic lethality insrc-1/FYNis an essential gene in regulating embryogenesis and morphogenesis, whereassrc-2/FRKis largely dispensable for normal embryonic development, consistent with prior workC. elegans. We find that the developmental defects observed in thesrc-1(RNAi)condition are largely due to asymmetric mis-patterning of ectodermal tissues, including both the epidermis and neurons. The putative pathogenic variantssrc-1orsrc-2caused only subtle phenotypes, which suggests that these mutations are not sufficient to explain the facial deformities observed in the Goldenhar Syndrome patient. However, we cannot rule out the possibility that the humanFYN/FRKcould have acquired new molecular interactions that are only required in human facialtissues that are specifically disrupted by the mutation. Nevertheless, thesrc-2mutantexhibits reduced brood size and moderately enhanced embryonic developmental phenotypes in thesrc-1(RNAi)condition. Thus, we cannot rule out that thesrc-2locus could play a supportive role during developmental processes.Our studies indicate thatOD1719,OD1689,AG605,AG606, andAG607) were either not injected or were injected with dsRNA, manufactured in house, directed atsrc-1 orsrc-2 20-22 hours prior to imaging. The injected dsRNA were used in the panel (J-M). Embryos were dissected from gravid adults in ice cold M9 + 0.1mg/ml tetramisole hydrochloride and transferred to 384-well glass bottom plate, which was kept on ice until wells were loaded and ready to be imaged. Embryos were imaged at 18°C by 2 micron steps, every 20 minutes for approximately 10 hours on a CV1000 confocal scanner box, equipped with a microlens-enhanced dual Nipkow spinning disk (Yokogawa Electric Corporation) and a 60×1.35NA U-PlanApo objective. Imaging was performed in a temperature-controlled room and data collection was done using CellVoyager software (Yokogawa Electric Corporation). Following overnight imaging, a whole well brightfield scan was performed at 10× to capture embryonic lethality and larval phenotypes. For more details on image acquisition see Wang, Ochoa, Khaliullin 2019. Individual embryos were cropped from the broader field using a custom embryo cropping algorithm  and developmental arrest points and phenotypic defects were manually scored. Embryonic lethality was assessed by scoring unhatched embryos from 10×post-imaging whole well scans as previously described . The feeding constructs for RNAi targetingsrc-2was selected from the Ahringer library, while thesrc-1(RNAi) feeding construct was kindly gifted by Dr. Erin Cram. The targeting insert of thesrc-1(RNAi)feeding plasmid were amplified by two primers: F400 5' aggctagcgagagtgaagaatggtacg 3', and R1449 5' agagacagcattggcatacggtagcc . Thesrc-1(RNAi)feeding construct was used in the panel . RNAi bacteria were cultured until log phase was reached, and then spread onto MYOB plates supplemented with 1mM IPTG and 25 μg/ml carbenicillin. The seeded plates were incubated overnight before conducting the brood size assay. To knock down the expression of the target genessrc-1andsrc-2, mid-L4 hermaphrodites were transferred onto the IPTG-induced RNAi plates. Animals were then grown on RNAi plates at 20°C for 60 hours for brood size and other assays. All DIC images were acquired using a spinning disk confocal system equipped with a Photometrics Prime 95B EMCCD camera, a Yokagawa CSU-X1 confocal scanner unit, and a Nikon 60X 1.2 NA water objective.4D imaging was conducted as previously described . Briefly, L4 stage animals (strains usedN2: Bristol (wild type)AG538:src-2(av233) [Val170Ile] IRB936:src-2(ok819) IAG605:src-2(av233) [Val170Ile] IltSi249I;ltSi511IIAG606:src-2(av233) [Val170Ile] I;stIs10389[pha-4::TGF(3E3)::GFP::TY1::3xFLAG inserted into fosmid WRM0617dE06 as C-terminal protein fusion];ltSi539II;ltSi507IVAG757src-1(av298) [Val190Ile] I:AG625:src-1(av298) [Val190Ile] Isrc-2(av233) [Val170Ile] IAG607:src-1(av298) [Val190Ile] Isrc-2(av233) [Val170Ile] I;ltSi249I;ltSi511IIOD1689:ltSi249I;ltSi511IIOD1719:stIs10389[pha-4::TGF(3E3)::GFP::TY1::3xFLAG inserted into fosmid WRM0617dE06 as C-terminal protein fusion];ltSi539II;ltSi507IVN2andRB936were obtained from the CGC.


Human ACE2 is heavily glycosylated and some of its glycans impair binding to the SARS-CoV-2 spike protein. Therefore, glycan-engineered recombinant soluble ACE2 variants might display enhanced virus-neutralization potencies. Here, we transiently co-expressed the extracellular domain of ACE2 fused to human Fc (ACE2-Fc) with a bacterial endoglycosidase in Nicotiana benthamiana to produce ACE2-Fc decorated with N-glycans consisting of single GlcNAc residues. The endoglycosidase was targeted to the Golgi apparatus with the intention to avoid any interference of glycan removal with concomitant ACE2-Fc protein folding and quality control in the endoplasmic reticulum. The in vivo deglycosylated ACE2-Fc carrying single GlcNAc residues displayed increased affinity to the receptor-binding domain (RBD) of SARS-CoV-2 as well as improved virus neutralization activity and thus is a promising drug candidate to block coronavirus infection.SARS-CoV-2 infects human cells via binding of the viral spike glycoprotein to its main cellular receptor, angiotensin-converting enzyme 2 (ACE2). The spike protein-ACE2 receptor interaction is therefore a major target for the development of therapeutic or prophylactic drugs to combat coronavirus infections. Various engineered soluble ACE2 variants (decoys) have been designed and shown to exhibit virus neutralization capacity in cell-based assays and  SARS-CoV-2 has evolved continuously which resulted in the emergence of several virus variants that show increased infectivity. However, all known SARS-CoV-2 variants utilize angiotensin-converting enzyme 2 (ACE2) as host cell receptor  which mar (FcRn) . Moreover (FcRn) . Differer (FcRn) .in vitro enzymatic removal of N-glycans from mammalian cell-produced soluble ACE2 resulted in a SARS-CoV-2 decoy with enhanced neutralizing activity  and virus neutralization (Wuhan-Hu-1 isolate) . Moreoveactivity . To examN. benthamiana) DNA fragment coding for the barley α-amylase signal peptide and amino acids 18-740 of human ACE2 including the collectrin domain was synthesized  and cloned into the AgeI and XhoI sites of plant expression vector pEAQ-HT (BsaI sites of the magnICON vector pICHα26211 (N. benthamiana) synthetic fragment  coding for amino acids 43-313 of endo-β-N-acetylglucosaminidase H (EndoH) from Streptomyces plicatus (P04067) was cloned into the BamHI site of plant expression vector p20  (XbaI/BamHI sites of p20-EndoH to generate p20-ST-EndoH.A codon-optimized (for  pEAQ-HT  or the BCHα26211 . For exproteins)  to generroteins)  was clonN. benthamiana were infiltrated with agrobacterium suspensions (OD600 = 0.1) carrying the p20-ST-EndoH plasmid. Confocal images were acquired 48 h after infiltration using a Leica SP5 confocal microscope (Leica Microsystems). Samples were excited using a 488 nm laser line and collected from 500 to 530 nm as described (Leaves of 5-week-old wild-type escribed .N. benthamiana ΔXT/FT (Agrobacterium tumefaciens carrying plasmids for transient expression of ACE2-Fc and ST-EndoH. ACE2-Fc was purified by affinity chromatography using a 5 mL HiTrap Protein A column (Cytiva) and 0.1 M glycine-HCl (pH 3.5) for elution. Eluate fractions were immediately neutralized using 2 M Tris (pH 12.0), pooled, and dialyzed against PBS (pH 7.4) at 4°C overnight using SnakeSkin Dialysis Tubing (Thermo Fisher Scientific). To isolate the dimeric fraction, affinity-purified ACE2-Fc was subjected to size exclusion chromatography (SEC) using a HiLoad 16/600 Superdex 200 pg column (Cytiva) equilibrated with PBS (pH 7.4). The purified protein was concentrated  and stored at −80°C until further use.ACE2-Fc was produced by agrobacterium-mediated transient expression in leaves of a ΔXT/FT  or wild-a ΔXT/FT . For expin vitro deglycosylation, purified ACE2-Fc proteins were incubated with Endoglycosidase H (EndoH) or Peptide-N-glycosidase F (PNGase F) (New England Biolabs) according to the manufacturer’s instructions and subjected to SDS-PAGE. ST-EndoH-GFP was detected on immunoblots using an anti-GFP-HRP antibody (Miltenyi Biotec).ACE2-Fc separated by SDS-PAGE under reducing conditions was detected by Coomassie Brilliant Blue staining or immunoblotting with anti-human IgG (H + L)-horseradish peroxidase antibody (Promega). For D) were calculated with Biacore T200 evaluation software using a 1:1 binding model. Two independent experiments were performed for two different batches of each ACE2-Fc variant (n = 4).Binding experiments of ACE2-Fc variants to RBD were performed using a Biacore T200 instrument (Cytiva). SEC-purified monomeric RBD  produced in HEK293 cells was available from a previous study . All mea2. SARS-CoV-2 Delta variant isolate  propagated in Vero E6 cells was preincubated with ACE2-Fc for 30 min at 37°C and subsequently used to infect Calu-3 cells with a multiplicity of infection (MOI) of 0.002. Untreated infected cells were used as controls in the assay. After incubation for 24 h at 37°C in MEM supplemented with 2% FBS, viral RNA was extracted from the culture supernatant of infected cells using the QiaAmp Viral RNA Minikit , according to the manufacturer’s protocol and quantified by RT-qPCR as described in detail previously   N-glycan structures lacking plant-specific β1,2-xylose and core α1,3-fucose residues  from ase (ST) . In addiase (ST) . Confocaal cells . We co-ial cells  together removal . When STan occur . These iresidues . To showthamiana .in vitro with EndoH or PNGase F. Upon SDS-PAGE, the migration position of the in vitro digested ACE2-Fc proteins was comparable to the in vivo digested ACE2-Fc implying that the majority of ACE2 N-glycans can be removed in planta by ST-EndoH-GFP co-expression (N. benthamiana ΔXT/FT plants carried predominantly GnGn N-glycans on six of the seven ACE2 N-glycosylation sites (1Man3GlcNAc2 (MGn) appeared as the most abundant N-glycan. Except for site N432 which was only glycosylated to approximately 50%, all other sites were efficiently glycosylated. By contrast, single GlcNAc residues were detected almost exclusively on all seven ACE2 N-glycosylation sites when ACE2-Fc was co-expressed with ST-EndoH-GFP in the presence of the α-mannosidase inhibitor kifunensine   . Overexpin planta further increased the affinity to RBD. Moreover, we showed here that the ACE2-Fc with “single GlcNAc” N-glycans displays increased virus neutralization. While enzymatic deglycosylation of purified ACE2 decoys in vitro is in principle feasible (in planta N-glycan removal procedure appears more straightforward and could lead to a more affordable product.The increased virus-neutralization activity of plant-produced ACE2-Fc carrying GnGn N-glycans as compared to its counterpart derived from human cells  is likelfeasible , the in in planta by co-expression of a Golgi-targeted endoglycosidase.Deglycosylated soluble ACE2 decoys with enhanced properties have also been generated in mammalian cells by mutating N-glycosylation sites like N90 or N322 or combinations thereof . Of note


An ideal therapeutic antibody–oligonucleotideconjugate(AOC) would be a uniform construct, contain a maximal oligonucleotide(ON) payload, and retain the antibody (Ab)-mediated binding properties,which leads to an efficient delivery of the ON cargo to the site oftherapeutic action. Herein, [60]fullerene-based molecular sphericalnucleic acids (MSNAs) have been site-specifically conjugated to antibodies(Abs), and the Ab-mediated cellular targeting of the MSNA–Abconjugates has been studied. A well-established glycan engineeringtechnology and robust orthogonal click chemistries yielded the desireduniform MSNA–Ab conjugates (MW ∼ 270 kDa), with an oligonucleotide(ON):Ab ratio of 24:1, in 20–26% isolated yields. These AOCsretained the antigen binding properties , studied by biolayerinterferometry. In addition, Ab-mediated endocytosis was demonstratedwith live-cell fluorescence and phase-contrast microscopy on BT-474breast carcinoma cells, overexpressing HER2. The effect on cell proliferationwas analyzed by label-free live-cell time-lapse imaging. During recentyears the development of therapeutic AOCs has accelerated and alsoentered clinical trials.2 In addition totherapeutics, AOCs can be used as diagnostic agents, e.g., in immuno-PCR,6 various proximity assays,12 and protein arrays.13 Covalent Ab conjugationutilizes typically reactive Lys,14 Cys,15 Tyr,16 and Arg17 residues. These approaches produce conjugatesthat are heterogeneous regarding conjugation sites and degree of labeling.Unspecific conjugation may also lead to poor reproducibility and complicatedanalytics due to the promiscuous conjugation.19 The ideal therapeutic AOC would be a uniform construct, retain theAb-mediated binding/delivery properties, and contain a maximal ONpayload, which will be finally released to the site of action in thecytoplasm of target cells. The retained delivery properties may bea hard task, as the negatively charged ON payload may, via electrostaticinteraction, mask the active binding sites or alter the native conformationof the Ab constituent. Successful targeted delivery has been demonstratedwith AOCs of an average Ab:ON ratio of 1:6.20Antibody–oligonucleotide conjugates(AOCs) offer a versatiletool for diagnostic and therapeutic applications.28 are nanostructures consisting of an appropriate core unit and adense layer of ONs. The dense layer of ONs can activate ScavengerA receptors that lead to an enhanced cellular uptake. This ratheruniversal receptor-mediated endocytosis cannot distinguish healthycells, and therefore SNAs are applicable for topical delivery primarily.29 However, depending on the particle size, multivalency of ONs, andstructural design of SNAs, the radial formulation may hide negativedistribution properties of ONs and offer more possibilities for thetargeted delivery, when these nucleic acid derivatives are integratedwith tissue-specific ligands. Conjugation of SNAs to Abs would resultin AOCs of high ON payload that are applicable for cell-/tissue-specificgene regulation. A noncovalent conjugate of a gold nanoparticle-basedpolydisperse SNA and an Ab has previously been reported.30 A monoclonal Ab–DNA conjugate was hybridizedto an SNA, which gave an average Ab–SNA ratio of 2:1. Thesepolydisperse macromolecular hybrid constructs exhibited cell typeselectivity with an enhanced uptake and gene knock down compared withnaked SNAs.Spherical nucleic acids (SNAs)1, 31 was selected for the assemblyof MSNAs. Its synthetic availability via Bingel cyclopropanation,32 radially symmetric structure, and in particulara controlled monofunctionalization technique33 have made it an attractive core structure for MSNAs.33 Furthermore, a 12-arm [C60]-based MSNA, consisting of an antisenseON that targets HER2 , hasbeen shown to present sufficient oligonucleotide density to activatethe Scavenger receptor A mediated cellular uptake, resulting in enhanceddownregulation of HER2.27 The MSNAs, whichconsisted of either an anti-HER2 ON sequence or the correspondingscrambled one (34 between the azide-modified [60]fullerenecore 1 and bicyclononyne (BCN)-modified ONs. Our publishedstepwise-SPAAC method,33 followed by aselective amide coupling, yielded mono-trans-cyclooctene(TCO)-modified [60]fullerene-based MSNAs. Trastuzumab (Tra) and an isotype control monoclonal Ab (IgG) were usedas Ab constituents. Tra’s (brand name Herceptin)35 therapeutic action is based on binding to HER2,and it is broadly used as a model in the development of antibody–drugconjugate technologies.36 Abs were site-specificallymodified by the azide group using an established glycan engineeringtechnology (GlyCLICK)38 and exposed then to SPAAC witha bifunctional DBCO/tetrazine linker. Inverse electron-demand Diels–Alderreaction (iEDDA)40 between the TCO-modified MSNAsand the tetrazine-modified Abs afforded the desired AOCs with an Ab-to-ONratio of 1:24. The AOCs were purified by size-exclusion chromatography(SEC), and their authenticity was verified by reducing polyacrylamidegel electrophoresis (SDS-PAGE) and size-exclusion chromatography equippedwith a multiple-angle light-scattering detector (SEC-MALS). The bindingaffinity of Tra–MSNA conjugates to HER2 protein was studiedby biolayer interferometry. The receptor-mediated endocytosis on breastcarcinoma cells (BT-474) was studied with Cyanine5-labeled conjugates.The effect of the Tra–MSNA conjugate on BT-474 cell proliferationwas analyzed by label-free live-cell time-lapse imaging. In all assaysthe corresponding IgG–MSNA conjugate was used as a negativecontrol to affirm that the target recognition was specific. Overall,this study demonstrates that uniform and site-specific MSNA–Abconjugates can be synthesized in a controlled manner. Furthermore,these conjugates, despite the high ON payload (Ab–ON ratioof 1:24), can retain the antigen binding properties (binding of Trto HER2 protein) and undergo Ab-mediated endocytosis. Such constructsmay be applied to the targeted delivery of the MSNAs and in an idealcase find the synergistic effect between the Ab and ON constituents.In the present study, uniform covalent conjugatesof molecularspherical nucleic acids, MSNAs, and Abs have been synthesized forthe first time 1, and thbled one 1, were aE,3E,5E)-5-penta-1,3-dienyl]-3H-indolium-5-sulfonate), TCO-PEG4-NHS ester 3-[2-[2-[2-[2-[[(4Z)-cyclooct-4-en-1-yl]oxycarbonylamino]ethoxy]ethoxy]ethoxy]ethoxy]propanoate),and Tz-DBCO ,4,6,8,12,14-hexaen-10-yn-2-yl)-4-oxobutanoyl]amino]-1-[3-[4-phenoxy]propylamino]-1-oxopropane-2-sulfonicacid) were purchased from Jena Biosciences . Reagentsused in oligonucleotide synthesis were purchased from Glen Research, LGC Bioresearch , and Cytiva . Other reagents were purchased from Sigma-Aldrich . All reagents were used as received.Sulfo-cyanine-5-NHS -6-oxohexyl]-3,3-dimethyl-2--3,3-dimethyl-2--3H-indolium-5-sulfonate, 7.5 μmol in 200 μL ofDMSO). The reaction mixture was gently shaken for 4 h at rt and subjectedto RP-HPLC for purification (25% isolated yield for the labeling).The authenticity of the oligonucleotides was verified by MS (ESI-TOF)(electrospray ionization time-of-flight) (Table S1).For the assembly of MSNAs1, BCN-moON1 or ON2 (0.2 μmol in 100 μL of H2O) were added to a mixture of [60]fullerene core 1 . Isolatedyields (45–50%) of C1 and C2 weredetermined by UV absorbance at 260 nm.BCN-modified oligonucleotides ne core 1,32 (0.8 C1 or C2 (100 nmol in 200 μL of H2O) was mixed with BCN–ON, ON3, ON4, or ON5 (1200 nmol in 400μL of H2O), and 257 μL of 5 M NaCl was added.The reaction mixture was gently shaken for 3 days at room temperatureand subjected to RP-HPLC. An analytical RP-HPLC column Phenomenex,Aeris 3.6 μm WIDEPORE XB-C18 200 Å, 150 × 4.6 mm,linear gradient from 5% to 35% MeCN in 50 mmol L–1 of triethylammonium acetate over 25 min, a flow rate of 1.0 mL min–1, and detection at 260 nm were used for purification.The product fractions were collected and lyophilized to dryness. Isolatedyields (40–50%) of the products were determined by UV absorbanceat 260 nm. The homogeneity of [NH2]MSNA(antiHER2), [NH2]MSNA(scramble), and [NH2]MSNA(antiHER2-Cy5) was evaluated by PAGE.MSNAs were assembled as follows: trans-cyclooctene-PEG4 3-[2-[2-[2-[2-[[(4Z)-cyclooct-4-en-1-yl]oxycarbonylamino]ethoxy]ethoxy]ethoxy]ethoxy]propanoate,1 μmol in 10 μL of DMSO) was added to a mixture of [NH2]MSNA(antiHER2/scramble/antiHER2-Cy5) (20 nmol each) in aqueous 0.1 M sodium borate , and the mixture was gently shaken for 4 h at rt. Phosphate-bufferedsaline PBS (450 μL) was added, and the excess TCO-PEG4-NHS was removed by centrifugal filtration . The PBS addition and centrifugation were repeatedfive times. [TCO]MSNA(antiHER2), [TCO]MSNA(scramble), and [TCO]MSNA(antiHER2-Cy5) were recovered in 95%yield after the final centrifugation (based on UV absorbance at λ= 260 nm). The authenticity and homogeneity of [TCO]MSNA(antiHER2), [TCO]MSNA(scramble), and [TCO]MSNA(antiHER2-Cy5) were verified by PAGE  andSEC-MALS .Succinimidylester of Tra or IgG) glycan chains were first azidefunctionalized using a Genovis GlyClick Azide Activation kit followingthe manufacturer’s protocol. The azide-activated Ab (2 nmolin 50 μL of PBS) and Tz-DBCO ,4,6,8,12,14-hexaen-10-yn-2-yl)-4-oxobutanoyl]amino]-1-[3-[4-phenoxy]propylamino]-1-oxopropane-2-sulfonicacid, 200 nmol in 2 μL of DMSO) were mixed and incubated for4 h at rt. PBS (450 μL) was added, and the excess Tz-DBCO wasremoved by a centrifugal filtration . The PBS addition and centrifugation wererepeated five times. Tra-Tz and IgG-Tz wererecovered in ca. 90% yield after final centrifugation (based on UV-absorbanceat 280 nm).General procedure: Ab(–1). Fractions were analyzed with SDS-PAGE,and those containing the favored 1:2 ratio Ab–MSNA conjugatewere pooled. Tra-MSNA(antiHER2), Tra-MSNA(scramble), Tra-MSNA(antiHER2-Cy5), IgG-MSNA(antiHER2), and Tra-MSNA(antiHER2-Cy5) were obtained in 20–26%yield (based on UV absorbance at λ = 260 nm) after SEC purificationand analyzed with SDS-PAGE  and TCO-modifiedMSNA (8 nmol in 80 μL of PBS) were mixed and incubated at roomtemperature for 4 h. The mixture was purified with size exclusionchromatography using a Superdex 200 Increase 10/300 GL column -5-penta-1,3-dienyl]-3H-indolium-5-sulfonate, in DMSO, 10 nmol, 20 equiv) wasadded to a mixture of Tra (0.5 nmol) in 0.1 M aqueoussodium borate (pH 8.5), and the mixture was incubated for 4 h at rt. Tra-Cy5 was purified twice with a spin desalting column (7K MWCO) and recovered in 85% yield based on absorbance at 280 nm.UV–vis absorbance at 280 and 646 nm was used to determine theratio of Tra/Cyanine5 (degree of labeling = 5.8).Succinimidyl ester ofsulfo-cyanine 5 1-[6--6-oxohexyl]-3,3-dimethyl-2-fullerene 1 and BCN-modified ONs was used for the assembly of amino-modifiedMSNAs ([NH2]MSNA(antiHER2), [NH2]MSNA(scramble), and [NH2]MSNA(antiHER2-Cy5), 33 First,3′-amino- and 5′-BCN-modified ONs  were treated with an excess of 1 (4 equiv)in DMSO to give [60]fullerene–ON conjugates C1 and C2 in 45% and 50% isolated yield. Then, C1 and C2 were exposed to a slight excess (1.2 equiv/azidearm) of 5′-BCN-modified ONs (ON3–ON5) in 1.5 mol L–1 aqueous NaCl to yield [NH2]MSNA(antiHER2), [NH2]MSNA(scramble),and [NH2]MSNA(antiHER2-Cy5) in 40–50% isolated yield. The homogeneity of [NH2]MSNAs was confirmed by PAGE , and it was introduced to amide coupling with an N-hydroxysuccinimidyl (NHS) ester-activated TCO reagent (for 4 h atrt). Spin column filtration (30 kDa molecular weight cutoff) affordedthe products: [TCO]MSNA(antiHER2), [TCO]MSNA(scramble), and [TCO]MSNA(antiHER2-Cy5) in 95% recovery. The homogeneityand authenticity of the products were confirmed by PAGE  and SEC-MALS . The SEC-MALS-based molecular weightanalysis of the products matched well with the expected molecularmasses: [TCO]MSNA(antiHER2), [TCO]MSNA(scramble), and [TCO]MSNA(antiHER2-Cy5): 59.6 ± 0.9 kDa (expected61.5 kDa), 59.2 ± 0.9 kDa (expected 61.5 kDa), and 78.1 ±0.6 kDa (expected 70.2 kDa), respectively.BCN-modified ONs usedfor the assembly of MSNAs were synthesized with an automated synthesizerusing commercial phosphoramidite building blocks and conventionalcoupling chemistry. Tra and IgG were azide-modified with an establishedand commercially available kit  that covers twosuccessive enzymatic reactions: First, the glycan chain is deglycosidatedfrom the innermost N-acetylglucosamine (GlcNAc) residuewith a specific endoglycosidase. Second, uridine diphosphate N-azidoacetylgalactosamine  as a substrate isattached to the exposed GlcNAc residue using galactosyltransferase,yielding the site-specifically azide-modified Ab (two azides/Ab).Azide-modified Abs  (for 4 h at rt) and purified by spin column filtration(50 kDa molecular weight cutoff) to yield tetrazine-modified Abs: Tra-Tz and IgG-Tz.Glycan chains of Tra-Tz and IgG-Tz have two reactivetetrazine groups, one in both heavy chains. An excess (8 equiv) ofthe [TCO]MSNAs was used to drive the iEDDA conjugation to 1:2 Ab–MSNAspecies. The [TCO]MSNAs and the tetrazine-modified Abs were incubatedin PBS for 4 h at rt; the reaction mixtures were subjected to SEC;and the eluted fractions were analyzed by SDS-PAGE (Tra-Tz + [TCO]MSNA(antiHER2) as an example). Each reaction mixturecontained the 1:2-Ab–MSNA conjugate as a major product andthe 1:1 Ab–MSNA conjugate as a minor product. The 1:2 Ab–MSNAconjugates were isolated by SEC in 20–26% yields. As seen inSDS-PAGE (Tra-MSNA(antiHER2), Tra-MSNA(scramble), Tra-MSNA(antiHER2-Cy5), IgG-MSNA(antiHER2), and IgG-MSNA(antiHER2-Cy5).The tetrazine-modifiedAbs SDS-PAGE 1B,homogSDS-PAGE 1A. SDS-PSDS-PAGE 1B. SEC-MSDS-PAGE 2, but it42 However, weexpected that the sterically demanding negatively charged MSNA payloadmight have an effect on the binding properties of the Ab constituent.To evaluate how the MSNA constituent affects Tra’s affinitytoward the HER2 protein, biolayer interferometry (BLI) experimentswere carried out. Biotinylated HER2 protein was immobilized in a streptavidinsensor. Tra, Tra-MSNA(antiHER2), Tra-MSNA(scramble), IgG-MSNA(antiHER2), and [TCO]MSNA(antiHER2) were used as analytes in 100, 33, and 11 nM concentrations (Tra and its MSNA conjugates were found to bind to the sensor in a concentration-dependentmanner. IgG-MSNA(antiHER2) and [TCO]MSNA(antiHER2) did not bind to the sensor, indicating that the binding was Tra-mediated.To further investigate this matter, we reversed the assay and immobilized Tra, Tra-MSNA(antiHER2), and Tra-MSNA(scramble) to anti-hIgG Fc capture sensors and used free HER2 protein as ananalyte in 75 25, 8.3, 2.8, and 0.93 nM concentrations  and Tra-MSNA(scramble) was approximately one-third of that of Tra. Despitethe reduced response, which is likely to occur with high ON-payload–Abconjugates, BLI experiments demonstrated that the Tra–MSNAconjugates retained the Tra-mediated activity toward HER2.In general, site-specific conjugation to an Ab’s Fc regiondoes not alter the immuno-reactivity.trations 3A. Tra atrations 3B. This Tra-MSNA(antiHER2-Cy5), IgG-MSNA(antiHER2-Cy5), [TCO]MSNA(antiHER2-Cy5), and Tra-Cy5.After 30 min incubation, unbound compounds were washed off with icecold PBS. Cells were then imaged for 16 h in 2 h intervals in a 37°C, 5% CO2 live-cell imaging instrument. Digital phasecontrast was used to visualize cells. At low temperatures, only thespecies with affinity toward HER2 are bound to the cell surface.43 Increased temperature (37 °C) initiatesendocytosis of the HER2–Tra-MSNA complex. With Tra-MSNA(antiHER2-Cy5) and Tra-Cy5, the fluorescence signal is seen on theedges of the cells at early time points. As the study advanced, thefluorescence signal moved toward the middle of the cell, indicatingthat compounds were internalized. No fluorescence signal was observedwith IgG-MSNA(antiHER2-Cy5) and [TCO]MSNA(antiHER2-Cy5). Quantitation of fluorescent spots per cell (Tra-Cy5 and Tra-MSNA(antiHER2-Cy5) produced different kinetic profiles as the MSNA conjugate seemedto be internalized more rapidly , Tra-MSNA(scramble), Tra, IgG-MSNA(antiHER2), and [TCO]MSNA(antiHER2) and imaged for 72 h in 4 hourintervals in a 37 °C, 5% CO2 live-cell imaging instrument.As seen in [TCO]MSNA(antiHER2) and IgG-MSNA(antiHER2) had no effect on cell proliferation.A marginal difference between profiles of Tra, Tra-MSNA(antiHER2), and Tra-MSNA(scramble) demonstratesthat the ON constituent had virtually no effect on the proliferation,which indicates that all activity comes from the binding of Tra toHER2. Half maximal effective concentration (EC50) of thecompounds was calculated from the 72 h time point; EC50 for Tra-MSNA(antiHER2) was 1.78 nM (SEM = 0.68 nM),for Tra-MSNA(scramble) 2.24 nM (SEM = 0.80 nM), and for Tra 1.28 nM (SEM = 2.13 nM), respectively. The similarityof dose–response profiles could be attributed to readily strongantiproliferative effects of Tra and furthermore to thecell’s limited capacity to regenerate HER2 receptors on itssurface following Ab binding and endocytosis.44 In addition, the stable covalent linkage of the model conjugate Tra-MSNA(antiHER2) might interfere with delivery of the antiHER2-ONpayload to cytoplasm and, consequently, downregulation of HER2.45 Despite the lack of improvement in the antiproliferationproperties, the proliferation assay supported the findings of theBLI and internalization experiments: The model high ON–payloadAb conjugates Tra-MSNA(antiHER2) managed to retain theTra-HER2 response.To investigate the effect of theconjugates on the proliferation of the BT-474 breast cancer cells,label-free live-cell time-lapse imaging was utilized. Cells were treatedwith 0.62–50 nM solutions of 46 or, alternatively, evaluation of more activeoligonucleotide payloads for more potent antiproliferative effectwith, e.g., G3139, which is an antisense oligonucleotide drug thattargets Bcl-2 mRNA and induces cell apoptosis.47 This framework could also be utilized for conjugating antibodiesto dendritic oligonucleotides without the full architecture of sphericalnucleic acids.48 Overall, this work introducesnovel uniform and site-specific high payload oligonucleotide antibodyconjugates that could find diagnostic and therapeutic applications.The scope of this article was to describemolecularly uniformconjugates of molecular spherical nucleic acids and antibodies forthe first time. The conjugates were synthesized in a molecularly definedand site-specific manner via biocompatible iEDDA-based conjugationbetween [60]fullerene-based molecular spherical nucleic acids anda glycan-engineered antibody. The desired conjugates (antibody–oligonucleotideratio of 1:24) were isolated in relatively high yield and purity.The conjugates were found to bind to the target antigen in a concentration-dependentmanner, as studied by biolayer interferometry. Receptor-mediated internalizationon BT-474 breast carcinoma cells was demonstrated with fluorescentlylabeled conjugates, and the effect of the Trastuzumab-containing compoundson BT-474 cell proliferation was shown. This promising data on retainedantibody-mediated targeting potential and the antiproliferation effectof these high oligonucleotide payload–antibody conjugates wouldsuggest further studies with a cleavable linker design


Ion channels play an important role in the cellular functions of various organ systems, such as the nervous, cardiovascular, immune, and endocrine systems, and are potential therapeutic targets for treatments of their dysfunctions, via ‘channelopathy’ . Ion chaInternational Journal of Molecular Sciences (Biochemistry section), entitled “Recent Developments in Ion Channel and Ion-Related Signaling”, included a total of nine contributions, comprising seven original articles and two reviews, which provided new information on the recent advances in research on ion channels and their regulatory molecules.This Special Issue of the 2+, leading to the modulation of motility and chemotaxis in sperm, and the Ca2+ influx is a prerequisite for sperm capacitation. They indicated the interactions between heparin and OR2C1, and determined the heparin-binding sites of OR2C1 through molecular dynamics simulation: Arg3, Ala6, Thr7, Asn171, Arg172, Arg173, and Pro287. OR2C1 was up-regulated in the heparin stimulation, and a knockdown of OR2C1 inhibited sperm capacitation. They concluded that OR2C1 plays a potential role in heparin-induced sperm capacitation.Yuan et al.  investigCiona NFAT5 (Ci-NFAT5) increased during notochord lumen formation and expansion, (2) a knockdown of NFAT5 in Ciona embryos impaired the notochord lumen expansion, and (3) the Ci-NFAT5 transferred from the cytoplasm into nuclei under the hyperosmotic stimuli. Importantly, Ci-NFAT5 regulated a SLC26 family anion transporter, SLC26A6. Taken together, Ci-NFAT5 responding to hypertonic stress regulates notochord lumen formation and expansion via SLC26A6.He et al.  investig2+-permeable cation channel. Matsubara et al. [The chemical compound JT010 (2-chloro-N-(4-(4-methoxyphenyl)thiazol-2yl)-N-(3-methoxypropyl-acetamide) is known as a potent and selective activator of TRPA1 , a Caa et al.  showed t2+-activated K+ channel (KCa3.1) in tumor-associated macrophages (TAMs) regulated the expression of pro-tumorigenic cytokines and angiogenic growth factors. In THP-1-derived M2 macrophages, the expression levels of interleukin (IL)-8 and IL-10 were significantly decreased by pharmacological activation of KCa3.1, without changes in those of vascular endothelial growth factor (VEGF) and transforming growth factor (TGF)-β1. Furthermore, under in vitro experimental conditions that mimic extracellular K+ levels in the tumor microenvironment, IL-8 and IL-10 levels were both significantly elevated, and their increases were reversed by KCa3.1 activation. Among several signaling pathways potentially involved in the transcriptional regulation of IL-8 and IL-10, respective treatments with extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) inhibitors significantly repressed their transcriptions, and activation of KCa3.1 significantly reduced the phosphorylated ERK, JNK, c-Jun, and cAMP response element binding protein (CREB) levels. Taken together, the KCa3.1 activator may suppress IL-10-induced tumor immune surveillance escape and IL-8-induced tumorigenicity and metastasis by inhibiting their production from TAMs through ERK-CREB and JNK-c-Jun cascades.Ohya et al.  investigSolorza et al.  reported2+-permeable cation channel, namely transient receptor potential vanilloid 4 (TRPV4), plays a key role in endocytosis. Li et al. [2+ entry through activated TRPV4. The enhanced TRPV4-mediated activation of folding and vesicle trafficking proteins promoted exocytosis. Taken together, this study uncovered the role of TRPV4 in mediating exocytosis and ferroptosis.A Cai et al.  investig2+-activated K+ (BK/BKCa/MaxiK/KCa1.1) channel is activated by nitric oxide (NO) and NO-donors in vascular smooth muscle cells, by direct action of NO and cGMP-dependent protein kinase (PKG)-mediated action. Gagov et al. [The large-conductance Cav et al.  investigThe review by Tureckova et al.  summarizDysregulated TRP channels , TRPV (Vanilloid), TRPM (Melastatin), TRPP (Polycystin), TRPML (Mucolipin), TRPN , and TRPA (Ankyrin)) are linked to many hereditary human diseases . The rev


Correction: BMC Cancer 22, 1335 (2022)10.1186/s12885-022-10451-4.Following publication of the original article , the autBelow is the link to the electronic supplementary material.Additional file 4. Chemokine receptors mediating chemotactic influences of CXCL12 and CXCL11 in A767 and A772 cells.


This overexpression is associated with increased susceptibility to the small brown planthopper (SBPH), an economically significant insect pest affecting rice crops. SDG703 is constitutively expressed in multiple tissues and shows substantial upregulation in response to SBPH feeding. SDG703 demonstrates the activity of histone H3K9 methyltransferase. Transcriptomic analysis revealed the downregulation of genes involved in effector-triggered immunity (ETI) and pattern-triggered immunity (PTI) in plants overexpressing SDG703. Among the downregulated genes, the overexpression of SDG703 in plants resulted in a higher level of histone H3K9 methylation compared to control plants. Collectively, these findings indicate that SDG703 suppresses the expression of defense-related genes through the promotion of histone methylation, consequently leading to reduced resistance against SBPH. The defense-related genes regulated by histone methylation present valuable targets for developing effective pest management strategies in future studies. Furthermore, our study provides novel insight into the epigenetic regulation involved in plant-insect resistance.Plant defense responses against insect pests are intricately regulated by highly complex regulatory networks. Post-translational modifications (PTMs) of histones modulate the expression of genes involved in various biological processes. However, the role of PTMs in conferring insect resistance remains unclear. Through the screening of a T-DNA insertion activation-tagged mutant collection in rice, we identified the mutant  Nilaparvata lugens Stål, BPH), the white-backed planthopper , and the small brown planthopper   Tween 20) for 1 h at room temperature. The target protein bands were sequentially detected using antibodies against H3K9me , H3K9me2 , H3K9me3 , and H3 , diluted at a ratio of 1:5000 with the dilution buffer . Anti-H3  antibody was used as an internal reference. Anti-IgG (H+L chain) (Rabbit) pAb-HRP (H+L)  and Anti-IgG (H+L chain) (Mouse) pAb-HRP  were employed as secondary antibodies. Finally, an enhanced chemiluminescence (ECL) immunoblotting detection kit  was used for signal detection. The experiments were repeated three times, and ImageJ software was utilized to quantify the relative protein levels.Two-week-old rice seedlings, cultured in a growth chamber under LD conditions, were ground to powder, and histones were extracted following the manufacturer’s protocol . The supernatant was boiled in 5 × SDS sample buffer . Protein extracts from plants were prepared as described above. Protein samples were separated on 10% SDS-PAGE gel and subsequently transferred to polyvinylidene fluoride fluoropolymer (PVDF) membranes . The membranes were blocked with 5% skimmed milk powder in PBST buffer  for RNA extraction, library construction, and RNA sequencing. High-throughput sequencing was performed using the BGISEQ-500 platform  with 150 bp paired-end reads. Data analysis was conducted using the Dr. Tom system from BGI http://report.bgi.com (accessed on 10 July 2020). Differentially expressed genes were identified based on an adjusted p-value < 0.05 and fold-change > 2. The results were further validated by RT-qPCR.Two-week-old seedlings of the wild type (Dongjin) and Chromatin immunoprecipitation (ChIP) assays were conducted according to a previously published protocol . For thet-tests. For multiple comparisons, when standard deviations were equal, two-way ANOVA (nonparametric) tests with Tukey’s multiple comparisons were employed.Statistical analyses were conducted using GraphPad Prism (Version 8.3.0) software. Differences between two groups were assessed using Student’s unpaired two-tailed D53 (Os11g0104300), OsWAK2-like (Os04g0370100), OsWAK3-like 1 (Os11g0690332), OsWAK3-like 2 (Os04g0367000), OsRLP7 (Os12g0222900), OsRLP23 (Os12g0221700), OsLecRK IX.1 (Os08g0124100), OsXa21-like (Os11g0692500), OsJRL9 (Os04g0330200), OsCRK44-like (Os04g0369033), OsRSR1 (Os11g0229500), OsRGA5-like 1 (Os11g0685600), OsRGA5-like 2 (Os11g0588600), OsPIK-2-like (Os11g0598300), OsPIK-5-like (Os11g0689000), OsRPP13-like 1 (Os01g0781600), OsWRKY53-like (Os11g0685700), and UBQ10 (Os02g0161900). The sequence data generated in this study have been deposited in the SRA database at NCBI with the corresponding accession numbers.Sequence data from this article can be accessed at the National Center for Biotechnological Information (NCBI) with the following accession numbers: In conclusion, our findings unveil a novel role of SDG703 in regulating rice resistance against SBPH. Our results indicate that SDG703 is involved in mediating histone H3K9 methylation at the loci of PRRs and NLRs in rice, resulting in decreased resistance to SBPH. This study provides compelling evidence for the epigenetic regulation of plant–insect interactions, thereby enhancing the comprehension of the intricate regulatory mechanisms involving NLRs and PRRs in the resistance response against insect infestation.


C. elegans NHR-23is a nuclear hormone receptor transcription factor involved in molting, apical extracellular matrix structure, and spermatogenesis. To determine NHR-23 expression dynamics, we previously tagged the endogenousnhr-23locus with aGFP::AID*::3xFLAG tag. To allow co-localization ofNHR-23with green fluorescent protein-tagged factors of interest, we generated an equivalent strain carrying anmScarlet::3xMyc tag to produce a C-terminal fusion. Similar to theGFP::AID*::3xFLAGknock-in,NHR-23::mScarlet::3xMyc was expressed in seam and hypodermal cells, vulval precursor cells, and the spermatogenic germline. We also observed a diffuse NHR-23::mScarlet expression pattern in spermatids and residual bodies after NHR-23 ceased to localize on chromatin. Further examination of this novel localization may provide insight into NHR-23 regulation of spermatogenesis. NHR-23) is an important regulator of both of these processes as well as spermatogenesisNHR-23is homologous with the mammalian circadian rhythm regulator RORα and the insect molting regulator DHR3nhr-23::GFP::AID*::3xFLAGknock-in to produce a C-terminal fusion that labels all isoforms and allows for conditionalNHR-23depletion using the auxin-inducible degron system . To allow for co-localization analysis ofNHR-23with green fluorescent protein-tagged factors we created an equivalentnhr-23::mScarlet::3xMycstrain using CRISPR/Cas9-mediated genome editing . Nuclear Hormone Receptor-23 . This work found thatNHR-23andNHR-85cooperated to promote the expression of thelin-4microRNA at specific points in development, and reported thatNHR-23::mScarlet::3xMyc peaks in expression between L4.2 and L4.3NHR-23::mScarlet::3xMyc in L4.2 vulval precursor cells .NHR-23animals , and itanimals . This etocytes . This dtocytes . The NHanimals . One ca nuclei .NHR-23expression patterns in the GFP and mScarlet fusions? One possible explanation is that gut autofluorescence, which is prominent in the GFP excitation/emission range, could be masking diffuse NHR-23 expression. Use of specific filter sets or genetic perturbations to reduce or eliminate autofluorescence will be an important future directionNHR-23::GFP and NHR-23::mScarlet patterns need to be closely compared, the diffuse NHR-23::mScarlet::3xFLAG expression may challenge the apparent rapid removal of NHR-23 from the germline that the NHR-23::GFP expression pattern suggested. Thenhr-23::mScarlet::3xMycwill be a useful reagent to study the role ofNHR-23in regulating gene expression during spermatogenesis, developmental timing, and apical extracellular matrix remodeling.One outstanding question is what might account for these differences inC. elegans strains and cultureC. elegansstrains (see table in Reagents) were cultured as originally describedStrain generationU6p::sgRNA(F+E) vector (pJW1856) targeting the 3’ end ofnhr-23was generated by annealing oligos 3488+3489 and SapTrap cloning into pJW1838nhr-23::30 amino acid (aa) linker::mScarlet-I^SEC^3xMycrepair template (pJW1877) was assembled through SapTrap cloning with pMLS257 (backbone), pJW1776 (nhr-235’ homology arm), pJW1781 (nhr-233’ homology arm), and pJW1816 (30aa linker::mScarlet-I^SEC^3xMyc)EG9615N2animals to remove the Cas9 transgene and theunc-119(ed3)allele. The loss of theoxSi1091 Cas9 transgene was confirmed by genotyping with oligos 5934+5935 (detects unmodified locus) and 5237+5238 (detects Cas9 transgene in locus). Loss of theunc-119(ed3)allele was confirmed by phenotyping. Thenhr-23::mScarlet::3xMycinsertion was genotyped with oligos 1586+1587  and oligos 1586+4120 (knock-in specific). Genotyping reactions were performed using a 63ºC annealing temperature.Knock-ins were generated by the self-excising cassette (SEC) CRISPR methodMicroscopyN2control animals andnhr-23::mScarlet::3xMycanimals, we set the exposure conditions to avoid pixel saturation of the brightest sample and kept equivalent exposure for imaging.Animals were picked into a 5 µl drop of M9+5 mM levamisole on a 2% agarose pad on a glass slide and secured with a coverslip. Animals were imaged using a Plan-Apochromat 63×/1.4 Oil DIC lens on an AxioImager M2 microscope (Carl Zeiss Microscopy) equipped with a Colibri 7 LED light source and an Axiocam 506 mono camera. We used Fiji software (version: 2.0.0- rc-69/1.52p) to process images


C. elegansmodel for three pathogenic VPS45 variants. We found that wild-type human VPS45 functionally complemented the loss ofC. elegansVPS-45, andthe pathogenic human VPS45 variants functioned almost normally with respect to larval development and endocytosis inC. elegans. These results suggest that SCN5-associated mutations have little effect on the core function of VPS45, and/or that the degree of VPS45 requirement varies, depending on the cell/tissue.VPS45, one of the essential membrane trafficking factors, has been identified as a cause of severe congenital neutropenia 5 (SCN5), but its pathophysiological role remains unknown. Here, we developed a humanized Severe congenital neutropenia (SCN) is a primary immunodeficiency disorder, which is genetically heterogeneous and characterized by the decreased number and dysfunction of neutrophils, suffering from early onset of severe bacterial and fungal infectionsC. elegans,Drosophila, zebrafish, and mammalsin vivo.However, the functional significance and pathological role of the VPS45 variants remain largely unknown.VPS45 belongs to the Sec1/Munc18 (SM) family of proteins and regulates SNARE-mediated membrane fusion in the endosomal/lysosomal system in yeast,vps-45(tm246), a null mutant ofC. elegans VPS45 homologue, exhibits endocytic defects andts(temperature-sensitive) lethal phenotype. We previously showed that mouse VPS45 can rescuetslethality invps-45(tm246), whereas yeast VPS45 cannotC. elegans models by substituting endogenous VPS45 protein (VPS-45) with human VPS45 protein (hVPS45) (wild-type or SCN5-associated variant forms).in silicostructure prediction of human andC. elegansVPS45 . That is, like other SM proteins,C. elegans VPS-45had a conserved topology of a three-domain structure with a central cleft between domains 1 and 3a. All three wild-type residues , which are mutated in SCN5, are conserved inC. elegansand other species . They are clustered in a hinge region ofVPS-45. These structural features are consistent with structural modeling studies of hVPS45The SM family proteins are composed of four subfamilies  and share a conserved topology with three domains sVPS45 . We conn VPS45 , but alin vivo, we conducted knockout-and-rescue approach. The lethal phenotype ofvps-45(tm246)can be rescued by aVPS-45minigene, which expressesC. elegansVPS-45under the ubiquitouseft-3promoter . Therefore, we generated transgenic lines carrying hVPS45 minigenes that express wild-type (WT) or variant  hVPS45 under theeft-3promoter in thevps-45tm246). Newly hatched L1 larvae (0-4 hours after hatching) were collected and incubated for 48 hours at the restrictive temperature (25°C). In this condition,vps-45(tm246)arrested at the early larval stage (L1/L2) with complete penetrance, while the humanized transgenic animals expressing hVPS45-WT developed into adult worms comparable in length to the wild-typeN2strain  . Notably, some hVPS45 mutant worms showed a slight delay in their development as compared to the control .pvalues of hVPS45-T224N, -E238K, and -P468L to hVPS45-WT were p=0.066, p=0.053, and p<0.05, respectively.Next, to examine the functional effects of the SCN5-associated VPS45 variants, n=52] . This iC. elegans has scavenger cells called coelomocytes in the pseudocoelom. These cells take up a marker for fluid-phase endocytosis, such as a secreted form of GFP (ssGFP) from muscle cellsvps-45(tm246)mutants exhibit a Coelomocyte uptake-defective (Cup) phenotype at 20℃; inability to endocytose secreted GFP, resulting in accumulation of GFP in the pseudocoelom  . As expected from the above rescue experiments, the SCN mutant strains were found to restore the endocytosis defect almost indistinguishably from the wild type .Next, we examined the functional effects of hVPS45 variants on endocytosis..The human andC. elegansVPS45 are structurally similar and human VPS45 can functionally complement C. elegansVPS45 depletion. Human VPS45 with SCN5-associated missense mutations functions normally in terms of larval development and endocytosis, except for a slight delay in development. This implies that these variants are simply weak alleles and/or that the degree of VPS45 requirement could vary depending on the cell/tissue, while overexpression from a high copy array with a strong promoter (eft-3p) might mask the reduced ability of the VPS45 mutants and their differences in functionality. The humanized worm model we developed here would provide a useful platform with which to analyze detailed functions of pathogenic VPS45 variantsin vivo.In summary, we developed an efficient and economical animal model for pathological analysis of the human genetic disease SCN5C. elegansstrains and maintenance:C. elegans strains were cultured using standard techniquesN2andarIs37[myo-3p::ssGFP +dpy-20(+)]were obtained from the Caenorhabditis Genetics Center.vps-45(tm246)andvps-45(tm246);arIs37were maintained at 15℃ as previously describedPlasmid construction: The cDNA sequence encoding human VPS45 (hVPS45-WT) was amplified from a U937 cDNA library using primers hvps45#F1: 5'-TGCGGCCGCATGAACGTGGTTTTTGCTGTG-3' and hvps45#R1: 5'-TAGATCTTCATCTTCTGCTTGCTGACC-3' and cloned into pGEMTeasy (Promega). The cDNAs encoding hVPS45-T224N, hVPS45-E238K, and hVPS45-P468L were generated using the KOD-plus mutagenesis kit (Toyobo). Primers used for mutagenesis are as follows; hvps45_T224N#F1: 5'-AaCCCATTGCTAAACCAGTGGAC-3', hvps45_T224N#R1: 5'-GATGGCATCATCACAGCGAT-3'; hvps45_E238K#F1: 5'-aAACTACTAGGCATAAACAACAATC-3', hVPS45_E238K#R1: 5'- GTGGACCATGGCCTGATATG-3', hvps45_P468L#F1: 5'- CtTTTCCTACATGAAACCCTGGA-3', hVPS45_P468L#R1: 5'- TTGATGCTGTGTATATACATTTTC-3'. To generate hVPS45 minigenes, EGFP sequence in a pFX_EGFPT vectoreft-3promoter was removed by NotI/BglII digestion and then replaced with the NotI/BglII fragments containing wild-type or mutant hVPS45. All constructs were verified by DNA sequencing.Generation of humanized strains: To generate transgenic animals, DNA construct encoding hVPS45 (wild-type or variant) was co-injected into the gonads ofN2hermaphrodites at 20 ng/µl each, along withgcy-10p::RFPat 180 ng/µl as an injection marker, using a standard protocolvps-45(tm246), and thenvps-45(tm246)homozygous mutants carrying the hVPS45 transgenes were obtained (QJ4126-QJ4133). Thetm246allele of each humanized strain was confirmed by PCR amplification using primers that span the deletion invps-45(tm246)Body length: Animals were observed and photographed under an SZX16 stereomicroscope (Olympus) equipped with a DP28 digital camera (Olympus). The cellSens software  was used to measure the length of the worm body. The developmental stage of each animal was assessed by worm length and vulval morphology as previously describedEndocytosis assay: The strainarIs37constitutively expresses ssGFP, which is secreted from muscle cells into the body cavityQJ4134,QJ4135,QJ4136, orQJ4137were obtained by crossingQJ4126,QJ4128,QJ4130, orQJ4132into thestrainarIs37, respectively. The resulting hVPS45strains carryingarIs37were cultured at 20°C.vps-45(tm246);arIs37was incubated at 20 °C for 24 hours prior to assay.Adult worms were mounted on agarose pads and viewed on a fluorescence microscope , using a 10× objective  or 100x objective . Images were taken with DP28 digital camera (Olympus). Animals were immobilized with 5 mM levamisole.Statistics: One-way ANOVA followed by Dunnett's test was performed using GraphPad Prism 10.0.3 (GraphPad Software) (ftware) . Hypothftware) .3D model: Prediction of 3D structure ofC. elegansVPS-45and human VPS45 were performed using the I-TASSER server (https://zhanggroup.org/I-TASSER/)C. elegans VPS-45and human VPS45 were based on the three-domain structure of SM proteins defined by Misura et al


Drosophila affects various Myc-dependent phenotypes and delays the growth of a Myc-dependent brain tumor, thereby significantly extending the longevity of tumorous animals.Knockdown of Spt5 in  Drosophila to address the relevance of this interaction in a living organism. Spt5 displays moderate synergy with Myc in fast proliferating young imaginal disc cells. During later development, Spt5-knockdown has no detectable consequences on its own, but strongly enhances eye defects caused by Myc overexpression. Similarly, Spt5-knockdown in larval type 2 neuroblasts has only mild effects on brain development and survival of control flies, but dramatically shrinks the volumes of experimentally induced neuroblast tumors and significantly extends the lifespan of tumor-bearing animals. This beneficial effect is still observed when Spt5 is knocked down systemically and after tumor initiation, highlighting SPT5 as a potential drug target in human oncology.The transcription factor SPT5 physically interacts with MYC oncoproteins and is essential for efficient transcriptional activation of MYC targets in cultured cells. Here, we use  MYC isoforms , defining MYC genes as the most frequently amplified oncogene family across human cancers (Drosophila model to (i) validate the genetic interaction between MYC and its binding partners in vivo and (ii) to estimate effects on healthy tissue of animals and thus the potential therapeutic window.Expression of MYC oncogenes is deregulated in most human tumors. Up to 28% of all tumors exhibit gene amplification of one of the  cancers . Indeed, cancers , genetic cancers  or exoge cancers , and exp cancers . MYC can cancers . At the  cancers . Instead cancers . In rece cancers . To be c cancers , but theDrosophila genome encodes a single MYC homolog that accomplishes the functions of its vertebrate counterparts in normal cells, and it also acts as an oncogene in Drosophila tumor models. Here, we focused on a brain tumor model derived from neural stem cells (type II neuroblasts = NB II), which allows to study proliferation and tumorigenesis during brain development. Briefly, NB II produces intermediate neural progenitors (INPs) with a restricted proliferation potential, which in turn generate ganglion mother cells as the precursors of neurons and glia cells (The ia cells . NB II eia cells . In caseia cells . Brat beia cells . Brat taia cells . We explSPT) genes, including SPT5, were discovered, because their mutation reactivated the transcription of an auxotrophy gene that was silenced by proximal insertion of a Ty transposon  altered Spt5 transcript levels to 62% ± 24% and 750% ± 90% of control, respectively. When driven with GMR-GAL4 in control eye discs, siRNA-mediated Spt5-knockdown had no discernible effect on adult eyes  and Asense (Ase), which are transcription factors that serve as markers for neuroblasts: NB II (of which there are eight per brain hemisphere) express Dpn but not Ase (Dpn+ Ase−), in contrast to type I NBs where both proteins are present (Dpn+ Ase+). NB II then generate INPs which pass through a maturation process (from Dpn− Ase− to Dpn− Ase+ to Dpn+ Ase+), before producing ganglion mother cells. As reported previously, Brat knockdown causes a massive expansion of NB II-like cells (Dpn+ Ase−) at the expense of INPs . Brain hTo identify the molecular basis of the observations described above, we isolated NB II from 96 h-old larvae and analyzed their transcriptomes. As shown in Comparison of control with Brat-depleted neuroblasts revealed several alterations of uncharacterized genes (shown in grey) and expected changes in gene expression : brat leNext, we analyzed the impact of Spt5 knockdown on tumors caused by brat knockdown. Brat levels themselves were not altered, but Myc targets were significantly down-regulated, in line with observations in mammalian cancer cells . The othDespite the massive brain overgrowth upon brat knockdown in NB II lineages, the tumor-bearing animals reached adulthood at expected frequencies . HoweverStatistical significance of differences in longevity. Pertains to Fig 5A and C.Table S1. Raw read counts from control NB II or Brat knockdown NB II ± Spt5 knockdown. Pertains to Fig 4B and C.Table S2. P-values, and FDR Q-values from pairwise comparisons of genotypes. Pertains to Fig 4B and C.Normalized expression values, Table S3. Nevertheless, Brat-/Spt5-knockdown animals did not live as long as control flies, apparently because their tumors recur . In contDrosophila brain tumor models  and Spt5 specifically in NB II. In this setting, Spt5-knockdown almost completely reverted the tumorous tissue overgrowth in larval brains and more than tripled adult animal survival. However, most of the larval NB II-derived cells retained neuroblast characteristics and expressed Imp and Chinmo. These cells apparently did not finally exit the cell cycle and terminally differentiate during metamorphosis, but instead regained a high proliferative potential after adult eclosion. We do not know whether these relapsing tumor cells somehow recovered their Spt5 expression or whether they proliferated despite continually low levels of Spt5.It was previously shown that Chinmo/Imp cells are the major drivers for NB II tumorigenesis; they propagate by repeated self-renewing divisions, but at low frequencies also spawn Chinmo/Imp-negative cells which have a reduced proliferative capacity . In lineDrosophila-based approach allows a simple pre-screening of these candidates to filter for the best targets that can subsequently be funneled into more laborious analyses in mice.Importantly though, knockdown of Spt5 in selected neuroblasts of control animals without brain tumors had mild effects on brain development, and did not negatively impact adult survival, demonstrating the potential value of Spt5 as a therapeutic target. However, in clinical settings, it is typically not possible to direct a therapy exclusively at transformed cells and therapeutic intervention cannot be initiated at early tumor development. For this reason, we developed a second system that allowed temporal separation of tumor initiation and Spt5 knockdown, and that targeted Spt5 not only in NB II but throughout the organism. Whereas this approach relied on the same system to deplete Brat and the same Spt5-targeting siRNA as the first approach above, the latter was induced by a heat shock and directly driven by the Actin5C promoter rather than being amplified by a GAL4/UAS loop, resulting in lower siRNA expression and less efficient Spt5 depletion in NB II. Nevertheless, Spt5 knockdown had a strong therapeutic benefit for tumorous flies, as it almost doubled their survival time. Importantly, ubiquitous Spt5-knockdown did not impair the survival of tumor-free control animals, nor did heat-shocks per se have any deleterious effect on longevity. A molecular explanation for this tumor-suppressive effect is provided by our analysis of NB II transcriptomes: Spt5-knockdown resulted in strong down-regulation of several genes associated with NB II transformation, and an up-regulation of genes opposing uncontrolled proliferation. Most of these expression changes can be ascribed to a reduction of Myc:Spt5 complexes, whereas some probably reflect a functional interaction of Spt5 with the Mediator complex, which itself plays a role in NB II tumor formation. As expected, Myc knockdown also extended the longevity of tumor-bearing flies, but this effect was less pronounced than for Spt5 knockdown. This difference might indicate that Myc:Spt5 complexes are more critical for transformed cells than for normal tissues. In any case, our experiments demonstrate that targeting a protein, Spt5, which was selected based on its physical interaction with Myc, can reduce tumor mass and provide a survival benefit for tumor-bearing animals, even though this protein is essential for normal development. It is open, though, whether Spt5 is the best-suited target in Myc-dependent cancers, as many additional proteins have been shown to bind Myc. Our Sources of flies: “GMR-GAL4” and “GMR-GAL4 3x(UAS-Myc)” were characterized by P0” additionally carry the hypomorphic allele MycP0 on the same X chromosome, whereas “ctr” flies are WT for Myc and only carry the two described transgenes. Hence, eye imaginal discs of the MycP0 flies described in Myc allele only reduces the amount of Myc protein, but does not alter its amino acid sequence.Eye-specific reduction of Myc levels as used for Type II neuroblasts were targeted by a combination of worniu (wor)-GAL4, which is expressed in type I and II NBs, and asense (ase)-GAL80 to repress GAL4 activity in the type I NBs .To knock down Spt5 ubiquitously after the onset of tumor generation, the system above (wor-GAL4 ase-GAL80 UAS-Brat-KD) was combined with the transgenes “hs-FLP” and “pACT5C-FRT-stop-FRT-siSpt5.” siSpt5 expression was initiated by transferring larvae at 120 h after egg deposition for 1 h to a water bath at 37°C.2HPO4, 2 mM KH2PO4, 2.7 mM KCl, 137 mM NaCl) and fixed on ice for 25 min in PLP solution . All washings were done in PBT (PBS plus 0.3% Triton X-100). After blocking in PBT containing 5% normal goat serum for 1 h, tissues were incubated overnight at 4°C with combinations of the following primary antibodies: rabbit anti-Ase , mouse anti-Bruchpilot , rat anti-Chinmo , rabbit anti-Dcp-1 , guinea pig anti-Dpn , chicken anti-GFP , rabbit anti-Imp , guinea pig anti-Lamin DmO . Secondary antibodies conjugated with AlexaFluor 488, Cy3 or Cy5-conjugated were purchased from Molecular Probes and Dianova.For immunostainings, brains from late third instar larvae or adults were dissected in PBS (10 mM NaFor 5-ethynyl-2′-deoxyuridine (EdU) labeling, brains from third instar larvae or adults were dissected in PBS and incubated with 20 μM EdU in PBS for 90 min. After fixation in 4% PFA for 15 min, followed by immunostaining, before EdU incorporation into replicating DNA was detected with the Click-iT Alexa Fluor 647 EdU imaging kit (Thermo Fisher Scientific [Invitrogen]).Embedding of brains was done in Vectashield (Vector Laboratories) and confocal images were collected with a Leica SPE or SP8 microscope (Leica Microsystems). Image processing was carried out with the ImageJ distribution Fiji .To measure adult eye sizes, adult males were collected at 1–7 d after eclosion and killed by freezing. One eye per individual fly was photographed on a Zeiss Discovery.V8 stereomicroscope fitted with a 1.5x lens and processed with Axiovision Extended Focus software and the ImageJ distribution Fiji.g. 10 μl of the supernatant was transferred into a black 96-well plate and assayed for luciferase expression using the Dual Luciferase Reporter Assay System in an automated luminometer. Note that the tumorous brat–KD flies contain a “UAS-FireflyLuciferase” transgene, whereas the non-tumorous flies without the brat–KD carry a “UAS-RenillaLuciferase” transgene  and homogenized with ∼10 steel beads in a “Bullet Blender Blue” Homogenizer at speed 10 for 2 min, followed by a 4′ centrifugation at 12,000gene see . Hence, gene see  and the gene see .For weighing flies, 1–4 d old adult flies were dried for 20′ at 95°  and then stored at RT. Before weighing on a Mettler UMT5 Comparator scale (Mettler Toledo), the flies were allowed to equilibrate with ambient atmosphere for at least 30′.To determine duration of development, timed egg lays (5–14 h) were performed and eclosion was monitored two to three times a day.Parents were transferred to a fresh food vial every 3 d. Offspring was collected within 1 d of adult eclosion, and subsequently transferred to fresh vials every 2 d. The number of living flies was monitored daily for a period of 60 d.2PO4, 1 g/liter NaHCO3, 1 g/liter glucose). After two washes, Rinaldini’s solution was replaced with a dissociation solution , and the brains were stirred up by pipetting. After 1 h incubation at 30°C with occasional mixing, the brains were washed twice with Rinaldini’s solution and with Schneider’s medium, and then mechanically dissociated by pipetting. The resulting cell suspension was filtered through a 30-μm mesh 5-ml FACS tube, which was then filled up with Schneider’s medium to a total volume of 10 μl per dissected larval brain and sorted in a BD FACSAria III sorter. Type II Neuroblasts were identified based on side scatter (SSC), forward scatter (FSC) and GFP intensity, collected into 96-well microtiter plates, containing 1 μl β-mercaptoethanol and 100 μl Lysis Buffer (Agilent Technologies Absolutely RNA Nanoprep Kit) per well, and subsequently stored at −80°C until use.Processing of larvae for next-generation sequencing was carried out as described by To induce expression of siSpt5 or of an Spt5-cDNA, flies of the genotype “hs-FLP act-FRT-stop-FRT-GAL4 UAS-siSpt5,” “hs-FLP act-FRT-stop-FRT-GAL4 UAS-Spt5” or “hs-FLP act-FRT-stop-FRT-siSpt5” were exposed to a 1-h heat shock at 37°. 1 d later, single wandering larvae were processed for total RNA extraction, reverse transcription, and qRT-PCR as described in RNA was isolated using Agilent Technologies’ Absolutely RNA Nanoprep Kit (including DNase I digestion). RNA concentration and quality were determined on 2100 Bioanalyzer Instrument (Agilent Technologies) using the Agilent RNA 6000 Pico Kit (Agilent Technologies). Library preparation was performed using the Poly(A) mRNA Magnetic Isolation Module (New England Biolabs) and the NEBNext Ultra II Directional RNA library Prep Kit for Illumina (New England Biolabs). For library amplification, 17 or 24 PCR cycles were used. Library size distribution and concentration were analyzed on the Fragment Analyzer (Agilent Technologies) using the NGS Fragment High Sensitivity Analysis Kit . The libraries were sequenced on Illumina instrument (NEXTSeq500).Bliss synergy scores  were calDrosophila genome, using bowtie2 with the setting “very-sensitive-local” /+GMR-GAL4 3x(UAS-Myc)/+; UAS-siSpt5/+GMR-GAL4 3x(UAS-Myc)/+; UAS-Spt5/+GMR-GAL4 3x(UAS-Myc)/+; UAS-Spt5 UAS-siSpt5/++/Y+/Y; Spt5[SIE-27]/+dm[P0]/Ydm[P0]/Y; Spt5[SIE-27]/+tub-FRT-Myc-FRT-GAL4 ey-FLP/Ytub-FRT-Myc-FRT-GAL4 ey-FLP/Y; UAS-siSpt5/+tub-FRT-Myc-FRT-GAL4 ey-FLP/Y; UAS-Spt5/+tub-FRT-Myc-FRT-GAL4 ey-FLP/Y; UAS-Spt5 UAS-siSpt5/+dm[P0] tub-FRT-Myc-FRT-GAL4 ey-FLP/Ydm[P0] tub-FRT-Myc-FRT-GAL4 ey-FLP/Y; UAS-siSpt5/+dm[P0] tub-FRT-Myc-FRT-GAL4 ey-FLP/Y; UAS-Spt5/+dm[P0] tub-FRT-Myc-FRT-GAL4 ey-FLP/Y; UAS-Spt5 UAS-siSpt5/+wor-GAL4 ase-GAL80 UAS-mCD8::GFPwor-GAL4 ase-GAL80 UAS-mCD8::GFP UAS-Brat-KDwor-GAL4 ase-GAL80 UAS-mCD8::GFP UAS-siSpt5wor-GAL4 ase-GAL80 UAS-mCD8::GFP UAS-Brat-KD UAS-siSpt5wor-GAL4 ase-GAL80 UAS-RLucwor-GAL4 ase-GAL80 UAS-RLuc UAS-siSpt5wor-GAL4 ase-GAL80 UAS-RLuc UAS-Spt5wor-GAL4 ase-GAL80 UAS-RLuc UAS-siSpt5 UAS-Spt5wor-GAL4 ase-GAL80 UAS-FLuc UAS-Brat-KDwor-GAL4 ase-GAL80 UAS-FLuc UAS-Brat-KD UAS-siSpt5wor-GAL4 ase-GAL80 UAS-FLuc UAS-Brat-KD UAS-Spt5wor-GAL4 ase-GAL80 UAS-FLuc UAS-Brat-KD UAS-siSpt5 UAS-Spt5wor-GAL4 ase-GAL80 UAS-RLucwor-GAL4 ase-GAL80 UAS-RLuc UAS-siSpt5wor-GAL4 ase-GAL80 UAS-RLuc UAS-Spt5wor-GAL4 ase-GAL80 UAS-RLuc UAS-siSpt5 UAS-Spt5hs-FLP wor-GAL4 ase-GAL80hs-FLP wor-GAL4 ase-GAL80 UAS-Brat-KDhs-FLP wor-GAL4 ase-GAL80 act-FRT-stop-FRT-siSpt5hs-FLP wor-GAL4 ase-GAL80 UAS-Brat-KD act-FRT-stop-FRT-siSpt5wor-GAL4 ase-GAL80wor-GAL4 ase-GAL80 UAS-Brat-KDwor-GAL4 ase-GAL80 UAS-Myc-KDwor-GAL4 ase-GAL80 UAS-Brat-KD UAS-Myc-KDGSE220110.RNA expression data are available at the Gene Expression Omnibus under the accession number GEO: