This study aimed to quantify the total cost of violent firearm-related offenses in British Columbia in 2016 Canadian dollars over a five-year period, 2012 to 2016. The purposes of this study were to estimate the direct costs to the health care system and indirect costs to society for violent firearm injuries and deaths; and to estimate criminal justice system costs pertaining to firearm incidents.Human and economic costs to the health care system and productivity losses were calculated using health administrative datasets such as B.C. Vital Statistics and Discharge Abstract Database. Criminal justice system costs pertaining to firearm incidents were estimated by applying weighted average costs to aggregate expenditures using methodology consistent with that used by Statistics Canada.There was a total of 108 deaths and 245 hospitalizations resulting from violent firearm injuries. The total estimated cost of all violent firearm crime averaged $294,378,985 per year; human costs averaged $188,416,841 per year, where health care costs averaged $3,910,317 per year, productivity losses from workforce and household averaged $17,299,054 and $4,559,470 per year, respectively, and loss of life averaged $162,648,000; and $105,021,145 in criminal justice system costs, and $941,000 in programming costs.This study clearly demonstrates the significant cost of violent firearm injury in British Columbia and the impacts on the health care system, criminal justice system, and to society at large, particularly within the criminal justice system where the costs were significantly higher than health care. Firearms are a lethal means to homicide, suicide, and unintentional injuries, highlighting an important worldwide public health issue with social and economic costs that extend beyond life lost. Firearm injuries are a major concern in the United States (US), with assaults being the most common overall mechanism . Canada The impact of firearm violent crime can be wide reaching and long lasting, requiring considerable societal resources to address the problem. Estimating the cost of firearm incidents is complicated and challenging, with the outcomes and consequences varying widely from case to case. Individuals, families, and communities bear hefty costs due to firearm injury, and these incidents have the potential to devastate lives, to incite fear, and to impose heavy social, psychological, and financial burden . In addiTo our knowledge, this study is the first to estimate the total cost of violent firearm injury in B.C. This study aimed to quantify the total cost of violent firearm-related offenses in B.C., with the objectives to: i) estimate the direct human costs to the health care system (treatment and care), as well as indirect costs to society (productivity losses) for violent firearm injuries and deaths; and, ii) estimate criminal justice system costs related to firearm incidents. This study was undertaken with a societal, or population, perspective. It included costs that have an impact on society as a whole, including individuals, families, employers, and the government. The application of a societal perspective supports a public health viewpoint and supports policies aimed at minimizing losses or maximizing gains to society.Firearm incidents, including those that result in injury or death, are reported by B.C. police forces as crimes against the person. Data for the five-year period January 1, 2012 to December 31, 2016, inclusive, were used to determine firearm-related incident information, associated injuries and deaths, and population statistics. These years were selected as 2016 was the most recent year with available hospitalization data at the time of this study, and five years of data were aggregated to allow for adequate case counts, and to “smooth” higher- and lower-than-average annual counts. Estimated average costs across the five-year period were applied to calculate costs in 2016 Canadian (CAN) dollars. All datasets used to calculate costs were not linked and therefore the incidents were not mutually exclusive and chances of double counting remain.The categorization of firearm violent crime incident costs is illustrated in Human costs were calculated using an incidence costing, human capital approach covering the lifetime of the injured individual. Human costs were primarily calculated using the ERAT—Electronic Resource Allocation Tool , a spreaDirect  costs comprise all goods and services used for the diagnosis, treatment, and care of people experiencing injury. Indirect costs (productivity losses), derived using the human capital approach, represent the injured individual's inability to perform their normal activities of daily living and are defined as lost resources resulting from inability to work, premature death, and costs of informal care . For deaIndirect costs are affected by age, the base year wage rate, the probability of participating in the labor market, and having participated, the probability of being employed, the real wage growth rate, and the discount rate. The value of time lost from work due to morbidity, disability, and premature death was measured using B.C. earnings data. Both direct and indirect costs were discounted to a present value in 2016 at 1.5% per annum . The effDeath and hospitalization data were extracted from the BC Vital Statistics and the Discharge Abstract Database (DAD), respectively , using tThe incidence of deaths, hospitalizations, ED visits, and disability; values for population and length of stay; resource intensity weights for resources allocated to the hospital stay; the average cost of hospital stay; and the unemployment rate, participation rate, and average weekly earnings were all entered into ERAT to obtain the corresponding direct and indirect costs. As the physician costs and ambulance costs for hospitalizations and ED visits were not included, they were appended to the hospitalization and ED costs derived from ERAT.Direct death costs were estimated on a complete episode of events due to a firearm death. These events range from costs incurred as a result of deaths occurring at the scene, ambulance transportation costs, treatment occurring in the ED and hospital prior to the death, coroner and autopsy costs, and funeral costs. As ERAT only included indirect death costs, total death costs were calculated by appending direct death costs to the indirect death costs from ERAT.Unpaid labor includes caregiving, volunteer work, household activities, or any other activity that occurs outside of the standard labor market. ERAT only included estimates of production losses of paid work as a result of morbidity and premature death. The value of lost production was estimated for the working-age population comprising individuals aged 15–64 years. For this study, it was assumed that all individuals contribute to society. Caregiving, defined as care received by the injured individual within the year of injury and the main condition for which the injured individual sought help, can be classified as either a direct cost or an indirect cost depending on whether a formal, or direct, payment was made . InformaThere is significant quality of life lost due to deaths, or a Value of a Statistical Life (VSL), the additional cost that individuals would be willing to bear for improvements in health and safety. As recommended by the Government of Canada, VSL was calculated for violent firearm deaths by applying an average VSL value of $6.5 million in 2007 Canadian dollars, converted using the Statistics Canada consumer price index , 27, whiVision care and prescription drug expenditures outside of hospital care were excluded as data were not available. Previous studies included personal costs, such as economic dependence and social isolation; intangible costs, such as pain and suffering; and third-party costs such as loss of affection to, or enjoyment of, family members, as well as social services operating costs . The curCriminal justice system costs were estimated by applying weighted average costs to aggregate expenditures using methodology consistent with that used by Zhang and Qin and Statistics Canada , 28. DatPolice and court costs were calculated using aggregate costs available from federal government official sources , howeverIn estimating the police costs for firearm incidents reported to police as crimes against the person, total costs of police expenditures were assigned to the various crimes according to their severity weights using methodology developed by Statistics Canada , with seViolence prevention and intervention programming costs were internally retrieved from the B.C. Ministry of Public Safety and Solicitor General.For the purposes of this study, violent crime included all person-related offenses processed within the judicial system that may or may not have resulted in injury. Incidents classified as undetermined were included with violent crime.Human, criminal, and justice costs, and violence prevention and intervention programming costs were summed to obtain the total firearm violent crime costs.The data sources used in this study are largely reliant on existing administrative data, where the quality of the data is reliant on the expertise of professional data coders and thus may exhibit inherent biases and inaccuracies. Injuries treated in doctor's offices or walk-in clinics are not accounted for as data were unavailable. There is a large data gap for hospitalized injuries requiring ongoing care outside of the hospital setting, ranging from short periods to long-term permanent disability.Unpaid labor was not included, such as volunteer work, or any other activity that is outside of the standard labor market. Indirect costs did not include transfer payments made by government or social services, as they are a reallocation of resources and the net effect of the transfer to society is zero. Furthermore, indirect costs for those aged 65 years and over were not included under the assumption that 65 is the age at which people stop working. This approach is not reflective of the continuing participation of older adults in the workforce, where the proportion of adults aged 65 and over continues to grow. The human capital approach uses lost past and future earnings and household work as a lower bound on the value of a human life. Because of labor market distortions and this article's omission of earnings after age 64, it undervalues women and minorities and places minimal value on the injury and death of people age 65 and older.The violence prevention and intervention programming costs were restricted to costs internally retrieved from the B.C. Ministry of Public Safety and Solicitor General and are not a comprehensive picture of the existing firearms programming landscape. City-funded programs or funding from other ministries or branches of government were not included.There was a total of 108 deaths and 245 hospitalizations resulting from violent firearm injuries in B.C. between 2012 and 2016. The incidence counts for injuries, deaths, offense cases, and offenders during this time period are presented in The total estimated cost of all violent firearm injuries and deaths averaged $294,378,985 per year, with a per capita cost of $67 for an average population of 4,404,917; human costs averaged $188,416,841 per year, where health care costs averaged $3,910,317 per year, productivity losses from workforce and household averaged $17,299,054 and $4,559,470 per year, respectively, and loss of life averaged $162,648,000 . HospitaconservativeThis study clearly demonstrates the significant cost of firearm injury in B.C. and the impacts on the health care and criminal justice systems, and to society at large. This is particularly noted within the criminal justice system, where costs were significantly higher than within the health care system. These There have been a number of studies conducted in other jurisdictions that have identified the costs of firearm injuries , 38–43, Similar to the findings of Zhang and Qin , who repEffective action to prevent firearm incidents can alleviate the human and economic costs that British Columbians currently bear. The B.C. government is addressing firearm violence through a new firearm violence prevention act that was received in 2021 and is anticipated to be regulated in 2022 . This acIn specifying the multiple cost centers related to firearm injury and death in B.C., this study highlights the scope of opportunities available to policy makers and government for investment in prevention programs to reduce the rate of the firearm injuries and deaths, and decrease costs. Previous research indicates that almost all injuries and injury-related deaths can be prevented , and invwicked problem—a social/cultural issue difficult to solve—(via strong community partnerships, collaboration with public health and health care systems, and an approach encompassing three core and two implementing components (via the Canadian Border Services Agency, and is addressing firearm and gang violence at the provincial/territorial level.While the prevention of firearm violence may represent a to solve—, preventmponents . It has mponents . The Fedmponents . It is aAs the complexity of policing and public health issues increases over time, it is anticipated that the costs associated with firearm injury will continue to rise in parallel. Police and emergency responders continue to experience heightened demands for service and are often inadequately equipped or resourced to respond to these demands. This is particularly evident for cases that require significant investigative resources, man hours, and specialized expertise. In order to effectively respond to and prevent these incidents, it is critical that initiatives to combat firearm violence and injury are appropriately funded, and grounded in evidence-based approaches.The original contributions presented in the study are included in the article/Conceptualization: VC. Data curation: FR, TN, and EK. Writing—original draft preparation and visualization: FR. Writing—review and editing: FR, KT, AZ, NP, TN, EK, VC, and IP. Supervision: IP. All authors have read and agreed to the published version of the manuscript.


Tsw, a Nod-like resistance gene from Capsicum chinense conferring resistance against Tomato spotted wilt virus (TSWV), belongs to the small group of Nod-like receptors with unusually large LRR domains. While typical protein domain dimensions rarely exceed 500 amino acids due to stability constraints, the LRR of these unusual NLRs range from 1,000 to 3,400 amino acids and contain over 30 LRR repeats. The presence of such a multitude of repeats in one protein is also difficult to explain considering protein functionality. Interactions between the LRR and the other NLR domains  take place within the first 10 LRR repeats, leaving the function of largest part of the LRR structure unexplained. Herein we discuss the structural modeling limits and various aspects of the structure-function relation conundrums of large LRRs focusing on Tsw, and raise questions regarding its recognition of its effector NSs and the possible inhibition on other domains as seen in other NLRs.Plant disease immunity heavily depends on the recognition of plant pathogens and the subsequent activation of downstream immune pathways. Nod-like receptors are often crucial in this process.  R) genes, or nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs), that can detect effectors and trigger an immune response , are recognized by pattern recognition receptors (PRRs) on the cell surface. However, effectors secreted by pathogens can suppress this pathway and lead to an infection. Plants in turn have acquired cytoplasmic resistance  adaptor shared by human Apaf1, plant resistance proteins, and nematode CED-4 (ARC) . The NB-Pseudomonas syringae  and the Nicotiana benthamiana TNL Roq1 (resistance against Xanthomonas sp. and Pseudomonas sp.)  studies showed that the Arabidopsis CNL HOPZ-ACTIVATED RESISTANCE 1  transfoerforans . This reesponses . Followinas sp.) .Roq1: 516) and number of LRR repeats  that form the archetype horseshoe shape , has an LRR domain of an estimated 1,652 aa , RPP1: 462, oe shape . The CNL1,652 aa , and thu1,652 aa , while thora sp. . However of 1 aa . The proTsw (A0A1C9TCM9_CAPCH) published by Kim et al. were used to search the C. chinense ‘PI159236’ scaffolds and assembled genome (Chinense.v.1.2 from the Pepper Genome Platform) for the full-length sequence of Tsw. The amino acid sequence of Tsw was also used to search the at the time most recent version of the genome of the susceptible C. annuum cv. CM334 version 1.55 for the full-length non-functional variant of tsw (The amino acid and coding sequences of t of tsw .https://nlrscape.biochim.ro) and analyzed with NLRscape webserver tools. These were further curated by eliminating CC/NBS incomplete and motif missing sequences, and by retrieving their secondary structure propensities and LRR repeat profiles precomputed with LRRpredictor (Tsw homologs sharing more than 30% identity and 50% coverage were retrieved from the NLRscape database (redictor  and NLReredictor  from NLRredictor  and secohttps://nlrscape.biochim.ro) and further processed for analysis. All sequences containing annotated LRR domains at 90% redundancy were collected using NLRscape provided online selection tools. Sequence data, domain annotations, and individual LRR repeat predictions computed with LRRpredictor  originating from plant NLR sequences were retrieved from NLRscape  variant of Tsw in C. annuum  proposed by AlphaFold were in the open ATP active state and none in the inactive ADP resting state, which seems unrealistic. Furthermore, we found that some of the delineation of LRR repeats as well as the local 3D structure in the LRR region proposed by AlphaFold is problematic. This prompted us to develop alternate models allowing to explore a larger configurational space consistent with the local sequence propensity as well as the overall architecture of the LRR domain.Due to some problems that will be clarified below, homology models were built herein by two distinct methods: (1) by using the recently developed AI-based AlphaFold  and (2) Tsw-CC, -NBS and -LRR domains were built using Modeller v10.1  primarily take place within the first four to eight LRR repeats  shares 30% identity to ZAR1 and it preserves all the functionally significant regions: the P-loop, Walker-B, RNBS motifs A through D, the GLPL motif, and the MHD motifs Figure 5C. chinense Tsw variant spans over ~1,550 residues . LRRpredictor delineates 57 LRR repeats, with several lower probability motifs discussed in detail below. Briefly, starting from the N-terminal side, the LRR domain is structured in a core section of 15 LRR repeats (aa 511-901), followed by seven consecutive quasi-identical blocks each consisting of six LRR repeats named A-F. The last repeat, 7-F, is incomplete and followed by a highly acidic tail with high propensity for an intrinsically disordered structure   Figure 6In Tsw, the NB-ARC-LRR linker corresponding to the region between the end of the ARC2 subdomain (aa 495) and the first proper LRR motif (aa 512 - YSHMSI) is nearly double in size to its equivalent region in ZAR1: 17 aa vs. 9 aa. In the cryo-EM structure of ZAR1, this 9 aa linker folds as an improper LRR repeat that is loosely attached to the rest of the LRR domain. In Tsw, its 17 aa equivalent has notable similarities with proper LRR repeats, both in length and secondary structure propensity. It was therefore modeled as such and referred herein as the LRR 0 repeat Figure 6In Tsw, the first seven LRR repeats, with which interactions with CC and NBS are expected to occur, show significant similarities with ZAR1. LRR motif predictions become problematic in the region aa 740-780. The motif probability (aa 761 - SEVVHS) is not very high here, thus this could be modeled either as a single long 44 aa repeat, or as two shorter repeats. Such a motif has a very low LRRpredictor probability. However, the propensity of this stretch for extended secondary structure and forming beta sheets is high. Hence, this was locked as a proper motif in both AlphaFold and adapted-JFRHM models.The AlphaFold oddly models the orientation of LRR 9 in reverse, i.e. with its dorsal side oriented towards the inside of the horseshoe. None of the structurally solved LRR domains contains such an odd repeat orientation. Moreover, this orientation is inconsistent with overall NLR architecture in the resting ADP state, as such a reverse repeat would clash with the NBS domain. Therefore, most likely the local solution adopted by AlphaFold for LRR 9 is an artefact derived from fusing two templates and was corrected in the adapted-JFRHM model.In each of the following seven blocks, LRRpredictor identifies six repeats termed herein A-F. This in contrast to AlphaFold, which merges repeats E and F in a single 53 aa strand. In our A-F delineation, all motifs on the ventral side of the horseshoe are of one of the following extended types: L-x-x-(L-x-L-x-x-L)-x-x-L-x-x/L-L-L-x/L, suggesting a structural reinforcement that was taken into consideration in the adapted models. Interestingly, all the A-F repeats display in addition a stretch of two to three consecutive hydrophobic residues on the dorsal side of the horseshoe in the 14–18 aa region of each repeat. This could potentially form a continuous hydrophobic ‘band’ over the entire structure.C. annuum lacks blocks 3–6, while the core section of block 1 and 7 follows the same LRR organization as the functional variant with only minor sequence variations (94.0% identity at nucleotide level). Apart from the missing LRR blocks, the last 23 aa at the C-terminal end – within the second half of the acidic tail – of tsw has a completely different, highly positively charged, aa sequence due to a frameshift resulting from a 1 nucleotide deletion. This makes a striking distinction between the functional vs susceptible variant, i.e. the C-terminal tail of Tsw being entirely acidic, while the tail of tsw is half acidic, half basic. Such differences could be responsible for the loss of its functionality as a resistance gene.The susceptible variant tsw from Solanaceae clade, the lengths of the LRR domains varies significantly, with most homologs not exceeding two or three repetitive blocks , CC-NBS linker (-7), NBS (-7), and the acidic C-terminal tail (-17), the total net charge of the overall protein becomes -57, which has to be compensated by the environment, or the folding of the protein. Tsw’s total net charge is quite extreme in comparison to other R proteins, for which it generally ranges around -10 to +10. When compensated for by length, the total net charge remains below average.While both the AlphaFold and the JFRHM adapted models display the LRR domain as a continuous solenoid supercoil, it cannot be excluded that the continuity of the LRR domain might be disrupted or altered at the less reinforced regions of the LRR pattern due to the molecular environment or on the large biological time scale.AlphaFold only models Tsw starting from the ZAR1 ATP active state. Moreover, the five best AlphaFold models display the CC domain in a completely different configuration and opposite orientation to that seen in ZAR1-ATP active state Figure 2To further investigate this incompatibility, we analyzed the corresponding CC-NBS and CC-LRR interface on the JFRHM models generated starting from the inactive ADP conformation of ZAR1. As can be seen in 1,-  in contact with the positively charged region at the end of α3 and the outset of the α4 helix of the CC domain . In silico models revealed a niche formed by CYR1 in which MYMIV-CP fitted neatly upon their interaction , and its effector, the coat protein of eraction . Modelineraction . HoweverC. annuum that confers resistance against potyviruses  (IG and RK) and the Romanian Academy programs 1 & 2 of IBAR (EM and A-JP).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Umbelopsis strains with next-generation sequencing (NGS). The identified viruses were named Umbelopsis ramanniana virus 5 (UrV5), 6a (UrV6a); 6b (UrV6b); 7 (UrV7); 8a (UrV8a); 8b (UrV8b); Umbelopsis gibberispora virus 1 (UgV1); 2 (UgV2) and Umbelopsis dimorpha virus 1a (UdV1a), 1b (UdV1b) and 2 (UdV2). All the newly identified viruses contain two open reading frames (ORFs), which putatively encode the coat protein (CP) and the RNA-dependent RNA polymerase (RdRp), respectively. Based on the phylogeny inferred from the RdRp sequences, eight viruses  belong to the genus Totivirus, while UrV5, UrV6a and UrV6b are placed into a yet unclassified but well-defined Totiviridae-related group. In UrV5, UgV1, UgV2, UrV8b, UdV1a, UdV2 and UdV1b, ORF2 is predicted to be translated as a fusion protein via a rare +1 (or −2) ribosomal frameshift, which is not characteristic to most members of the Totivirus genus. Virus particles 31 to 32 nm in diameter could be detected in the examined fungal strains by transmission electron microscopy. Through the identification and characterization of new viruses of Mucoromycota fungi, we can gain insight into the diversity of mycoviruses, as well as into their phylogeny and genome organization.The presence of viruses is less explored in Mucoromycota as compared to other fungal groups such as Ascomycota and Basidiomycota. Recently, more and more mycoviruses are identified from the early-diverging lineages of fungi. We have determined the genome of 11 novel dsRNA viruses in seven different  Totiviridae family [Giardiavirus, Leishmaniavirus, Trichomonasvirus, Totivirus and Victorivirus have been discerned, among which Totivirus and Victorivirus contain the majority of mycoviruses. Recently, two other genera have been proposed, i.e., Artivirus, which contain viruses from arthropods and fishes, and Insevirus, which contain viruses from insects [Totiviridae family have bicistronic, nonsegmented dsRNA genomes of 4.6–7.0 kilobases (kb), which are packaged into icosahedral virus particles with an average diameter of 40 nm [Viruses have been identified in all main groups of fungi ,2. Howeve family ,5. Withi insects . Further insects ,5. Virusof 40 nm . Their gof 40 nm .Umbelopsis belongs to the order Umbelopsidales, a sister group of Mucorales within the basal fungal phylum Mucoromycota [The genus romycota . The genromycota . Some ofromycota ,9,10. Thromycota ,12.Mucor, during which four dsRNA fragments were detected in an isolate of Mucor ramannianus, which is now considered U. ramanniana [Totiviridae family were described in the same isolate [Umbelopsis species, suggesting that virus harboring might be common in this fungal group [Vágvölgyi et al.  were the first to investigate the virus harboring of the genus manniana ,14. LateU. ramanniana were examined for dsRNA carrying. Among them, two contained dsRNA elements. Thus, altogether, seven virus-harboring fungal strains were included in a detailed examination, during which the genome sequence of the identified mycoviruses were determined using whole genome sequencing (WGS), and the presence of virus particles were proven.In the present study, six further isolates of Umbelopsis strains involved in the study are presented in The 30 To screen the strains for the presence of dsRNA elements, the total nucleic acid extraction method of Leach et al.  was usedFor synthesis and amplification of cDNAs from dsRNA fragments, the FLAC method was used as described by Maan et al. 2007  and modiU. gibberispora CBS 109328, U. angularis CBS 603.68, U. dimorpha CBS 110039 and U. versiformis CBS 473.74 samples, the libraries were amplified for 10 PCR cycles, and fragment sizes of 200–300 bp were selected. In the case of U. ramanniana CBS 478.63 and U. ramanniana CBS 243.58 samples, 12 PCR cycles were applied and fragment sizes of 200–500 bp were selected. Sequencing library concentrations and fragment size distributions were determined by capillary gel electrophoresis with a 2100 Bioanalyzer  instrument using an Agilent DNA 1000 Kit. Libraries of shorter  or longer (U. ramanniana CBS 478.63 and U. ramanniana CBS 243.58) fragment sizes were pooled separately. After denaturing pooled libraries were sequenced in an Illumina MiSeq instrument , the pool of shorter (200–300 bp) libraries were sequenced using the MiSeq Reagent Kit v3-150, the pool of longer (200–500 bp) libraries were sequenced using the MiSeq Reagent Nano Kit v2-500, generating 2 × 75 bp or 2 × 250 bp paired-end sequence reads, respectively.Indexed sequencing libraries were prepared from 80 ng viral genomic RNA samples using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina  with NEBNext Multiplex Oligos for Illumina following the manufacturer’s protocol for use with purified mRNA or rRNA-depleted RNA. The main steps of this protocol, in short, are: fragmentation of RNA followed by synthesis of double-stranded cDNA, repair of cDNA ends, the addition of indexed adaptors to cDNA ends by ligation and limited PCR, and purification of the amplified libraries with NEBNext Sample Purification Beads. In the case of Umbelopsis sp.-specific sequences available from the National Center for Biotechnology Information (NCBI) using the BLASTn algorithm.FASTQ sequence files were generated by GenerateFASTQ 1.1.0.64 application of Illumina BaseSpace. Adapter trimming and quality-dependent sequence trimming were done using TrimGalore with parameters: —paired —length 36 —q 20. To filter out host sequences, reads were aligned to subject sequence databases generated from corresponding http://blast.ncbi.nlm.nih.gov/Blast.cgi (accessed on 12 September 2022)). Putative amino acid sequences of the viral proteins were analyzed using the tools of the Expasy Bioinformatics Resource Portal (https://www.expasy.org/ (accessed on 12 September 2022)). Molecular weights were predicted using the Protein Molecular Weight program (https://www.bioinformatics.org/sms/prot_mw.html (accessed on 12 September 2022)). Possible RNA H-type pseudoknots were predicted using the DotKnot (https://dotknot.csse.uwa.edu.au/ (accessed on 12 September 2022)) [http://pseudoviewer.inha.ac.kr/ (accessed on 12 September 2022)) [https://www.ebi.ac.uk/Tools/msa/clustalo/ (accessed on 12 September 2022)) was used.The sequences obtained were compared with those in NCBI nucleic acid and protein databases using the NCBI BLAST tool r 2022))  and visu22)  and Totiviridae, Chrysoviridae and Partitiviridae were obtained from viruSite (http://www.virusite.org/index.php (accessed on 12 September 2022)). The dataset was supplemented by homologous hits of U. ramanniana RdRp sequences [For the phylogenetic analysis, representative RdRp sequences of equences . Sequencequences  with theequences  based onequences . The maxequences  in 5000 Umbelopsis phylogeny was inferred using sequences of the ITS region and part of the Mini-chromosome maintenance complex component 7 (MCM7). Alignments corresponding to the two regions were concatenated and partitioned. The dataset consisted of three partitions . Model testing was performed on the partitioned dataset using the built-in model selection software of IQ-TREE v. 1.6.12. Best-fit models for the partitions were TPM2+F+G4 for the rDNA, TIM2e+G4 for the ITS region and TPM2u+F+G4 for the MCM7 region. Statistical support of the phylogenetic tree that best fits the dataset was tested with ultrafast bootstrap sampling with 5000 replicates.The dsRNA molecules and control plasmids were separated by electrophoresis on 1.0% agarose/TAE  horizontal gels. Separated dsRNAs were denatured by rinsing the gel slides in 0.05 M NaOH and 0.15 M NaCl buffer for 30 min and neutralized in 1 M Tris-HCl and 1.5 M NaCl buffer (pH 7.5) for 2 × 20 min, as described by Hong et al. (1998) . DNA samw/v) sucrose in PBS) centrifugation as described earlier [Virus particles were isolated from 30 g frozen mycelium using the method of Lot et al. (1972)  followed earlier . The pur earlier  and examU. ramanniana strains  of the viral genome assemblies was the following: CBS 109328: 4629 bp contig 216/215/1/471, 4622 bp contig 48.38/46/1/158; CBS 603.68: 4613 bp contig 31/16/1/318, 3846 bp contig 408.2/48/1/7219; CBS 110039: 4753 bp contig 3894/3345/1/11,020, 4629 bp contig 344.6/287/1/1038; CBS 473.74: 221.1/48/1/3224; CBS 478.63: 4977 bp contig 117.2/111/1/224, 4619 bp contig 32.58/34/1/63, 4614 bp contig 18.66/19/1/38 and CBS 243.58: 31.12/32/1/66. The identified sequences were deposited to the NCBI database .Umbelopsis strains , Umbelopsis ramanniana virus 7 (UrV7) and Umbelopsis ramanniana virus 8a (UrV8a), respectively  and Umbelopsis gibberispora virus 2 (UgV2), respectively. For both viruses, ORF1 and ORF2 were predicted to be translated as a fusion protein via a +1 (or −2) ribosomal frameshift .In termined B. The idU. dimorpha CBS 110039, two mycovirus genomes were revealed, and the corresponding viruses were named as Umbelopsis dimorpha virus 1a (UdV1a) and Umbelopsis dimorpha virus 2 (UdV2) (In the sample of 2 (UdV2) B.U. versiformis CBS 473.74 strain, NGS identified a 4624-nt viral genome, which showed 91.4% (CP) and 80.6% (RdRp) identity with the corresponding UdV1a sequences at the amino acid level. Based on this, we named it Umbelopsis dimorpha virus 1b (UdV1b) and regarded as a variant of the UdV1 virus  hybridized to the 5.0-kb fragment . We also identified two genome variants of the same virus (UrV6a and UrV6b) in U. ramanniana CBS 478.63 and CBS 243.58 isolates, which are more closely related to each other than to the other U. ramanniana isolates. In other cases, however, we found that, despite the close phylogenetic relationship of the virus-carrying isolates, the identified virus genomes differed to a greater extent. This can be seen in the case of U. angularis CBS 603.68 and U. gibberispora CBS 109328 isolates. Moreover, the UrV8b virus was identified in the U. angularis CBS 603.68 strain, while UrV8a was detected in the more distantly related U. ramanniana CBS 478.63 isolate.To get an idea about the viral diversity of the genus, the virus harboring feature was compared with the phylogeny of the examined  strains . In thisU. ramanniana NRRL 1296 was already reported [Umbelopsis strains  in reported . In this strains . VLPs abUmbelopsis strains representing 11 species were screened for the presence of mycoviruses and 23% of the tested isolates proved to be virus-harboring. This proportion corresponds to the virus carriage frequencies found within Mucoromycota fungi by other studies. Myers et al. (2020) examined 71 isolates from 30 Mucoromycota genera, and the presence of viruses was detected in 19.7% of the tested isolates [Rhizopus isolates [In our studies , the similarity between the two genomes was minimal, which is supported by their location on the phylogeny (UgV1 is located away from the former group and forms another subclade of d toti 3  and Xantd toti 3 . Althoughylogeny .When virus carriage was compared with the phylogeny of the fungal isolates , it was U. ramanniana NRRL 1296 [Aspergillus fumigatus [Exobasidium sp. [Among the seven tested strains, four isolates contain multiple viruses . MoreoveRRL 1296 . The preumigatus , or fromdium sp. . In agredium sp. ,4,5, ourTotivirus, i.e., an undivided dsRNA genome with two partially overlapping ORFs [Totiviridae and has been reported mainly in the genera Trichomonas and Leishmaniavirus [Totivirus [Genome organization of the identified viruses show the typical features of the genomes in the genus ing ORFs . This aring ORFs . Generaling ORFs . Howeverniavirus ,41. It iotivirus .Umbelopsis strains. Morphology and size of the detected VLPs are typical for the Totiviridae family [U. ramanniana CBS 478.63 and U. gibberispora CBS 109328 with multiple mycoviruses, we detected one-sized VLPs, with 31 or 32 nm in diameter (U. dimorpha CBS 110039, we did not detect any VLPs, which could be explained by the very low attendance of VLPs in this fungus.Approximately the same-sized VLPs, about 31 to 32 nm in diameter, could be observed in all examined e family . In the diameter . This codiameter B. In U. Totiviridae-related viruses from five Umbelopsis species and could obtain an insight into the mycoviral diversity of this fungal genus. In four strains, the presence of multiple viruses was proven. Our results also suggest that a +1 ribosomal frameshift might not be so rare a process in the Totiviridae family as previously reported.In conclusion, we identified and described 11 novel


Genetic recombination is used as a tool for modifying the composition of poxvirus genomes in both discovery and applied research. This review documents the history behind the development of these tools as well as what has been learned about the processes that catalyze virus recombination and the links between it and DNA replication and repair. The study of poxvirus recombination extends back to the 1930s with the discovery that one virus can reactivate another by a process later shown to generate recombinants. In the years that followed it was shown that recombinants can be produced in virus-by-virus crosses within a genus  and efforts were made to produce recombination-based genetic maps with modest success. The marker rescue mapping method proved more useful and led to methods for making genetically engineered viruses. Many further insights into the mechanism of recombination have been provided by transfection studies which have shown that this is a high-frequency process associated with hybrid DNA formation and inextricably linked to replication. The links reflect the fact that poxvirus DNA polymerases, specifically the vaccinia virus E9 enzyme, can catalyze strand transfer in in vivo and in vitro reactions dependent on the 3′-to-5′ proofreading exonuclease and enhanced by the I3 replicative single-strand DNA binding protein. These reactions have shaped the composition of virus genomes and are modulated by constraints imposed on virus–virus interactions by viral replication in cytoplasmic factories. As recombination reactions are used for replication fork assembly and repair in many biological systems, further study of these reactions may provide new insights into still poorly understood features of poxvirus DNA replication. Most of us learn about recombination in introductory genetics. There we studied the pioneering work of T.H. Morgan and his students and how that led to the discovery that inherited traits (genes) reside on chromosomes and can be mapped into linear arrays by analyzing the frequency of recombination between fruit fly genes. In subsequent years, such studies along with H. Muller’s new insights into the origins of mutations and J.B.S. Haldane’s work on population genetics were to form the foundations of what J. Huxley called the “Modern synthesis”. The modern synthesis united Darwin’s theory of natural selection with these and other new 1940s) discoveries in genetics. It served to explain how selection acting upon a population of organisms, each encoding (and sometimes expressing) different combinations of recombining traits, could drive the evolution of a species. Although Eugene Koonin has argued that “The edifice … has crumbled, apparently, beyond beyond repair” [940s discFrom the author’s perspective, a problem with the way introductory genetics is taught is that few of us instructors have time to explain why peas and flies are having sex and recombining genes. The knowledge vacuum can lead students to adduce that recombination mostly exists to eliminate bad gene combinations and generate good ones . HoweveE. coli recA gene was the first recombination gene ever found, using a screen for mutagenized strains that did not recombine in Hfr crosses [recA mutants were also UV-sensitive as are many of the other recombination-defective mutants that were later identified in different organisms. These many disparate observations have been assembled into now coherent models linking replication, repair, and recombination whose development has been well-summarized in helpful reviews [This understanding emerged in the last few decades, although the links between recombination and DNA repair had been known for many years. The  crosses . Even th reviews ,5,6,7. D reviews ,9. Inter reviews , and lam reviews , replicaFar more comprehensive descriptions of the poxvirus infection cycle can be found in many excellent reviews ,19,20,21Collectively, these features of virus biology offer the tools and impetus to look for virus-encoded recombination activities.Poxvirus research has long been associated with smallpox research and dates back to Jenner’s pioneering discovery that one could vaccinate against smallpox using a safer agent than the variola virus (VARV) than in current use . The speThese experiments were complicated by a paucity of genetic markers and the difficulties of assaying them. For example, the phenotypes scored by Fenner and Comben in VACV-rabbitpox crosses included pocks on chorioallantoic membranes, virulence in mice, rabbit lesions, haemagglutin activity, and heat resistance. Despite the challenges, recombinants were still easily recovered leading these authors to offer the conclusion that “Recombination was not an uncommon event…” . These pIn the absence of sequence data, the fact that these methods could be used to delineate the relationships between poxvirus strains was adapted to try and clarify the still uncertain relationships between VARV and other Orthopoxviruses. The pursuit of that goal led Bedson and Dumbell to test whether hybrid viruses could be recovered using chorioallantoic membranes as a culture environment and VARV to reactivate heat-inactivated cowpox and rabbitpox viruses. Using semi-permissive growth temperatures to modulate VARV growth, hybrids were recovered between rabbitpox and VARV minor (a.k.a. Alastrim), and between cowpox and VARV major ,44,45. Tf) in a time-dependent manner [The adoption of tissue culture methods in the 1960s facilitated the production and characterization of new poxvirus mutants; these and other advances in virus biology and biochemistry were reviewed by Joklik in 1968 . Recombit manner . Althougt manner ,52,53 bet manner . In addit manner ,56.HindIII restriction map, which also hinted at what were later shown to be inverted duplications in the telomeres [HindIII-based naming convention that is still used to label VACV genes and proteins [The later 1960s and 1970s was an extraordinarily interesting and productive period as the molecular biology revolution swept through the field of virology. It was shown that VACV infection induced the expression of many other enzymatic activities relating to nucleic acid metabolism including a thymidine kinase , DNA-depelomeres . A legacproteins .During this period there were also some continuing efforts to assemble and genetically map collections of temperature-sensitive and/or drug-resistant mutants ,71,72,73f and physical distance is obscured by experimental noise in virus-by-virus crosses. At distances <20 kbp, these and other experiments detect ~1.5% recombinants per 1 kbp, but the variance in intra- and inter-genic crosses [f-based genetic maps. Linkage is lost at longer distances, but even in long-distance crosses one never recovers 50% recombinants. Since these studies incorporated the necessary two-fold correction for there being two (assumed) reciprocal classes of recombinants, it suggests that other factors operate to limit recombinant production. From a practical perspective, these problems meant that the marker-rescue technologies described in the following sections became the tool of choice for mapping VACV mutations.These studies were followed by more extensive efforts to generate recombination-based maps of VACV genes or genomes ,79,80. T crosses , is geneThe end of the 1970s also marked a significant event, the eradication of smallpox as an endemic disease . As a reThe development of methods for transfecting DNA , led resE. coli beta-galactosidase [Since these initial discoveries, marker rescue has been widely used to map poxvirus genes -loops, or simpler models dependent on single-strand annealing reactions. The models that predicted the existence of a strand invasion step led to the discovery that these reactions were catalyzed by  protein , as well protein . Figure  protein , that inrecombination deficient”) recombination pathway, which employs a 5′-to-3′ exonuclease and Redβ “annealase”, serves as the prototype for these two-component systems [E. coli recA [However, as I discuss below, poxvirus infections do not induce an ATP-dependent strand transferase , nor do  systems . The her systems . Figure oli recA , and humoli recA , pathwaynon-homologous recombination in a much rarer process, although there is no doubt of the biological importance as horizontal gene transfer offers a route by which viruses can acquire and transmit novel traits [In contrast to homologous recombination, poxvirus l traits . In one l traits . Anotherl traits ,128. Thel traits . Curiousl traits ) by mixil traits ,130. Tral traits . Becausel traits , kinds otrans [The observation that any DNA transfected into a poxvirus-infected cell is replicated in trans , providetrans . Similartrans ,134,135)trans .−1 is comparable to what is measured in T4 and λ crosses [A convenient feature of this process is that it generates long concatemers, and so one can transfect λ phage DNA and later convert the extracted recombinant products into phage particles by in vitro packaging. All the genetic tools devised by phage researchers can then be used to study a poxvirus problem. Using these assays, several additional features could be adduced about poxvirus (in this case SFV) recombination systems. Perhaps the most striking feature was that linkage is lost in the transfected DNAs at distances >350–500 bp. This is a high-frequency process where the 0.02% recombinants bp crosses . Even co crosses . Transfe crosses . Some ofE. coli mismatch repair systems acting on the hybrid DNA delivered by the packaged phage. These studies used pairs of λ cI mutations since the clear versus turbid plaque phenotype provided a simple assay for recombinant molecules. Interestingly, ~1% of the phage plaques showed a mottled phenotype, and the effect was enhanced 2–3-fold when the phages were plated on an E. coli mutS strain [High-negative interference is characteristic of systems that produce hybrid DNA and, in the aforementioned experiments, it is caused by S strain . This ofThese data provided the rationale for experiments designed to detect and purify an activity catalyzing the requisite strand transfer reactions. Using a gel-based assay ,141 Fig, panel Ats42 E9L mutation destabilized the recombinase activity in vivo and in vitro. With this highly purified form of VACV DNA polymerase, one can show that the enzyme also catalyzes another reaction  strand was transiently attacked by the 3′-5′ exonuclease and then additional portions of that 3′-ended strand were displaced by the enzyme tracking in a 5′-to-3′ manner relative to the duplex (+) strand. The displaced 3′-ended strand would then be free to hybridize with either the (+) stranded single-strand circle (in the M13 assay) or with sequences exposed by another enzyme attacking a second homologous duplex (in the duplex–duplex assay) . A critiA314T drug-resistant mutation in the exonuclease domain, which enhances excision of newly incorporated CDV molecules [A314T-mutant virus. Many other of the reaction features observed in vitro were subsequently documented in vivo using plasmids as substrates in transfection assays [These I3·E9 catalyzed single-strand annealing reactions clearly belong to the two-component category of recombination schemes while differing from systems like λ Red in that the poxvirus reaction is initiated by a 3′-rather than 5′-exonuclease. The model is unusual and proving that poxvirus recombination employs such a scheme in vivo presents a challenge. A polymerase is required, but is that only because it is needed to replicate the molecules that are being recombined and analyzed? The 3′-5′ exonuclease is special in one sense in that unlike other proofreading exonucleases  the VAColecules . Using bn assays ,155. ThiAlthough a strand-transfer step is needed to catalyze the initial formation of recombination intermediates, other gene products would still be required to process the joint molecules into mature recombinants . For exaThe application of sequencing and genomics technologies has provided further molecular insights into how recombination affects poxvirus biology. An early example is illustrated by the sequencing of portions of the malignant rabbit virus, which showed that it was a natural hybrid . The vir14 doublings (or perhaps more as only a portion of the DNA is packaged into the infectious particles measured in plaque assays) and was associated with at least 18 ± 11 exchanges per genome (The polymorphic sites (SNP’s) that have accumulated during passage and through geographical isolation of smallpox vaccines provide a tool for exploring how much recombination any given genome has been exposed to. This is illustrated by an experiment in which cells were co-infected with strains cloned from North American Dryvax and Chinese TianTan vaccines . These gr genome . BearingThese experiments do also illustrate a still poorly understood feature of poxvirus recombination, regarding how much sequence drift it takes to genetically isolate one family or strain of viruses from another. One SNP per ~140 bp is of little consequence as illustrated by the preceding study. At the other end of the spectrum, hybrids are never recovered when SFV is used to reactivate VACV, where even the most highly conserved gene homologs  are only ~75% identical . PlasmidSequencing has also provided insights into another related genetic phenomenon involving gene amplification under selection for increased gene expression. This was first discovered by Mathews and colleagues while studying the VACV F4·I4 ribonucleotide reductase complex. VACV ribonucleotide reductase is inhibited by hydroxyurea, but continued exposure to the drug selects for resistant mutants. Many of the drug-resistant viruses encoded multiple copies of the F4L gene arrayed as a tandem repeat and flanked by novel joints . The mecIt is conceivable that these tandem duplications are a common but ephemeral feature of poxvirus genomes. One possible explanation for their origin arises from the hypothesis that the primary purpose of the E9-catalyzed reactions might be to promote repair of broken replication forks . Under nf) is calculated using the formula Rf = 100% × NR/(NR + NP), where N equals the numbers of parental or recombinant viruses, any extra contribution of viruses to the Np class ensures Rf will be less than 50%.A characteristic feature of poxvirus-catalyzed recombination reactions is that one never recovers equal numbers of parental and recombinant viruses , even if the markers are located far enough apart to lose the genetic linkage. This is a classical property of phage and virus systems where various phenomena conspire to reduce the maximum recombination frequency below the 50% seen in classical meiosis. Broadly it reflects the fact that some of the input genomes are replicating in isolation from others, for various reasons, and so can yield only progeny of the input parental (P) class. Isolated viruses can, of course, still recombine with replicating duplicates of themselves, but these “invisible” interactions cannot yield genetic recombinants (R). Since the recombinant frequency  or mCherry . These pR relative to NP and thus decrease Rf. Because all these parameters are stochastic in nature, they would also combine to create greater experimental variation. This effect can well explain the difficulties encountered trying to accurately map poxvirus genes using classical genetic crosses to measure recombination frequencies.First and foremost, these methods illustrate the impact that the multiplicity of infection (MOI) has on the likelihood that VACV recombinants might be formed. Of course, MOI will affect the likelihood that a cell gets co-infected with two different parental strains in the first place, but it also affects the likelihood that at some point in the infection two different factories will collide, fuse, and mix their viroplasm. For example, in cells initially infected with five factories only 2–3 collisions and fusions are observed in the first 4 h of the infection . As the trans. These studies recapitulate what is clear from genetic analysis. Intragenomic recombination is a far more rapid and efficient process than what is seen in virus-by-virus crosses.These methods also illustrate something regarding the kinetics of recombinant gene formation . This caFrom these studies one can take away several conclusions. First, there are several impediments which collectively restrict recombination between co-infecting poxviruses. These can be detected when one examines virosome dynamics and the timing of recombinant formation. Secondly, because co-transfected DNAs are delivered in a premixed format into poxvirus factories , these Dtrans-dominant selection” which employs a knock-in, select, and excise-out strategy to produce marker-free recombinants [Recombination reactions provide a well-established tool for genetically modifying poxviruses and are widely used to mutate virus-encoded genes or introduce new ones. The products find uses in discovery research and as vaccines and, more recently, as experimental oncolytics for treating cancers. Many improvements on the technology were devised in the years subsequent to the original descriptions of marker rescue ,89,90. Smbinants ,194. It mbinants , and to mbinants .Another interesting application of these technologies exploits the duplex-to-duplex joining reaction catalyzed by VACV DNA polymerase , panel BE. coli to engineer a bacmid encoding a full-length copy of VACV, and then used fowlpox to rescue the clone as infectious virus [Reactivation and recombination reactions have also found several applications, principally as a tool for more efficiently genetically modifying these viruses. One early example used λ Red recombination in us virus . The utius virus , (they dus virus ). Recombus virus . An SFV us virus . CRISPR us virus .Finally, reactivation reactions have also been used to reconstruct and reactivate poxviruses using DNA fragments obtained through wholly synthetic approaches. The first such example was a 220 kbp horsepox virus , but synThere remain many opportunities for further research into the biology behind poxvirus recombination systems. The most obvious questions concern the relationship between replication and recombination, although, that is greatly complicated by lingering uncertainty about the mechanism of poxvirus replication itself and by the fact that the two processes seem to be inextricably interlinked. A replication-proficient but recombination-deficient mutant has never been identified through a mutant screen , or by ais basically correct, then it remains to be tested whether the strand transfer reactions catalyzed by enzymes like VACV E9 play any role in this process. In particular, can they provide a way to assemble or repair the broken replication forks that would inevitably form when a virus tries to replicate a 150–220 kbp genome? Without some such repair capacity it would be difficult to explain how poxviruses can exhibit particle-to-PFU ratios approaching 1:1 [Ter traps [If this hypothesis hing 1:1  and a mihing 1:1 . Similarhing 1:1 , absent hing 1:1 . Such lier traps ,215,216.There also remain unanswered questions regarding how poxvirus DNA polymerases can catalyze strand transfer reactions. A random selection of commercially available B-family polymerases  will not catalyze the same reactions. The evidence suggests that E9 catalyzes only a limited attack on 3′-ended duplex substrates, and can then melt enough of the remaining duplex end to promote strand annealing if a complementary sequence is present. I3 stimulates the strand transfer reaction, perhaps by transiently trapping greater quantities of exposed single strands and then favouring a search for homology. One clue to how all of this might be accomplished is suggested by the presence of an unusually long β-hairpin “finger” within the 3′-5′ exonuclease domain . This elUltimately any such investigations should be complemented with efforts to assemble an in vitro replication system catalyzed by highly purified proteins. Although it is likely that the polymerase preparation that we first used to demonstrate strand transfer activity was purified in a form complexed with A20 and D4 , better-


Infections by viruses including severe acute respiratory syndrome coronavirus 2 could cause organ inflammations such as myocarditis, pneumonia and encephalitis. Innate immunity to viral nucleic acids mediates antiviral immunity as well as inflammatory organ injury. However, the innate immune mechanisms that control viral induced organ inflammations are unclear.To understand the role of the E3 ligase TRIM18 in controlling viral myocarditis and organ inflammation, wild-type and Trim18 knockout mice were infected with coxsackievirus B3 for inducing viral myocarditis, influenza A virus PR8 strain and human adenovirus for inducing viral pneumonia, and herpes simplex virus type I for inducing herpes simplex encephalitis. Mice survivals were monitored, and heart, lung and brain were harvested for histology and immunohistochemistry analysis. Real-time PCR, co-immunoprecipitation, immunoblot, enzyme-linked immunosorbent assay, luciferase assay, flow cytometry, over-expression and knockdown techniques were used to understand the molecular mechanisms of TRIM18 in regulating type I interferon (IFN) production after virus infection in this study.We find that knockdown or deletion of TRIM18 in human or mouse macrophages enhances production of type I IFN in response to double strand (ds) RNA and dsDNA or RNA and DNA virus infection. Importantly, deletion of TRIM18 protects mice from viral myocarditis, viral pneumonia, and herpes simplex encephalitis due to enhanced type I IFN production in vivo. Mechanistically, we show that TRIM18 recruits protein phosphatase 1A (PPM1A) to dephosphorylate TANK binding kinase 1 (TBK1), which inactivates TBK1 to block TBK1 from interacting with its upstream adaptors, mitochondrial antiviral signaling (MAVS) and stimulator of interferon genes (STING), thereby dampening antiviral signaling during viral infections. Moreover, TRIM18 stabilizes PPM1A by inducing K63-linked ubiquitination of PPM1A.Our results indicate that TRIM18 serves as a negative regulator of viral myocarditis, lung inflammation and brain damage by downregulating innate immune activation induced by both RNA and DNA viruses. Our data reveal that TRIM18 is a critical regulator of innate immunity in viral induced diseases, thereby identifying a potential therapeutic target for treatment.The online version contains supplementary material available at 10.1186/s12929-022-00840-z. Innate immunity is the first line of defense against invading pathogens including RNA and DNA viruses. Activation of innate immunity requires the recognition of pathogen-associated molecular patterns (PAMPs), such as atypical viral nucleic acids, by pattern-recognition receptors (PRRs) on innate immune cells . RecogniViruses such as severe acute respiratory syndrome coronavirus (SARS-CoV) and influenza virus are major threats to human health , since tIn the present study, we found that the E3 ligase TRIM18 served as a negative regulator of viral myocarditis, pneumonia, and encephalitis by downregulating innate immune activation against both RNA and DNA viruses. TRIM18 was shown to recruit protein phosphatase 1A (PPM1A) to dephosphorylate TBK1, which inactivates TBK1 and blocks its interactions with upstream adaptors MAVS and STING, thereby dampening type I IFN mediated antiviral signaling during viral infections. Given the critical role of TRIM18 in controlling viral pathogenicity and antiviral innate immunity against RNA and DNA viruses, TRIM18 may serve as an important therapeutic target for controlling viral-induced diseases including COVID-19.Trim18 knockout (KO) mice were originally generated as described previously [eviously  and cryoThe high molecular weight poly I:C (Cat: tlrl-pic), 5’triphosphate double-stranded RNA , HSV-60 (Cat: tlrl-hsv60n), 2′3’-cGAMP , and LPS (Cat: tlrl-3pelps) were from Invivogen. Lipofectamine 3000 (Cat: L3000015) was from Invitrogen. The following antibodies were used for immunoblot analysis: anti-TRIM18 , anti-MAVS , anti-STING , anti-IRF3 , antibody to phosphorylated IRF3 at Ser396 , anti-TBK1 , antibody to phosphorylated TBK1 , anti-MAVS , anti-GAPDH , anti-β-actin , anti-HA , anti-Myc , peroxidase affinipure goat anti-mouse light chain specific IgG , and peroxidase mouse anti-rabbit light chain specific IgG . Anti-HA and anti-Myc agarose beads were from Sigma. Lentiviral vectors for shRNA were from Dharmacon Inc. (Horizon Discovery Group company): TRIM18 (clone TRCN0000019813); MAVS (clone TRCN0000146651); STING (clone TRCN0000161052). The IFN-β and IFN-α enzyme-linked immunosorbent assay (ELISA) kits were from PBL Interferon Source. The IL-6, TNF-α and IL-1β ELISA kits were from R&D systems. The Dual-Luciferase Reporter Assay System (E1910) was from Promega. Influenza A virus (PR8 A/Puerto Rico/8/1934(H1N1)), Coxsackievirus B3 , human adenovirus (Type 5) and herpes simplex virus type I  were from ATCC .Human THP-1 cells (ATCC® TIB-202™) were differentiated to macrophages (THP-1 macrophages) with 60 nM phorbol 12-myristate 13-acetate  for 16 h, and cells were cultured for an additional 48 h without PMA –40.The pLKO.1 lentiviral vector carrying a scrambled shRNA or target gene sequences (Open Biosystems) were co-transfected into HEK 293FT cells  with packaging plasmids psPAX2 (Addgene 14858) and pMD2.G (Addgene 12259) using lipofectamine 3000 (ThermoFisher Scientific) for producing lentivirus. Human THP-1 macrophages were infected by lentivirus as previously . After 2Trim18+/+ (WT) and Trim18−/− (KO) male mice  were injected intraperitoneally with CVB3 (1 × 107 PFU/mouse). The survival of mice was monitored daily for 14 days after CVB3 infection. At day 2 after infection, mice were euthanized, and whole hearts were excised into PBS and homogenized for determining viral titers and concentrations of cytokines by ELISA. Additionally, the heart weight was determined by scale before and after CVB3 infection for 5 days.For the in vivo CVB3 infection study, age-matched Trim18+/+ (WT) and Trim18−/− (KO) mice  were infected intranasally with Flu PR8 (1 × 105PFU/mouse) in 50 µl PBS after anesthesia [ g, 5 min) to isolate cells and supernatants were centrifuged again  to completely remove remaining cells. The concentration of cytokines in BALF were measured by ELISA.For the in vivo influenza PR8 virus (Flu PR8) infection study, age- and sex-matched esthesia . The surTrim18+/+ (WT) and Trim18−/− (KO) mice  were infected with HSV-1 (5 × 106PFU/mouse) by intravenous injection [For the in vivo HSV-1 infection study, age- and sex-matched njection . The surTrim18+/+ (WT) and Trim18−/− (KO) mice  were infected intranasally with adenovirus (1 × 108PFU/mouse) in 50 µl PBS after anesthesia [For the in vivo adenovirus infection study, age- and sex-matched esthesia . At day After CVB3 or Flu PR8 infection in mice, total heart or lung tissues were removed and homogenized to prepare heart or lung extracts in 1 ml of PBS (pH 7.4). The supernatants from the homogenized heart or lung tissues were diluted and then used to infect confluent HeLa cells (ATCC® CCL-2™) or Madin-Darby canine kidney (MDCK) cells (ATCC® CCL-34™) cultured on 12-well plates, respectively. At 1 h post-infection, the supernatant was removed, and 2% low melting-point agarose was overlaid. At 3-days post-infection, the overlay was removed, and cells were fixed with methanol: acetic acid solution (3:1 of methanol: acetic acid) for 20 min and stained with 0.2% crystal violet. Plaques were counted, averaged, and multiplied by the dilution factor to determine viral titer , 42–44.After HSV-1 or adenovirus infection in mice, total brain or lung tissues were removed and homogenized to prepare brain or lung extracts in 1 ml of PBS (pH 7.4). The supernatants from the homogenized tissues were diluted and then used to infect confluent Vero cells (ATCC® CCL-81™) or A549 cells (ATCC® CCL-185™) cultured on 12-well plates, respectively. At 1 h post-infection, the supernatant was removed, and 2% low melting-point agarose was overlaid. At 3-days post-infection, the overlay was removed, and cells were fixed with methanol: acetic acid solution (3:1 of methanol: acetic acid) for 20 min and stained with 0.2% crystal violet as described above for the HeLa and MDCK cells. Plaques were counted, averaged, and multiplied by the dilution factor to determine viral titer , 45, 46.To obtain differential cell counts in BALF samples, a 100 µL aliquot of cells was subjected to cytocentrifugation , air dried, and stained with a Hema 3 staining kit (Fisher Scientific) and a modified Wright–Giemsa stain . DiffereTransthoracic echocardiography was performed at day 4 after CVB3 inoculation. Mice were anesthetized by isoflurane inhalation. A comprehensive echocardiographic study was performed, including 2-dimensional imaging and M-mode imaging using the Vevo 2100 system  .Trim18+/+ (WT) and Trim18−/− (KO) mice respectively, while lungs were removed from mock as well as Flu PR8 or adenovirus infected Trim18+/+ (WT) and Trim18−/− (KO) mice. These removed tissues were washed using PBS before being fixed with 10% formaldehyde for 24 h at room temperature. The tissues were embedded in paraffin and processed by standard techniques. Longitudinal 5-μm sections were stained with Hematoxylin & Eosin (H&E) [Hearts and brain were removed from mock and CVB3 virus or HSV-1 infected in (H&E) , 47, 49.The severity of myocarditis in the hearts of CVB3 infected mice was scored as follows: 0, no lFor immunohistochemistry (IHC) staining, paraffin-embedded hearts were cut transversely into 5-μm sections. Following a 5-min high-pressure antigen retrieval process in citrate buffer with a pH of 6.0, the heart sections were blocked with 10% bovine serum albumin for 60 min and were subsequently incubated overnight at 4 °C with the primary antibodies including anti-neutrophil Rat antibody , anti-macrophage Rat antibody , anti-NK cell marker mouse antibody , anti-T cell CD3 Rabbit antibody , anti-TRIM21 Rabbit antibody  and anti-TRIM18 Rabbit antibody . Binding was visualized with the appropriate peroxidase-conjugated secondary antibodies (Horseradish Peroxidase AffiniPure Goat Anti-Rat or Anti-Rabbit IgG (H + L), 111-035-003, Jackson ImmunoResearch) for 30 min at 37 °C.For the preparation of purified TRIM18 and PPM1A, HEK293T cells were transfected with expression plasmids encoding full-length or truncated versions of HA- or Myc-tagged TRIM18 or PPM1A. Lysates were prepared from the transfected cells, followed by incubation with anti-HA or anti-Myc beads. Proteins were eluted from the beads after beads were washed six times with PBS. For precipitation with anti-HA or anti-Myc beads, purified HA-tagged wild-type PPM1A or truncations of PPM1A were incubated for 2 h with purified Myc-tagged TRIM18 or purified HA-tagged TRIM18 or truncations of TRIM18 were incubated for 2 h with purified Myc-tagged PPM1A. Beads were added; after 2 h of incubation, the bound complexes were pelleted by centrifugation. Proteins and beads were analyzed by immunoblot analysis with anti-HA or anti-Myc Abs. For immunoprecipitation of endogenous proteins, the whole-cell lysates of WT and TRIM18 KO BMDM that were either mock infected or infected with CVB3 or adenovirus were incubated with anti-PPM1A, anti-TBK1 or immunoglobulin G antibodies. After 2 h of incubation, the protein A/G beads were added for another 4 h incubation, and the bound complexes were pelleted by centrifugation. Proteins and beads were analyzed by immunoblot analysis with anti-TRIM18, anti-PPM1A, anti-TBK1, anti-MAVS, and anti-STING antibodies. WT and TRIM18 KO BMDM were either mock infected or infected with CVB3 or adenovirus for 2 h, and were then lysed in 1% NP-40 lysis buffer  supplemented with protease inhibitor (ThermoFisher Scientific) followed by centrifugation. Supernatants were collected and incubated with SDS sample buffer by boiling of samples for 8 min followed by SDS-PAGE and immunoblot analysis. Immunoblot films were scanned by CanoScan 9000F mark II and images were processed with Adobe Photoshop Creative Cloud (CC) 2019 (version 20.0.10).HEK 293 T cells were seeded in 24-well plates and transfected the following day with the IFN-β firefly luciferase reporter vector , TK-renilla luciferase reporter vector (5 ng), Flag-MDA5 (100 ng), Flag-MAVS (50 ng), Flag-TBK1 (100 ng), Flag-IKKi (100 ng), Myc-cGAS (50 ng) and Myc-STING (50 ng) together with increasing doses of HA-TRIM18 plasmid  or HA-vector by Lipofectamine 3000 transfection (ThermoFisher Scientific) as per the manufacturer’s instructions. At 24 h after transfection, cells were lysed and then measured using a dual-luciferase reporter assay system (Promega) according to the manufacturer’s instructions.RNA was isolated using the RNeasy Kit (Qiagen) according to the manufacturer’s instructions. The isolated RNA was used to synthesize cDNA with the iScript cDNA Synthesis Kit (Bio-Rad). The quantitative RT-PCR (qRT-PCR) was performed on a CFX-96 real-time PCR detection system (Bio-Rad) with iTaq Universal SYBR Green Supermix (Bio-Rad). All qRT-PCR primers were listed in Additional file Trim18+/+ (WT) and Trim18−/− (KO) mice. The cells were then fixed and stained with Zombie Aqua fixable viability kit , FITC anti-mouse CD45 antibody , Brilliant Violet 785 anti-mouse CD11b antibody  and PE/Cyanine7 anti-mouse F4/80 antibody  (1 μl antibody for 2 million cells) for analyzing composition of CD11b+F4/80+ macrophages. Flow cytometry data were acquired on an LSR-II flow cytometer (Beckton Dickinson) and analyzed using FlowJo v10 software (Tree Start) [Mouse spleen cells were isolated from e Start) , 38, 44.https://www.genecards.org/cgi-bin/carddisp.pl?gene=MID1&keywords=TRIM18). Mouse TRIM18 expression was evaluated from Immunological Genome Project (ImmGen) ULIRNASeq dataset (GSE127267) obtained using skyline data viewer from ImmGen website (http://rstats.immgen.org/Skyline/skyline.html).For metadata analysis, human TRIM18 expression was evaluated using the publicly accessible database Genecards  and Microsoft Office Excel 2016. Statistical significance is calculated using Student’s two tailed unpaired t test. The log-rank (Mantel-Cox) test is used for survival comparisons. NS, not significant (p > 0.05); *p < 0.05; **p < 0.01; ***p < 0.001.Previously, we screened all 70 members of the TRIM family proteins in the mouse alveolar macrophage cell line MH-S by a small-interfering-RNA approach and identified TRIM29 as a crucial negative regulator in antiviral innate immunity . MeanwhiTo further assess the role of TRIM18 in antiviral innate immunity against virus infection, we analyzed protein expression of TRIM18 in normal tissues from GeneCards . TRIM18 Trim18 gene deletion was confirmed by PCR assisted genotyping analysis  mice. The To further investigate the role of TRIM18 in response to RNA viruses, we prepared mouse BMDM from WT and TRIM18 KO mice, and stimulated BMDM with dsRNA poly I:C and 5′-triphosphate RNA (5’pppRNA), and measured type I IFN proteins (IFN-α and IFN-β) by ELISA as well as mRNA levels of interferon stimulated gene 15 (ISG15) and ISG56 by qRT-PCR. The results showed that TRIM18 KO BMDM produced much more IFN-α and IFN-β proteins Fig. a, b and To further determine the role of TRIM18 in response to DNA viruses, we prepared mouse BMDM from WT and TRIM18 KO mice, and stimulated BMDM with dsDNA HSV60 and cGAMP (STING stimulator in DNA sensing pathway), and measured type I IFN proteins by ELISA and mRNA levels of ISG15 and ISG56 by RT-qPCR. Consistent with the earlier results, TRIM18 KO BMDM produced much more IFN-α and IFN-β proteins Fig. e, f and Viral myocarditis has been recognized as a cause of congestive heart failure and CVB3 infection is the main cause of viral myocarditis , 21. SinViral pneumonia is an inflammation of the lungs caused by respiratory viruses, such as influenza virus, adenovirus and SARS-CoV-2 causing the ongoing pandemic of COVID-19 , 24. IntNext, we evaluated the importance of TRIM18 in controlling pneumonia following infection by respiratory DNA virus adenovirus in vivo. Both WT and TRIM18 KO mice were infected intranasally with adenovirus, which is normally transmitted by the nasal route and targets the lungs for pneumonia. Lung histopathology revealed much-reduced edema, alveolar hemorrhage, alveolar wall thickness, and neutrophil infiltrations in TRIM18 KO mice compared to the lung pathology in WT mice Fig. h, i, indHerpes simplex encephalitis (HSE) is caused by HSV-1 infection of the brain and is the most common cause of sporadic fatal encephalitis worldwide. Given tTo determine the molecular mechanisms by which the enhanced production of type I IFN and innate immune activation were achieved in BMDM from TRIM18 KO mice, we isolated BMDM from WT and TRIM18 KO mice and infected the cells without or with CVB3 and adenovirus for 1 h, then assessed activation of the transcription factors IRF3 by immunoblot analysis. We found that phosphorylation of IRF3 in TRIM18 KO BMDM was enhanced relative to WT BMDM after infection with CVB3 or adenovirus Fig. a. TypicaNext, we investigated if TRIM18 could interact with PPM1A in BMDM at the endogenous protein level. The anti-PPM1A antibody, but not control IgG, precipitated TRIM18 in WT BMDM, but not in TRIM18 KO BMDM Fig. b, indicaBecause TRIM18 is an E3 ubiquitin ligase and less PPM1A is seen in TRIM18 KO cells, we surmised that TRIM18 may mediate the stability of PPM1A. Consequently, we next determined whether TRIM18 was responsible for the ubiquitination of PPM1A ex vivo. We isolated BMDM from WT and TRIM18 KO mice and infected those cells with CVB3 or adenovirus for 6 h. Cell lysates were then prepared and analyzed for the ubiquitination of PPM1A. As results, PPM1A was modified via K63-mediated ubiquitination in BMDM from WT mice but not TRIM18 KO mice Fig. a. Additi.PPM1A contains twenty-two lysine residues. Thirteen of these  were predicted to be possible ubiquitination sites with high scores by the BDM-PUB program (Additional file The elucidation of immune regulatory mechanisms is critical to understanding how the host constrains adventitious inflammation to maintain immune homeostasis. In the present study, we demonstrate an essential role of E3 ubiquitin ligase TRIM18 in serving as negative regulator of antiviral innate immunity against organ inflammations induced by RNA and DNA viruses (Fig. Accumulating evidence suggests that members of the TRIM family play versatile roles in antiviral immunity , 58. A pTBK1 is a key adaptor protein shared by both RNA and DNA sensing pathways and is crucial for the activation of IRF3 and subsequent type I IFN induction , 72. TBKIn summary, we have identified TRIM18 as a novel negative regulator of viral myocarditis, lung inflammation and brain damage by downregulating innate immune activation against both RNA and DNA viruses. Our results underscore the pivotal role of TRIM18 in controlling both RNA and DNA virus infections, where TRIM18 provides a safeguard against aberrant and excessive type I IFN production as well as potential tissue damage and autoimmunity diseases. Importantly, the ongoing COVID-19 pandemic serves as a reminder that the new emerging RNA viruses remain a significant public health threat . Thus, oAdditional file 1: Table S1. PPM1A is in the TRIM18-binding protein complex. Table S2. The potential ubiquitination sites at the PPM1A molecule. Table S3. Primers for qRT-PCR and PCR used in this study.Additional file 2: Fig S1. TRIM18 inhibits production of type I IFN by human THP-1 macrophages after stimulation with dsRNA and dsDNA, but not LPS. Fig S2. TRIM18 negatively regulates IFN-α production in human THP-1 macrophages after infection with RNA and DNA viruses. Fig S3.Trim18 gene targeting and TRIM18 expression in mouse macrophages and different tissues. Fig S4. TRIM18 does not affect expression of differentiation markers CD11b and F4/80 in mouse splenic macrophages. Fig S5. Knockout of TRIM18 enhances production of ISG15 and ISG56 in BMDM in response to dsRNA and dsDNA stimulations or infection with RNA and DNA viruses. Fig S6. TRIM18 is induced in human patients with SARS-CoV infection. Fig S7. TRIM18 inhibits IFN-β reporter activation mediated by overexpression of MDA5, MAVS, TBK1 and cGAS/STING, but not IKKi.


Although the feasibility of ultrasound stimulation has been reported in preclinical and clinical settings, its mechanistic understanding remains limited. While optical microscopy has become the “gold standard” tool for investigating population-level neural functions in vivo.We aimed to develop a transparent acoustic transducer based on a glass coverslip called the acousto-optic window (AOW), which simultaneously provides ultrasound neuromodulation and microscopic monitoring of neural responses The AOW was fabricated by the serial deposition of transparent acoustic stacks on a circular glass coverslip, comprising a piezoelectric material, polyvinylidene fluoride-trifluoroethylene, and indium-tin-oxide electrodes. The fabricated AOW was implanted into a transgenic neural-activity reporter mouse after open craniotomy. Two-photon microscopy was used to observe neuronal activity in response to ultrasonic stimulation through the AOW.The AOW allowed microscopic imaging of calcium activity in cortical neurons in response to ultrasound stimulation. The optical transparency was in vivo.The AOW is an implantable ultrasonic transducer that is broadly compatible with optical imaging modalities. The AOW will facilitate our understanding of ultrasound neuromodulation  However, most conventional ultrasound transducers are barely compatible with microscopy systems. First, the form factors of conventional ultrasound transducers are typically large compared to the several millimeter-scale working distances of a typical high numerical aperture objective lens.,,Two-photon microscopic imaging on a cranial window model is a well-established approach to observe spatiotemporal activities of specific neuronal and glial subtypes longitudinally in vivo using two-photon microscopy.Here, we propose a solution to this technical challenge by introducing an optically transparent acoustic transducer, termed the acousto-optic window (AOW). The AOW simultaneously allows ultrasound stimulation over a defined brain area and two-photon fluorescence imaging of neuronal activity through the transducer. We describe the fabrication of an AOW based on a transparent piezoelectric material and its surgical implantation on a mouse brain under anesthesia and demonstrate the feasibility of recording ultrasound-evoked functional activity in a genetically specified neuronal subtype 22.1Surface of glass coverslip was cleansed using a series of solutions of acetone , degenerative alcohol , 1% water dissolved detergent , and deionized water. After masking the glass coverslip with a Kapton masking tape  excluding the central region  with an electrical transmit pulse of 1 ns of The acoustic beam profile of the AOW was measured using a needle hydrophone  in a tank filled with deionized water. The needle hydrophone was installed in a custom-made motorized three-axes stage. The beam profile of the AOW was scanned with a resolution of spatial translation along the axial and lateral directions of 100 and 2.3All mice were housed with littermates in groups of two to five in a reverse day/night cycle and provided ad libitum access to food and water. Male or female C57BL6J wild-type mice aged 8 to 16 weeks (Orient Bio) were used to image the cortical vasculature. Male or female Gad2-GCaMP6-tdTomato mice aged 8 to 16 weeks were obtained by crossing Gad2-IRES-Cre  and CAG-floxed-GCaMP6f-tdTomato . All animal experiments were performed in compliance with institutional guidelines and were approved by the Subcommittee on Research Animal Care of Seoul National University.2.4The AOW was implanted over the somatosensory cortex following the procedures previously described for the cranial window model.l cement . After t2.5The imaging procedure was initiated 2 h after AOW implantation under isoflurane anesthesia. For the vascular imaging, a wild-type mouse received retro-orbital injection of tetramethylrhodamine isothiocyanate-dextran  and high-power LED . Two-photon microscopy images were processed using ImageJ FIJI (NIH) and MATLAB (MathWorks). Image processing included adjustment of brightness and contrast, averaged projection of 2.7Statistical and regression analyses were performed using the Prism software (GraphPad). Data are expressed as mean ± Standard Error of the Mean (SEM), unless otherwise indicated. A one-way ANOVA (Dunnett’s multiple comparison test) is shown in 33.1–in vivo. Considering the small form factor of the glass coverslip, we designed the transducer with an area of To fabricate an AOW capable of being implanted on mouse cranium, we formed transparent acoustic stacks on a circular glass coverslip with a diameter of As shown in  1000 nm . In two-3.2To characterize the acoustic performance of AOW, we first simulated the pulse-echo impulse response [center frequency of 24 MHz with As shown in the impedance curve in Because the use of higher frequencies -dextran [For -dextran . Owing t-dextran , the inp3.4Finally, we tested whether the AOW allows the microscopic observation of ultrasound-mediated neuromodulation. We used a Gad2-GCaMP6-tdTomato mouse line to measure functional calcium activity in a subset of inhibitory interneurons. Two-photon imaging through AOW provided near-artifact-free microscopic imaging of subcellular neuronal structures in Gad2-GCaMP6-tdTomato mice .The relatively bright tdTomato signal helped us to confirm the minimal influence of movement artifacts from ultrasonic waves or physiologic motions. Upon ultrasound stimulation at an operation frequency of 10 MHz, a subset of fluorescent interneurons showed a reliable increase in calcium signal with the 10-s stimulation . To furt4in vivo. However, the simultaneous monitoring of neuronal responses with ultrasound stimulation is hindered by the conventional transducers and the glass coverslip used in a cranial window model, which increases the attenuation of ultrasonic waves. To apply ultrasound stimulation at the same position as the optical field of view, the stimulant transducer needs to be positioned to introduce an acoustic beam between the optical lens and cortical surface. We presently observed that the incidence of lower frequency along the normal direction reduced the postglass intensity by only 27% and that the ultrasound beam intensity introduced at an angle of 45 deg was reduced by 97% in the postglass region . Therefore, a transparent transducer loaded directly on a glass coverslip is necessary for studies that simultaneously combine confocal microscopy monitoring with ultrasound brain stimulation.There is an increasing need for two-photon microscopic imaging in ultrasound neuromodulation investigations of neuronal functions Presently, we describe the AOW, an optically transparent acoustic transducer that can be implanted into the mouse brain under anesthesia for monitoring ultrasound-mediated neuromodulation with microscopic precision. An optical transmittance of Supplemental Material. The opacity of the piezoelectric could be increased with an increase thickness because of the inherent low transparency of the PVDF-TrFE ,For more generalized applications, several technical improvements in AOW remain to be addressed. The current version of AOW provides a limited tuning range for ultrasound parameters, such as the frequency and input voltage range. The operational frequency can be further lowered by changing the thickness of the glass coverslip and PVDF-TrFE.,,Unlike the instantaneous excitatory responses to the short ultrasound stimulations nse peak . The del,Finally, the compatibility of the AOW with optical imaging allows the monitoring of neuronal activity in response to ultrasound stimulation. The fusion of advanced imaging technologies with the AOW would help understand the extensive effect of ultrasound on neuromodulation, as the performance was demonstrated using two-photon microscopy. Although two-photon microscopy allowed the monitoring of neuronal activities from a single in-plane imaging slice, the activities from out-of-focus planes could not be acquired. Robust volumetric imaging techniques, such as the Bessel-beamClick here for additional data file.


The attachment or entrapment of microbial cells and enzymes are promising solutions for various industrial applications. When the traps are beads, they are dispersed in a fluidized bed in a vessel where a pump guarantees fresh liquid inflow and waste outflow without washing out the cells. Scientific papers report numerous types of cell entrapment, but most of their applications remain at the laboratory level. In the present research, rigid polymer beads were manufactured by two different additive manufacturing (AM) techniques in order to verify the economy, reusability, and stability of the traps, with a view toward a straightforward industrial application. The proposed solutions allowed for overcoming some of the drawbacks of traditional manufacturing solutions, such as the limited mechanical stability of gel traps, and they guaranteed the possibility of producing parts of constant quality with purposely designed exchange surfaces, which are unfeasible when using conventional processes. AM proved to be a viable manufacturing solution for beads with complex shapes of two different size ranges. A deep insight into the production and characteristics of beads manufactured by AM is provided. The paper provides biotechnologists with a manufacturing perspective, and the results can be directly applied to transit from the laboratory to the industrial scale. It has led to considerable research for industrial applications, such as food industries, enzyme biosynthesis , hydrocaEven though the literature reports on numerous types of matrices and beads being locked up in a fluidized bed, as early as 2004, Junter and Jouenne  reportedThe complex shapes, dimensions, and mechanical resistance required by the beads make AM a viable manufacturing solution for their production. Despite this potential, only a few papers have investigated the bead manufacturing process and the accuracy/deviation versus the nominal shape of such beads. A majority of the literature deals with the efficiency and biological mechanisms involved in using the beads. Dong  and BelgThe experimental plan included two different lines:3.0, 2.0, and 1.5 mm-diameter beads manufactured by vat photopolymerization  using unreinforced and charged (10 wt.% and 20 wt.% hydroxyapatite) acrylic acid ester resin (Vitra DL375)15 and 30 mm-diameter lattice beads manufactured by PBF  using polyamide 12, with three different repetitive units and three strut dimensionsTwo material/process combinations were thus considered, both of which were within the optimal size range. The two AM systems are sketched in washing in 96% ethyl alcohol with the aid of compressed air to remove the unpolymerized resin from the as-built partfurther ultrasonic washing in ethyl alcohol to ensure the unpolymerized resin removal from the bead cavityUV oven post-curing for 20 minutesremoval of the supports followed by a surface finishThe fragile and sensible-to-tensile stress of acrylic acid ester has a 1% polymerization shrinkage that causes residual stresses; therefore, it requires a specific manufacturing strategy for avoiding deformations and ruptures. The manufacturing strategies consisted of arranging the beads in continuous chains of balls either built along a spiral  or suppoPreliminary tests were performed to verify the feasibility of using 200 µm holes. The results showed that this diameter was at the lower boundary of the machine’s capability. Some of the holes were thorough and well-defined, while occasionally, the holes were closed. Therefore, in the design of beads, the hole dimensions were increased to 500 µm for the 3 and 2 mm-diameter beads and to 350 µm for the 1.5 mm-diameter beads.c indicates the energy required to overcome the critical threshold of the polymerization activation process (mJ/mm2). The penetration coefficient Dp is an experimentally determined characteristic of the resin, and it expresses the distance at which the irradiance decreases by 1/e2 (37%). Therefore, the energy E required to reach the critical value  at a depth zp is provided by:In the simplified relationship reported in Equation 2 , Ec indiEleven different types of beads were produced using PBF while ten types of beads were produced by vat photopolymerization.2) for each specimen using the confocal head on a Nikon Eclipse LV150N  optical microscope and using a 200× magnification objective with a Z scanning interval of 0.20 mm. The point data were post-processed by Mountains Map software using a Gauss filter set of 250 mm. The surface roughness has been recognized to have an effect on the first steps of the interaction between the cells and the entrapment media [The diameter of the beads was measured by a Nikon SMZ1270  optical microscope. With respect to previous studies on alginate beads of simple shapes , in thisThe hole replicas were obtained using the STC SHAPE-IN injection method. This method consists of pouring bicomponent silicone material (poly-vinyl siloxane) at room temperature, and after curing for approximately 50 min at room temperature, a silicone rubber replica (which has high elasticity) is obtained. It is important to ensure that the silicone material completely fills the bead at the holes, and for this purpose, the injection is continued until the silicone material fills half of the bead. As a consequence, to allow the extraction of the replica, the bead is broken. The optical measurement procedure was also applied to the replicas in order to verify the hole accuracy. This approach allowed for a dimensional verification of the hole along its entire length as direct measurements of the beads would only provide information about their outer surfaces.Both bead typologies produced by PBF and vat photopolymerization were investigated using scanning electron microscopy (SEM) ,29 and XThe measurements of the hole diameters showed a different shape from the nominal circular one. In fact, the hole had a more elliptical shape, with values ranging from a minimum of 0.43 ± 0.04 mm and a maximum of 0.57 ± 0.04 mm.00 laser). Typically, the photopolymer resin requires a UV inhibitor, and its function is to prevent UV rays from penetrating excessively into the already-consolidated layers, which would cause solidification of the resin in undesired areas. If Equation (2) is considered, the presence of HA could partially diminish the energy available for the photopolymer, and therefore, at the periphery of the spot, where the incident energy was lower because of the Gaussian distribution, the reinforced resin could not reach an energy value sufficient to trigger polymerization. This would turn into a different degree of polymerization in the core and contour areas. Around the hole, the material under the peripheral spot area toward the center of the hole would not be completely polymerized, while that at the antipodes (but facing the fill zone) would undergo complete polymerization due to the adjacent passes in the core region. It can be conjectured that, in the washing phase, the non-polymerized material would be removed, whereas the HA may remain attached to the solidified resin. The post-processing phase in the UV oven would then consolidate the bond. Therefore, the observed higher concentration of HA around holes may have been due to the subtraction of the matrix from the boundary of the hole (The SEM observations highlighted that when the sizes of the uncharged beads decreased, a localized wall subsidence could occur in the anchoring point during the support removal. An example of this is shown in the hole .The observation by the XmCT scan and the reconstruction of the bead model showed that although an internal alteration of the surface characteristics appeared in the zone where the staircase effect was most remarkable, the wall was substantially intact . The XmCThe production rate depends on the bead size and post-treatment time. Vat photopolymerization allows for the manufacture of approximately 100–150 uncharged beads per hour, with a cost of approximately 0.3–0.4 euros per part. The production rate of charged beads decreases by approximately 15–20% due to the preliminary mix phases.The diameters of the thicker struts were nearly half of the nominal value, while the thinner struts were produced with better accuracy. The closed porosity increased in accordance with the thickness of the strut.The images reconstructed by XmCT showed that some struts were broken and others had non-constant sections and discontinuities closed to the nodes . The defTo summarize the feasibility of the beads made using PBF, the manufacturing limits are primarily attributable to the unconsolidated powder-emptying. The poor removal repeatability depended on the structure geometries and the partial sintering of the powder inside the beads. The widespread presence of defects, such as the strut thickening or the ruptures induced by residual stresses, depended on the heat distribution during the process. The tests showed that the production limit was not due to the minimum achievable definition, but rather, to the emptying capacities of the shapes produced.The measured surface roughness of the beads on the upskin surface was between 5.66 and 9.06 μm.The production capacity of the beads built by PBF ranged between 6 and 60 parts per hour, depending on the bead dimensions, with a cost of between 0.50 and 4 euros per part.Overall, the production of beads for cell immobilization by AM, as conjectured in the previous literature , proved Vat photopolymerization proved to be a viable method for the production of beads with a minimum hole size of 200 µm and with a satisfactory surface roughness. By using resin reinforced with HA, a higher accuracy was obtained as the filler acted as a UV blocker, but the HA concentration was not uniform at the hole boundaries. With unreinforced resin, the bead accuracy declined, especially for the smallest bead sizes.PBF, as a solution for the manufacture of beads, evidenced several limitations. The most critical aspect was the difficulty with unsintered powder emptying as a consequence of thermal accumulation and partial powder sintering for some bead geometries. The widespread presence of defects such as strut-thinning and ruptures was also observed. For PBF, the production capacity ranged between 6 and 60 parts per hour, depending on the bead dimensions, with a cost of between GBP 0.50 and 4 per part. Vat photopolymerization proved to have better feasibility, with a production capacity of approximately 100–150 parts per hour and a cost of approximately GBP 0.3–0.4 per part. In the scenario of an expected decrease in AM costs in the near future, the economic viability of the studied solutions may soon be achieved.