Exophiala that were recovered from dryland biological soil crusts. Despite notable differences in colony and cellular morphology, both fungi appear to be members of the same species, which has been named Exophiala viscosa (i.e. E. viscosa JF 03-3 Goopy and E. viscosa JF 03-4F Slimy). A combination of whole genome sequencing, phenotypic experiments, and melanin regulation experiments have been performed on these isolates to fully characterize these fungi and help decipher their fundamental niche within the biological soil crust consortium. Our results reveal that E. viscosa is capable of utilizing a wide variety of carbon and nitrogen sources potentially derived from symbiotic microbes, can withstand many forms of abiotic stresses, and excretes melanin which can potentially provide ultraviolet resistance to the biological soil crust community. Besides the identification of a novel species within the genus Exophiala, our study also provides new insight into the regulation of melanin production in polyextremotolerant fungi.Black yeasts are polyextremotolerant fungi that contain high amounts of melanin in their cell wall and maintain a primar yeast form. These fungi grow in xeric, nutrient depletes environments which implies that they require highly flexible metabolisms and have been suggested to contain the ability to form lichen-like mutualisms with nearby algae and bacteria. However, the exact ecological niche and interactions between these fungi and their surrounding community are not well understood. We have isolated 2 novel black yeasts from the genus  The production of melanin is a key functional trait observed in many fungi spanning the fungal kingdom . PyomelaUnfortunately, due to the unique characteristics of melanins and their association with larger biomolecules, we do not know the complete structure of any type of melanin . NotablyPolyextremotolerant fungi are a paraphyletic group of melanized fungi that can be divided morphologically into black yeast and microcolonial/meristematic fungi types  in British Columbia, Canada. These novel fungi are of the genus  in BSCs . They ar in BSCs . Once th in BSCs . As a re in BSCs . AccordiAn extensive amount of research has been dedicated to certain members of the BSC community, but one such less-studied microbe has been the “free-living” fungal taxa. These fungi are nonlichenized , and no Despite the importance of microbial interactions in enabling the successful formation of BSCs in a niche characterized by poor nutrient and water availability, the fungal components of BSCs and their relative functions within the interaction network remain poorly understood. Here, we combine genome sequencing with computational tools and culture-based phenotyping to describe 2 strains of a new species of black yeast fungi associated with BSCs. We report on their carbon and nitrogen utilization profiles, stress responses, and lipid accumulation patterns. In addition, we characterize their capacity for melanin production and generate valuable insight into mechanisms that might be involved in regulating the synthesis of these compounds so that melanin production contributes to the BSC community.Exophiala viscosa JF 03-3F Goopy CBS 148801 and E. viscosa JF 03-4F Slimy CBS 148802. E. viscosa is typically grown in malt extract medium  strain ATCC 34100. S. cerevisiae strains are grown in yeast peptone dextrose medium , Canada, Latitude: 52.94997°, Longitude: −119.41919°. Soil samples were taken from the top 2 cm of the BSC. A 0.1 g portion of the crust was re-suspended in 1 mL of water, ground with a sterile micropestle, and diluted with a dilution factor (DF) of 10 till they reached 10,000× dilution. Each dilution was then spread out onto 2 different MEA petri plates containing either no antibiotics or a plate containing: ampicillin , chloramphenicol (50 mg/L), gentamycin (10 mg/L), and cycloheximide (100 mg/L). The plates were then incubated in a Percival light incubator at 23°C with a 12 hr light/dark cycle and examined daily using a dissection microscope to check for small black colonies. Once a potential black colony was seen, half of it was removed and transferred to a new MEA petri plate with no antibiotics. It was vital to examine the plates daily because even in the presence of antibiotics many unwanted fast-growing microbes would grow on the plates and cover up the slower-growing polyextremotolerant fungal colonies. Once a pure culture of each isolate was grown up (approximately 2 weeks), they were preserved in 30% glycerol and stored in the −80°C freezer.DNA sequencing of amplified internal transcribed spacer (ITS) sequences was used to initially identify the isolates. DNA was extracted using the Qiagen DNeasy Powersoil DNA extraction kit. Primers used to isolate the ITS region were: ITS1- (5′-TCC GTA GGT GAA CCT GCG G-3′) and ITS4- (5′-TCC TCC GCT TAT TGA TAT GC-3′) (A cetyltrimethylammonium bromide (CTAB) based DNA extraction method was performed to obtain high molecular weight DNA for whole genome sequencing. The DNA extraction method used was derived from (RNA was obtained using the Qiagen RNeasy Mini Kit (Cat. No. 74104). Cells were grown in 25 mL of 3 different liquid media types ; see https://support.illumina.com/sequencing/sequencing_kits/truseq-stranded-mrna.html, and with the following conditions: total RNA starting material was 1 μg per sample and 8 cycles of PCR was used for library amplification. The prepared libraries were quantified using KAPA Biosystems’ NGS library qPCR kit and run on a Roche LightCycler 480 real-time PCR instrument. The libraries were then multiplexed and prepared for sequencing on the Illumina NovaSeq sequencer using NovaSeq XP v1 reagent kits, and S4 flow cell, following a 2 × 150 indexed run recipe.Both genomes and transcriptomes were sequenced using Illumina technology. For transcriptomes, a plate-based RNA sample prep was performed on the PerkinElmer SciClone next-generation sequencing (NGS) robotic liquid handling system using Illumina's TruSeq Stranded mRNA high throughput sample prep kit utilizing a poly-A selection of mRNA following the protocol outlined by Illumina in their user guide: https://sourceforge.net/projects/bbmap/). Raw reads were evaluated for artifact sequence by k-mer matching (k-mer = 25), allowing 1 mismatch, and detected artifacts were trimmed from the 3′ end of the reads. RNA spike-in reads, PhiX reads, and reads containing any Ns were removed. Quality trimming was performed using the phred trimming method set at Q6. Finally, following trimming, reads under the length threshold were removed . Filtered reads were assembled into consensus sequences using Trinity ver. 2.3.2  and then the selected fragments were end-repaired, A-tailed, and ligated with Illumina compatible sequencing adaptors from Integrated DNA technologies (IDT) containing a unique molecular index barcode for each sample library. The prepared libraries were quantified using KAPA Biosystems’ NGS library qPCR kit and run on a Roche LightCycler 480 real-time PCR instrument. The quantified libraries were then multiplexed with other libraries, and the pool of libraries was then prepared for sequencing on the Illumina HiSeq sequencing platform utilizing a TruSeq paired-end cluster kit, v4, and Illumina's cBot instrument to generate a clustered flow cell for sequencing. Sequencing of the flow cell was performed on the Illumina HiSeq2500 sequencer using HiSeq TruSeq SBS sequencing kits, v4, following a 2 × 150 indexed run recipe.https://github.com/lh3/wgsim) using “-e 0 -1 100 -2 100 -r 0 -R 0 -X 0 -d 3000 -s 30”. 25× of the simulated long read mate-pair was then co-assembled together with 125× of the original Illumina filtered fastq with AllPathsLG release version R49403  with Velvet version 1.2.07  using thhttp://www.repeatmasker.org/) using both the manually curated RepBase library (http://www.softberry.com) and COMBEST (The assembly scaffolds were masked by RepeatMasker ( library  and a li COMBEST  and FGEN COMBEST  seeded bhttp://www.micans.org/mcl/) into a first draft of candidate multigene families for each genome. Percent GC content for the genomes of various Exophiala spp. was determined by uploading each fungi's “Masked assembly scaffolds” file from JGI's Mycocosm to the program Geneious  and the MycCosm Black Yeasts EcoGroup Portal (https://mycocosm.jgi.doe.gov/black_yeasts/). The assemblies and annotations of both genomes are available at the fungal genome portal MycoCosm  and in the DDBJ/EMBL/GenBank repository under accessions JALDXI000000000 and JALDXH000000000.All genomic scaffolds, RNA contigs, gene models and clusters, and functional predictions thereof, may be accessed through MycoCosm Herpotrichiellaceae PhyloGroup Portal (E. viscosa JF 03-3F and JF 03-4F reside. We created this tree using the pipeline described by N*(N–1)/2 pairs) and then use BBBs as an input computed for each pair of genomes. Another script finds BBBs that are present in all N genomes, so these are sets of N genes (one from each species or strain) that are BBB to each other in all N genomes. The resulting defined homologous proteins across all 24 genomes were then used as our sequences for the rest of the pipeline. These sequences were aligned with Mafft v7.123b with no alterations . The alignments were then uploaded to Geneious Prime 2023.0.4 /bioNJ, the resulting Bayesian tree was used as the initial tree file, and no branch swap filter. The resulting tree in newick (.nwk) form with branch lengths and bootstrap values was then imported into the interactive tree of live program ver. 6.6 for visualization, and edited in Adobe Illustrator to add combined posterior probabilities and bootstrap values and highlight the new species (Phylogenetic estimates were performed using Bayesian inference via the Mr. Bayes plug-in ver. 2.2.4 in Geneious . For the species .https://bioinfo.mnhn.fr/abi/public/asap/) using the Kimura (K80) model on our aligned ITS-only, 18S-28S-ITS gene concatenation alignment, and our 18S-28S-ITS-RPB1 gene concatenation alignment. The resulting lowest ASAP score  was chosen to be the most likely delimitation of species of that dataset.We used assemble species by automatic partitioning (ASAP) to confirm our species delimitation that was observed in our phylogenetic analyses  and protein-protein BLASTed using JGI's Mycocosm. Once the genes were found, analysis of upstream and downstream flanking genes was performed until the mating gene MAT1-1 was located. Genes close to MAT1-1 and within the span of genes listed above were considered part of the MAT locus.Mating loci for E. sideris, we created synteny dot plots using their genome assemblies. The genome assembly scaffolds of E. viscosa JF 03-3F and E. viscosa JF 03-4F were used to create the synteny dot plots comparing these 2 genomes. The assembled genome of E. sideris was downloaded from NCBI accession number KN846951. Dot plots were created using the program D-genies (https://dgenies.toulouse.inra.fr/) (To further elucidate the similarities between our 2 strains and their relatedness to nra.fr/) . SettingPhenotyping experiments of E. viscosa JF 03-3F and E. viscosa JF 03-4F were performed to characterize the new species’ capabilities and differences between the strains. Experiments performed were carbon utilization, nitrogen utilization, UV resistance, metal resistance, growth temperature range, budding patterns, lipid profiles, and growth capabilities on various fungal media as described below.Protocols for observing the budding patterns of these new strains were derived from methods in N-acetyl glucosamine (NAG), potato dextrose agar (PDA), spider, YPD, YPD + trace elements (TE), and V8 , MEA, minimal media (MN), MNV, MN + , and V8 . Both isCryptococcus neoformans (The carbon utilization of each isolate was determined using a BioMerieux ID C32 carbon utilization strip (Cat. No. 32200-1004439110). These strips have 30 different carbon sources in individual wells, one well with no carbon source for negative control, and one well with esculin ferric citrate. The esculin  ferric citrate assay is a colorimetric assay originally used to identify Enterobacteria via the production of a dark color in positive cultures for esculin degradation  and below (25% ≥ % Dilu.), OD to percent dilution at 50% dilution (DF = 2) and below (50% ≥ % Dilu.), OD to cell count at 2.19 × 106 cells/mL (DF = 8) and below, OD to cell count at 4.38 × 106 cells/mL (DF = 4) and below, and the accuracy that the ODs quantified the actual percent dilutions at 12.5% dilution (DF = 8) and below , were compared across all wavelengths to determine the most optimal wavelength. The most optimal wavelength would have the highest R2 values for all comparisons except OD to the consistency of the actual cell counts percent dilution at 12.5% dilution and below which would have the lowest R2 value. OD reads were done using the BioTek Synergy H1 hybrid spectrophotometer.The proper wavelength for optical density (OD) readings of these new strains was determined by creating a standard curve. We performed a full spectrum analysis of serially diluted cells of these fungi at the end of their exponential growth phase. Cells were allowed to grow in 25 mL of MEA in a 250 mL Erlenmeyer flask for 5 days. 200 μL of culture was removed, washed 3× with water, and diluted 1:1 in MEA (100 μL of cells to 100 μL of MEA) 9 times to reach a maximum dilution of 1:256 . OD was measured at wavelengths from 300 nm–700 nm in 10 nm increments. Then the Nitrogen utilization tests were performed using 10 different nitrogen conditions in MN base media. 100 mM was the concentration used for all compounds that contained one nitrogen atom per molecule: proline, ammonium tartrate dibasic, serine, sodium nitrate, glycine, glutamate, and aspartate; 50 mM was the concentration used for urea because it has 2 atoms of nitrogen per molecule; 1% w/v of peptone was used as a positive control and no nitrogen was added as a condition for negative control . This coTo determine the temperature resistance range and optimal growth temperature for each isolate, we grew both fungi at 4°C, 15°C, and 23°C (i.e. ambient room temperature), 28°C, 37°C, and 42°C. Isolates were first grown up in 25 mL of MEA for 5 days at room temperature to maximum density. Then 1 mL of cells was removed, and a 10× serial dilution was made from 0× to 100,000×, using prefilled 1.5 mL tubes with 900 µL of MEA and adding 100 µL of the previous tubes each time. Then 5 µL of each serial dilution was spotted onto a square MEA plate which allowed us to determine the carrying capacity of each isolate at the different temperatures. Plates were kept at their respective temperatures for 7 days before observations were made, however, the 37°C and 42°C plates were only kept at those temperatures for 2 days and the incubators required cups of water inside of them to prevent the plates from dehydrating. Plates grown in 42°C and 37°C were then allowed to grow at room temp for up to a week to determine if the isolates died at these temperatures or if their growth was just arrested.Resistance to UV light was observed to determine if these black fungi, with highly melanized cell walls and constant exposure to sunlight in their natural habitat, were UV resistant. To determine this, we used the UVP HL-2000 HybriLinker UV crosslinker as our source of UV light, which has a UV wavelength of 254 nm. Lower wavelengths (100–280 nm) are in the UV-C range, they are considered ionizing radiation and are the most detrimental to living organisms, but are completely blocked by the ozone layer (2 (254 nm)  . After 2E. viscosa JF 03-3F, E. viscosa JF 03-4F, E. dermatitidis, and S. cerevisiae ML440) was done in 25 mL of MEA, shaking at 200 rpm for 5 days at room temperature. We then spread 100 μL of each fungus onto 100 mm-sized MEA plates using a glass spreader to create a lawn. Using flame sterilized tongs our metal paper discs were placed onto the center of the petri dish on top of the fungal lawn and lightly pressed down to ensure the metal disc was touching the plate completely. These plates were then placed in the Percival light incubator at 23°C with a 12 hr light/dark cycle for up to 2 weeks. Once a zone of the clearing was clearly visible amongst the fungal growth (1–2 weeks), the zone of clearing was then measured in cm. Generally, large zones of clearing indicated sensitivity to the metal, whereas zones of reduced size were consistent with resistance to the metal.Metal resistance is a relatively universal trait in many polyextremotolerant fungi. Due to the under-studied nature of this particular characteristic in BSCs and fungi, we decided to test if any of our isolates were resistant to any heavy metals which would indicate possible bioremediation capacity. In order to test metal resistance, we used the antibiotic disk method by aliquoting metal solutions onto paper discs and observing zones of clearance. The metals and concentrations used are listed in S. cerevisiae, E. dermatitidis, E. viscosa JF 03-3F, and E. viscosa JF 03-4F was performed in the presence of fermentable vs nonfermentable sugars in high and low nitrogen. To test these conditions, we grew all 4 fungi in 4 different media types: (1) MEA; (2) MEA + 20 g/L of peptone instead of 2 g/L; (3) MEA with the dextrose replaced with the same weight amount of glycerol; and (4) MEA with glycerol instead of dextrose and 20 g/L of peptone instead of 2 g/L. All 4 fungi were first inoculated in 25 mL of liquid MEA in a 250 mL Erlenmeyer flask and shaken at 200 rpm for 5 days at room temperature to reach peak density. Then 100 μL was inoculated into 5 mL of each media in a size 25 mm tube, placed in a roller drum, and allowed to grow at room temperature for 5 days.Comparison of the lipid production of To observe their lipid profile, we performed a standard Bligh Dyer lipid extraction . Equal wS. cerevisiae, 7 μL of the lipid suspension was used to dot the TLC. For S. cerevisiae, 10 μL of the lipid suspension was needed. The solvent systems used for TLC were chloroform: methanol: glacial acetic acid: water 85:12.5:12.5:3 for the polar lipid solvent system, and petroleum ether: diethyl ether: acetic acid 80:20:1 for the neutral lipid solvent system. The TLC plates were loaded with 7 or 10 μL of the re-suspended samples, and they were placed in the polar solvent system for approximately 30 minutes  before transferring to the neutral lipid solvent system in a separate container till the solvent front reached just a few cm below the top of the plate. The plate was then removed and dried for 15 minutes, until the solution on the plate was no longer volatile by smell, and the plate was placed in the presence of iodine (Sigma-Aldrich cat. No. 207772) in a glass chamber for 5 minutes until all the lipids were visible. The plates were then immediately placed in plastic covers and scanned and photographed for visualization and documentation.For all samples except the Aspergillus and Exophiala, which are capable of producing 2 or all 3 forms of melanin   and were tested using protein-protein BLAST against the filtered model proteins database of E. viscosa JF 03-3F and E. viscosa JF 03-4F on Mycocosm. Since A. niger contains paralogs for some melanin biosynthetic genes, all genes listed in A. niger and A. fumigatus , e-values (<10−6), and bit scores (>50) of the protein-protein BLASTs followed standards for homology laid out by Melanin biosynthesis in fungi occurs via 3 different pathways: the 1,8-DHN pathway which creates allomelanin, the L-DOPA pathway which creates pheomelanin, and the tyrosine degradation (HGA) pathway which creates pyomelanin . Most fuOnce it was established that both isolates contain the potential for the production of all 3 fungal melanins, we wanted to determine which melanin was actively being produced using chemical blockers of the 1,8-DHN and L-DOPA-melanin production pathways. DHN-melanin blocker phthalide and the L-DOPA-melanin blocker kojic acid were both used to determine which melanin was being actively produced. Stock solutions were made according to  and changed the concentration of peptone to 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 5%. We then spotted 5 μL of both strains onto the plates after performing a 10× serial dilution up to 10,000× dilution. The plates were grown at 23°C for 10 days with a 12 hr light/dark cycle.To assess the role of organic nitrogen levels in melanin excretion, we initially switched the concentration of peptone added to YPD and MEA media such that the new media were YPD + 0.2% peptone and MEA + 2% peptone. E. viscosa JF 03-3F and E. viscosa JF 03-4F were grown on “Tyrosine media”  were the source of the increased melanin excretion in the increased peptone experiment. Growth of 5 μL spots of e media” ): withouet al.'s method for extraction and purification of melanin from culture filtrate was adapted and used for all future melanin extractions, mainly supernatant melanin extractions. Adjustments to the Pal et al. method included: the 6 M HCl precipitation taking multiple days instead of overnight for melanin to precipitate, and the protocol was stopped when 2 M NaOH was added to the extracted melanin. We did not continue to re-precipitation and drying of the melanin as this product did not reprecipitate in any solvents used.Extraction of melanin from a variety of sources has been performed using 2 different approaches: chemical extraction and enzymatic extraction  were discarded, leaving behind the dark layer. 2 mL of water was added to the sample for washing, and the tubes were centrifuged at 4,000× g for 1 minute, and the entire supernatant was discarded. Finally, 1 mL of 2 M NaOH was added to each sample to allow for a standard volume added even if the melanin amount and, therefore, the final volume varied.The exact methods are as follows. 10 mL of culture was centrifuged at 3,000× g for 5 minutes, and the resulting supernatant was filter sterilized through a 2 μm filter to ensure all cells were removed. The filtered supernatant was then transferred into a 50 mL centrifuge tube, and 40 mL of 6 M HCl was added to the tube. The filtrate was then allowed to precipitate out for up to 2 weeks. Precipitated solutions were then centrifuged at 4,000× g for 3 minutes, and the resulting supernatant was discarded. The pellet was washed with 2 mL of dd HExtracted melanin samples suspended in 1 mL of 2 M NaOH were then diluted 5 μL into 195 μL of 2 M NaOH into a 96-well plate, with a 200 μL 2 M NaOH blank well. These diluted samples were then read using the BioTek Synergy H1 hybrid spectrophotometer. The settings were for a full spectrum read from 230 to 700 nm, with 10 nm steps. However, the machine could not read OD values above 4.0, and therefore, only data from 300 to 700 nm was used.E. viscosa JF 03-3F and E. viscosa JF 03-4F were actively excreting melanin, as opposed to dead cells lysing and releasing it, we grew up both strains and took daily aliquots for melanin extraction. We use the term excrete/excreting instead of secrete/secreting for the terminology of melanin being released into the media by the fungi because we have no evidence currently that this melanin release mechanism is an active process  warranting active secretion. Additionally, we wanted to compare the melanin excretion capabilities of these strains to E. dermatitidis for a baseline comparison. All 3 fungi were grown in liquid MEA shaking at room temperature for 5 days. Then 2 mL of cells were washed with water 3 times. 500 μL of washed cells were then inoculated into 100 mL of MEA and YPD in 500 mL flasks. We let the cells grow at 23°C shaking at 200 rpm for 7 days, removing 11 mL of cells and supernatant daily and pipetting them into 15 mL centrifuge tubes. The tubes of cells were then centrifuged at 3000× g for 5 minutes, the supernatant was removed, filter sterilized through a 2 μm filter, and placed into a new tube. We filter sterilized the supernatant to ensure that no cells remained in the supernatant, therefore, all of the melanin extracted came only from excreted melanin. Melanin was then extracted using the chemical method explained above. The resulting pure melanin for all samples was read with the full spectrum as stated above, and both standard OD and log scale graphs were created to confirm the presence of melanin with the proper R2 value above 0.9  to induce G:C to A:T mutations EMS mutagenesis was performed using the method by https://www.migscenter.com/; 355 Fifth Avenue, Suite 810 Pittsburgh, PA 15222, now called SeqCenter  for genome re-sequencing and base-called against the WT genome sequences. Resulting mutations were then manually annotated using the JGI Mycocosm genome “search” tool to identify affected genes.Genetic modification of E. dermatitidis, and L-DOPA is needed for certain fungi to produce melanized structures, including the albino mutant form of E. dermatitidis WdPKS1 , L-DOPA, 1,8-DHN, and tyrosine. HU has been shown to enhance melanin production in Additionally, we also tested the presence of tyrosine, the starting compound for L-DOPA and HGA melanins on the albino mutant. Growth of 5 μL spots of EMS 2-11 were grown on “tyrosine media” ): withouExophiala, and later apparent sequencing errors created nucleotide differences in their ITS sequences which initially placed them as different species. Further whole genome sequencing using Illumina-based sequencing allowed for more accurate sequences of the ITS region for these isolates.Two fungi were isolated from BSCs located near Jackman Flats Provincial Park in BC, Canada . BSC samples were diluted in water, crushed with a plastic micropestle, and plated out onto MEA with antibiotics and cycloheximide, and grown at 23°C for 2 weeks to obtain the isolates. Once colonies began to form, they were picked and struck out onto regular MEA to obtain pure cultures. DNA extractions were performed on liquid cultures of the isolates that had been grown in liquid MEA for 5 days at 23°C and 160 rpm of shaking. PCR amplification of the isolates’ ITS region was performed and then sequenced using Sanger sequencing. The resulting ITS sequences placed the fungal isolates within the genus Exophiala nigra strain CBS 535.95” accession number: MH862481.1, which was performed on September 29th, 2022. UNITE's fungal species identification delineation typically separates species on ITS sequence similarity with ≥98% as the cutoff  were a separate species from E. nigra. However, since that date, the UNITE database has been further curated with more strict parameters and now only contains 19 Exophiala species, removing E. nigra and E. sideris as species in Taxon name searches. Repeating this analysis on December 12th, 2022, resulted in a BLAST result of Capronia acutiseta UDB035414 86.16% identity as the highest match.Using the Illumina-based ITS sequences of the isolates, it was determined that they have 100% identity to each other's ITS sequence . BLAST rE. viscosa JF 03-3F and E. viscosa JF 03-4F are 2 strains of the same species indicated via their marker gene alignment, their cellular morphology and budding patterns differ drastically as described below. Both strains form smooth, shiny, sooty black colonies on PDA after 9 days of growth at 23°C. When grown on high organic nitrogen media such as YPD, they will begin to excrete melanin into the agar by day 7 when grown at 23°C, and by day 3 in liquid YPD , 28S rDNA (548 bp), and ITS rDNA (547 bp), with a combined sequence length of 2,685 base pairs. These genes were chosen because while they are predominant marker genes, they are also the only genes available on NCBI for the species E. viscosa within the monophyletic clade composed of E. nigra, E. sideris, E. bergeri, and P. elegans with a posterior probability of 100%  is added to the gene concatenation, creating the combination of 18S, 28S, ITS, and RPB1 , E. nigra is again placed on its own node below E. bergeri maintaining a separate position to E. viscosa and the E. sideris clade with 100% posterior probability and 79 bootstrap value , the percent identical base pairs between E. viscosa and E. sideris are 98.436%, and 99.07% identical bases between E. viscosa and E. nigra. However, when RPB1 is added to the concatenation, the percent identical bases between E. sideris and E. viscosa remain similar at 98.551%; whereas the percent identical bases between E. viscosa and E. nigra becomes 97.03%. Percent identity matches between all species represented in the phylogenetic trees of Exophiala species and related fungi who have been whole genome sequenced, and whose genomes are available in JGI's Mycocosm. With their whole genomes, we were able to perform single-copy homologous protein sequence approximate maximum likelihood phylogenetic analyses, providing additional support for E. viscosa's intraspecific variation from specifically the fungus E. sideris. Our approximate maximum likelihood single-copy homologous protein phylogenetic tree shows with a 100 bootstrap value, that E. sideris is separate from E. viscosa , with both strains of E. viscosa as one species and E. sideris and P. elegans as one species. However, with the three-gene concatenation, the distance threshold decreased to 0.75%. Finally, the concatenation of 18S-28S-ITS-RPB1 provided an ASAP score of 2.5 which suggested that there be 16 species with a distance threshold of 2.5%, combining both strains of E. viscosa into one species, and combining E. aquamarina and E. pisciphila into one species (E. crusticola and P. elegans are not in this dataset). All 3 ASAP analyses suggest that both strains of E. viscosa remain as one species and that they are a distinct species from the other Exophiala spp.ASAP values of the marker genes allowed us to confirm our phylogenetic inferences of separating of 1.32% . Both stE. viscosa, JF 03-3F and JF 03-4F, are very similar to each other in genomic content and have 100% similarity across the 4 marker genes 18S, 28S, ITS, and RPB1, they vary in gene synteny, they contain multiple single nucleotide polymorphisms across their genomes, and their morphologies differ. E. viscosa JF 03-3F is found primarily as a yeast morphology on all media tested, with ellipsoidal cells 4.4 μm ± 0.34 μm wide and 5.36 μm ± 0.37 μm long . E. viscosa JF 03-4F is found primarily in a pseudohyphal form on all media tested, with more elongated cells 3.6 μm ± 0.38 μm wide and 6.0 μm ± 0.67 μm long . E. viscosa JF 03-3 will form pseudohyphae when grown for longer than 2 weeks and only on solid media, whereas E. viscosa JF 03-4F readily form pseudohyphae in liquid media and on solid media as a primary growth from and will form a few true hyphae after 3 weeks of growth on MEA  and the abundance and make-up of microbial neighbors    .E. viscosa E.C. Carr, S.D. Harris, sp. nov.MycoBank MB845198Typification: Canada. British Columbia: Adjacent to Fraser-Fort George H, Jackman Flats Provincial Park , in BSC sample, sampled June 2015, S. Harris, isolated July 2015, E. Carr. Holotype culture strain JF 03-3F Goopy: CBS 148801. Additional strains JF 03-4F Slimy: CBS 148802.GenBank: Whole genome = JALDXI000000000.1NCBI Taxonomy ID: 2486360Etymology: E. viscosa was colloquially referred to as “Goopy” due to the nature of its yeast morphology lending to a very goopy colony. Accordingly, we have formally named it E. viscosa for the Latin term for viscous.Sexual and asexual morph: not observed.Culture characteristics: Hyphae not observed in liquid culture, aerial pseudohyphae in colonies observed by day 30 in MEA, primarily found in yeast form in liquid MEA and YPD mediums (23°C) but will form pseudohyphae as well. Cells in MEA liquid culture are ellipsoid in shape, 3.9 μm ± 0.55 μm wide and 5.66 μm ± 0.62 μm long (n = 40 cells measured). Cell walls start off thin and become thicker over time. In the pseudohyphal form on solid MEA, budding occurs at 180° or 90° from the mother cell's origin, or at acute angles from the previous bud site. Cells either maintain the size and shape of yeast form as pseudophyphal cells bud out for 4 buds and then begin to elongate, or the buds begin to elongate from the first bud and continue to elongate with each linear budding. Colony morphology all at 23°C with a 12 hour light/dark cycle: on MEA colonies are initially shiny dark grayish brown and smooth to curled, then develops a rainbow sheen with a colony size of 5.2–6.6 mm by day 14, additionally dark supernatant is observed in liquid MEA culture starting on day 3; on malt extract agar with vitamins and trace elements (MAG) colonies are sooty black and glossy with smooth colony edges forming a dome shape, and 6.3–8.1 mm growth is observed on day 14; on MN + N-acetyl glucosamine as the carbon source, the colony is matte sooty black smooth edged and grows to 2.7–2.8 mm in 14 days; on MNV, the colony is glossy raisin black with smooth edges and smooth top, colonies grow to 4.7–5.3 mm in 14 days; on PDA colonies are glossy sooty black with smooth edges and grow to 5.6–5.8 mm in 14 days; on Spider media, the colonies are sooty black and irregularly shaped with a size of 3.0–4.5 mm in 14 days, additionally a red pigment Is observed surrounding the colonies in the media; on V8 medium colonies are glossy sooty black and irregularly shaped, growth reaches 7.5–8.1 mm in 14 days; on YPD colonies are blackish brown (1) and matte with round to irregular side morphologies and undulate top morphology, dark excretions are observed coming from the colonies and excessively dark supernatant is observed in liquid culture starting on day 2, colony growth reaches 6.1–6.2 mm in 14 days. Color descriptions used above come from Cardinal temperatures: Growth is capable at temperatures from 4°C to 28°C, and optimal growth is at 23°C, growth is not capable at or above 37°C.Additional specimen examined: Canada. British Columbia: Adjacent to Fraser-Fort George H, Jackman Flats Provincial Park, in BSC sample, sampled June 2015, S. Harris, isolated July 2015, E. Carr. Culture E. viscosa Slimy JF 03-4F CBS 148802.Notes: Support for E. viscosa as a novel species stems from molecular multi-locus phylogenetic analyses both for marker genes and single-copy genes, morphological comparisons of micro- and macromorphology to phylogenetically related species, ecology, and biogeography of the novel species. E. viscosa is phylogenetically placed on all analyses as related to but separate from E. sideris with 100% posterior probability in all Bayesian analyses, and bootstrap values above 95 in all Maximum likelihood analyses . Their predicted gene contents are 11,344 for E. viscosa JF 03-3F and 11,358 for E. viscosa JF 03-4F  amongst the Exophiala species listed in Genome sequencing, assembly, and annotation were performed by DOE JGI, and their genomes and transcriptomes are deposited on the Mycocosm website under their genus species names. All accession numbers for both strains are listed in JF 03-4F . These sJF 03-4F . E. sideferences . E. viscE. viscosa are seemingly much less complex compared to other Eurotiomycete fungi (e.g. Aspergillus nidulans), there are few differences in the complement of genes that are assigned to these processes when these fungi are compared. For example, compared to A. nidulans, differences include losses of a velvet regulator (VosA) as well as a class of hydrophobins , along with the gains of additional cryptochrome/photolyases (CryA) and an NADPH oxidase (NoxB)  compared to other sequenced and annotated genomes from the members of Chaetothyriales .Although the morphology and development of e (NoxB) . This paE. viscosa JF 03-3F vs E. viscosa JF 03-4F was performed to determine if these strains had nonsyntenic regions which could potentially contribute to their morphological differences. The resulting dot plot showed that most of their genomes are syntenic and 98.31% of their genomes are greater than 75% matching  in E. viscosa JF 03-3F and E. viscosa JF 03-4F were identified via BLAST searches. We found that APN2 and SLA2 flank the MAT locus of both species, and they also contain the Herpotrichielleaceae-specific mating gene MAT1-1-4 . When the MAT protein sequences of E. viscosa JF 03-3F and E. viscosa JF 03-4F are aligned with the MAT gene of E. sideris, we see 83.79% homology between their protein sequences indicating that these fungi also contain a fused MAT gene  presumably confers homothallic mating behavior, although this has not been confirmed experimentally in any of these 3 fungi. Additionally, neither E. viscosa JF 03-3F nor E. viscosa JF 03-4F contain an additional gene between APN2 and MAT1-1-4 as is found in E. sideris  of both 0–420 nm . To reduE. viscosa JF 03-3F and E. viscosa JF 03-4F can utilize a wide range of carbon sources. Triplicates were performed on these strips to ensure the results were uniform and representative. Overall, a variety of carbon sources supported the robust growth of both strain , and there are only a few quantitative differences in utilization patterns , ammonium tartrate, sodium nitrate, urea, peptone (mixed short chain amino acids) as a positive control, and no nitrogen as a negative control. Both strains are incapable of utilizing aspartate and glutamate as nitrogen sources; E. viscosa JF 03-3F has a P-value of 0.012 (n = 6) for aspartate and 0.0005 (n = 6) for glutamate; E. viscosa JF 03-4F having a P-value of 0.0003 (n = 6) for aspartate and.00005 (n = 6) for Glutamate  , P-value for E. viscosa JF 03-4F is 0.0012 (n = 6), over peptone. However, they differ in that E. viscosa JF 03-3F most prefers urea P-value 0.009 (n = 6), while E. viscosa JF 03-4F had additional significantly preferred nitrogen sources including serine ), ammonium ), and nitrate  (We were particularly interested in patterns of nitrogen utilization for peptone) . E. visc (n = 6) .E. viscosa JF 03-3F and E. viscosa JF 03-4F were isolated from an environment that experiences wide seasonal and diurnal temperature changes. As such, we determined the optimal growing temperature for these strains, as well as the limits at which they are capable of survival . Both isolates were serially diluted and spotted onto MEA plates to determine the carrying capacity at different temperatures. Both E. viscosa JF 03-3F and E. viscosa JF 03-4F were capable of growth at 4°C, 15°C, 23°C, and 27°C, but could not grow at 37°C and 42°C  that was lethal to S. cerevisiae and E. dermatitidis (data not shown). The same level of exposure did not kill either E. viscosa JF 03-3F or E. viscosa JF 03-4F  proteins and their homologs in both E. viscosa JF 03-3F and E. viscosa JF 03-4F are summarized in E. viscosa JF 03-3F and E. viscosa JF 03-4F have the genetic capability to produce all 3 forms of known fungal melanins: DOPA-melanin (pheomelanin), DHN-melanin , and L-tyrosine/HGA-derived pyomelanin. Two A. fumigatus proteins, hydroxynaphthalene reductase (Arp2) and scytalone dehydratase (Arp1) were also separately BLASTPed against the sequences of E. viscosa JF 03-3F and E. viscosa JF 03-4F because those protein sequences are not homologous between A. niger and A. fumigatus  method of melanin-blocking, we added the highest dosage (100 µg/mL) of phthalide and kojic acid individually and combined to agar-solidified MEA, and both spread a lawn of cells and spotted serially diluted cells onto the plates. Neither assay resulted in blockage of melanin production in E. viscosa JF 03-3F or E. viscosa JF 03-4F  in the peptone that was inducing melanin excretion at higher peptone levels, we tested differing tyrosine amounts on our fungi. When 0.5 g/L of tyrosine is added to the media,  strains . AdditioTo confirm that the dark pigment in culture supernatants is indeed melanin, we performed a melanin extraction using previously described methods . TherefoE. viscosa JF 03-3F, E. viscosa JF 03-4F, and E. dermatitidis on YPD and MEA were observed daily for melanin excretion; 11 mL aliquots of their supernatant were removed daily, and the melanin was extracted from each sample. As the experiment progressed, it was obvious that E. viscosa JF 03-3F and E. viscosa JF 03-4F began excreting melanin on day 3 and excreted more in YPD than in MEA  displayed the typical results from a full spectrum read on pure melanin , 4 distinct phenotypic classes of mutants were recovered: pink/albino, crusty, brown, and melanin over-excretion (data not shown). At this time, the most important of these phenotypes was the pink/albino phenotype found in a mutant of E. viscosa JF 03-4F, a mutant we named EMS 2-11 . Further investigation of these ideas will require demonstration that these Exophiala species undergo sexual development. Additional investigations will also be performed to determine the environmental factors for triggering sexual or asexual development in E. viscosa, as past attempts of established methods have failed but do not constitute evidence for the absence of development.The organization of the E. viscosa were unable to be elucidated using their automatic gene annotations. However, their gene synteny dot plots could indicate locations of the genome where gene synteny has influenced their phenotype. The dot plot comparing the 2 E. viscosa strains showed high % sequence similarity, but very distinct regions of nonsyntenic genes. If the genes responsible for cell polarity are found within the nonsyntenic regions, then it could be a reason for the morphological differences we have observed. However, we were also able to see gene transcription direction differences between the strains in some genes, including the gene COX13 of the MAT locus which is forward in the strain JF 03-3F and reverse in JF 03-4F. Between the differences in gene synteny and transcriptional direction, there could be a reason to re-evaluate the intraspecific variation of these strains once it becomes standard practice for fungal taxonomic naming systems to compare their whole genomes, as is done with bacteria.Phenotypic differences between the 2 strains of E. viscosa JF 03-3F and E. viscosa JF 03-4F are capable of using a diverse array of carbon sources. These include mannitol, ribitol, and glucose, which are considered the main carbon sources exchanged between photobionts and their mycobiont partners in lichens , some of which could be public goods provided by other constituents of the BSC community, or an enhanced capacity to repair damaged DNA. This will ultimately be an interesting topic to investigate in the future.As for resistance to UV radiation, E. viscosa JF 03-3F and E. viscosa JF 03-4F, but we did not necessarily observe was higher metal resistance. Melanin is known to enhance resistances to metals (E. sideris is primarily isolated from arsenate-contaminated soils (3 and CuCl2 showed a decreased zone of clearing (higher resistance) in E. viscosa JF 03-3F and E. viscosa JF 03-4F, compared to S. cerevisiae and E. dermatitidis. A potential explanation for the latter observation is that melanin has a higher affinity for copper due to the phenolic compounds within the polymer , pyomelanin (HGA/L-tyrosine-derived), and pheomelanin (L-DOPA-derived); but it is still unknown how these melanin biosynthetic pathways are regulated. We were able to confirm through genetic means that they are producing DHN-derived allomelanin, but we were unable to definitively confirm the production of the other 2 forms of melanin. Additionally, we can speculate that tyrosine-derived melanins are not being directly produced for excretion under conditions we have tested, since using tyrosine as a sole nitrogen source did not induce melanin excretion. However, tyrosine used as an additive did induce melanin excretion at 0.5 g/L but not at 1 g/L, implying a possible tyrosine-dependent regulation of melanin production/excretion that is not entirely understood. We hope to eventually elucidate the melanin(s) that are produced by these fungi by combining genetic approaches with mass spectrometry and NMR in the future.E. viscosa JF 03-3F and E. viscosa JF 03-4F, we tried blocking individual pathways using known chemical blockers of DHN and L-DOPA-melanin, phthalide, and kojic acid, respectively. Neither blocker nor their combination at the highest amounts used was able to block the production of melanin in either strain. If this species has the capability to produce all 3 melanins, then it is possible that they are producing more than one at the same time. This could be the case here since the combination of both kojic acid and phthalide still resulted in melanized cells which would indicate that tyrosine-derived pyomelanin was the cause of melanization. Alternatively, since these fungi are fully melanized, it could be that the concentrations used on fungi that use melanin in asexual structures only were not strong enough to block melanin production in these fungi. Additionally, our melanin gene annotation revealed that both fungi contain homologs to A. niger and A. fumigatus’ Arp1 and Arp2, which could be causing a bifurcation of the DHN-melanin pathway and prevent phthalide from being an effective blocker of melanin production  E. viscosa JF 03-3F and E. viscosa JF 03-4F only produce DHN-derived allomelanin and disruption of pks1 causes all melanin production to shut down, (ii) pks1  is essential for the regulation of other melanin productions, or (iii) pheomelanin and pyomelanin are only produced under specific conditions that were not tested during analysis of the mutants.Linked regulation of potentially multiple forms of melanin, was observed with an EMS-induced mutant in which the E. viscosa JF 03-3F and E. viscosa JF 03-4F actively excrete melanin when they are viable and growing. We observed that melanin began to accumulate starting as early as day 2 (by OD reads), therefore, suggesting live cells are excreting melanin and not melanin coming out of dead lysed cells. However, at this time we do not have a mechanism for how melanin is being released from the cells and something to be investigated later. We also observed a decrease in melanin concentrations after day 6 in both YPD and MEA, which raises the intriguing potential for melanin uptake or degradation by E. viscosa JF 03-3F and E. viscosa JF 03-4F. Additionally, we observed that increasing organic nitrogen in the media-induced melanin excretion from both fungi, but we do not know how this phenomenon is regulated or which melanin is being excreted.Our experiments also showed that E. viscosa JF 03-3F and E. viscosa JF 03-4F might be transiently associated with autotrophs in response to specific environmental conditions. Members of the genus Exophiala (Class Eurotiomycetes) are phylogenetically related to the lichen-forming Verrucariales and reside between Lecanoromycetes and Lichinomycetes (E. viscosa JF 03-3F and E. viscosa JF 03-4F, and other related fungi have lichen-like qualities, which allow them to interact with algae, cyanobacteria, and other bacteria to form lichen-like symbioses. The idea that polyextremotolerant fungi can engage in lichen-like associations with microbes has been postulated (Fungal residents of BSC communities include lichenized mycobionts, lichenicolous fungi, endolichenic fungi, and free-living fungi. Although the latter is likely not directly associated with lichens themselves, they are surrounded by algae, cyanobacteria, and other bacteria in the same way that lichen-associated fungi are surrounded by a similar community of microbes. This raises the intriguing possibility that fungi such as omycetes , which aomycetes . As suchE. viscosa JF 03-3F and E. viscosa JF 03-4F instead represents the optimal sharing of an expensive public good for the overall betterment of the BSC community in which they reside.Correlation between environmental organic nitrogen availability and excreted melanin amounts could have strong implications for interactions between these fungi found in BSCs and their nitrogen-fixing community partners. The presumed source of nitrogen used to enhance the excretion of melanin would be nitrogen-fixing prokaryotes such as cyanobacteria, other bacteria, and archaea in the community. Additionally, organic nitrogen was found to be more abundant than inorganic nitrogen in BSC soils, and more abundant than in other soil ecosystems (E. viscosa JF 03-3F and E. viscosa JF 03-4F will enable more detailed investigations of the mechanisms that support the adaptation of these fungi to extreme environments. This in turn should provide greater insight into the roles that these fungi play in the BSC community. Of particular interest will be the regulatory mechanisms that coordinate the production of different types of melanin in response to environmental and chemical inputs. Moreover, the broad applicability of melanin as a bioproduct has triggered growing interest in microorganisms that excrete this polymer. In this context, E. viscosa JF 03-3F and E. viscosa JF 03-4F could potentially serve as excellent platforms for additional engineering focused on optimizing or tailoring the type of melanin produced in responses to specific applications.The availability of complete, annotated genome sequences for jkad110_Supplementary_DataClick here for additional data file.


The low BLL group was associated with increased TNF-α levels, and the high BLL group was associated with reduced TNF-α levels in female subjects. No associations between BLL and the levels of IL-8 and TNF-α cytokines were observed in either females or males, respectively. A negative correlation between BLL and TNF-α was found in female subjects, suggesting that an increase in BLL accompanied by a reduction in TNF-α. The reduced levels of circulating TNF-α in female subjects suggest that chronic Pb exposure could predispose females to immune and inflammation-related disorders than their male counterparts. Further studies are recommended to ascertain the impact of chronic Pb exposure on immunomodulatory cytokines, especially in females.Lead (Pb) poisoning remains a great public health challenge globally known to induce a wide range of ailments in both children and adults. The current study investigated the association of chronic environmental Pb exposure and immunomodulatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin-8 (IL-8) in adult males and females living in Kabwe, Zambia. The standard human cytokine/chemokine Milliplex assay was used to quantify plasma cytokines from four groups categorized as low (<10 μg/dL) and high (>10 μg/dL) blood lead level (BLL) groups, namely, low BLL female ( Lead (Pb) poisoning remains a great public health challenge globally and accounts for approximately 0.6% of the world’s total disease burden . DespiteLead poisoning may take two forms, namely, acute and chronic poisoning with the former being less common than the latter . Acute POn the other hand, chronic Pb poisoning in adults has been reported to elicit personality changes and other maladies including cardiovascular, hematological, and renal-related diseases . MoreoveA classical scenario of chronic Pb poisoning in males and females of reproductive age has been reported in Kabwe, Zambia . Based oThe study targeted adult males and females of reproductive ages from Kabwe town in Zambia. Huge piles of Pb contaminated soils dispersed by natural elements such as wind and water runoffs from the abandoned tailings and mine dumps have extensively polluted the town with Pb . In the TM Trace Elements Whole Blood L-2  with recoveries of 95–105%. The sampling during the survey was approved by the University of Zambia Biomedical Research Ethics Committee , and all participants were volunteers that gave written informed consent. The sex, age, body mass index (BMI), and smoking status were extracted from the questionnaire and screening information that were recorded from participants during the survey. Only healthy males and females of reproductive age of 18 years and above and with no history of smoking were included in this study. Based on the Kabwe residents’ BLLs characteristics, the BLLs were divided into two groups, namely, low BLL and high BLL. The low BLL group represented the low exposure threshold in adults of less than 10 µg/dL, while BLLs above 10 µg/dL were classified as the high BLL group [n = 47; low BLL male: n = 43; high BLL female: n = 21; and high BLL male: n = 18) were used for cytokine analysis, as shown in The plasma samples used in this study were randomly drawn based on the BLLs, age, and sex clustering. The BLLs data used were based on the venous blood samples of adult Kabwe residents  sampled during the July–August 2017 KAMPAI human broad survey that had been previously analyzed using an inductively coupled plasma mass spectrometry  with a limit of detection of 0.0001 μg/dL, courtesy of Yamada et al. [LL group . As show® MAP Kit-Human Cytokine/Chemokine Magnetic Bead Panel (HCYTMAG-60K)  with strict adherence to the manufacturer’s instructions. All reagents and standard solutions (3.2 to 2000 pg/mL) were stored and prepared for the assays according to the guidelines outlined in the manufacturer’s manual. The frozen plasma samples that were kept at −20 °C were thawed prior to analysis at room temperature. As an initial step, the 96-well plates were conditioned using 200 μL wash buffer on a plate shaker for 10 min at room temperature. Then, the wash buffer was decanted, and all residual amounts were removed by inverting and tapping onto clean absorbent towels. The standards, as well as the positive and negative control samples, were added to appropriate wells, followed by the addition of 25 μL assay buffer to the background and sample wells, respectively. The plasma samples (25 μL) were then added to the sample wells, followed by 25 μL of premixed beads. The plate was then sealed with a plate sealer and wrapped with aluminum foil and incubated with mild agitation overnight for 18 h at 4 °C. Following incubation, the well contents were decanted, and the plate was gently washed with 200 μL two times. The detection antibodies were then added to each well and the plate contents were incubated for 1 h at room temperature. This was followed by the application of the fluorescent conjugate streptavidin-phycoerythrin to each well and incubation for 30 min at room temperature. The well contents were gently decanted, and the plate was washed two times using the wash buffer. Finally, 150 μL sheath fluid was added before running the samples on Luminex® 200TM with xPOTENT® software  using a standard cytokine protocol.Based on unpublished pre-experiment cytokine assays we conducted on several cytokines , only interleukin-8 and tumor necrosis factor-alpha were consistently detected in Kabwe human samples. Therefore, only TNF-α and IL-8 were assayed for the current study. The two plasma cytokines were assayed using MILLIPLEXp-value < 0.05 were considered significant.The statistical analyses were all performed using GraphPad Prism software . The data were reported as mean ± SD (standard deviation). Due to the lack of normality in the data following the application of the Kolmogorov–Smirnov test, the Kruskal–Wallis test and Dunn’s Multiple Comparison test or Mann–Whitney U test were applied to analyze the data. Correlation analyses were performed using the Spearman non-parametric correlation coefficient test. The principal component analysis (PCA) was performed to explore the association between plasma cytokines (TNF-α and IL-8) and age, BMI, and BLL using JMP Pro version 15 . All statistical analyses with p = 0.51). The BMI between the low BLL and high BLL groups in females was statistically similar between the two groups. In males, the age range for the low BLL was 19–72 years, and it was 21–66 years in the high BLL group. The mean age between the low BLL and the high BLL, as shown in p < 0.01). The BMI mean values were not different among the groups.The general characteristics of the studied subjects are listed in p < 0.05) reduction in the circulating TNF-α was observed, as shown in The plasma cytokine concentration in females ranged between 1.5 and 36.7 pg/mL, with the mean of 17.7 ± 1.45 pg/mL for TNF-α in the low BLL group. In the high BLL group, the TNF-α range was between 1.88 and 39.1 pg/mL with a mean of 11.7 ± 1.80 pg/mL. A statistical significance  between TNF-α and IL-8 levels was observed in female subjects (Following Spearman’s correlation analyses, a positive correlation (r = 0.288, subjects . A non-sThe Spearman’s correlation analyses for the plasma cytokines and other variables in males are shown in The comparative effect of chronic exposure on plasma cytokines based on sex is shown in In the current study, the first baseline findings that could be associated with chronic environmental Pb exposure on immunomodulatory cytokines, namely, tumor necrosis alpha and interleukin-8 in adult male and female residents of Kabwe, Zambia, are reported. Immunomodulatory cytokines play a vital role in the ability of the body to combat both communicable and non-communicable diseases through adaptive innate immune responses . The dysIn general, the levels of both TNF-α and IL-8 detected in plasma samples of adult males and females of reproductive ages under chronic Pb environmental exposure in our study were similar to the levels that have been reported in occupationally Pb-exposed subjects . The plaThe differences observed in the levels of the plasma cytokines between males and females have also been reported by other authors who attributed the differences to the inherent differences in the genetic makeup and hormonal profiles . EstradiThe plasma TNF-α and IL-8 levels were positively correlated in the female subjects in the current study, a pattern that agreed with other authors that demonstrated that an increase in TNF-α was accompanied by a corresponding increase in IL-8 levels . MoreoveOn the other hand, a negative correlation between BLL and the two immunomodulatory cytokines observed suggests that female Kabwe residents with high BLL will have low levels of these cytokines. Considering the age of female participants in the current study, the findings imply that the deleterious effect of Pb on immunomodulatory cytokines could be among the factors that may negatively impact on fertility and the wellbeing of the fetuses in addition to potential exaggerated immune-induced diseases . The lacThis study has some limitations. The first limitation was the small sample size, although the selection represented a broad section of Kabwe residents. The lack of other analyses on the general blood and hormonal profiles is another limitation. This being a cross-section study naturally entails that follow-up studies must be conducted to further explore the impact of chronic environmental Pb exposure with a large sample size. Notwithstanding, the study has established an association between chronic environmental Pb exposure and some plasma cytokines. We, therefore, recommend that further studies be conducted to ascertain the impact of the long-standing environmental Pb pollution on plasma cytokines and other immune mediators in adult residents in Kabwe, Zambia.Chronic environmental lead exposure as it has been ascertained in Kabwe, Zambia, could be associated with negative effects on the levels of the circulating plasma TNF-α and IL-8 cytokines in adult female and male subjects. The female subjects showed increased levels of TNF-α in the low blood lead level group and a reduction in the TNF-α levels in the high blood lead level group. In male subjects, the impact of lead exposure on the levels of TNF-α and IL-8 was not significant between the low blood and high blood lead level groups. A negative correlation between BLLs and TNF-α was found, suggesting that an increase in BLLs could lead to a reduction in TNF-α and IL-8 levels. Therefore, our findings suggest that adult females under Pb exposure may be more vulnerable and susceptible to TNF-α and IL-8 dysregulation-linked diseases than their male counterparts. Further studies are recommended to investigate the impact arising from the effect of lead on immunomodulatory cytokines, especially in females.


Tumor necrosis factor (TNF) binding to endothelial TNF receptor-I (TNFR-I) facilitates monocyte recruitment and chronic inflammation, leading to the development of atherosclerosis. In vitro data show a heightened inflammatory response and atherogenic potential in endothelial cells (ECs) from African American (AA) donors. High laminar shear stress (HSS) can mitigate some aspects of racial differences in endothelial function at the cellular level. We examined possible racial differences in TNF-induced monocyte adhesion and TNFR1 signaling complex expression/activity, along with the effects of HSS. Tohoku Hospital Pediatrics-1 (THP-1) monocytes were used in a co-culture system with human umbilical vein ECs (HUVECs) from Caucasian American (CA) and AA donors to examine racial differences in monocyte adhesion. An in vitro exercise mimetic model was applied to investigate the potential modulatory effect of HSS. THP-1 adherence to ECs and TNF-induced nuclear factor kappa B (NF-κB) DNA binding were elevated in AA ECs compared to CA ECs, but not significantly. We report no significant racial differences in the expression of the TNFR-I signaling complex. Application of HSS significantly increased the expression and shedding of TNFR-I and the expression of TRAF3, and decreased the expression of TRAF5 in both groups. Our data does not support TNF-induced NF-κB activation as a potential mediator of racial disparity in this model. Other pathways and associated factors activated by the TNFR1 signaling complex are recommended targets for future research. Tumor necrosis factor (TNF) is a potent, pro-inflammatory cytokine produced predominately by activated leukocytes. One of the main targets of TNF is the endothelium, where it initiates a cascade of events leading to endothelial dysfunction ,2,3,4. TThe efficacy of exercise training in improving endothelial function by increasing NO bioavailability and reducing inflammation is well established ,17,18. FHigh laminar shear stress (HSS) mediates functional and structural vascular adaptations associated with exercise training . EvidencAA individuals have the highest prevalence of cardiovascular disease (CVD) . Recent Racial differences in EC responses to stimuli, particularly TNF, could play an important role in the promotion of endothelial dysfunction, plaque development, and consequent CVD. Despite the compelling data supporting a higher prevalence of endothelial dysfunction and CVD in AA, it is predominantly of an observational nature ,34,35,36In this in vitro study, we hypothesized that AA-derived ECs would exhibit greater THP-1 monocyte adhesion and higher expression/activity of the TNFR1 signaling complex that can be attenuated by HSS. Using naïve ECs such as HUVECs, a valid model to mechanistically investigate EC function, minimizes the influence of pre-existing factors and enables race/ethnicity-related EC profiling. The aims of our study were to examine the following: (1) racial differences in TNF-induced monocyte adhesion; (2) racial differences in TNFR1 signaling complex expression/activity, TNFR1 shedding, and downstream effects; and (3) the effects of HSS on mitigating expected racial differences.p < 0.001, p = 0.77, p = 0.12, TNF treatment increased THP-1 monocyte adhesion in both racial groups  TNF-induced monocyte adhesion, (2) TNF-induced activation of the TNFR1 signaling complex, or (3) the modulatory effects of HSS on TNFR1 signaling. Additionally, HSS did not mitigate TNF effects on NF-κB binding activity, diminished shedding of TNFR1, and unexpectantly modulated TRAF5 protein expression (a novel finding in this study). Although our results do not support our proposed hypothesis of higher TNFR1 signaling complex expression/activity and downstream effects in AA-derived ECs, HSS-induced atheroprotective effects mitigated TNFR1 signaling in both racial groups. A summary of the results is illustrated in TNFR1 activation by TNF binding induces endothelial dysfunction manifested by suppressed eNOS activity, higher oxidative stress levels, and amplified expression of adhesion molecules. Adhesion molecules  play a key role in recruiting monocytes and initiating atherogenesis ,11. PlasResearch implementing co-culture systems to investigate molecular mechanisms prompting racial differences in CVD is scarce. In our experiments, we used THP-1 cells, a validated monocyte cell model, to investigate atherogenesis and other vascular inflammatory conditions . NonetheContrary to our hypotheses, our results do not support differential expression/activity of TNFR1 signaling between races. Both CA and AA HUVECs responded similarly to all experimental conditions. NF-κB activation was numerically higher in AA HUVECs in response to TNF; however, it was not statistically significant. This may be explained by two factors. First, we did not measure the expression or activity of MAP kinases or other transcription factors such as AP-1 that may play a bigger role in prompting racial differences than NF-κB signaling. Second, in addition to mediators of inflammation, NF-κB induces transcription of TNFR1 signaling negative regulators that may be differentially expressed, such as IκBα and A20. It has been shown that, despite the well-established suppressive effect of A20 on TNF-induced genes regulated by NF-κB, it does not interfere with the nuclear translocation of the NF-κB heterodimers or DNA binding . If suchSeveral clinical reports have demonstrated higher shedding of TNFR1 with aerobic exercise and its association with better vasodilation and functional outcomes ,20,21,432 of shear for 18 h, we used 20 dyne/cm2 for 24 h. In vivo data suggest a negative regulatory role for TRAF5, as knocking out TRAF5 accelerates atherosclerosis in animal models. Additionally, higher systemic levels of TRAF5 are associated with recovery in patients with coronary heart disease [TRAFs are a family of intracellular adaptor proteins that function as signal transducers for the TNF receptor superfamily ,45,46,47 disease . TherefoDifferent concentrations of serum were necessary to use in our culture media for different conditions. Serum deprivation is recommended for prime cells when examining proinflammatory cytokine signaling. Therefore, we used serum-free culture medium for the static conditions and serum culture medium for the shear stress conditions. This is mainly because our cone and plate shear stress model is optimized for a limited range of medium viscosities. Nonetheless, we used only 2% fetal bovine serum, which is considered to be in the lower range . Additio2 atmosphere and cultured in gelatin-coated 100 mm tissue culture dishes. When cells reached 80–90% confluency, they were washed with Hank’s Balanced Salt Solution (HBSS) buffer and serum starved for 2 h before stimulation or application of shear stress. A concentration of 30 ng/mL of TNF (cat. No. 210-TA-020/CF) from R&D Systems  was used and incubated for 6 h. This dose was chosen based on preliminary experiments.HUVECs were purchased from Lonza  and cultured in the endothelial basal medium-2 medium containing 2% fetal bovine serum and growth supplements and used between passages 6 and 7. Cells from three AA and three CA donors were grown in parallel, and the experiment was repeated three times. This experimental design has been validated in our previous work ,29,33. C® TIB202™, Manassas, VA, USA) were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum , 1% penicillin/streptomycin , and 2 mM/L GlutaMAX (Gibco). Cells were cultured in T-75 flasks and maintained at 37 °C in a 5% CO2 atmosphere.THP-1 monocytes  or left untreated. THP-1 monocytes were labeled with 5 µM of Calcein-AM (cat. no. 354216) from Corning  and suspended in serum-free RPMI 1640 medium. After activation of HUVECs, labeled THP-1 cells were added to the HUVEC monolayer (5 × 105 per well) and incubated at 37 °C with 5% CO2 for 1 hr. Labeled THP-1 cells were added to empty wells as a background control. Nonadherent THP-1 monocytes were removed and washed with 1X phosphate-buffered saline (PBS) three times. Adherent cells were then lysed with 200 µL of RIPA lysis buffer. To quantify cell adhesion, fluorescence intensity was measured with a SpectraMax M2 plate reader  at an excitation wavelength set at 494 nm and an emission wavelength of 517 nm. Cell adhesion was determined as a percentage of the control (untreated HUVECs).HUVECs were seeded in gelatin-coated 24-well plates at a density of 5 × 102) and HSS (20 dyne/cm2) followed by TNF  incubation.After 2 hrs of serum deprivation, confluent monolayers were exposed to unidirectional shear stress for 24 h with a rotating cone-in-plate instrument at a 0.5° angle, designed for 100 mm tissue culture dishes, as we have used previously . Cone-anCell lysate was fractionated using the Nuclear Extraction Kit  following the manufacturer’s instructions with some modifications. Cytosolic extract was used for Western blotting. For each experimental condition, 10–15 μg of protein was loaded into wells, with the amount kept constant within each gel. All samples were run on duplicate gels. Proteins were separated using SDS-PAGE  according to molecular weight and transferred to PVDF membranes, which were blocked with 5% non-fat dry milk (NFDM) for 1 h at room temperature. This was followed by incubation with primary antibodies of interest with gentle agitation overnight at 4–8 °C. Primary antibodies used were as follows: TNFR1 (cat. no. 3736), TACE (cat. no. 6978), TRAF2 (cat. no. 4724), TRAF3 (cat. no. 4729), and TRAF5 (cat. no. 41658) from Cell Signaling Technology . β-actin (cat. no. 3700) from Cell Signaling Technology was used as a loading control. Protein was visualized by chemiluminescent detection using an HRP illumination substrate, Luminata Forte . The UVP ChemiDoc-It2 imaging system  was used to image each PVDF membrane, and VisionWorks software (8.19) was used to quantify protein expression by band densitometry analysis. Densities of selected proteins were expressed relative to a loading control for normalization.Cell culture supernatant was collected, centrifuged, aliquoted, and immediately stored at −80 °C until analysis. The sTNFR1 assay kit (cat. no. DRT100) from R&D Systems  was used to detect sTNFR1 production in HUVEC cell culture supernatant. The NF-κB factor assay kit (cat. no. 43296) from Active Motif  was used to quantify NF-κB DNA binding activity in nuclear extracts. p ≤ 0.05.A two-way ANOVA was performed to examine any race-by-condition interaction effect on any of the outcome variables. Violations of assumptions were examined. Non-normally distributed data were natural log-transformed. Post hoc adjustment for multiple comparisons was done using Bonferroni’s test. Analysis was performed using SPSS version 26 . Data are expressed as mean ± SE and the level of significance was set at Our results do not support racial differences in the expression of the TNFR1 complex or its signaling activity, particularly NF-κB activity, in this model. While HSS can mitigate some aspects of TNFR1 signaling similarly in ECs from both racial groups, its modulatory effect did not attenuate NF-κB binding activity. Given the higher burden of subclinical atherosclerosis in AA and the central role that TNF plays in atherogenesis, the TNFR1 signaling complex may still have a role in promoting racial differences through additional pathways not observed or measured in this study . Therefore, we propose other aspects of TNFR1 signaling, such as MAP kinase signaling and their associated downstream effects, as recommended targets for future research.


COVID-19 is a disease caused by a virus named SARS-CoV-2. SARS-CoV-2 is a single-stranded positive-sense RNA virus. Reverse transcription quantitative PCR (RT–qPCR) assays are the gold standard molecular test for detection of RNA viruses. The aim of this study was to construct an RNA-positive control based on MS2 phage-like particles (MS2 VLPs) to detect SARS-CoV-2 RNA. pCDFDuet-1 was used as a one-plasmid double-expression system to construct MS2 VLPs containing ssRNA of SARS-CoV-2. The sequence encoding one copy of maturase, His-tag and coat protein dimer was cloned and inserted into MCS1 of the plasmid; the fragment encoding protein N and ORF1ab from SARS-CoV-2 was cloned and inserted into MCS2. The prepared plasmid was transformed into Escherichia coli strain BL2 (DE3), and expression of the construct was induced by 1 mM isopropyl-L-thio-D-galactopyranoside (IPTG) at 30°C for 12 hours. MS2 VLPs were purified and collected with Ni-NTA affinity chromatography columns. The size and shape of the MS2 VLPs were verified by transmission electron microscopy, and the stability of MS2 VLP packaged RNA was evaluated by treatment with RNase A. Effects of storage temperature and buffer on MS2 VLP stability were also investigated. The results showed that SARS-CoV-2 MS2 VLPs could be successfully produced by this one-plasmid double-expression system. MS2 VLPs showed high stability and may be used as a positive control in molecular diagnosis of COVID-19.  The number of COVID-19 cases will again climb as immunity from vaccines and infections wanes. It has been reported that in China, an infection cycle will occur every six months , and repSARS-CoV-2 is a positive-sense single-stranded RNA (ssRNA) virus . For det3 solution as a storage buffer.MS2 is a type of positive-sense ssRNA virus with an icosahedral capsid, which is built from one copy of maturase  and 178 copies of coat protein . Assembl22.1Escherichia coli BL21 (DE3) was obtained from TransGen Biotech Co., Ltd. Viral RNA extraction kits were purchased from Omega Bio-Tek Co., Ltd., and peptone, yeast powder and agar powder were purchased from Beijing Solarbio Biotechnology Co., Ltd. In addition, Isopropyl-β-D-thiogalactopyranoside (IPTG) and limidazole were supplied by Sigma–Aldrich, and Ni-NTA agarose resin was obtained from Thermo Fisher. Real-time PCR kits were purchased from TOYOBO Life Science Co., Ltd.2.2E. coli BL21(DE3). The bacteria were grown overnight on LB agar plates containing streptomycin (10 µg mL-1).A 2003 bp DNA sequence encoding one copy of maturase and two copies of the coat protein with His-tag modification was synthesized by Genscript Biotech Co., Ltd. The pCDFDuet-1 plasmid was treated with NcoI and NotI at 37°C for 2 hours in a 50 µL reaction mixture containing 500 ng of plasmid DNA, 5 µL of buffer solution, and 10 U each of endonucleases. The cleaved vector DNA was purified with a Gel Extraction Kit (Omega). Ligation of the fragment encoding maturase and coat protein with MCS1 of pCDFDuet-1 was performed with a One Step Cloning Kit (Vazyme) according to the manufacturer’s instructions. Then, the recombinant plasmid (pCDFDuet-MS2) was transformed into de novo synthesized by Genscript Biotech Co., Ltd. The pCDFDuet-MS2 plasmid was then digested by NdeI and AvrII, and the cleaved plasmid DNA was purified with a Gel Extraction Kit (Omega). Ligation of the positive control sequence to MCS2 of the cleaved pCDFDuet-MS2 vector was performed with a One Step Cloning Kit (Vazyme) according to the manufacturer’s instructions. Positive clones were selected by DNA sequencing. After sequencing identification, the confirmed recombinant pCDFDuet-MS2-SARS-CoV-2 plasmid was extracted, purified and transformed into E. coli BL21(DE3) to screen for the recombinant strain at a concentration of 50 μg mL-1 streptomycin on LB plates.To meet a variety of different targeted amplicons of RT–qPCR methods for detecting SARS-CoV-2, the coding sequence of protein N and ORF1ab from SARS-CoV-2 was used as the positive control sequence. The chimeric sequence is 1857 nt in length, with one C-variant pac site of MS2 at the 3’-end. The sequence of the C-variant pac site of MS2 is ACATGAGGATCACCCATGT. The chimeric sequence was 2.3E. coli BL21(DE3) cells harboring the pCDFDuet-MS2-SARS-CoV-2 plasmid were cultured in LB broth medium containing 10 μg mL−1 streptomycin at 37°C for 4 hours. Cultures of the recombinant BL21(DE3) cells were diluted 1:100 in fresh 100 mL LB medium. When the optical density (OD) 600 reached 0.4–0.6, isopropyl-β-d-thiogalactoside (IPTG) was added to a final concentration of 1 mM, and the culture was incubated at 30°C for 12 hours. The cell suspension was centrifuged at 10000 rpm for 10 minutes at 4°C, and then the pellet was washed three times with PBS.The engineered The pellets were resuspended in PBS and then sonicated. To eliminate cell debris, the bacterial suspension was briefly centrifuged at 10000×g for 15 minutes at room temperature. Twenty microliters of the supernatant was mixed with 5 μL of 5×SDS sample buffer, and 15 μL of this mixture was separated by 12.5% SDS–PAGE electrophoresis.2.4The supernatant from the above step was mixed with equilibrium buffer  for purification of the His-tagged MS2 VLPs with a Ni-NTA column. In brief, the column was preequilibrated with 100 mL binding buffer  at a flow rate of 0.5~1.0 mL/min. The His-tagged MS2 VLPs were bound to the columns, washed with washing buffer  until OD280=0, and eluted from the column using elution buffer  at a flow rate of 0.5 mL/min.Ten microliters of the solution containing MS2 VLPs was dropped onto carbon-coated copper grids and then negatively stained with 2% uranyl acetate. After drying, the MS2 VLPs were observed by transmission electron microscopy (TEM) at 150,000 × magnification.2.5RNA packaged into MS2 VLPs was extracted using a viral RNA extraction kit (Qmega) according to the manufacturer’s instructions and then reverse transcribed to cDNA. qPCR was performed using the following protocol: 1 cycle at 95°C for 10 min, followed by 30 cycles of 95°C for 30 seconds, 62°C for 60 seconds, and 72°C for 60 seconds. The primers and probes were synthesized by Genscript Biotech Corporation  and are listed in 2O). qPCR was performed in duplicate for each sample.To determine the amount of contaminating DNA in the MS2 VLPs, qPCR was performed without reverse transcriptase  were stored in PBS solution (pH 8.0) at 37°C for 90 days or -20°C for 60 weeks. Then, total RNA was extracted with a viral RNA extraction kit, and RT–qPCR was performed to determine the number of copies of RNA at different time points.MS2 VLPs (containing 7.85×102.83-Tris (pH 8.0) buffer at 37°C for 90 days or -20°C for 60 weeks. RT–qPCR and qPCR were performed to determine the concentration and purity of the positive control RNA, respectively.MS2 VLPs were also stored in NaNO33.1The plasmid pCDFDuet-1 was linearized by NotI digestion, and pCDFDuet-MS2-SARS-CoV-2 was treated with NdeI and AvrII endonucleases at 37°C. The results of 1% agarose gel electrophoresis analysis are shown in 3.2E. coli BL21 (DE3) cells without the pCDFDuet-MS2-SARS-CoV-2 plasmid. To confirm expression of the MS2 coat protein, western blotting was performed using an anti-His-tag antibody. The results are shown in After induction by IPTG, the bacterial cells were centrifuged and the pellets collected. SDS–PAGE (12.5%) was performed to analyze expression of MS2 coat protein and maturase. The results showed that both MS2 coat protein dimer (44 kD) and maturase (29 kD) were expressed by IPTG induction in BL21 cells Figure 23.3After purification with a Ni-NTA affinity chromatography column, the MS2 VLPs were observed by TEM. The results confirmed that the MS2 VLPs had assembled into intact capsids of approximately 30 nm in diameter. Moreover, the MS2 VLPs were not damaged by the purification process and did not form any aggregates Figure 43.412 copies/μL. The purity of the RNA in MS2 VLPs was 99.99%.RT–qPCR was performed to detect the copy numbers of RNA in MS2 VLPs. The copy number of contaminating DNA in the MS2 VLPs was determined by qPCR. The results showed that the copy number of RNA in MS2 VLPs was 6.47×103.58 copies/μL packaged RNA, with 2.24×107 copies/μL naked RNA , HIV-1, avian influenza A (H7N9), ebolavirus (EBOV), measles virus (MeV), hepatitis E virus (HEV), and dengue virus (DENV). However, the length of the exogenous RNA packaged by MS2 VLPs is limited, resulting in two or more armored RNAs being expressed for an assay of one kind of virus . The one5 copies/μL. In this study, we prepared MS2 VLPs containing SARS-CoV-2 RNA fragments using the double expression system pCDFDuet-1. The results showed that the positive control RNA sequence (1857 nt) was successfully encapsulated into MS2 VLPs, which were stable at 37 and -20°C. These results are consistent with previous studies  buffer as the storage solution for SARS-CoV-2 MS2 VLPs, and the stability of the MS2 VLPs was obviously enhanced when stored in NaNO3-Tris solution.PBS, HEPES, and Tris buffers, alone or mixed with electrolytes such as MgSO storage . The pH  storage . It has  storage . In the 5SARS-CoV-2 MS2 VLPs prepared using the one-plasmid double-expression system were studied, and their stability in ribonuclease challenges or time–temperature tolerance was evaluated. The results demonstrated that SARS-CoV-2 MS2 VLPs meet the requirements for application as a positive control material in molecular diagnosis of COVID-19. In the last two decades, coronaviruses (CoVs) have caused major emerging infectious diseases, which have greatly threatened public health. To date, 7 species of CoVs have been identified as infecting humans. In the future, it is likely that high-risk CoV species will appear, jump to humans and cause widespread infection. Hence, it is essential to establish methods for rapid detection and diagnosis. One or more consensus RNA sequences from α-CoV or β-CoV can be packaged into MS2 VLPs, which may be used as the positive control of RT–qPCR assays for new high-risk CoV species.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.LQ drafted the manuscript. ZZ, MW and GD performed protein expression, purification and sample preparation. ZK and HM performed the data collection and analyzed the data. JW designed the study. All authors discussed the results and commented on the manuscript. All authors contributed to the article and approved the submitted version.


The vanilla federated learning is made for a trusted environment, while in contrast, its actual use cases require collaborations in an untrusted setting. For this reason, using blockchain as a trusted platform to run federated learning algorithms has gained traction lately and has become a significant research interest. This paper performs a literature survey on state-of-the-art blockchain-based federated learning systems and analyzes several design patterns researchers often take to solve existing issues through blockchain. We find about 31 design item variations throughout the whole system. Each design is further analyzed to find pros and cons, considering fundamental metrics such as robustness, efficiency, privacy, and fairness. The result shows a linear relationship between fairness and robustness in which, if we focus on improving fairness, it will indirectly become more robust. Furthermore, improving all those metrics altogether is not viable because of the efficiency trade-off. Finally, we classify the surveyed papers to spot which designs are popular among researchers and determine which areas require immediate improvements. Our investigation shows that future blockchain-based federated learning systems require more effort regarding model compression, asynchronous aggregation, system efficiency evaluation, and the application for cross-device settings. Federated Learning (FL)  has gainRecent advances in blockchain have revolutionalized existing architectures to lean more towards decentralization. Bitcoin , in partOur argument on naming blockchain as a suitable candidate for FL is backed up by the fact that many researchers in the literature are also integrating blockchain into their FL system ,8. HowevWe investigate research papers from the literature and find about 31 design variations researchers often take to solve FL issues in their system.We analyze the pros and cons of committing to each design pattern concerning the fundamental FL metrics such as robustness, efficiency, privacy, and fairness.We classify the surveyed papers to seek trends of popular design patterns.Based on our analysis, we derive several research directions to enhance the quality of future blockchain-based FL systems.This paper examines the state-of-the-art blockchain-based FL systems to analyze their progress, trends, and possible future research directions. We study the surveyed papers in detail to extract and analyze specific system-design parameters. In summary, we made the following contributions:Several surveys related to the FL in general  or blockThe rest of this paper is organized as follows. Vanilla FL aims to train a global model collaboratively in devices’ machines. Instead of sending private data to a server for remote training , and a given entity may play multiple roles simultaneously. The While FL guards user private data by design, the vanilla FL system also opens novel privacy, security, and trust issues as consequences. Leakage: The trained models become more susceptible to theft in FL than in conventional machine learning (ML) architecture. FL design encourages model sharing among entities, which generates a high probability of model leakage. Adversaries can quickly obtain a trained global model by spoofing or acting as valid clients. They do not need to compromise the server to get the model, as is usually required in ML. Moreover, even though attackers cannot obtain the private data, they can guess the trained data using a membership inference attack )In conclusion, the most robust FL system is achievable through the combinations of contribution-based rewards, restricted-trainer, blockchain storage, data encryption, peer-reviewed model verification, deposit, reputation system, and multiple on-chain aggregators design. The most efficient FL system relies on model compression and asynchronous aggregation design. The most privacy-preserving FL system should include homomorphic encryption or differential privacy. Finally, for the fairest FL system, we can use the same features as in the most robust FL but replace the restricted-trainer with an open-trainer design.After introducing the design patterns of blockchain-based FL and analyzing their pros and cons, this section classifies the surveyed papers to find trends of design and implementation that are popular among the papers. This section also discusses the future research directions for blockchain-based FL research and mentions the limitations of our survey.We perform two classifications from surveyed papers regarding their designs and implementations.✓). Meanwhile, the cross (✗) indicates otherwise. From this classification, we can identify which designs gain lots of traction and which ones lack adoptions to be solved as future research directions.We read the surveyed papers and classified them based on found design patterns in How to attract trainers?: Among all papers with a reward system, the majority (66.67%) implement contribution-based rewards, with the most frequently-used method being dataset (37.5%) and accuracy measurement . With the importance of incentives to motivate trainers, we think a reward system is a must-have feature, especially for blockchain-based FL, where the incentive mechanism can be built quickly by leveraging blockchain tokenization.How to choose trainers?: The open-trainer design is favorable among surveyed papers (about 56.67%), while the rest uses the restricted-trainer method (43.33%). The deposit mechanism takes the lead (41.18%) in the latter design since, similar to incentive, the deposit can be easily implemented with blockchain tokenization. On the other hand, building a reputation system is the most complex approach; therefore, few papers (only 17.65%) employ this strategy despite its vast benefits.How to prevent model leakage?: Only about half (53.33%) of our surveyed papers utilize encryption algorithms to protect the secrecy of the shared models in their system. Most use a public-key mechanism (about 45.83%), with HE implemented by 29.17%.How to distribute models?: Because all the surveyed papers are blockchain-based systems, most (about 90.32%) leverage blockchain as a medium for model transfer, specifically, 48.39% of papers save the model directly in the blockchain, while 41.94% use IPFS as model storage and save the corresponding model hash in the blockchain.How to prevent data leakage?: Among all the surveyed papers, only 36.67% apply DP or HE to prevent membership inference attacks. The membership attacks and their defenses are complex [How to make communication efficient?: Model compression has not been used widely in surveyed papers; only about 6.67% of the papers implement such a compression scheme. Thus, this info can be seen as an opportunity window for future FL systems to focus more on the efficiency of the FL system.Who should become reviewers?: Not many papers apply a verification mechanism to the trained local models before the aggregation (only about 53.33%). This may indicate that most papers are vulnerable to data or model poisoning attacks. Nevertheless, when deploying a future FL system, it is necessary to include model verification because it can still be effective as a safeguard to protect the quality of the global model, even when run in a trusted environment.How to select models for aggregation?: The contribution score calculation over the trained models is used in most papers (about 60%) that employ the model verification scheme. Furthermore, most papers that apply model verification reward the clients based on their contribution with a contribution-based reward design.How to punish malicious actors?: Unfortunately, most surveyed papers (66.67%) have no punishment for malicious behavior. Among all papers with punishment, deposit confiscation is used 66.67%Who can become aggregators?: Researchers often choose a single aggregator (56.67%) over multiple aggregators (43.33%). A possible reason for this is that a single aggregator is closely similar to the vanilla FL. Hence, it is easier to implement than multiple ones.How to aggregate models?: Most of the surveyed papers apply synchronous aggregation (93.33%), an aggregation process similar to vanilla FL. Therefore, future research is needed to apply asynchronous FL  for the blockchain-based system.Where does aggregation happen?: Because the off-chain aggregation approach is straightforward and not different from the vanilla FL, many of the surveyed papers (about 66.67%) take this approach.We analyze the trends of implementation procedures that the surveyed papers take in their experiment and derive possible research directions from this analysis. Our findings are summarized in Most surveyed papers (about 86.67%) have provided prototype implementation and evaluations. Based on their results alone, we can safely note that blockchain-based FL is highly viable.FL algorithms: The vanilla Federated Averaging (FedAvg) algorithm [lgorithm  is used lgorithm , Model dlgorithm , DANE [7lgorithm , CDW Fedlgorithm , Model clgorithm , and Siglgorithm . This stFL nodes: Regarding the number of FL nodes used during the experiments, about 57.69% of papers employ up to 10 nodes, while 34.62% of papers run their experiments using 10 to 50 nodes, and 15.38% evaluate using 50 to 100 nodes. Meanwhile, only 7.69% leverage more than 100 nodes. Hence, future blockchain-based FL research should focus on using more nodes (in order of hundreds or even thousands) better to evaluate the feasibility and scalability of the system. Especially considering that a cross-device FL environment is expected to have an enormous number of nodes involved [involved .FL library: Most papers (about 26.92%) use Tensorflow, while 11.54% use PyTorch or Keras. Syft (7.69%) is also worth looking at in the future since this library focuses heavily on the FL environment. This library diversity indicates the versatility of the blockchain-based FL system.FL datasets: Based on our observations, most papers evaluate their system for the image classification tasks using MNIST (46.15%) and CIFAR-10 (23.08%) datasets. Meanwhile, the rest of the papers evaluate diverse tasks such as health-related  a,60 aFL dated , scalability (in terms of latency or throughput), and efficiency . 88.46% of papers have widely evaluated the accuracy, proving that the accuracy of blockchain-based FL is on par with the vanilla FL. Meanwhile, only 50% of papers have scalability evaluation, and only 38.46% mention their gas cost measurements. Based on this statistic, future blockchain-based FL research should stop measuring the model accuracy unless they modify the FL-related parts in their system. Instead, more research is required to boost the system throughput and minimize the latency and gas cost.Availability: Only 19.23% of papers open their implementation in the public repository. We think that more blockchain-based FL research should open their codes to the public to help future research better.This part elaborates on the ideal architecture for future blockchain-based FL systems and strategic choices on which design to use.contribution-based reward design, which fairly rewards participants based on their performance. Similarly, deposit and/or reputation system should also be employed to protect against Sybil attacks.First, the incentive is a mandatory feature to encourage honest behavior from participants. Especially the homomorphic encryption, we do not need to add differential privacy and other encryption schemes. Employing HE also does not reduce the trained global models’ accuracy as opposed to using DP. Therefore, HE should be the goal for future FL systems because it can solve security and privacy issues in one go.Second, when using asynchronous aggregation should be preferable over the synchronous ones. It provides participants flexibility regarding when they should download, train, review, and aggregate models. The process feels more seamless without time-gated operations as in the synchronous approach.Third, the model compression increases if we use many trainers and reviewers in the system because those designs generate many back-and-forth model transfers between clients.Fourth, the value of open channel, third-party distributed storage , off-chain aggregation becomes more appealing than the alternatives. Otherwise, we can process most things on-chain if using a private network for a better integrity guarantee.Finally, if using a public blockchain network, we want to limit on-chain operations such that the use of FL and blockchain need a separate research direction on their own. First, FL research needs to produce more accurate, communication-efficient, and bias-tolerant algorithms . Based oThe cross-silo and cross-device FL systems have their trade-offs. Applying cross-silo makes the FL system more efficient, but the training is more likely to be centralized. Meanwhile, the cross-device solution produces much overhead, but the training can be more decentralized. As demonstrated in , cross-sFinally, the ideal goal for a fair FL system has been described in , where , making it difficult to classify as no standardized blockchain architecture is available. Similarly, other blockchain platforms that support compatibility with Ethereum Virtual Machine (EVM), such as Polygon, Avalanche, and Klaytn, might also be overlooked. However, since they support EVM, design patterns from this paper can be applied to their networks out of the box when needed.This paper performed a literature survey on many blockchain-based federated learning systems. First, we drew out about 31 design patterns from each research study in various FL workflows, such as registering clients, distributing models, training models, validating models, and the aggregation process. Second, we analyzed the pros and cons of committing to a particular design concerning its robustness, efficiency, privacy, and fairness. From our analysis, we found a linear relation between robustness and fairness in which, when we focused on improving the fairness of the FL system, we would end up with a robust system. We also discovered that designs that make the system more robust, private, and fair would suffer inefficiency due to the additional processing required. Therefore, pursuing excellent values on all metrics might not be feasible due to such trade-offs. Third, we classified the surveyed papers to find which designs were popular among surveyed papers. We found that more efforts must be made to improve system efficiency since only two designs  improve it, and only four papers adopt those designs. Finally, we investigated the surveyed papers based on their proof-of-concept implementations. The result shows diverse choices of FL algorithms, libraries, and datasets used in their experiments. Therefore, we can conclude that blockchain and FL are loosely coupled and can be used in various use cases .