Hippoboscid flies (Diptera: Hippoboscidae), also known as louse flies or keds, are obligate blood-sucking ectoparasites of animals, and accidentally of humans. The potential role of hippoboscids as vectors of human and veterinary pathogens is being increasingly investigated, but the presence and distribution of infectious agents in louse flies is still unknown in parts of Europe. Here, we report the use of molecular genetics to detect and characterize vector-borne pathogens in hippoboscid flies infesting domestic and wild animals in Austria.n = 25), sheep (n = 3), and red deer (n = 12) across Austria between 2015 and 2019. Individual insects were morphologically identified to species level and subjected to DNA extraction for molecular pathogen screening and barcoding. Genomic DNA from each louse fly was screened for Borrelia spp., Bartonella spp., Trypanosomatida, Anaplasmataceae, Filarioidea and Piroplasmida. Obtained sequences of Trypanosomatida and Bartonella spp. were further characterized by phylogenetic and haplotype networking analyses.Louse flies were collected from naturally infested cattle (Hippobosca equina (n = 62) collected from cattle, Melophagus ovinus (n = 100) from sheep and Lipoptena cervi (n = 120) from red deer (Cervus elaphus). Molecular screening revealed pathogen DNA in 54.3% of hippoboscids, including infections with single (63.39%), two (30.71%) and up to three (5.90%) distinct pathogens in the same individual. Bartonella DNA was detected in 36.9% of the louse flies. Lipoptena cervi were infected with 10 distinct and previously unreported Bartonella sp. haplotypes, some closely associated with strains of zoonotic potential. DNA of trypanosomatids was identified in 34% of hippoboscids, including the first description of Trypanosoma sp. in H. equina. Anaplasmataceae DNA (Wolbachia spp.) was detected only in M. ovinus (16%), while < 1% of the louse flies were positive for Borrelia spp. and Filarioidea. All hippoboscids were negative for Piroplasmida.A total of 282 hippoboscid flies corresponding to three species were identified: Bartonella spp.) and the first report of Trypanosoma sp. in H. equina, suggesting a potential role of this louse fly as vector of animal trypanosomatids. Experimental transmission studies and expanded monitoring of hippoboscid flies and hippoboscid-associated pathogens are warranted to clarify the competence of these ectoparasites as vectors of infectious agents in a One-Health context.Molecular genetic screening confirmed the presence of several pathogens in hippoboscids infesting domestic and wild ruminants in Austria, including novel pathogen haplotypes of zoonotic potential (e.g. The online version contains supplementary material available at 10.1186/s13071-023-05810-4. Melophagus spp., Lipoptena spp. and Hippobosca spp. have been described to commonly infest domestic and wild ungulates in Europe [Lipoptena cervi on the late neolithic human mummy “Ötzi” in the Ötztal Alps [Hippoboscid flies (Diptera: Hippoboscidae), also known as louse flies or keds, are obligatory blood-sucking ectoparasites infesting mammals and birds worldwide . To daten Europe –11, and n Europe –15. Indetal Alps . Considetal Alps .Anaplasma spp., Babesia spp., Bartonella spp., Borrelia spp., Mycoplasma spp., Rickettsia spp., Theileria spp. and Trypanosoma spp. [Hippoboscid flies have been investigated for their role as vectors of animal pathogens for over a century , 19, witoma spp. –31. Despoma spp. , 33 and The aim of the present study was to detect the presence of vector-borne pathogens in hippoboscid flies infesting domestic and wild ruminants in Austria using molecular techniques. In addition, DNA barcoding of the hippoboscid flies was performed to confirm and characterize their identity.Cervus elaphus;n = 12), sheep  and cattle  in various locations in Austria, between 2015 and 2019  in 1.5-ml Eppendorf tubes, and two 1.4-mm ceramic beads  were added per tube, followed by mechanical homogenization in a TissueLyser II  at room temperature for 6 min. Then, 20 μl proteinase K was added, and the tubes were vortexed and incubated at 56 °C overnight. After incubation, total DNA was extracted from insect material using the QIAGEN DNeasy Blood & Tissue Kit , following the manufacturer’s instructions.COI by conventional PCR [COI sequences were used for the taxonomical characterization of the hippoboscid species by comparison with available sequences on the GenBank nucleotide database for organism identification using BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and BOLD .To confirm the species identity and to explore the genetic diversity of the collected hippoboscids, selected specimens were subjected to DNA barcoding analysis. Total DNA extracted from 21 hippoboscids was used to amplify a region within the insects’ mitochondrial onal PCR  as descr16S ribosomal RNA), the genus Borrelia  and the genus Bartonella (citrate synthase gene—gltA), as well as for nematodes of the superfamily Filarioidea . In addition, the DNA was screened for parasites of the orders Trypanosomatida  and Piroplasmida  using nested PCRs. The PCR methodologies were based on previously published protocols [18S sequences of Trypanosomatida available on GenBank and allow the amplification of all strains. All PCR reactions were performed in an Eppendorf Mastercycler Pro . The PCR products were stored at 15 °C until confirmation of the amplified regions of interest by electrophoresis in 2% agarose gels stained with Midori Green Advanced dye . PCR products positive for the investigated pathogens were sequenced at LGC Genomics GmbH  using amplification primers. The sequences were assembled with BioEdit [https://blast.ncbi.nlm.nih.gov/Blast.cgi) using multiple BLAST searches.The obtained DNA from each hippoboscid fly was screened for the presence of several vector-borne pathogens by targeting selected genes using primers and PCR protocols summarized in Table rotocols –42, exce BioEdit  and compBartonella spp. isolated from the investigated hippoboscids were subjected to phylogenetic analyses as previously described [Bartonella spp. sequences, which suggested a co-infection with two distinct strains of Bartonella sp. in the same insects. In those cases, the two strains were unphased to obtain single sequences and uploaded as individual sequences to GenBank. Each strain was separately uploaded to GenBank  and used for phylogenetic analysis.Selected sequences of Trypanosomatida and escribed , with moBartonella spp. strains, maximum likelihood (ML) and Bayesian inference (BI) trees were calculated for each of the two groups based on alignments including 409 sequences (991 nucleotide positions) for Trypanosoma spp. and 582 sequences (338 nucleotide positions) for Bartonella spp. Gaps in the alignments were removed using TrimAl v.1.3 [Trypanosoma spp. and 261 haplotypes for Bartonella spp. As outgroup of the Trypanosoma spp. tree, a sequence of Belchomonas wendygibsoni (KF054126) was used. No suitable sequence was available as outgroup for Bartonella spp., and this tree was instead mid-point rooted. The ML bootstrap consensus trees (1000 replicates) were calculated using the W-IQ-TREE web server [Trypanosoma spp. and JC for Bartonella spp., which were suggested as best fit for the data set in the model test according to the corrected Akaike information criterion. The BI trees were calculated using MrBayes v.3.2.7 [Trypanosoma spp. and JC for Bartonella spp. The analyses were run for 10,000,000 generations (number of chains: 4), sampling every thousandth tree. The first 25% of trees were discarded as burn-in, and a 50% majority-rule consensus tree was calculated based on the remaining 7500 trees. The sequences for the DNA haplotype network analyses were selected based on well-supported clades in the phylogenetic trees  Trypanosomatida and Al v.1.3 , and theAl v.1.3 , leavingb server  and appl v.3.2.7  applyingprop.test) and pairwise comparison for proportions with Holm-Bonferroni method (pairwise.prop.test). The risks of each hippoboscid species to be infected with only one or with two concurrent pathogens in the same individual (positive/negative) were evaluated with logistic regression models  using louse fly species as explanatory variable to calculate odds ratios (OR) and 95% confidence intervals (95% CI). A level of P < 0.05 was considered significant.Data processing and descriptive statistics were performed in Microsoft Excel and GraphPad Prism 7. Statistical analyses were implemented in R version 4.0.3 . Differen = 25), sheep (n = 3) and red deer (n = 12) in different regions of Austria. The hippoboscids were identified as Hippobosca equina  revealed that the COI sequences of each louse fly species clustered within a respective Barcode Index Number with sequences previously reported from Europe, Northern Africa and Asia for H. equina (BOLD:AAX0882), M. ovinus (BOLD:AAX4771) and L. cervi (BOLD:ABX1452). Obtained COI sequences from the barcoded hippoboscids were submitted to GenBank under the following accession numbers: ON129173, ON129175, ON129176, ON129178, ON129181 (H. equina); ON129174, ON129177, ON129179, ON129180, ON129182 (M. ovinus); ON341137 – ON341147 (L. cervi).A total of 282 louse flies were collected from naturally infested cattle  and H. equina , whereas no difference in total infection rate was observed between L. cervi and H. equina . The percentages of hippoboscids from each species infected with single or multiple pathogens in the same individuals are illustrated in Fig. M. ovinus had significantly higher odds to be infected with at least one pathogen compared with H. equina , whereas L. cervi had slightly higher odds to be infected with a single pathogen compared with H. equina . Melophagus ovinus was significantly most likely to be infected with two concurrent pathogens compared with H. equina . No differences were observed in the risk of carrying two pathogens between L. cervi and H. equina  of the collected hippoboscids, with substantial differences between louse fly species (Table Bartonella spp. was detected in 36.9% (104/282) of the investigated hippoboscids, followed by Trypanosomatida in 34.0% of all louse flies (96/282). Individuals of M. ovinus were found more frequently infected with Bartonella spp. in comparison with H. equina  and with L. cervi . The sheep ked M. ovinus were also more often infected with Trypanosomatida compared to H. equina  and to L. cervi . Only M. ovinus individuals were positive to Anaplasmataceae in 5.7%  and less than 1% of the louse flies were positive for Borrelia spp. (one M. ovinus specimen) and Filarioidea (one L. cervi specimen). All investigated hippoboscids were negative for Piroplasmida. The sequences of the pathogens screened in the present study were deposited in GenBank under the following acc. no.: ON668330 (Borrelia spp.), ON678056 (Filarioidea), ON637624 – ON637640 (Trypanosomatida) and OP198738 – OP198806 (Bartonella spp.).Bartonella spp. were the most common infectious agents detected in H. equina and L. cervi, and the second most frequently identified pathogen in M. ovinus after trypanosomatids  sequences showed 100% identity with a Bartonella sp. strain isolated from the bat Miniopterus schreibersii in Hungary  and from L. cervi , which was 100% identical to sequences of Bartonella chomelii reported from Spain, France and New Caledonia  showed 100% identity to sequences of CandidatusBartonella melophagi from M. ovinus in Peru, the USA and China and from a European hedgehog (Erinaceus europaeus) in Czechia . Most sequences of B. chomelii, CandidatusB. melophagi and Bartonella spp. detected in the present study clustered in one subclade with B. chomelii, B. schoenbuchensis, B. capreoli and Bartonella spp. sequences  was placed in a separate sister clade together with B. bovis and Bartonella spp. .ds Table , Fig. 3.18S rRNA) were detected in all hippoboscid species and represented the most common pathogens in M. ovinus with 87% positive individuals . The DNA haplotype network analysis revealed two distinct strains of T. melophagium: a new strain (ON637626) and a second one (ON637624) identical to T. melophagium sequences isolated from M. ovinus in Croatia, UK and Czechia  were positive for trypanosomatids, featuring a Trypanosoma sp. (ON637634) that clustered together with other trypanosomatids of ruminants, including Trypanosoma theileri, T. trinaperronei, T. melophagium, T. cervi and Trypanosoma spp. , Trypanosoma sp. from horse flies in Russia (MK156792-MK15794) and T. theileri obtained from tsetse flies in the Central African Republic (KR024688). While the trypanosomatid sequences isolated from L. cervi showed  > 99% identity to sequences of non-parasitic kinetoplastids of the genus Bodo from the UK and USA .Trypanosomatid sequences  were only detected in 16 M. ovinus individuals, featuring sequences identical to those of several Wolbachia strains, including a strain previously isolated from M. ovinus . Borrelia spp. (16S rRNA) was detected in a single M. ovinus and had a 93.7% similarity with a reported Borrelia sp. (CP043682) isolated from ticks associated with passeriform birds. Finally, one L. cervi individual featured the COI sequence of an unknown onchocercid nematode (Filarioidea), most similar (95.1%) to Mansonella perforata isolated from Sika deer (Cervus nippon) in Japan (AM749265).Anaplasmataceae sequences , to Bartonella spp. and to trypanosomatids. Sheep keds also had a higher risk of being infected with two concurrent pathogen groups compared with L. cervi and H. equina. However, detailed molecular analyses revealed different pathogens (within each pathogen group) infecting each louse fly species; therefore, a comparison of the prevalences of the same pathogen between hippoboscids and their animal hosts is not possible. The different pathogens identified in the three hippoboscid species, and the probable role of these hippoboscids as vectors of the identified pathogens, are discussed below.Here, we confirmed the molecular presence of various pathogens in blood-sucking hippoboscid flies infesting domestic and wild ruminants in Austria. The three louse fly species collected and investigated, n Europe , 50. In ve hosts . The thrBartonella spp. were the most frequently detected pathogens in H. equina and L. cervi, and the second most in M. ovinus. All Bartonella spp. in our study corresponded phylogenetically to species of the Bartonella linage II associated with strains that infect domestic and wild ruminants [Bartonella spp. were first described in H. equina, L. cervi and M. ovinus almost 20 years ago [Bartonella vectors [Bartonella spp. strains in L. cervi collected from red deer. Seven of these Bartonella spp. strains were highly similar (> 99%) to B. schoenbuchensis, a widespread pathogen infecting the midgut of deer keds [Bartonella schoenbuchensis has been molecularly detected in blood and tissues samples from various wild ungulates, including red deer, roe deer and moose , all natural hosts for L. cervi [. Our results suggest that B. schoenbuchensis and related Bartonella spp. strains are common in L. cervi in Austria and may also be circulating in the local wild red deer populations. This is noteworthy in a One-Health context, considering that B. schoenbuchensis can be transmitted to humans, as described by a report of bacteremia in a patient suffering from fatigue, muscle pain and fever following a tick bite [B. schoenbuchensis has been suggested as the etiological agent of deer ked dermatitis in humans bitten by L. cervi [Bartonella henselae [B. schoenbuchensis in wild deer, deer keds and potentially other arthropod vectors in Austria warrant confirmation. Additionally, one Bartonella sp. strain isolated from deer keds in our study matched with a previously reported Bartonella sp. sequence detected in the common bent-wing bat M. schreibersii [Bartonella sp. and the B. schoenbuchensis-like strains identified in our study clustered together with B. schoenbuchensis and B. chomelii in the DNA haplotype network analysis. The other two Bartonella spp. strains detected in L. cervi clustered in a separate subclade and were highly similar to sequences of CandidatusB. melophagi reported from M. ovinus [Bartonella sp. isolated from Sika deer [Bartonella spp. lineages detected in deer keds in the present study and the presence of co-infections with two different Bartonella spp. lineages in several individuals indicate that L. cervi are reservoirs for a wide range of Bartonella spp. strains in Austria. Recent studies have also reported the recovery of several Bartonella spp. strains with zoonotic potential in deer keds (Lipoptena cervi and L. fortisetosa) and in cervids across Europe [Bartonella spp. in deer keds and cervid populations.uminants . Bartoneears ago , with gr vectors , 52–57. eer keds , 54. BarL. cervi , 58–61. ick bite . Moreovehenselae . Therefoeibersii . This Ba. ovinus  and to Bika deer . The divs Europe , 33, 67,s Europe , 68, it Bartonella chomelii was the sole Bartonella species detected in H. equina collected from cattle in the present study. Bartonella chomelii was first described as a distinct Bartonella species from blood samples of cows in France [H. equina [B. chomelii in ticks collected from rodents and dogs [B. chomelii has not been detected in horses or other equids (the primary hosts of H. equina) or in H. equina parasitizing horses [B. chomelii, which may be more closely related to Bartonella spp. from wild ruminants than strains isolated from domestic cattle such as B. bovis [B. chomelii in Austria, further studies are needed to understand the occurrence and potential impact of this pathogen in cattle and wild ruminant populations. Previous work has suggested a higher prevalence of B. chomelii in older cattle (> 2 years old) and livestock managed in mountain pastures (> 600 m above sea level) [B. chomelii-positive H. equina were collected from cattle grazing on mountain grasslands , located in the Hohe Tauern Alps of Salzburg [B. chomelii, although this remains to be confirmed. To date, B. chomelii has not been demonstrated to induce disease in cattle, but infections with the related species B. bovis have been associated with bovine endocarditis [n France , and subn France , 70, 71 . equina , 56, 57.and dogs , 73. In g horses , 71. It B. bovis . ConsideSalzburg , which scarditis .Bartonella spp. sequences detected in M. ovinus belonged to Candidatus B. melophagi, one of the most common pathogens in sheep ked populations [Candidatus B. melophagi in sheep, including its successful culture from ovine blood, thus suggesting that sheep can serve as a host reservoir for this pathogen, with M. ovinus as its likely vector [Candidatus B. melophagi can cause clinical disease in sheep and whether M. ovinus is a competent vector transmitting these bacteria. Importantly, Candidatus B. melophagi was isolated from blood samples of two human patients presenting non-specific symptoms such as cardiovascular problems, pain and fatigue [Candidatus B. melophagi infections and the clinical symptoms [Candidatus B. melophagi and the role of M. ovinus as its vector require further elucidation.Our results confirmed that the ulations , 53, 56.ulations , new eviy vector , 75. How fatigue . Despitesymptoms . TherefoT. melophagium was the sole trypanosomatid species detected in M. ovinus, with high infection rate among the investigated sheep keds. Trypanosoma melophagium has been known to infect sheep keds and sheep for over a century [M. ovinus collected from sheep in Scotland [T. melophagium is a single species restricted to M. ovinus and sheep; it is a member of the subgenus Megatrypanum, which includes other host-restricted pathogens infecting domestic and wild ruminants such as Trypanosoma theileri in cattle [T. melophagium in sheep infested by M. ovinus carrying this pathogen, and it has been suggested that sheep may become infected with T. melophagium merely by oral ingestion of sheep keds [T. melophagium can cause disease in sheep or be transmitted to other ruminant or mammalian species, and it has been proposed that it is non-pathogenic for infected M. ovinus [M. ovinus in Austria could be infected with T. melophagium, and this should be confirmed, particularly in sheep farms with low or no use of ectoparasiticides for the control of sheep keds (e.g. organic farms), as previously described [In the present study,  century , 77, andScotland , CroatiaScotland  and receScotland . Phylogen cattle , 79. Eareep keds , 25. To . ovinus . NeverthTrypanosoma sp. strain in three H. equina individuals collected from cattle in two different states of Austria . To the best of the authors’ knowledge, this is the first report of Trypanosoma sp. in H. equina, thus pointing at a potential role of this hippoboscid as a novel vector of animal trypanosomatids. In our phylogenetic analysis, this Trypanosoma sp. strain clustered together with sequences of the T. theileri group and was highly similar to Trypanosoma cf. cervi sequences isolated from white-tailed deer (Odocoileus virginianus) in the USA [T. theileri-like strains from the horse flies Hybomitra tarandina, Chrysops divaricatus and Hybomitra muehlfeldi in Russia [T. theileri obtained from the tsetse fly Glossina fuscipes in the Central African Republic [T. theileri/cervi complex in mosquitoes, suggesting a widespread distribution of these pathogens in animal hosts, potentially wild ruminants [Trypanosoma sp. strain detected in H. equina in the present work might belong to the T. theileri group/complex, which may be elucidated by future molecular studies monitoring trypanosomatids in H. equina and cattle using various target genes. Furthermore, considering that T. theileri has not yet been reported to infect cattle in Austria but is present in neighboring countries [T. theileri in vectors such as H. equina and tabanid flies, as well as in Austrian cattle, is warranted. Molecular genetic studies on trypanosomatids such as T. theileri-like strains have also been reported in the deer keds Lipoptena fortisetosa and L. cervi in Poland and Czechia [L. cervi, we identified sequences highly similar to Bodo sp., which are non-parasitic kinetoplastids (suborder Bodonina) present in soil and water. Although Bodo spp. were isolated from bat ectoparasites and from the woylie Bettongia penicillate, an Australian marsupial, these findings were associated with environmental contamination of the mammalian hosts rather than infection [Bodo sp. in hippoboscids.Our work identified a  the USA , with T.n Russia  and withRepublic . A previuminants . Therefoountries , 86, the Czechia , 78, butnfection , 88. TheWolbachia spp. strains, which are known endosymbionts of nematodes and arthropods, including hippoboscids such as H. equina and M. ovinus [16S sequences of Wolbachia were identical to those of strains previously isolated from M. ovinus [Wolbachia on M. ovinus and other hippoboscid flies are unknown [M. ovinus and L. cervi can be infected with Anaplasma ovis [A. phagocytophilum [A. phagocytophilum has been confirmed to infect wild cervids and bovids in Austria [Borrelia-positive hippoboscid detected was a M. ovinus individual infected with a previously unreported strain 93.7% similar to a Borrelia isolate denominated A-FGy-1 and to Candidatus Borrelia mahuryensis isolated from neotropical passerine-associated ticks [. An early work confirmed the presence of Borrelia burgdorferi sensu lato, one of the causative agents of Lyme disease in humans, in M. ovinus [. The Borrelia sp. 16S sequence obtained in our study was 92.9% similar to two B. burgdorferi sequences uploaded to GenBank (AJ224138 and AJ224134). Considering the potential zoonotic risk of B. burgdorferi, new studies should characterize the identity and distribution of Borrelia spp. in sheep keds in Austria. Finally, only one L. cervi individual was positive for Filarioidea, featuring the COI sequence of an unknown onchocercid with a genetic similarity of 95% to the dermal filaroid Mansonella perforata, previously isolated from Sika deer [M. perforata or to another, not previously sequenced Mansonella species needs to be confirmed, as well as their potential distribution in red deer and deer ked populations in Austria.In the present study, Anaplasmataceae were only detected in sheep keds and identified as . ovinus , 89, 90.. ovinus . To datesma ovis , 92 and tophilum , respect Austria , 93, 94,ed ticks . An earl. ovinus . The Borika deer , 98. WheRickettsia spp., Theileria ovis, Acinetobacter spp., Bacillus spp., Staphylococcus spp., Blue-tongue virus, Border Disease virus and Corynebacterium pseudotuberculosis [Anaplasma phagocytophilum and Babesia spp. in wild ungulates [Since the hippoboscid flies collected in the present study were isolated following a convenience sampling in selected farms with expected high infestation prevalence (sheep/cattle) and hunted animals as part of a tuberculosis surveillance program (deer), our results do not allow an accurate estimation of the country-wide prevalence of hippoboscid-associated pathogens in Austria. However, our data describe the widespread presence of pathogens in louse flies infesting ruminants in various geographic regions. Therefore, our findings support the need for the monitoring of hippoboscids and hippoboscid-borne diseases infecting domestic and wild ruminants in Austria, and potentially in other European regions, on a wider scale. Other pathogens previously reported in hippoboscid flies but not surveyed in the present study could also be included in future molecular surveys such as rculosis . Furtherngulates .Bartonella spp. can be vertically transmitted in L. cervi, with the pathogen being detected in adult deer keds blood feeding on ruminants, in pupae and in unfed adult flies that had not yet started to feed on blood [L. cervi can transmit Bartonella spp. to a vertebrate host remains to be confirmed. Moreover, it should still be clarified whether the pathogens detected in L. cervi (e.g. Bartonella spp.) have zoonotic potential, how widespread these infectious agents are in wild ruminant populations in Austria and whether these animals may act as reservoirs for pathogens that could be vectored by deer keds. This is critically important considering the potential exposure of humans to bites by Bartonella-infected L. cervi during working or leisure activities .This work contributes to the ongoing research efforts towards clarifying the role of blood-sucking hippoboscid flies as vectors of infectious agents of veterinary and medical importance. However, the presence of hippoboscid-associated pathogens confirmed by PCR and sequencing does not prove the vector competence of the investigated louse fly species for these pathogens, and only suggests their vector potential. Vector competence is the ability of arthropods to acquire and transmit the infective stage of a pathogen to a vertebrate host, including the pathogen’s replication within the vector. It requires conclusive experimental, epidemiological and clinical evidence of the pathogen’s transmission from the vector to the host, and the subsequent infection , 100. Foon blood . HoweverBartonella spp. We report several novel pathogen sequences in hippoboscids that can contribute to the ongoing research efforts to understand the vector role of louse flies, including the first detection of Trypanosoma spp. in H. equina. Expanded monitoring of hippoboscids and hippoboscid-borne pathogens is warranted to clarify the distribution and impact of these ectoparasites as vectors of emerging infectious agents of public and animal health importance in a One-Health context.In conclusion, molecular genetic screening confirmed the presence of several pathogens in three hippoboscid species infesting domestic and wild animals in Austria, with some potentially representing emerging zoonotic risks such as Additional file 1: Figure S1. Bayesian inference tree featuring gltA sequencesof selected Bartonella spp. Nodes are marked with Bayesian inference posterior probabilities and maximum likelihood bootstrap values. Clades which are marked with a red bar were used for calculation of the median-joining haplotype networks containing the sequences obtained in this study. Scale bar indicates the expected mean number of substitutions per site according to the model of sequence evolution applied. Figure S2. Bayesian inference tree featuring 18S rRNA sequencesof selected Trypanosoma spp. Nodes are marked with Bayesian inference posterior probabilities and maximum likelihood bootstrap values. Clades which are marked with a red bar were used for calculation of the median-joining haplotype networks containing the sequences obtained in this study. Scale bar indicates the expected mean number of substitutions per site according to the model of sequence evolution applied.Additional file 2: Table S1. Blast analysis of Bartonella spp. sequencesobtained from kedscollected from domestic and wild ruminants in Austria.


Euterpe oleracea palm, endemic to the Amazon region, is well known for açai, a fruit violet beverage with nutritional and medicinal properties. During E. oleracea fruit ripening, anthocyanin accumulation is not related to sugar production, contrarily to grape and blueberry. Ripened fruits have a high content of anthocyanins, isoprenoids, fibers, and proteins, and are poor in sugars. E. oleracea is proposed as a new genetic model for metabolism partitioning in the fruit. Approximately 255 million single-end-oriented reads were generated on an Ion Proton NGS platform combining fruit cDNA libraries at four ripening stages. The de novo transcriptome assembly was tested using six assemblers and 46 different combinations of parameters, a pre-processing and a post-processing step. The multiple k-mer approach with TransABySS as an assembler and Evidential Gene as a post-processer have shown the best results, with an N50 of 959 bp, a read coverage mean of 70x, a BUSCO complete sequence recovery of 36% and an RBMT of 61%. The fruit transcriptome dataset included 22,486 transcripts representing 18 Mbp, of which a proportion of 87% had significant homology with other plant sequences. Approximately 904 new EST-SSRs were described, and were common and transferable to Phoenix dactylifera and Elaeis guineensis, two other palm trees. The global GO classification of transcripts showed similar categories to that in P. dactylifera and E. guineensis fruit transcriptomes. For an accurate annotation and functional description of metabolism genes, a bioinformatic pipeline was developed to precisely identify orthologs, such as one-to-one orthologs between species, and to infer multigenic family evolution. The phylogenetic inference confirmed an occurrence of duplication events in the Arecaceae lineage and the presence of orphan genes in E. oleracea. Anthocyanin and tocopherol pathways were annotated entirely. Interestingly, the anthocyanin pathway showed a high number of paralogs, similar to in grape, whereas the tocopherol pathway exhibited a low and conserved gene number and the prediction of several splicing forms. The release of this exhaustively annotated molecular dataset of E. oleracea constitutes a valuable tool for further studies in metabolism partitioning and opens new great perspectives to study fruit physiology with açai as a model. Euterpe oleracea Martius is the most commercialized species , k-mer additive methods provided a transcriptome that is based on a considerable increase in both N50 and CUSCOpB percent value. The best N50 and CUSCOpB values were obtained with TransABySS assembler  that performs a transcript classification based on CDS-DNA local alignment identity, dropping contigs with perfectly identical CDS sequences and maintaining contig with the difference in CDS sequences. The UTR sequences are not considered for assemblies combining step and consequently allows to remove many mis-assemblies contigs but decreases the number of reads mapped back to the assembled transcriptome. The high-quality de novo assembly generated by Trans-ABySS produced 18 Mb of data, 22,486 transcripts with an N50 value of 959 bp and 19,458 predicted proteins. Approximately 67% of the E. oleracea transcripts showed a high confidence homology with E. guineensis proteome. RMBT was approximately 61%, and the mean read coverage was approximately 70x , using the subset Viridiplantae of Uniprot database and an e-value of 1 × 10−3 (Musa acuminata (7431 hits), Ananas comosus (4469 hits), Anthurium amnicola (1780 hits), V. vinifera (500 hits) and E. guineensis (403 hits). The Gene Ontology (GO) as a standard system provides a classification of genes in several categories: Biological Process, Molecular Function, and Cellular Component. For a given organism, GO provides a prediction of the function of a gene and its product(s) [E. oleracea transcripts were assigned to one GO term at least. The most critical GO categories were Biological Process , followed by Cellular Components  and Molecular Functions .The 1 × 10−3 c. From toduct(s) . ApproxiE. oleracea transcriptome was annotated using Blast2GO and comparing to the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Enzyme Codes database to have a global view of metabolic pathways [E. oleracea fruit pigmentation. Twenty-three subcategories were annotated, of which the top 10 was represented in E. oleracea fruits including flavonoids, phenolic acids and anthocyanins [pathways . A totalpathways . The “mepathways . Furtherocyanins .E. oleracea, E. guineensis, and P. dactylifera fruit transcriptomes into GO terms at level 2. Datasets of genes expressed in E. guineensis and P. dactylifera fruits were obtained by mapping published NGS read datasets during fruit development [elopment  and consE. oleracea transcripts, predicting gene orthology and paralogy by mapping these predicted protein sequences to documented orthology and paralogy groups. Due to the high computation time to infer HOG and OMA groups [E. oleracea fruit proteome was compared to five complete proteomes: P. dactylifera, E. guineensis, S. lypersicum, V. vinifera and A. thaliana. P. dactylifera, E. guineensis are two palm trees with a complete and annotated genome  [E. oleracea singletons compared to the whole-fruit transcriptome  . The sincriptome . In the election  and werel plants ,62. Phenl plants ,64. E. oocyanins ,66, and E. oleracea orthologs in A. thaliana, Vitis vinifera, Solanum lycopersicum, E. oleracea, P. dactylifera, and E. guineensis proteomes . In fact, transcriptome analyses have been successful in the identification of genes involved in anthocyanin biosynthesis in plants [E. oleracea fruit has a high content of vitamin E due to an active tocopherol pathway. Along these lines, all five enzymes involved in the biosynthesis of tocopherols were identified in açai, based on clear, direct orthology with A. thaliana enzymes  and CCG/CGG in other palms, approximately 25%  as small in size, with a thin skin, black with a substantial UV-absorbing anthocyanin content, and covered by a thin wax layer at the late mature stage  is preferred for the assembly of poorly expressed transcripts, whereas a higher value (50 or more) helps well the accurate reconstruction of highly expressed transcripts [k-mer size, increasing then the completeness of a unique predicted transcriptome [k-mer strategies exhibit superior metrics (such as the N50) than a transcriptome assembled at one k-mer value only. Post-processing steps reducing the total transcript number from 100,000 to 20,000–30,000 consolidated transcripts in agreement with the gene number predictions offers the considerable advantage to carry out a functional annotation of a non-model organism on very solid bioinformatic analysis.Interestingly, we found that the chnology . Other sue of 25 ,89. The er value –23 is prcriptome ,21,90. Tcriptome . SpecifiViridiplantae section of UniProt [A. thaliana or Oryza sativa, are the most informative to obtain sound functional inferences for non-model plant annotation, and this independently from the evolutionary distance between species [E. oleracea, using an extended timeframe of two months of calculation to obtain series of pairwise orthology relationships . A total of sixteen E. oleracea drupes were selected according to the four different ripening stages described by Rogez et al., 2011 [l., 2011  and origl., 2011 . ImmediaThe mRNA-Seq library was constructed following Ion Total RNA-Seq Kit v2 specifications and using kits and reagents from Thermo Fisher Scientific . Briefly, Poly (A)+ transcripts were isolated from 15 μg of total RNA using a Dynabeads mRNA Direct Micro Purification Kit. After this step, the samples were subjected to enzymatic fragmentation with rNase III for 1 min and 50 s at 37 °C in a thermo-block. The fragment purification was performed using a Magnetic Bead Clean-up Module as described in the Ion Total RNA-Seq v2 kit protocol. The cDNA preparation was made on the AB Library Builder System, respectively. The template preparation, the PI chip loading and sequencing step were automated using the Ion Chef system and Ion Proton sequencer, respectively. Libraries were sequenced using four PI chips (version 3), and all runs were merged and deposited in SRA-NCBI under SRR5330846 accession number. http://www.clcbio.com/products/clc-main-workbench/), accessed on 12 February 2022. Multiple additive k-mer approach was based on a two-step protocol. First, assemblies were generated with a specific assembler using k-mer size from 19 to 99 and secondly, assembly results were concatenated and combined with the tr2aacds script of mRNA Transcript Assembly Software  to remove redundancy and extend transcripts in super-transcripts, as described by Nakasugi et al. (2014) [The pre-processing step was performed with Trimmomatic and cd-hit-EST tools ,96. Trim. (2014) . Transcr. (2014) . AdditioE. oleracea TSA functional annotation was performed using Blast2Go software and an initial BLASTX homology search with an e-value of 1 × 10−3 and the Viridiplantae section of the UniProt database [E. oleracea proteome was predicted using Transdecoder tool (https://transdecoder.github.io/), accessed on 12 February 2022. The orthology assignment between proteomes was performed using OMA [Arabidopsis thaliana, Solanum lypersicum, Vitis vinifera, E. guineensis and P. dactylifera were retrieved from TAIR, under version 10.36, and NCBI, under accession GCF_000188115.3_SL2.50, GCF_000003745.3_12X, GCF_000442705.1_EG5, GCF_000413155.1_DPV01, respectively. In-house shell scripts and the FamilyAnalyzer tool , accessed on 18 February 2022 were used to convert OMA results into annotation tables and to generate orthology statistics to identify identical, duplicate, novel and lost genes between taxa and ancestral nodes. For the different metabolic pathways, candidate genes were automatically retrieved with a shell script from OMA prediction and by comparison with the list of A. thaliana genes that were already functionally characterized. The detection of positive selection with site models was performed using the POTION pipeline based on CodeML and PAML tools [database ,102. Adddatabase . E. olersing OMA . ProteomML tools . The Ks ML tools ,105.E. oleracea transcriptome was mined to identify polymorphic genic-SSR as described by Gordo et al. 2012 [P. dactylifera and E. guineensis, respectively.al. 2012 . The SSRal. 2012 . The traEuterpe oleracea palm is endemic to the Amazon region, and its fruits possess nutritional and medicinal properties. Approximately 255 million single-end-oriented reads were generated on an Ion Proton NGS platform combining fruit cDNA libraries at four ripening stages. The de novo transcriptome assembly using the multiple k-mer approach showed that TransABySS assembler had best results, with an N50 of 959 bp, a read coverage mean of 70x, a BUSCO complete sequence recovery of 36% and an RBMT of 61%. The fruit transcriptome dataset included 22,486 transcripts representing 17.9 Mbp, of which 87% had significant homology with other plant sequences. The GO classification of transcripts from E. oleracea showed similar categories to that in P. dactylifera and E. guineensis fruit transcriptomes. The pipeline AMAZONE was developed whose was observed the annotation of putative genes from anthocyanin and tocopherol pathways. In the anthocyanin pathway was showed a high number of paralogs, such as in grape, whereas the tocopherol pathway showed conserved gene number and the prediction of several splicing forms. The annotated molecular dataset of E. oleracea constitutes a valuable tool for further studies in metabolism partitioning and opens new great perspectives to study fruit physiology with açai as a model in several areas such agriculture to develop açai varieties resistant to diseases and pests, in molecular biology to identify genes involved in biological processes, and in biotechnology to develop genetic improvement techniques for produce bioactive compounds on a large scale through genetic engineering.


Mycobacterium smegmatis (M.sm) is frequently used as an alternative model organism in Mycobacterium tuberculosis (M.tb) studies. While containing high sequence homology with M.tb, it is considered non-pathogenic in humans. As such it has been used to study M.tb and other infections in vivo and more recently been explored for potential therapeutic applications. A body of previous research has highlighted the potential of using genetically modified M.sm displaying rapid growth and unique immunostimulatory characteristics as an effective vaccine vector. Novel systems biology techniques can further serve to optimize these delivery constructs. In this article, we review recent advancements in vaccinomics tools that support the efficacy of a M.sm-based vaccine vector. Moreover, the integration of systems biology and molecular omics techniques in these pioneering studies heralds a potential accelerated pipeline for the development of next-generation recombinant vaccines against rapidly developing diseases. Mycobacterium smegmatis (M.sm) by Lehmann and Neumann in 1889. M.sm is a saprophytic, biofilm-forming bacterium that is non-infectious in mammals. M.sm is commonly found in soil, water, and plants endemic to sixteen states, Australia, Russia, Canada, and Switzerland [Mycobacterium species, and it grows in non-pigmented, velvety, yellow-colored colonies. The surface morphology of this microbe is classically described as “shiny, smooth, and finely wrinkled or coarsely collapsed” [M.sm is a strict aerobe, like other Mycobacterium species, it may survive through short anaerobic periods during certain stages of its disease course. There are various similarities between M.sm and other harmful, infectious Mycobacterium species, such as Mycobacterium tuberculosis (M.tb). The most immediately apparent similarity between the two species a common mycothiol biosynthesis pathway for the production of basic thiol, which is required for intra-bacterial homeostasis [M.tb and M.sm at multiple functional levels.In 1884, Sigmund isolated a novel species of the mycobacterium family which was designated llapsed” . Althougeostasis . The shaAlmost twenty years ago, Dr. Trey Ideker defined systems biology as the use of systematic genomic, proteomic, and metabolomic technologies to acquire data for developing models of complex biologic systems and pathologies . By inteM.sm vaccine has gained a new significance [M.sm vector vaccines. We further highlight the potential for future integration of omics technology in improving M.sm recombinant vaccines.Various techniques and methods based on systems biology have evolved to incorporate cross-disciplinary process analysis systems. These methods are integrative experiment systems that connect multiscale interactions such as molecular, cell, tissue, and organ systems level functions to predict a physiological outcome or organismal phenotypes based on quantitative reasoning, computational models, and high-throughput experimental technologies . A majorificance . In thisM.sm was made available to the public in November 2006 (https://www.ncbi.nlm.nih.gov/ accessed on 20 November 2006). M.sm is a saprophytic, fast-growing strain, although there have been a few cases of human and animal infection reported [M.tb specifically [M.sm as a useful model for studying the greater Mycobacterium genus based on its high homology. With regard to M.tb specifically, >2800 orthologs among its ~4000 protein-coding genes were identified in M.sm. These furthermore displayed >50% global amino acid identity [M.tb complex (MTBC). An estimated set of ~1150 core M.sm proteins were found to share >50% global amino acid identity with a number of other mycobacterial species including M. abscessus, M. marinum, M. avium, and M. leprae [The complete genome sequence of reported . This orreported , and hasifically ,13. In ridentity . The aveidentity . While t. leprae .M.sm have homologs in M.tb, 90% of which are comparably critical for survival in M.tb [M.tb and M.sm [These core proteins likely perform similar functional roles in each species, and transposon mutagenesis studies support this 96% of essential genes identified in  in M.tb . The greand M.sm .M.tb [M.sm genome has predicted the existence of 26 sigma factors; twice the number that have been identified in M.tb. Two-component regulatory systems (2CRSs) are another key player in the bacterial response to changing environmental conditions [Two conserved elements that are of key importance are termed sigma factors and two-component systems. Sigma factors, a family of extra-cytoplasmic signaling molecules, serve a function in adaptive responses to various environmental stressors and are responsible for some of the virulence traits of M.tb . A bioinnditions ,20. Thes2155 strain of M.sm has historically been utilized as a model organism. As such, its transcriptional landscape and genome information data have been extensively mapped and documented, laying a solid foundation for further related research [M.sm have been identified, such as PrrAB-mediated control of many metabolic, respiratory, and dormancy pathways [M.tb orthologous genes in M.sm. For instance, Rv2788 overexpression was shown to increase Mycobacterium endurance to stress [The non-pathogenic MCpathways . One como stress . FindingM.sm as a model organism, this methodology might be extended to gain insight on core protein functional pathways in other organisms [M.sm and M.tb is further affirmed by recent genomic advances like CRISPRi gene inhibition technology. Applying this yielded a hallmark study, which through knockdown analysis indicated 263 essential M.sm genes with precise orthologues in M.tb [Mycobacterium smegmatis as a model bacterium.Over the past decade, the proliferation of technology has allowed us to conduct breakthrough omics research at increasing rates. For example, high-throughput screening of conserved, fluorescently labeled core protein libraries produced in Msr (methionine sulfoxide reductase), yielded unprecedented mechanical data. For example, the relative position of ribosomal proteins which were revealed to be regularly clustered but specifically absent at the nucleoid and polar regions of the cell. As seen here, colocalization of proteins is an important measurable characteristic that could elucidate previously unknown functions or even help identify unclassified proteins. Utilizing rganisms . The bas in M.tb . Figure M.sm antigenicity is most specific for secreted, peptidoglycan-associated protein targets. Cell wall proteome analysis of M.sm strain MC2155 shows that type VII secretion (T7S) systems are used by M.sm in passing these proteins across the cell envelope [M.tb. [M.sm, only ESX-1, ESX-3, and ESX-4 are present [M.sm. M. smegmatis and M. tuberculosis in ESX gene clusters.Based on proteomics studies, envelope . Pathogee [M.tb. ,29. Amon present , Althoug present ,32,33, m present . As for  present . FurtherM.sm [The pathogenesis of mycobacterial infection relies heavily on immune non-dominant virulence factors, secreted by the T7S mechanism, which are involved in all stages of infection, from host colonization to persistence. These days, significant progress has been made in discovering T7S substrates and categorizing the general secretion motif. There are also an increasing number of studies on the expression of recombinant proteins in M.sm .M.tb antigens that are both recognized by the immune system in M.tb infected humans and that provide protective immunity in animal models has been a challenging task. M.tb with that of M.sm, most of which were isolated by traditional biochemical methods.Searching for M.tb 41 and M.tb 39a proteins were also achieved by T-cell expression cloning and serologic expression cloning, respectively [Until recently, most approaches to antigen discovery were based on traditional methods of separating and identifying antigens from complex mycobacterial protein mixtures. While antigens in culture filtrate were initially considered key candidate antigens, the focus has now shifted to protective T-cell antigens found among non-secretory protein isolates. This includes the biochemical identification of the 71-kDa cell wall protein ,38. Idenectively .M.tb genome has revealed that many low-mass antigens belong to common gene families, sharing genetic organization and thus similar antigenicity/immunogenicity. Due to difficulty with isolating specific low-mass antigens in significant amounts, this genetic basis has been considered for the isolation of functional target groups instead. The primary biochemical approach for target molecule identification has thus been replaced by direct T-cell antigen screening techniques based on genomic analysis.Interestingly, sequencing of the M.tb proteome to look for “hit” antigens. “Lead” CD4+T cells are commonly used in these proteomic approaches [Modern bioinformatics techniques of whole genome analysis e can quickly group genes into different families . Combined with the recognition of specific coding sequences, this allows for precise searches of other candidate molecules within the same gene family. With the development of high-throughput peptide and protein synthesis techniques it is, therefore, now possible to thoroughly analyze the entire proaches   in a rapidly growing non-pathogenic Mycobacterium species. This was the first time that a recombinant M.tb extracellular protein had been secreted in its native form [M.tb by the innate immune response.In a basic early study, G. Harth et al. performed heterologous expression and secretion construction of four major ive form . Other eM.sm vaccine derived from M.sm showed strong immunogenicity, promoted Th1 responses, and inhibited the Th2 response in mice [Sweeney et al. investigated the role of ESX-3 loci in the pathogenesis of mycobacteria. The study uncovered a previously unrecognized function of ESX-3 loci in innate immune evasion. They further found that M.sm with the deletion of the ESX-3 gene could serve as a new vaccine vector with enhanced innate immune activation properties. When engineered to express M.tb ESX-3, the vector was found to be an effective TB vaccine that was able to provide a level of protection superior to BCG when administered intravenously [M.sm can exert immunotherapy effects in M.tb infections.Miao Xu et al. found  in mice . Sweeneyvenously . Thus, rMycobacterium virulence; however, the ESX-5 gene in particular is absent in M.sm. Here, we see a more recent example of small differences between M.tb and M.sm that can be leveraged for insight. By serving as a surrogate host for M.tb viral genes, the normally non-pathogenic M.sm can help elucidate their pathogenic and immunomodulatory mechanisms. For instance, the PE/PPE protein family was revealed via recombination studies to be encoded by esx-5 genes and is likewise an essential component of the bacterial cell wall, indicating likely antigenicity and bringing it into focus as a potential viral vector. Kaisar Ali et al. explored this hypothesis by testing the PE subfamily member PE31 (Rv3477) function in virulence and host–pathogen interactions. By expressing the M.tb PE31 in the non-pathogenic M.sm strain (Ms_PE31), they isolated the interaction of this protein with the host immune system; any pathogenic response observed would be due to the presence of the protein. PE31 was thus identified as a functionally relevant virulence factor of M.tb and as a potential vaccine target for ESX-5 [M.sm. The addition of the protein was found to alter the production of host cytokines IL-6, IL-1β, IL-12p40, and IL-10, and also enhanced survival within macrophages by halting apoptosis [Rv0431, a membrane protein associated with glycosylation, finding it to be associated with mycobacterial virulence [M.sm. The recombinant strain effectively stimulated the proliferation and differentiation of T cells [M.sm expressing M.tb Rv1987, encoded by the region of difference (RD)-2 gene. Even though the region of difference (RD)-2 gene is not a virulence-related gene, like esx-5, the recombinant strain still effectively stimulated the proliferation and differentiation of T cells [The ESX-1 and ESX-5 genes have also been thoroughly studied to classify their role in or ESX-5 . Operatipoptosis . Guoyingirulence . T. Garb T cells . Shansha T cells . These rM.sm expressing M.tb ESAT-6 gene and found the strain had strong immunogenicity [M.sm-based vaccine expressing an ESAT6 and CFP10 fusion protein. The protective efficacy of the vaccine rM.S-e6c10 was found to be similar to that of the BCG vaccination, based on measurements of M.tb burden and lung pathology. The result suggests that the recombinant M.sm vaccine expressing the ESAT6-CFP10 fusion protein shows the potential for clinical application [Many of the proteins secreted by ESX-1 are immunodominant and, therefore, at the forefront of infection and disease research. The major secreted proteins of ESX-1 are antigenic target 6-kDa antigen (ESAT6) and culture filtrate protein 10 (CFP10). These essential heterodimeric complexes have become major candidates of recombinant studies, and a number of representative achievements in advancing the understanding of ESX-1 in this regard are listed below. From the recombinant construction of single antigens to the combined expression construction of multi-antigens, the evaluation of immune complexes has acquired the more multi-dimensional nature of a systems biology aproach. Yan Li et al. separately studied a recombinant genicity . H. Zhanlication .M.tb virulence genes mentioned above, M.tb has a series of genes that encode important antigenic proteins, such as the Ag85 complex and MPT64.In addition to the three crucial M.sm expressing three T cell epitopes from M.tb Ag85B  in a murine model. The results showed that the resistance of total immunoglobulin G and its subclasses to Ag85B epitopes in the serum of immunized mice was significantly increased [M.sm vaccine design that overexpresses Ag85B for treating immunodeficient patients. After successfully constructing the recombinant strain, they compared its immunogenicity with that of the wild type. The results confirmed that the recombinant strain could effectively stimulate and increase the number of CD4+ IFN-γ + T cells in the lung [Nur-Ayuni Kadir et al. evaluated the immunogenicity of ncreased . Devin Rthe lung .M.tb has been shown to release MPT64 (Rv1980c) protein in high amounts in patients with active tuberculosis. P. W. Roche et al. constructed a recombinant M.sm strain expressing MPT64 protein that was found to stimulate human rMPB64 T cell lines, establishing the T cell reactivity of the MPT64 protein. This result suggests that the recombinant strain has the potential to be used as a skin reagent for tuberculosis infection [M.sm strain expressing MPT64 protein by recombination with an M. avium-derived antigen gene rather than that of M.tb. Testing revealed immunization using above recombinant vaccine can confer equal protection against M.avium infection as the Mycobacterium bovis BCG, and strongly induced IL-17 to stimulate CD4+ and CD8+ T cell responses [esponses .M.sm. Valeria Falcone et al. used the genomic library of BCG to generate multiple recombinant M.sm strains. The strains produced showed a high splenic survival rate compared to attenuated M.tb, which further supports the use of M.sm for recombinant vaccine development [M.sm using recombinant techniques, would produce a potent vaccine construct.In studying the recombinant expression of single antigen genes, BCG is an excellent cloning candidate due to its homology with elopment . In addielopment . This neM.tb antigen 85B and ESAT-6 as recombinant fusion proteins in M.sm. This study serves as further proof that vaccine candidates from M.tb can be expressed in a surrogate mycobacterial host [M.sm (rMS) expressing Ag85B and ESAT6 fusion protein (AE-rMS). After antigen fusion expression, AE-rMS immunized C57BL/6 mice with strong stimulation of IFN-γ and IL-2, which produced mainly th1 type immune response. Specifically, this leads to robust production of spleen cells and increases antigen-specific cytotoxic T lymphocytes (CTL) activity [M.sm recombinant strain expressing a fusion protein (rMS-Hsp65/IL-2) that is composed of heat shock protein 65 (Hsp65) and human interleukin 2 (IL-2), and next explored the effect of this construct on lymphocyte function in mice. The rMS-Hsp65/IL-2 markedly enhances lymphocyte function. They considered the fusion protein generated by rMS-Hsp65/IL-2 to be of potential value in developing an effective tuberculosis vaccine [M.sm (rMS) to express the fusion protein of heparin-binding hemagglutinin (HBHA) and human interleukin-12 (il-12). The immune response induced by rMS in mice and its protective effect against M.tb were studied. The results showed demonstrated that compared with BCG vaccination, the novel rMS enhanced the th1 cell response (IFN-γ and IL-2) in mice and reduced the bacterial load in the lungs [Another achievement was made with the production of recombinant strains incorporating multiple genes, effectively producing multiple antigenic proteins. Anthony G. et al. first successfully expressed ial host . Wang Piactivity . In furt vaccine . Shanminhe lungs .M.sm (rMS) harboring a co-expression plasmid-encoding human granulysin (GLS) and mouse interleukin-12 (IL-12) on M.tb infection in mice. It was found to have an immunogenic effect by stimulation of Th1 response and GLS antimicrobial activity [M.sm. When introduced to macrophages in a BALB/c murine model, the vaccine was found to strongly induce specific Th1 responses against M.tb, with no observed side effects [M.sm recombinant strain with partial sequences containing M.tb genes Ag85c, MPT51, and HspX (CMX). A vaccine formulated with the recombinant strain induced significant immune responses in mice, and conferred greater protection compared to wild-type M.sm and BCG-based vaccine treatment groups [C. Yang et al. investigated the effect of recombinant activity . On this effects . In anott groups .M.sm vector vaccines, and although most of them are related to the development of anti-TB vaccines, the result has broad implications for vaccinomics. Based on the current success, the potential M.sm vector vaccine is becoming a more extensively researched direction.These are some of the initial applications of M.sm has the potential to be used in vaccine development not only against M.tb, but also other pathogens. As mentioned earlier, neoantigen epitopes for many viruses have been discovered in recent years using computational omics approaches. This technology has allowed for rapid candidate vaccine antigen identification and accelerated the design of recombinant vaccines with a broad range of applications in many pathologies. Mycobacterium smegmatis vector vaccines in the prevention of other diseases.Because of its excellent antigenicity as a vaccine carrier, Mycobacterium smegmatis vector vaccine is a representative application of Helicobacter pylori and Escherichia coli in the prevention of other bacterial diseases. Lin Lü et al evaluated the ability of H. pylori outer membrane protein 26kda antigen (Omp26) to induce therapeutic protection against H. pylori infection in mice. Omp26 was cloned and expressed in M. smegmatis, fusing with M. fowleri β-lactamase protein. They found that immunizing Helicobacter pylori infected mice with ms-omp26, by oral administration, significantly reduced bacterial colonization in the stomach [Mycobacterium smegmatis constructed to ex-press BfpA or intimin from enteropathogenic E. coli (EPEC). It was found that TNF-α and INF-γ were produced in vitro by splenocytes from mice immunized with recombi-nant BfpA, whereas only TNF-α was produced recombinant endomembrane immun-ized controls. Adhesion of EPEC (E2348/69) to HEp-2 target cells was blocked by IgA or IgG antibodies produced from mice immunized with recombinant BfpA or intimin [ stomach ,76. Vasc intimin .M.sm recombinant vaccines have many applications in the prevention and treatment of viral diseases, such as hepatitis B and AIDS. Qiaohong Yue et al. generated a recombinant M.sm strain expressing the CS1 antigen, a fusion protein consisting of the HBV truncated core protein (amino acids 1–155), and the PreS1 peptide (amino acids 1–55). Compared with DNA vaccines, they found that vaccination with the recombinant strain induced a more robust cellular immune response with a longer duration of humoral immune response [M.sm strains overexpressing genes encoding various Mycobacterium bovis BCG lipoproteins to determine the role of TLR2-stimulated lipoproteins on mycobacterial mediated HIV infection in CD4 + T cells [M.sm strain expressing the entire HIV-1 HXBc2 g120 envelope protein. In testing the strains’ immunogenicity, they found it could induce both effector and memory T lymphocytes. Furthermore, after repeated immunization, a stable virus-specific central memory pool was generated [response . In anot T cells . In a suenerated .M.sm vectors that expressed the HIV-1 group M consensus envelope protein (Env) as a surface, intracellular, or secreted protein. Although the vaccine does not induce an anti-HIV-1 envelope antibody response due to insufficient expression of the inserted protein, they suggested that recombinant M.sm immunization could initiate HIV-1 Env protein expression and induce an associated immune response by further improvement is needed to promote the induction of anti-HIV-1 antibody response. [M.sm. Analysis of its cellular and humoral immune responses against HIV gag proteins showed it elicited a more potent immune effect, determined by higher levels of HIV-1 Gag-specific CD4 and CD8 T lymphocyte proliferation, INF-γ ELISPOT cell induction, and antibody production by comparing with the recombinant M.sm using the pAL and pMV306 plasmids [On this basis, Jae-Sung Yu et al. generated and tested another set of recombinant esponse. . Byoung-plasmids .M.sm also has promising applications in some autoimmune diseases. Ling Chen et al. constructed a recombinant M.sm containing the fusion gene Ag85a-IL-17a, which expressed the fusion protein Ag85A-IL-17A displaying effective immunogenicity and that induced the production of specific IL-17A autoantibodies in mice [M.sm based vaccine expressing a chimeric protein that comprises the self-epitopes of proteolipid protein 139–151 (p139) and MPT64 to study autoimmune encephalomyelitis. They found a significant reduction in disease severity compared to immunization with p139 alone [M.sm vector to secrete recombinant tripeptide constructs  and found that when administered orally, it induced regulatory immune responses and reduced the development of atherosclerosis in a mouse model. The effect was comparable to the potency of common therapies delivered by purified AHC protein [Recombinant  in mice . Next, a in mice . Chiara  protein .M.sm has gradually emerged as a form of cancer vaccine. J. L. Haley et al. have successfully engineered M.sm to express and secrete biologically active human tumor necrosis factor-alpha (TNF-α). They found that the transgenic Mycobacterium was more effective than wild-type controls in inducing or upregulating a range of anticancer cytokines [M.sm and further demonstrated that the expression of mammalian cytokines is a significant basis for the antitumor properties of recombinant M.sm vaccines [M.sm recombinant strains by Wen Jian et al. and found to have significant antitumor activity [M.sm (rsmeg-hif-hil-7) vaccine that delivers fusion proteins of human macrophage migration inhibitor (MIF) and IL-7, which serve as target antigens and cancer vaccine adjuvants, respectively. Their data showed that rsmeg-hif-hil-7 exhibited a solid anti-tumor immune response in mouse models. They also found that the anti-cancer effects associated with immune checkpoint inhibitors were enhanced. These results suggest that rSmeg-hMIF-hIL-7 is a promising adjuvant for cancer immunotherapy [In recent years, ytokines . Sarah Lvaccines . To geneactivity . Hyein Jotherapy .Balancing efficacy and cost is a major issue in achieving widespread distribution of vaccines to combat disease in developing countries. Because of differences compared to developed countries, the very limited annual health budgets of many developing countries may hinder or delay the use of advanced and expensive vaccine technologies (such as primary/booster or multicomponent subunit vaccines), even if they are highly effective. As a result, we are faced with the paradox of the infectious disease market—a potentially huge market with a clear public health need, but a high-risk venture in terms of recouping the cost of vaccine development. Thus, getting new vaccines through clinical trials requires not only expanding research efforts, but also reasonably limiting research to ideal antigens and delivery systems with a higher likelihood of producing effective therapies.M.sm has been at the forefront of Mycobacterium research, not only revealing new strategies for addressing mycobacterial diseases but also providing unexpected insights and valuable approaches where other model bacteria and delivery systems have proved lacking. Because of its high homology and antigenicity with M.tb, it can be used as a new vaccine design vector. Defining the repertoire of antigenic targets is central to understanding the immune response against this pathogen. Due to limitations in information collection, data from clinical trials of M.sm vector vaccines are not mentioned above. We speculate that this information may not have been yet made publicly available due to the confidentiality of these studies.M.sm to design vaccines. Advances in proteome-wide screening methods now enable a more extensive and unbiased survey of antigenic targets for complex pathogens like M.tb. Some autoimmune diseases and cancers can also be treated via immunization with M.sm modified with related cytokines. The progress made in improving the development of M.sm vaccines is highly encouraging. In this light, novel licensed recombinant M.sm vaccines for preventive and therapeutic use in humans and animals may become a reality in the near future.In recent years, various omics methods based on systems biology have revealed high-titer antigens of some pathogens, which can be used as foreign genes for recombinant expression in


Leishmania donovani (strain HU3) genome using publicly available data from the deep sequencing of ribosome-protected mRNA fragments (Ribo-Seq). As a result of this analysis, we uncovered 70 previously non-annotated protein-coding genes and improved the annotation of around 600 genes. Additionally, we present evidence for small upstream open reading frames (uORFs) in a significant number of transcripts, indicating their potential role in the translational regulation of gene expression. The bioinformatics pipelines developed for these analyses can be used to improve the genome annotations of other organisms for which Ribo-Seq data are available. The improvements provided by these studies will bring us closer to the ultimate goal of a complete and accurately annotated L. donovani genome and will enhance future transcriptomics, proteomics, and genetics studies.Advances in next-generation sequencing methodologies have facilitated the assembly of an ever-increasing number of genomes. Gene annotations are typically conducted via specialized software, but the most accurate results require additional manual curation that incorporates insights derived from functional and bioinformatic analyses . In this study, we improved the annotation of the  Leishmania. Leishmaniasis ranks second after malaria among parasitic diseases, with an annual incidence of approximately 0.2 to 0.4 million cases of visceral leishmaniasis (VL) and 0.7 to 1.2 million cases of cutaneous leishmaniasis (CL) worldwide [Leishmania donovani and Leishmania infantum, the latter of which also affects dogs [Leishmaniasis refers to the spectrum of diseases caused by several species of the genus cts dogs .Leishmania parasites, substantial efforts have been made to determine their genome sequences in order to understand their molecular biology and to develop strategies to combat them. Classic Sanger sequencing methods were used to determine the genome sequence of Leishmania major (the main species causing CL), which was published in 2005 [L. major genome, a recent reassembly based on a combination of second and third next-generation sequencing (NGS) methodologies has introduced some improvements [Leishmania species  based on their homology with orthologous sequences. Consequently, the automatic annotation of Leishmania genomes, usually carried out via the Companion server [Leishmania transcriptomes has facilitated the revision of the translation start sites for hundreds of ORFs in which the predicted start site was found to exceed the transcript 5′-end boundaries [Given the medical relevance of  in 2005 . While tpository ). Howevetic tree . Thus, fn server , should undaries ,10,11. Sundaries .The sequencing of ribosome-protected mRNA fragments  allows for the detection of those transcripts that are actively being translated at a given time in a cell under defined conditions . The comTrypanosoma cruzi [Trypanosoma brucei [T. brucei, the Ribo-Seq data were used to curate genome annotations; in particular, 225 previously unannotated coding sequences were uncovered, and the proper start codon was found to be misannotated in more than 400 ORFs [L. donovani promastigotes as part of a study examining the role of HSP90 in regulating gene expression [L. donovani (HU3 strain) genome, which was previously assembled and annotated by our group from a combination of short- and long-read sequencing data [L. donovani mRNAs, suggesting a potential conserved role for uORFs in the translational regulation of kinetoplastids.Ribosome profiling studies have been conducted in the related kinetoplastid parasites ma cruzi  and Trypa brucei ,16,17. I400 ORFs ,17. In bpression . In thising data . Like thL. donovani strain 1SR (MHOM/SD/62/1SR) were cultured at 25 °C in supplemented Medium 199 (Sigma-Aldrich) and harvested at a cell density of 6.7 × 106 mL−1. After the samples underwent cellular lysis and RNase I-treatment, isolated ribosomes (monosomes) were purified via sucrose gradient (10% to 50% (wt/vol)) centrifugation. Footprint RNA from the monosome fraction was isolated and used for the construction of libraries, following the protocol of Ingolia et al. [L. donovani promastigotes and used as the control sample) were used.A detailed description of the samples and footprint methodology were published elsewhere . Brieflya et al.  with minL. donovani genome by using the BWA-MEM aligner [https://github.com/lh3/seqtk; accessed on 11 August 2023). The L. donovani genome used as reference is available from the ENA (SAMEA104389403), Leish-ESP , and/or Mendeley dataset  repositories. Thereafter, the BWA-MEM aligner was also used to map the remaining reads to the genome, and the resulting alignment files were sorted and indexed using SAMtools [https://data.mendeley.com/datasets/cgptcyhvd5/1 (accessed on 11 August 2023).The Trimmomatic tool  was used aligner , accordiSAMtools . The resThe tool htseq-count ,23 was uhttps://github.com/CBMSO-L302/Genomic-Tools; accessed on 11 August 2023). These scripts use a nucleotide genomic FASTA file as input. For this work, we used the L. donovani HU3 genome sequence [https://data.mendeley.com/datasets/b82fm2w2h9/2 (accessed on 11 August 2023). As output, the script coord_prot_to_gff.py creates a gff file with the coordinates for all possible ORFs , whereas the script DNA_to_proteins.py generates a FASTA file containing the amino acid sequences of the possible ORFs. The resulting files can be downloaded through the following link: https://data.mendeley.com/datasets/6b54424fgs/1 (accessed on 11 August 2023).Two Python scripts were developed to extract all the possible ORFs (longer than 60 triplets), considering all six reading frames, from any given genome sequence. Both scripts are publicly available in our GitHub repository . Subsequently, the 5′-UTR coordinates of each gene were extracted and processed using bedtools software [A database consisting of all the possible ORFs (between 11 and 100 triplets) was generated from the software  to identhttps://tritrypdb.org; accessed on 11 August 2023), included in the VEuPathDB bioinformatics resource center [Leishmania genomes.The TriTryDB resource  genome. This information is being deposited at EMBL-EBI/NCBI, and it will appear as an updated version of the entry ERS2007345|SAMEA104389403. Also, in the L. donovani (HU3) genome. Additionally, a FASTA file with the improved proteome annotations may be downloaded through the following Mendeley dataset: https://data.mendeley.com/datasets/mcm8wyr2k4/1 (accessed on 11 August 2023).The analyses described in this work have resulted in the correction of a significant number of protein sequences annotated from the L. donovani promastigote samples. At that time, the available genome assembly for L. donovani was quite incomplete and fragmented, and the authors opted to align the resulting sequence reads to the L. infantum reference genome, which has a remarkable sequence conservation with the L. donovani genome [Leishmania protein synthesis, comparing ribosome footprint data from inhibitor-treated and untreated control samples. In our studies, to avoid any unforeseen alterations in ribosome footprints linked to the inhibition of HSP90 (a protein which has pleiotropic effects in the cell), we analyzed only those datasets derived from control samples.In 2018, Bifeld et al. [i genome . The RibL. donovani genome (HU3 strain) [L. infantum genome by Bifeld and colleagues [L. donovani (HU3 strain) transcriptome [The Ribo-Seq reads were mapped to the  strain) . As was lleagues , the foocriptome .L. donovani (HU3) genome sequence . This file (gff format) was visualized using the IGV viewer tool [L. donovani genome . Those transcripts in which Ribo-Seq reads mapped evenly on a possible ORF were postulated to be potential protein-coding genes. In all, 71 transcripts were postulated to have protein-coding functions , Leishmania arabica (LARLEM1108_360036300), and Leishmania braziliensis (LBRM2903_350038200), among others; however, no orthologues were annotated in the genomes of other prototypical species such as L. major, L. infantum, and L. mexicana. A BLAST search using the LDHU3_36.4050 , a database consisting of all possible ORFs longer than 20 triplets  was created via the virtual translation of all six possible reading frames of the wer tool  togetherions see . Figure new CDS;  ORF agaialis see . Taken tLeishmania genomes  genome, we searched the L. mexicana (U1103 strain) genome sequence using the LDHU3_12.0020 ORF as a query. The BLAST algorithm identified a perfect match  at positions 2252–2965 of the L. mexicana chromosome 12  and L. donovani  mapped on the 3′-UTRs; however, for around two thousand genes, a significant number of reads mapped to the 5′-UTRs. These results support the possibility that some of the annotated CDSs might be extended at their 5′-ends. Using the all-possible-ORFs database see , the anaLeishmania species. LDHU3_17.0810 is currently annotated at TriTryDB.org as coding for a hypothetical protein (but is conserved among trypanosomatids) that is 2500 amino acids in length. However, the presence of a significant number of Ribo-Seq reads evenly distributed on the 5′-UTR was suggestive of a misannotation of the CDS. In fact, there was a theoretical ORF that fit perfectly in the genomic region covered by the mapped Ribo-Seq reads (Leishmania species deposited in TriTrypDB (release 62) showed that indeed, orthologous proteins of a similar size (around 3700 amino acids) to the new predicted sequence for LDHU3_17.0810 are currently annotated in most Leishmania proteomes (see L. infantum (strain JPCM5), L. donovani (strain CL-SL), Leishmania panamensis (strain PSC-1), L. amazonensis (strain M2269), and L. mexicana (strain U1103), the orthologous proteins are 150 amino acids shorter than those predicted from the re-annotated LDHU3_17.0810 CDS. Therefore, before working with the orthologous proteins from these species, a possible reannotation should be considered.eq reads A. The prL. aethiopica (L147) and L. major (Friedlin) genomes  is annotated in the genome of L. donovani . Similarly, in L. tropica (strain L590), the orthologous protein (gene ID: LTRL590_170015100) is 7028 amino acids in length with a sequence identify of 91%; in Leishmania turanica (strain LEM423), the orthologous protein has 7032 amino acids (91% sequence identity), and the orthologue in L. major (strain Friedlin) has 7020 amino acids and a sequence identity of 90%  upstream of the primary annotated CDSs in the transcripts. Ribo-Seq reads were then mapped on these small ORFs, and those with more than five mapped reads were considered putative uORFs. In total, 304 putative uORFs were found in 264 different transcripts  in L. donovani (HU3)  in some transcripts. This class of short ORFs has been identified in mRNAs from different organisms; for instance, over 40% of mammalian mRNAs contain uORFs . In gene see the B showed  see the .L. donovani (HU3 strain) genome annotation in three ways. First, alignment of ribosome-protected fragments to the genome uncovered 71 new protein-coding sequences that had escaped previous algorithm-based methods, bringing the total number of annotated proteins in this strain up to 8463. Second, our analyses led to extension of the 5′ ends of 588 CDSs that were improperly truncated in the original automated annotations. Third, we uncovered eight instances in which single transcripts had been misannotated as two separate transcripts. These improvements, in combination with our recent assembly of the L. donovani (HU3 strain) genome and transcriptome [L. donovani genome to date. These data can serve as a reference for comparative genomics to improve the genome annotations for other Leishmania species. Our understanding of Leishmania biology and pathogenesis will be enhanced by our discovery of expanded and new coding sequences, which will increase the accuracy of transcriptomic and proteomic analyses. In addition, accurate definitions of CDSs are essential for functional genetic screening platforms. For example, we are currently generating a genome-scale inducible overexpression library encoding the majority of protein-coding genes in L. donovani  . The updated annotations described herein will help ensure that PCR primers are designed to target the true boundaries of each ORF. The improved annotation will prove similarly useful for CRISPR/Cas9-mediated KO libraries and endogenous tagging strategies.In this work, we reanalyzed the Ribo-Seq data generated by Bifeld et al.  to improcriptome , have reL. donovani transcriptome, which is consistent with previous results found for T. brucei and T. cruzi [L. donovani.Finally, our analysis showed the presence of uORFs in 264 protein-coding transcripts in the T. cruzi ,32. Thes


Xenobiotics are primarily metabolized by hepatocytes in the liver, and primary human hepatocytes are the gold standard model for the assessment of drug efficacy, safety, and toxicity in the early phases of drug development. Recent advances in single-cell genomics demonstrate liver zonation and ploidy as main drivers of cellular heterogeneity. However, little is known about the impact of hepatocyte specialization on liver function upon metabolic challenge, including hepatic metabolism, detoxification, and protein synthesis.Here, we investigate the metabolic capacity of individual human hepatocytes in vitro. We assess how chronic accumulation of lipids enhances cellular heterogeneity and impairs the metabolisms of drugs. Using a phenotyping five-probe cocktail, we identify four functional subgroups of hepatocytes responding differently to drug challenge and fatty acid accumulation. These four subgroups display differential gene expression profiles upon cocktail treatment and xenobiotic metabolism-related specialization. Notably, intracellular fat accumulation leads to increased transcriptional variability and diminishes the drug-related metabolic capacity of hepatocytes.Our results demonstrate that, upon a metabolic challenge such as exposure to drugs or intracellular fat accumulation, hepatocyte subgroups display different and heterogeneous transcriptional responses.The online version contains supplementary material available at 10.1186/s13059-023-03075-9. Single-cell sequencing technologies have revealed a wealth of information regarding cellular heterogeneity in health and disease in multiple tissues –8. In paIn the liver, drug and xenobiotic metabolism is mainly performed by hepatocytes, the major and predominant cell type of the parenchyma –17. The An additional potential source of cellular heterogeneity is the intracellular accumulation of triglycerides in hepatocytes, known as hepatic steatosis , 32. ThiMoreover, the co-administration of five or more drugs (polypharmacy) is associated with a higher incidence of adverse drug reactions (ADR) and drug-induced liver injury (DILI) –40. ImpoHere, we report concealed cellular heterogeneity in primary human hepatocytes which are classically considered a seemingly homogenous population of cells and a “gold standard” for in vitro hepatic assays. The comparison between pseudobulk and single-cell transcriptomics reveals four distinct hepatocyte subgroups that can be found also in fourteen human donors from publicly available data sets. We present evidence that chronic accumulation of lipids and xenobiotics leads to transcriptional variability and the impairment of multiple metabolic pathways in a subgroup-specific manner. Our results suggest that liver steatosis combined with multiple drug intake differentially affects the transcriptional profiles of individual cells decreasing the expression of key cytochrome P450 genes.Liver function is compartmentalized along the liver lobule, and the maintenance of liver function is regulated by liver-enriched transcription factors , 45. PriCryopreserved and metabolically competent PHHs from four donors were used as in vitro model to characterize the metabolic profile of individual hepatocytes in response to a drug challenge and chronic accumulation of fat Fig. ”. The heA total of 38,232 high-quality hepatocytes with an average of 2550 genes per cell were analyzed. Lenient doublet filtering was performed to account for polyploid hepatocytes . The varLouvain clustering led to the detection of heterogeneity separating four subgroups of hepatocytes, independently of the treatment  ; 38.7% otlas.org . We founcyclone  were assigned to S-phase according to the cell cycle phase classification using the tool .2% were HNF4A . Am. Am99]. In summary, we observed that intracellular lipid accumulation led to an impairment of drug metabolism characteristics for each subgroup, in which multiple metabolic pathways are simultaneously affected. Therefore, primary human hepatocytes lose their drug-related metabolic specificity in the presence of chronic accumulation of lipids.The application of single-cell genomics technologies allows the dissection of subtle differences within a seemingly homogeneous population of cells, which has been demonstrated in a plethora of organs, tissues, and cell types, including the liver –102. TheMLXIPL (ChREBP), RXRA, NH1H4 (FXR), PPARA, HNF4A, and CEBPA .Additionally, 2-fold change of greater than 1 and a Bonferroni-adjusted p-value below 0.05. We used the venn package to visualize the overlap of significantly up-regulated genes between the subgroups. Furthermore, ShinyGO [After defining metabolically functional subgroups in our in vitro data, we first calculated the top 500 DEGs per subgroup under DMSO in comparison to each other. To assess what drives the basal functional specialization of the subgroups, these top 500 genes per subgroup were taken as input for the online tool ChEA3  which pr ShinyGO  was used2-fold change than when we compared Cocktail and DMSO. Therefore, to capture the subtler effects of fat accumulation on the cells, a gene was identified as significantly up-regulated if it had a Bonferroni-adjusted p-value below 0.05 and a log2-fold change greater than 0.75. We used the same marker genes as described above .When comparing FFA- with DMSO-treated cells, we observed that there were on average 3.5 times fewer genes with a positive log2 is the variation of gene j in the group of interest.It is generally assumed that lowly expressed genes have an inflated transcriptional variability. Therefore, genes with a mean normalized log-transformed expression smaller than 0.25 were removed, and the coefficient of variation per condition in each of the subgroups was calculated on 3434 genes. To obtain the coefficient of variation on normalized, log-transformed counts, we used the formula described in Canchola et al. .\documenU test was performed to check if the coefficient of variation differed significantly between treatment condition.In every subgroup, a Mann–Whitney Additional file 1: Fig. S1. Quality controls. Fig. S2. Clustering and cell annotation. Fig. S3. Comparison between cell cycle analysis using FACS and Cyclone. Fig. S4. ChEA3 and scATAC-seq analysis. Fig. S5.In vitro and in vivo comparisons of hepatocyte subgroups. Fig. S6. Transcriptional signatures in response to the phenotyping cocktail. Fig. S7. Transcriptional profiles upon intracellular lipid accumulation. Fig. S8. Transcriptional dysregulation of multiple metabolic pathways upon fat accumulation. Fig. S9. Transcriptomic changes on phase III transporter genes.Additional file 2: Table S1. Metadata donors.Additional file 3: Table S2. Marker genes.Additional file 4: Table S3. ChEA3 results per subgroup.Additional file 5: Table S4. Unique DEGs per subgroup upon Cocktail.Additional file 6: Table S5. Differential expression Cocktail vs. DMSO.Additional file 7: Table S6. Significance levels coefficient of variationAdditional file 8: Table S7. Differential expression FFA vs. DMSO.Additional file 9: Table S8. Differential expression FFA+Cocktail vs. DMSO.Additional file 10: Table S9. Genes up-regulated upon Cocktail with and without FFA.Additional file 11: Table S10. Drug Cocktail preparationAdditional file 12. Review history.


Oncolytic viruses have many anticancer strategies. Genetically modified viruses may more effectively infect cancer cells than normal cellsNumerous preclinical studies have shown the treatment’s safety, and evidence of both a local and systemic antitumor effect has emerged. Using CF33, it would be able to enhance the clinical advantages and quality of life for patients with malignancies that are now untreatable with standard therapy methods. Phase 1 clinical studies will use CF33-hNIS, a new chimeric orthopox virus, either as monotherapy or in combination with pembrolizumab to evaluate the safety and effectiveness of the treatment regimens and the immunological alterations in the tumor microenvironmentGene therapy Talimogene Laherparepvec (T-VEC) overexpresses granulocyte macrophage colony-stimulating factor by using an attenuated herpes simplex virus 1 virus, which is then used in dendritic cell-mediated antigen presentation studies. Patients with advanced, nonresectable melanoma who received intratumoral granulocyte macrophage colony-stimulating factor alone had a lower rate of long-term response compared to those who received T-VEC+ T-cell infiltration and interferon-gamma signaling and by upregulating PD-L1 in the microenvironmentOncolytic virus injection may enhance the effects of checkpoint inhibitors by enhancing CD8In addition to the encouraging findings from the preclinical studies, we are very excited to explore the possibilities presented by the oncolytic virotherapy platform comprised of VAXINIA and T-VEC.This case report has been reported in line with the Case Report (CARE) guidelines.Not applicable.None.R.A.: conceptualization, data curation, writing – original draft preparation, writing – reviewing and editing. B.R.S., P.P., and A.R.: data curation, writing – original draft preparation, writing – reviewing and editing. T.B.E.: writing – reviewing and editing, visualization, supervision. All the authors confirm the concept, design, data collection and analysis and interpretation, and writing are our own. The authors confirm there are no other contributors.The authors declare that they have no financial conflict of interest with regard to the content of this report.None.https://orcid.org/0000-0003-3188-2272. E-mail: talhabmb@bgctub.ac.bdTalha Bin Emran, PhD, Associate Professor, Department of Pharmacy, BGC Trust University Bangladesh, Chittagong 4381, Bangladesh. Tel: +880 303 356 193, Fax: +880 312 550 224. Not commissioned, externally peer reviewed.


Dear Editor,Myocardial infarction (MI) is one of the leading causes of cardiovascular-related mortality worldwide. Timely restoration of the blood supply to ischemic myocardium by thrombolysis or percutaneous coronary intervention is a common clinical practice and decreases the mortality risk for MI patients . However+ and pH3+ CMs in the infarct border zone of left ventricles, decreased Ki67+/vimentin+ fibroblasts, and increased CD31+ blood vessels  or 5SM immediately after IR (acute IR) or at 7 days post-IR (subacute IR). Adult rats were subjected to a 60-min occlusion of the left anterior descending coronary artery followed by reperfusion as previously described . We conf vessels . 5SM did vessels K and L.  vessels M. ECHO s vessels . Direct  vessels . Consist vessels Q. Our pr vessels . Similar vessels  and amel vessels . Notably vessels .It becomes well recognized that induction of CM reentry into the cell cycle and cytokinesis can promote heart regeneration. Our previous work demonstrated that 5SM promotes adult rat heart regeneration via induction of CM proliferation after acute MI . In thisbiorender.com.]We would like to acknowledge Prof. Iain C. Bruce, Guest Professor at Peking University, for his critical comments and revisions of this manuscript. This work is supported by grants from the National Key R&D Program of China (2018YFA0800501 and 2019YFA0801602), the National Natural Science Foundation of China , and Synogen Biopharma Co., Nanjing, China. L.Z. and Y.C. designed and performed experiments, analyzed data, and wrote the manuscript; Z.W. helped with ECHO; X.Z. supervised this work and revised this manuscript; J.-W.X. conceived, supervised, and designed this work, analyzed the data, and revised this manuscript. The featured image was created with mjad034_Supplemental_FileClick here for additional data file.