Bifidobacterium has been demonstrated to predominate in the gut microbiota of breastfed infants and to benefit the host by accelerating maturation of the immune response, balancing the immune system to suppress inflammation, improving intestinal barrier function, and increasing acetate production. In particular, Bifidobacterium longum subspecies infantis (B. infantis) is well adapted to the infant gut and has co-evolved with the mother-infant dyad and gut microbiome, in part due to its ability to consume complex carbohydrates found in human milk. B. infantis and its human host have a symbiotic relationship that protects the preterm or term neonate and nourishes a healthy gut microbiota prior to weaning. To provide benefits associated with B. infantis to all infants, a number of commercialized strains have been developed over the past decades. As new ingredients become available, safety and suitability must be assessed in preclinical and clinical studies. Consideration of the full clinical evidence for B. infantis use in pediatric nutrition is critical to better understand its potential impacts on infant health and development. Herein we summarize the recent clinical studies utilizing select strains of commercialized B. infantis.Since originally isolated in 1899, the genus  Bifidobacterium in the process of immune maturation early in life  delivery [Bifidobacterium spp. colonization between vaginal and C-section delivery diminish by 30 days of age [Bifidobacterium spp. in infants has been correlated to chronic diseases, including asthma and obesity [Bifidobacterium spp. in the infant gut in populations of developed countries is linked to increased incidence of allergic and autoimmune diseases [Bifidobacterium spp. abundance can be further influenced by nutrition, antibiotic use, and puberty [Bifidobacterium longum subspecies infantis (B. infantis) in particular, dominates the gut microbiota of breastfed infants and benefits the host by accelerating maturation of the immune response, balancing the immune system to suppress inflammation, improving intestinal barrier function, and increasing acetate production [B. infantis) to protect the full term neonate and nourish a healthy gut microbiota prior to weaning  health from infancy to advanced years (as reviewed ), includreviewed ), have breviewed . Genus Breviewed ). Bifidoreviewed ,5. Vertireviewed . Vaginaldelivery ,8; howevs of age , highlig obesity , as well obesity . Henrickdiseases . Bifidob puberty ,15,16. Boduction . This syB. infantis, including the preclinical data and clinical evidence.Here, we focus on the current status of knowledge for B. infantis metabolism  [B. infantis also contribute to protection against Escherichia coli O157:H7 [B. infantis strains [B. infantis preferentially consumes smaller HMO species (degree of polymerization <7) which are consistently produced over the course of lactation and represent the bulk of HMO present in pooled milk samples [B. infantis produces short chain fatty acids (SCFA), such as acetate, which provides an important role in nutrition and intestinal and immune development, facilitates direct binding to intestinal cells, and stimulates anti-inflammatory/inhibits pro-inflammatory cytokine release by intestinal cells [B. infantis becomes a carbon source that stimulates growth and function of butyrate-producing microbes [B. infantis can enter circulation and directly affect the adipose tissue, lungs, brain, and liver, inducing overall beneficial metabolic effects [Human milk oligosaccharides (HMO) are the primary substrate for reviewed ). HMO inescentis ,21. Onceinfantis  to activlidases) ,23,24 pr O157:H7 . Of impo strains . B. infa samples . HMO metal cells ,29. Acetal cells . Furthermicrobes . Thus, Hmicrobes . In addi effects . For exa effects , suggest effects . B. infantis may result in fecal pH changes and reduction of dysbiotic taxa in the gut. Preclinical models, including the necrotizing enterocolitis (NEC) model, have demonstrated that B. infantis could potentially decrease intestinal permeability and increase stabilization of tight junction proteins [B. infantis contributes to maintaining the intestinal barrier integrity through ILA, a metabolite of tryptophan [B. infantis could also potentially protect against excessive intestinal inflammation which often affects the premature infant and may be a contributing factor in NEC.Whereas other bacteria consume HMO , only Bifidobacteriaceae converts HMO to acidic end products that affect stool pH . Therefoproteins ,34. For yptophan , and mayB. infantis is highly specialized for the consumption of HMO and has a competitive advantage against other bacteria, allowing increased colonization and resulting in fewer luminal pathogens  assigned qualified presumption of safety (QPS) status to the bacterial species concerns . The QPSulations .n = 66) in a multicenter double-blind randomized controlled trial (DBRCT); participants received a standard infant formula (IF) or an IF with added probiotics ) and 10 million CFU Lactobacillus rhamnosus LCS742 per 100 mL formula [n = 97; <postnatal day (PND) three of age) received M63 and LCS742  for six months [p = 0.03) and at six months, atopic dermatitis was less frequently observed (p < 0.05). Overall, the results from these two studies suggest the potential beneficial effect in a population of children with colic as being improved tolerance and a protective effect against the development of atopic dermatitis.Term infants identified with colic were enrolled ( formula ) over a x months . After oB. breve M16 (1 billion CFU/day), and B. longum BB536 (3 billion CFU/day) has been studied in several clinical trials. For example, children  with functional constipation received polyethylene glycol  or PEG and the probiotic mix over an eight week period [n = 40; 9 ± 2.2 years of age) with allergic rhinitis and asthma received the same probiotic mix over a four week period [n = 48) and functional dyspepsia (n = 25) received the same probiotic mix over a six week period [n = 44, six weeks old), low-birthweight infants assigned to a modified probiotic mix of M63, M16, and BB536 (500 million CFU/day of each strain) over a six week feeding period demonstrated higher bifidobacteria GI colonization compared to those receiving M16 alone [B. infantis M63. Although the benefits cannot be directly attributed exclusively to B. infantis, the results are in agreement with several mechanisms of B. infantis described above. A probiotic mix of M63 (1 billion CFU/day), k period . The prok period  and showk period . Adminis16 alone . The abon = 12, five weeks old) received formula feedings and were randomized to receive B. infantis ATCC 15697 (4 billion CFU twice daily) or B. animalis subsp. lactis with doses increased over a five week feeding period. In phase two, nine premature infants receiving their mother’s milk received each of those two bifidobacteria for two weeks separated by a one-week washout period. Fecal bifidobacteria count was significantly higher and, in case of phase two, Proteobacteria were significantly lower in the ATCC 15697 group, with authors concluding that ATCC 15697 was more effective at colonizing the premature infants compared to B. animalis sbsp. lactis [B. infantis was most effective at “normalizing” the fecal microbiota confirming the specialized ability of B. infantis to metabolize HMO. A study by Underwood et al.  consiste. lactis . The stun = 24, gestational age at birth > 34 weeks) in a placebo-controlled pilot study. Participants were randomized to receive either ATCC 15697 (1 billion CFU) or a placebo twice daily for six weeks or until hospital discharge [B. infantis had higher fecal Bifidobacteriaceae, lower Clostridiaceae, and trends toward lower Enterobacteriaceae, Enterococcaceae, Staphylococcaceae, and Streptococcaceae. Clinical outcomes, including length of hospital stay did not differ between groups.Infants with confirmed gastroschisis were enrolled  and a history of chronic constipation, diarrhea, or IBS were enrolled in a crossover study; participants received a combination of B. infantis UCD272  and a prebiotic (bovine colostrum product) over a five-week period, followed by a two-week washout, and five weeks of prebiotic alone [Children with autism spectrum disorders  were randomized by parallel assignment to either lactation support  or LS + EVC001  over a 21-day feeding period [Bifidobacterium spp. counts from PND 6 to 28. Stool samples were collected through PND 60. In each group, 34 participants were included in the analysis. Stool Bifidobacterium spp. count was significantly higher and stool frequency significantly lower in infants receiving EV0001 from PND 6 to 28. No group differences in health and safety outcomes were detected. One month after discontinuing feeding EVC001, stool count of EVC001 persisted and was significantly higher compared to the control group [E. coli, Klebsiella, Clostridium and Staphylocossus were decreased by over 93% [n = 31; EVC001, n = 29) from PND 7 to 21 [Escherichia (which predominantly harbored ARG) were also significantly reduced [B. infantis) were measured in stool samples from the EVC001 group. The release of selectively fermentable N-glycans may increase the prebiotic activity and increase the growth of EVC001 in vivo. Finally, in stool samples from the same cohort, significantly lower calprotectin and proinflammatory cytokines were reported in infants receiving EVC001 [Clinical outcomes related to g period . EVC001 g period . Primaryol group . The domol group . In addiol group . Stool eover 93% . Shotgun 7 to 21 . The res reduced . It is p reduced . In contg EVC001  suggestin = 97) or IF with added B. infantis CECT 7210 at 10 million CFU per 100 mL formula  (n = 93) for a 12-week feeding period [n = 78; CECT 7210, n = 73). Overall diarrhea events per infant  were not significantly different between groups. Stool frequency was significantly lower in the control group after four weeks of study feeding. By study end, total stool Bifidobacterium spp. was similar between groups; however, B. infantis was significantly higher in the CECT7210 group. No differences in growth, formula intake, or other secondary outcomes were observed. Overall, the authors concluded that formula with added CECT was safe, effective, and well tolerated in healthy term infants.Term infants (<3 months of age) enrolled in a multicenter DBRCT were randomized to receive a standard IF  enrolled in a multicenter DBRCT. Infants were randomized to receive daily administration of a probiotic combination  or a placebo  [Bifidobacterium spp. count and reduced genus Enterococcus count in infants who received probiotics [B. infantis, the observed benefits cannot be attributed definitively to BB02. However, the observed increase in Bifidobacterium spp. and reduction in NEC are consistent with the mechanisms we described above, e.g., increase of beneficial microbiota and improvement of gut barrier integrity. Jacobs and colleagues studied a mix of three probiotics, including the strain n = 551) . The priobiotics . Considen = 208; 3 to 12 months of age) and received B. infantis R0033  over an eight week feeding period [L. helveticus and B. bifidum. R0033 was safe, well tolerated, and had no impact on growth . A significant decrease in fecal Blautia, Collinsella, Enterococcus and Klebsiella genera and increase in the IL-10/IL-12 ratio were demonstrated in infants receiving R0033, suggesting anti-pathogenic and anti-inflammatory activity [Healthy term infants were enrolled (g period . Other sactivity . The antn = 135) received a daily synbiotic preparation , and fructo-oligosaccharide (FOS)) over a three month feeding period [B. infantis, which include prohibition of pH-sensitive pathogenic organisms.In another DBRCT, children three to seven years of age  in a DBRCT; participants received a control IF or IF with an added probiotic mix of B. infantis BT1, B. bifidum BF3, B. breve BR3, and B. longum BG7  through 12 months of age to measure fecal microbiota diversity and composition [Bacteroides and Blautia spp. counts were lower after one month of feeding in infants receiving probiotics, no group differences in microbiome or metabolite profile were detected after 12 months of age. No significant differences were observed between infant feeding groups regarding growth, antibiotic uptake, or other health variables.Term infants were enrolled . As evidenced in clinical studies, the administration of B. infantis leads to successful colonization in the gut, where the highly selective and acidic fermentation of HMO results in increased production of lactate and acetate, and subsequent reduction of gut dysbiosis and lower pH. This potentially includes an important role in development and maturation of the immune system. Modern practices, including C-section and perinatal antibiotics have disrupted the mother-to-infant transfer of B. infantis at birth, leading to a loss of this key member of the infant gut microbiota, which has resulted in an increase of stool pH in infants [B. infantis strains within the infant gut will help us better understand the critical role of the infant gut microbiome in establishing long-term health.Studies reviewed in this manuscript suggest that colonization with  infants . Continu


Mycobacterium tuberculosis remains the leading cause of death attributed to a single infectious organism. Bacillus Calmette-Guerin (BCG), the standard vaccine against M. tuberculosis, is thought to prevent only 5% of all vaccine-preventable deaths due to tuberculosis, thus an alternative vaccine is required. One of the principal barriers to vaccine development against M. tuberculosis is the complexity of the immune response to infection, with uncertainty as to what constitutes an immunological correlate of protection. In this paper, we seek to give an overview of the immunology of M. tuberculosis infection, and by doing so, investigate possible targets of vaccine development. This encompasses the innate, adaptive, mucosal and humoral immune systems. Though MVA85A did not improve protection compared with BCG alone in a large-scale clinical trial, the correlates of protection this has revealed, in addition to promising results from candidate such as VPM1002, M72/ASO1E and H56:IC31 point to a brighter future in the field of TB vaccine development. Mycobacterium tuberculosis, is the leading cause of death attributed to a single infectious organism [Consigned to history in the minds of many, tuberculosis (TB) is a disease far from defeated. The pathogen responsible, organism . Adminisorganism , the vacorganism . The efforganism . It is eorganism , the cruorganism .Following the disappointing results from the first phase 2b efficacy trial with a new-generation subunit vaccine, MVA85A, the TB vaccine field had a period of review and reflection . Even wiM. tuberculosis [The derivation of BCG is a result of pathogenesis experiments carried out by Calmette and Guerin , who aftrculosis . Major trculosis  and USA rculosis , 13, therculosis . In contrculosis  showed arculosis . Carriedrculosis . Up to trculosis .Though declared by Calmette to be a ‘virus fixe’ , multiplAlthough administering BCG vaccination shortly after birth reduces the risk of prior NTM exposure, such timing is frequently not possible in resource poor settings, particularly rural ones, and in areas where HIV infection needs to be excluded prior to BCG administration. A study from The Gambia has previously demonstrated NTM exposure in BCG-naive 4-month-old infants . A studyM. tuberculosis results in rapid eradication of BCG, such that the amount of available BCG derived antigens is limited, diminishing immunity [There are two hypotheses proposed for the mechanism by which NTM exposure reduces BCG efficacy: masking and blocking , 24. Theimmunity .One implication of this for the development of novel vaccines is that a dominant blocking mechanism suggests that a new vaccine need only be as good as BCG to have measurable effect, as long as it is not blocked by prior sensitisation . ImportaM. tuberculosis infection is increasingly recognised [M. tuberculosis are capable of achieving sterilising immunity post-exposure, termed early clearance [M. tuberculosis infection (LTBI) is usually assessed via a tuberculin skin test (TST) or interferon-gamma release assay (IGRA), which when positive indicates immune sensitisation to M. tuberculosis, i.e. that an infection has occurred. In studies with a minimum of 2 years of longitudinal observation, the frequency of early clearance in household contacts of TB patients ranged from 3.4 to 26.8% when using TST conversion [The role of an early innate immune response in preventing or clearing early cognised . A subselearance . The prenversion –30, whilnversion . A numbenversion , antibodnversion  and lungnversion . Investinversion . NOD2 sinversion .M. tuberculosis exposure are outlined below. Whilst vaccine development has traditionally focussed on the induction of an adaptive immune response, adjuvants that modulate innate immune pathways and a vaccine delivered by aerosol to the respiratory mucosa might target these pathways [The different components of an innate immune response to pathways .M. tuberculosis enters the body via small aerosolised droplets, inhaled into the airways [M. tuberculosis activates a number of signalling pathways, inducing the production of cytokines such as tissue necrosis factor α (TNFα) and interferon-ɣ (IFN-ɣ), and chemokines such as IL-6 and IL-8 [ airways , 38. Tho airways . Airway  airways . This epand IL-8 –44. Furtand IL-8 . AECs arand IL-8 .M. tuberculosis, phagocytosing the bacilli. This phagocytosis is mediated principally by complement receptor 4 (CR4) [M. tuberculosis even with high bacterial loads [M. tuberculosis infection induces a phenotypic shift from oxidative phosphorylation  to aerobic glycolysis , resulting in increased IL-1β levels and decreased IL-10 levels [AMsare some of the first cells of the immune system to come into contact with  4 (CR4) ; howeveral loads . M. tube0 levels . This po0 levels . However0 levels , 49.M. tuberculosis improved survival [In a mouse model, the depletion of macrophages prior to a lethal infection with survival , yet spesurvival . The proM. tuberculosis exposure are neutrophils, among the first immune cells to migrate to the site of infection [M. tuberculosis primarily through the respiratory burst and phagocytosis [M. tuberculosis prior to granuloma formation [Another cell type implicated in the initial response to nfection . Neutropnfection , chemokinfection  and MCP-nfection  and cytonfection . Neutropocytosis . Whilst ocytosis . In miceormation , potentiormation .M. tuberculosis killing [M. tuberculosis, the evidence suggests a role for neutrophils in the pathogenesis of TB, a possible granulocytic Trojan horse [In humans, peripheral neutrophilia is a hallmark of TB disease and is a poor predictor of outcome , with ne killing . A neutr killing . As a pran horse .M. tuberculosis, recruited via a CXCL13-CXCR5 axis to inducible bronchus-associated lymphoid tissue (iBALT)-associated granulomas [There are many other innate cell types for which there is some evidence for a role in protection against mycobacterial infection, including NK cells , 64, ɣδ anulomas .M. tuberculosis infected humans [iBALT, like other lymphoid organs, are composed of segregated T and B cell areas . These hd humans , NHPs [7d humans  and miced humans . The absd humans , 77.M. tuberculosis infected lung cells [M. tuberculosis burden, resulting in a decreased formation of iBALT, whilst mice lacking ILC3 exhibit a reduction in the accumulation of early alveolar macrophages [M. tuberculosis infection, recruiting lymphocytes through CXCR5 to mediate their spatial organisation within iBALT [ILC3 produce IL-17 and IL-22 in response to IL-23 stimulation , the IL-ng cells . Early nrophages . CXCL13 in iBALT . IL-17 iin iBALT  and willUsing a mycobacterial growth inhibition assay, a new subset of innate cell has been found to be strongly associated with trained innate immunity . ControlTrained immunity describes epigenetic changes to the genes of the innate immune system, resulting in a memory like function. BCG vaccination has been shown to alter the acetylation and methylation of innate immune genes, amplifying the response to subsequent stimulus . This maM. tuberculosis antigens to T cells by dendritic cells and macrophages. There is a significant delay between infection and the induction of an adaptive immune response, which in humans is first detectable 18–20 days after infection [M. tuberculosis to the draining lymph nodes only occurs 7–9 days post-infection; this delay allows for a marked expansion in bacterial population [Activation of the adaptive immune response requires the presentation of nfection . Migratipulation .M. tuberculosis control [M. tuberculosis burden [Methods of activating dendritic cells (DCs) via adjuvants may improve the protective efficacy of vaccines. Amphiphilic-CpG (amph-CpG), a modified TLR9 agonist in mice, enhances the T cell response to peptide vaccination in addition to upregulating CD103 . When us control . The uses burden .Intravenous BCG vaccination is capable of priming HSCs, resulting in a polarisation towards the myeloid lineage, correlating with an upregulation of IFN-ɣ . This loM. tuberculosis has a variety of means to mitigate its destruction by macrophages, enabling it to survive in macrophages without destruction. An overview of this can be seen above in Fig. M. tuberculosis result in less effective antigen presentation, resulting in a less-effective adaptive immune response [M. tuberculosis to uninfected DCs, thus allowing antigen presentation and activating adaptive immunity [response . Increasimmunity .Fig. 2M.M. tuberculosis inhibits inflammasome activation and IL-1β processing through zinc metalloproteinase 1 (zmp1) [1 (zmp1) . A recom1 (zmp1) –107.M. tuberculosis to inhibit and neutralise reactive oxygen species (ROS) production by macrophages is yet another target for vaccine design. In doing so, macrophages would be better able to present antigens, thus improving immunogenicity and efficacy [M. tuberculosis stress-response pathway [M. tuberculosis∆sigH shows greater levels of apoptosis and a markedly stronger innate immune response [Negating the capacity of efficacy –111. Sig pathway . A dupli pathway . The vacresponse , with stresponse .M. tuberculosis is also capable of inhibiting the maturation and trafficking of MHC molecules [M. tuberculosis infected macrophages [In addition to inhibiting phagolysosomal maturation/fusion and ROS inhibition, olecules . The actolecules –95. A rerophages , associarophages . Additiorophages . BCG-Catrophages .M. tuberculosis inhibits autophagy through ESAT6, secreted by the ESX-1 secretion system [M.marinum, BCG::ESX-1Mmar has comparable efficacy and immunogenicity to BCG::ESX-1M. tuberculosis, yet has a safety profile comparable with BCG Pasteur in preclinical models [Another strategy may be to target the autophagy and apoptosis pathways. n system . As thisn system , autophan system . A recomn system . By utill models .Listeria monocytogenes [The most clinically advanced recombinant BCG (rBCG) strain in development is rBCG ∆ureC::Hly, also known as VPM1002. This construct replaces the urease C gene with that of listeriolysin O, a haemolytic (hly) pore forming protein originating from ytogenes , 121. Liytogenes , 123. Byytogenes . The netytogenes .In mice, VPM1002 was cleared faster than BCG  and was A phase IIb trial in South Africa, evaluating the safety and immunogenicity of VPM1002 in comparison with BCG in both HIV unexposed and HIV-exposed uninfected (HEU) BCG naïve newborns (NCT02391415), has now concluded with data awaiting public release. A phase III trial is also underway in India (NCT03152903), investigating efficacy against relapse in adolescents and adults who have been recently treated for active TB.M. tuberculosis [M. tuberculosis, thus indicating the importance of the Th1 response [M. tuberculosis infection, IFN-ɣ may not be sufficient [IFN-ɣ, the hallmark cytokine of a pro-inflammatory Th1 response, is critical for protection against rculosis , 132. Inresponse –135. Whifficient . Deficiefficient .hi/CD69hi CD4+ T cells, which are highly proliferative, in contrast with the more terminally differentiated KLRG1hi/T-bethi CD4+ cells resident in the vasculature [hi cells produce more IFN-ɣ [Most studies look at peripheral, systemic immune responses, but there is increasing interest in the lung environment. The pulmonary CD4+ T cell response can be divided into two subsets, one in the lung parenchyma, and one residing within the vasculature . The parculature –141. There IFN-ɣ  and are re IFN-ɣ ; howeverre IFN-ɣ .M. tuberculosis infection. IL-17 itself drives Th1 response by overcoming IL-10 inhibition [There is also evidence that a Th17 response may be implicated in protection. IL-17 is produced principally by Th17 cells, which require both TGF-β and IL-16 for initiation –145, in hibition , thus ILhibition . Th17 cehibition , 157. Alhibition .M. tuberculosis-specific mucosal immune response following intranasal vaccination [M. tuberculosis-specific IgA secretion seen in the respiratory airways and reduced lung expression of polymeric immunoglobulin receptor (pIgR), which translocates IgA into the airway lumen [In addition to their role in granuloma formation, there also appears to be a link between IL-17 and protective antibodies. Using the TB susceptible DBA/2 mouse strain, it was found that intranasal but not subcutaneously administered BCG conferred robust protection against pulmonary TB . This wacination . Neutralay lumen .There is increasing evidence from animal models that delivery of a vaccine direct to the respiratory mucosa may be a more protective route of vaccination. An understanding of the different T cell subsets within the lung would inform the design of vaccines targeting this route.EM), T central memory cells (TCM), and T resident memory cells (TRM). Much of the available data on this has come from murine studies. It is not always clear how these findings relate to NHPs and humans. Most of the lung resident T memory cells are of the TEM phenotype [hi CD62Llo CD127hi [EM subset act in the first line of defence [EM population induced is unable to maintain persistent memory [EM become terminally differentiated T effector (Teff) cells, losing the ability to proliferate and migrate into the lung parenchyma, expressing the KLRG1 marker [Thus far, three main subsets of lung resident memory cells have been defined: T effector memory cells , not TCM [CM to confer greater protection than TEM is possibly best shown by adoptive transfer of the separate T subsets , in which TCM markedly protected against TB in contrast with TEM and T follicular helper (TFH) cells [CM cells had characteristic CXCR5+ CCR7+ expression and CXCR5 expression correlating with decreased lung pathology [A recent study has challenged the conventional view that T not TCM . The abiH) cells . These Tathology .CM response compared with BCG, which was associated with improved protection after M. tuberculosis challenge [CM specific for M. tuberculosis conferred protection, whereas adoptive transfer of TFH alone did not [In mice, VPM1002 delivered subcutaneously induced a significantly increased Thallenge . Adoptiv did not .RM are CD44hi CD62Llo CD69+ CD103+ in phenotype [CM deriving from KLRG1− precursors [RM permanently reside in non-lymphoid tissue, making them strategically placed for a rapid recall response [RM are highly heterogeneous, with some CD4+ TRM displaying a regulatory profile (Foxp3hi IL-10hi) and others with a more effector profile (T-bet+) [RM cells are more homogenous, expressing predominantly Th1 cytokines [RM are also capable of activating bystander NK and B cells through IFN-ɣ, TNFα and IL-2 [RM may be reliant on the presence of live bacilli, as clearance of BCG in mice with chemotherapy abrogates the antigen-specific CD4+ T cell response [RM cells was associated with the most protection against M. tuberculosis challenge on a per-cell basis [Thenotype , like TCecursors . TRM perresponse . As a gr(T-bet+) . In contytokines . In addiand IL-2 . Maintenresponse . Of all ll basis .RM, mucosal boosting with a protein/adjuvant candidate vaccine, H56:CAF01, did not enhance protection [M. tuberculosis antigens Ag85B, ESAT-6 and Rv2660c [Despite promoting lung-localised Totection . H56 is  Rv2660c , which h Rv2660c . H56:IC3 Rv2660c  and is cIn summary, less-differentiated CD4+ T cells seem to provide greater protection than more-differentiated effector T cells. Vaccine strategies should therefore attempt to induce these cell populations, which appear to be related to dose and persistence of the vaccine construct , 177.The concept of delivering a TB vaccine direct to the respiratory mucosa is nothing new. Nebulised BCG was demonstrated to be safe and immunogenic in terms of tuberculin skin test conversion in 1968 . There aAerosolised MVA85A, a modified Vaccinia virus Ankara expressing Ag85A, was evaluated in a proof-of-concept phase 1 trial (NCT01497769) in BCG vaccinated healthy adults . In thisM. tuberculosis antigens Ag85A, Ag5B and TB10.4, AERAS-402, had demonstrated robust cellular immune responses in the lungs of rhesus macaques, however this failed to confer added protection [M. tuberculosis used in the macaque trial [Adenoviruses are another promising candidate for use in a mucosal TB vaccine due to their natural tropism for respiratory epithelium . Two adeotection . Whilst ue trial .RM and an enhanced proliferative capacity of lung parenchymal CD4+ T cells [M. tuberculosis infection [Mucosal BCG vaccination has been shown to confer superior protection in the lungs compared with intradermal BCG in mice , and par T cells , 141. Th T cells . CXCR3 e T cells , was onl T cells . A recennfection . The mucnfection . Though nfection , 192.M. tuberculosis infection in the acute phase was unaltered [The role of humoral immunity in protection against TB was for a long time discounted. When cynomolgus macaques were treated with rituximab, a B cell depleting agent (but not plasma cell depleting), the overall disease progression and outcome of naltered . Though naltered . IL-6 annaltered , 195, ILnaltered  and B cenaltered , 197. Innaltered , 199, thnaltered .M. tuberculosis [M. tuberculosis persistence or outcome of successful control are thus far unclear.By comparing the antibody profiles between those with active TB and LTBI, a functional role for antibodies is emerging. An unbiased systems serology approach found nine specific antibodies capable of distinguishing the two groups, LTBI or active TB . Latent rculosis . Fc glycrculosis . Furtherrculosis , 202. WhM. tuberculosis [M. tuberculosis-specific IgA than those with active TB, with 7/12 isolated IgA mAbs capable of restricting M. tuberculosis growth compared with 0/16 IgG mAbs [M. tuberculosis growth [Further clues as to a role for humoral immunity come from health care workers (HCWs) with occupational exposure to rculosis , 204. HCIgG mAbs . In anotM. tuberculosis infection in mice lacking activating FcɣR ɣ-chain results in more severe immunopathology during disease due to higher IL-10 levels, further supporting the importance of Ab-Fc function [M. tuberculosis to be internalised via phagocytosis into target macrophages [The protection shown by IgA but not IgG points to the importance of invariant antibody function in protection. function . FcRs cafunction . It is prophages .M. tuberculosis is frequently grown in detergent, stripping the capsule [Though the natural protective effect of antibodies appears small, this does not necessarily mean that a humoral vaccine would fail, only that different antigens need be found. In the laboratory,  capsule ; these c capsule . Carbohy capsule , 209.M. tuberculosis in addition to those not protected [The lack of validated immune correlates of protection is one of the greatest challenges in TB vaccine development. Identification and validation of such correlates is possible only when samples from successful placebo controlled efficacy trials become available, requiring a comparison of the immune responses in vaccinated and unvaccinated individuals protected against rotected .The greatest potential for immune insight would be if the leading candidate TB vaccines induced a diverse immune response. However, in a recent comparison of antigen-specific T cell responses from human clinical trials, the functional profiles suggested a lack of response diversity, with the main difference in the magnitude of response . This coM. tuberculosis infection [Due to the difficulties in identifying antigens capable of generating a protective response, whole cell derived vaccines have gained increasing interest . Whilst nfection .Results reported by Nemes et al. have raised interest in the use of BCG re-vaccination, rather than simply focussing on novel vaccines . This isM. tuberculosis immunodominant antigens such as rBCG30. rBCG30 overexpresses Ag85B [CM induction. Some examples of BCG-based vaccines have been described throughout this review, whether they work through a return of lost virulence factors as in the case of BCG::ESX-1 [Recombinant BCG strategies can broadly be divided into two camps, the first of which being those that overexpress es Ag85B  and was es Ag85B . The secG::ESX-1 , aiding G::ESX-1  or throuG::ESX-1 .The clinical development of one recombinant BCG strain, AERAS-422, an rBCG overexpressing three mycobacterial antigens and expressing perfringolysin, was terminated after 2/8 immunised healthy volunteers developed shingles after the reactivation of varicella zoster virus .M. tuberculosis itself. MTBVAC has deletion of the transcription factor phoP [Rather than creating a more immunogenic/virulent BCG, another tactic is to attenuate tor phoP , which wtor phoP , 221, antor phoP , 223. A tor phoP .Mycobacterium vaccae has been approved in China, yet there is little publicly available information from the Chinese trials [Mycobacterium indicus pranii (MIP) is a non-pathogenic mycobacterium, FDA approved as a leprosy vaccine [Heat-inactivated e trials , with the trials . Now re-e trials , it is c vaccine . MIP has vaccine .M. tuberculosis within liposomes, is an immunotherapeutic agent to reduce the extent and duration of required drug treatment of active TB [RUTI, detoxified and fragmented ctive TB  and is cCM formation [One means of retaining the protective effect of BCG is using a prime-boost strategy, in which BCG is still used, but with the addition of a heterologous vaccine booster . Subunitormation , 232.M. tuberculosis [M. tuberculosis32A and M. tuberculosis39A, the adjuvant also a component of the malaria vaccine RTS, S/AS01 [M. tuberculosis-uninfected subjects. If not, the efficacy will be lower in areas of the world where M. tuberculosis infection prevalence is lower. Preclinical studies demonstrate some level of protection in M. tuberculosis-uninfected NHPs and guinea pigs [The final analysis of the post-exposure phase IIb trial of the subunit vaccine M72/ASO1E has shown 50% efficacy against progression to TB relative to placebo in patients already latently infected with rculosis . M72 is , S/AS01 . This wanea pigs , 235.M. tuberculosis antigens  [The main candidate subunit vaccines are M72/ASO1E  and H56: Rv3620)  combined Rv3620) , which h Rv3620) .With their natural tropism, viral vectors allow greater targeting than subunit vaccines. The benefits of adenovirus-based vaccines have previously been discussed. TB/FLU-04l uses another respiratory epithelium tropic virus, the influenza H1N1 as a base, expressing the Ag85A and ESAT-6 antigens .MVA85 was the first TB vaccine to enter efficacy trials since 1968 . MVA  as the vector base . Results disease . The aut disease . CMV is EM cells –244, thoEM cells , suggestM. tuberculosis, one of the principal barriers to designing an effective vaccine, has slowly but surely been built up to the increasingly clear picture we have today. The classic dogma of the past, focussed solely on the adaptive response, has evolved into something far more complex, integrating the innate, adaptive and humoral systems. With this greater understanding, a variety of novel vaccine design strategies has been made possible. The recent M72/AS01E result gives renewed cause for optimism in this challenging field. It is critical to maintain the momentum that has been built up over the last two decades so that M. tuberculosis, a pathogen that has been with us for 3 million years [Whilst the field of TB vaccine development has experienced significant hurdles, it is important to recognise the great progress made of late, both in immunological understanding and in empirical learning from human clinical trials. An immunological understanding of the pathogenesis of on years , can fin


Calonectria species , Celoporthe dispersa, Sporothrix phasma and Alectoria sarmentosa are presented. Species of Calonectria are the causal agents of Eucalyptus leaf blight disease, threatening the growth and sustainability of Eucalyptus plantations in China. Celoporthe dispersa is the causal agent of stem canker in native Syzygium cordatum and exotic Tibouchina granulosa in South Africa. Sporothrix phasma was first discovered in the infructescences of Protea laurifolia and Protea neriifolia in South Africa. Alectoria sarmentosa is fruticose lichen belongs to the alectorioid clade of the family Parmeliaceae. The availability of these genome sequences will facilitate future studies on the systematics, population genetics, and genomics of these fungi.Draft genome sequences of five  Draft genome sequences of fiveCalonectriaspecies fromEucalyptusplantations in China.Calonectria have a global distribution, particularly in tropical and subtropical regions of the world .Calonectria crousiana: China: FuJian: isol. leaves of Eucalyptus grandis, Aug. 2007 M.J. Wingfield .Calonectria fujianensis: China: FuJian: isol. leaves of Eucalyptus grandis, Aug. 2007 M.J. Wingfield .Calonectria honghensis: China: YunNan: isol. soil collected in a Eucalyptus plantation, 14 Nov. 2014, S.F. Chen & J.Q. Li .Calonectria pseudoturangicola: China: FuJian: isol. soil collected in the campus of Fujian Agriculture and Forestry University (FAFU), 14 Dec. 2014, S.F. Chen .Calonectria isolates have been deposited at DDBJ/EMBL/GenBank under BioProject PRJ562676. The accession numbers for each of the species are presented in Table The draft genome data for the five tef1) for the extracted DNA was carried out on each isolate. The tef1 sequences were then aligned against the sequences which developed in previous studies . The sequences of representative isolates of the different species in this genus were obtained from GenBank, as reported by Liu and Chen   using the method described by Duong et al. . To veriand Chen , and aliThe genomic DNA was submitted to Macrogen (South Korea), where one pair-end library with 550 bp median insert size was prepared using TruSeq DNA PCR-free protocol, and sequenced on Illumina Hiseq 2500 platform to get 250 bp pair-end reads. The quality of the data obtained was assessed using the software FastQC v. 0.11.5  reflecting the position of these five species in relation to other Calonectria species was produced . The sequencing of this additional C. dispersa genome will be valuable for comparative genome studies within the Cryphonectriaceae family, and for improving disease management strategies, as well as preventing the threat to commercial plantations.The Cryphonectriaceae family includes several phytopathogens, and many of these pathogens cause severe damage to commercial plantations, especially Celoporthe dispersa: South Africa, Limpopo Province, Tzaneen, Syzygium cordatum, 2003, M. Gryzenhout .Celoporthe dispersa (isolate number CMW 9976) has been deposited in DDBJ/EMBL/GenBank databases under the accession number WAID00000000. The version described in this paper is WAID00000000.The genome sequence of Genomic DNA was extracted from freeze-dried mycelium of isolate CMW 9976 grown in malt yeast broth  using the Qiagen® Genomic-tip DNA extraction protocol for plants and fungi. Nanopore sequencing was conducted using the MinION sequencing device. The sequencing library was prepared using the Genomic DNA by Ligation (SQK-LSK109) protocol. The library was loaded on a MinION flowcell (R9.5.1) and sequencing was run for 48 h. Base calling was conducted using ONT Guppy basecalling software v 2.3.7.C. dispersa genome. The Fusarium graminearum augustus species model was used as this is the most closely related species to C. dispersa available. The assembled genome completeness was evaluated using the Benchmarking Universal Single-Copy Orthologs tool, BUSCO     (http://genome.jgi.doe.gov/Crypa2/Crypa2.home.html). The availability of the C. dispersa genome from this study will enable various genomic comparisons between species of Cryphonectriaceae to be conducted. Additionally, the genome can be used to study pathogenicity, mating type and other important factors in phytopathology. The availability of the C. dispersa genome will enable us to include this genome within the Cryphonectriaceae during genomic comparison studies.The estimated genome size and gene number for Authors:B.D. Wingfield*, T.A. Duong, M. Sayari, M. A. Ferreira, A. M. Kanzi.*Contact: brenda.wingfield@fabi.up.ac.zaDraft genome sequence ofSporothrix phasma.Sporothrix ; PREM 58941- dried culture).South Africa, isolated from Sporothrix phasma  has been deposited at DDBJ/EMBL/GenBank under the accession number WJIH00000000. The version described in this paper is version WJIH01000000.The genomic sequence of Sporothrix phasma isolate CMW 20676 was obtained from the culture collection (CMW) of the Forestry and Agricultural Biotechnology Institute (FABI), the University of Pretoria, South Africa. Genomic DNA was extracted using the method described by Duong et al.  of the nuclear ribosomal RNA (rRNA) gene, the Internal Transcribed Spacer (ITS) regions, β tubulin (BT) gene and calmodulin (CAL) gene. The sequences of representative isolates in this genus were obtained from GenBank, as reported by De Beer et al. , followed by S. globosa (33.5 Mb), S. brasiliensis (33.2 Mb) and S. schenckii (32.3 Mb) .The advent of DNA sequencing has advanced our understanding on biodiversity research  and microalgae, including cyanobacteria  to advance biodiversity research. Our data will also be helpful for accurate assessment of cryptic biodiversity. Alectoria sarmentosa is fruticose lichen belongs to the alectorioid clade of the family Parmeliaceae .Alectoria sarmentosa binned metagenome project has been deposited at DDBJ/ENA/GenBank under accession no. PRJNA588068.The Alectoria sarmentosa was collected from a bark tree in Telemak, Norway and a partial piece of thallus was deposited at the MAF Herbarium  with code MAF-Lich 21,536. The sample was identified using classical procedures as morphology and chemistry; as well as molecular technique with internal transcribed spacer (ITS) sequence of rDNA.Quick-DNA™ Fungal/Bacterial Miniprep Kit and following the manufacturers’ instruction. DNA concentration was calculated using the Qubit dsDNA dBR assy kit . Two paired-end libraries (300 bp and 900 bp) were built using a Illumina Tru-Seq library preparation kit. Sequencing was carried out the Unidad de Genómica  with Illumina Miseq platform (250 bp paired-end reads). Raw sequences were downloaded from Illumina BaseSpace application and were quality trimmed and filtered using Trimmomatic-0.36 . Sequences were aligned using the program MAFFT v. 7 . Nodal support was evaluated with 1000 bootstrap pseudoreplicates. Phylogenetic trees were drawn using the program FigTree v. 1.4.2 , commonly used for species delimitation in Parmeliaceae and the standard DNA barcode for fungi  assembled into 1788 contigs. Of these, 1046 were longer than 1000 bp and contained 46,193,838 bp (46.1 Mb) of genome. The largest contig was 400,628 bp and the N50 and L50 were 92,863 bp and 140 bp, respectively  markers, or mating types (Alors et al. The gene prediction conducted by MAKER2 (Holt and Yandell de Table . The aleosa Fig. . The draAuthors:D. Pizarro, A. Crespo, P.K. Divakar*.*Contact: pdivakar@farm.ucm.es


Entada phaseoloides (L.) Merr. is an important traditional medicinal plant. The stem of Entada phaseoloides is popularly used as traditional medicine because of its significance in dispelling wind and dampness and remarkable anti-inflammatory activities. Triterpenoid saponins are the major bioactive compounds of Entada phaseoloides. However, genomic or transcriptomic technologies have not been used to study the triterpenoid saponin biosynthetic pathway in this plant.Entada phaseoloides with three independent biological replicates and obtained a total of 53.26 Gb clean data and 116,910 unigenes, with an average N50 length of 1218 bp. Putative functions could be annotated to 42,191 unigenes (36.1%) based on BLASTx searches against the Non-redundant, Uniprot, KEGG, Pfam, GO, KEGG and COG databases. Most of the unigenes related to triterpenoid saponin backbone biosynthesis were specifically upregulated in the stem. A total of 26 cytochrome P450 and 17 uridine diphosphate glycosyltransferase candidate genes related to triterpenoid saponin biosynthesis were identified. The differential expressions of selected genes were further verified by qPT-PCR.We performed comparative transcriptome analysis of the root, stem, and leaf tissues of Entada phaseoloides.The dataset reported here will facilitate the research about the functional genomics of triterpenoid saponin biosynthesis and genetic engineering of  Entada phaseoloides (L.) Merr. is a liana belonging to Fabaceae family. It grows in Southern China and other tropical countries. The stem of Entada phaseoloides is popularly used in traditional medicine because of its significant pharmacological activities [Entada phaseoloides, also called “Guo Gang Long,” produces curative effects that dispel wind and dampness and exhibits remarkable anti-inflammatory activity. Its main bioactive ingredients are triterpenoid saponins compounds [E. phaseoloides. The representative saponins of Entada phaseoloides are oleanane-type triterpene saponins which contain seven sugar chains.tivities –3. The sEntada phaseoloides have not been identified.The mevalonic acid (MVA) pathway is an important metabolic pathway in plants , 5. TritPanax ginseng [Bacopa monnieri [Artemisia annua [Glycyrrhiza uralensis [Parthenium argentatum [, cardiac glycoside in Calotropis procera [Cinnamomum camphora [Cannabis sativa [Withania somnifera [Picrorhiza kurrooa [Taxus chinensis [Asparagus racemosus [High-throughput sequencing analysis is a useful method to clarify the molecular mechanism of plant secondary metabolism , 12. Rec ginseng , triterpmonnieri , artemisia annua , 16, flaia annua , glycyrrralensis , rubber gentatum , cardiac procera , terpenocamphora , cannabis sativa , withanoomnifera , picrosi kurrooa , paclitahinensis  and steracemosus .P. ginseng are higher in the early growth stage, while the content of triterpenes in the roots of old plants are higher [Hedera helix [Panax notoginseng [Asparagus racemosus [Entada phaseoloides was performed to identify genes related to triterpenoid saponin. We obtained thousands of putative genes, including a series of genes related to triterpene saponin biosynthesis. Moreover, different expression patterns of CYP450s and UGTs in the three tissues were analyzed. This work was established to functionally research the genes related to triterpene saponin biosynthesis and provide more information about this species.Because the synthesis and accumulation of specific metabolites in different tissues depends on the age of the plant, and are greatly affected by the different developmental stages. It was found that the content of triterpenes accumulated in the leaves of e higher . Comparara helix , Panax noginseng  and Aspaacemosus . The maxEntada phaseoloides, we sequenced nine cDNA libraries prepared from the root, stem, and leaf tissues with three biological repeats by using the Illumina Hiseq 2500 platform. A total of 53.26 Gb clean data were obtained after removing adaptors, poly-A tails, and primer sequences, short (< 50 bp), and low-quality sequences. A total of 57–60 million for each tissue were generated , 41,228 (35.3%), 28,126 (24.1%), 26,874 (23.0%), 15,119 (13.0%) and 11,812 (10.1%) unigenes showed significant similarity to known proteins in NR, Uniprot, GO, Pfam, KEGG and COG database, respectively. The result of BLASTX with different databases and their annotation were listed in Additional file Entada phaseoloides unigenes shared high similarity with the genes of Cicer arietinum, 13.46% similarity with Cajanus cajan, 12.84% similarity with Glycine max, and 9.07% similarity with Medicago truncatula , Uniprot, Kyoto Encyclopedia of Genes and Genomes (KEGG), Pfam, Gene Ontology (GO), and Clusters of Orthologous Groups (COG) databases using the BLASTx program with , 42,191 6.1%, 41,GO analysis included three main domains that describe biological processes, cellular components, and molecular functions. When GO was used to classify gene functions, 28,126 unigenes were assigned to 60 functional categories  could be assigned to five main categories and 31 sub-categories  program to analyze the DEGs among different tissues . The FraKEGG enrichment analyses were performed with the DEGs among the root, stem, and leaf tissues to investigate the genes regulating the distribution of triterpenoid saponin. These DEGs were evidently enriched in specific pathways. Meanwhile, the top 20 significant pathways were analyzed based on the FDR ≤ 0.01. Between the leaf and stem, plant hormone signal transduction, phenylpropanoid biosynthesis, photosynthesis, terpenoid backbone biosynthesis, and phosphatidyllnositol signaling system showed significant enrichment Fig. a. BetweeEntada phaseoloides transcriptome. Their expression value was monitored in three biological replicates along with their mean values  via the MVA pathway. All genes encoding the enzymes associated with the upstream regions of triterpenoid biosynthesis were successfully detected in the es Table , Fig. 5.Entada phaseoloides are oleanane-type triterpenoid saponins derived from β-amyrin. The Illumina sequencing of Entada phaseoloides revealed 21 OSC sequences, among which eight unigenes were putative β-amyrin synthases. A full-length OSC sequence (EpBAS) with high identity to β-amyrin synthase was obtained  and GiBAS in Glycyrrhiza inflata  of unigenes associated with the MVA pathway. Single copies of SQE were identified in mouse and yeast, and the destruction of SQE in these species is lethal . Howevernal SQEs . The exp ginseng . Thus, SEntada phaseoloides are oleanane-type triterpenoids. Oleanolic acid sapogenin was derived from β-amyrin after hydroxylation by CYP450s and glycosylation by UGTs [Entada phaseoloides. Similar tissue-specific concentrations of triterpenoid sapogenins have already been reported in other plants [Entada phaseoloides.The cyclization of 2,3-oxidosqualene is a branch point of saponin synthesis. The major saponins in the stem of  by UGTs , 42. A tr plants –45. MoreEntada phaseoloides was performed to investigate the putative genes involved in triterpenoid saponin biosynthetic pathway of an important medicinal plant. The differential expression pattern of pathway genes suggest tissue-specific synthesis. The identified data will help the further discovery and functional genomics and transcriptomics analysis of Entada phaseoloides.In the present study, the comparative transcriptome analysis of root, stem and leaf tissues of Entada phaseoloides plants were collected from the experimental farm of South China Botanical Garden, Guangzhou City, Guangdong Province, P.R. China, in May 2019. After cleaning with ultrapure water, the roots, stems, and leaves were collected separately, immediately frozen in liquid nitrogen, and stored at − 80 °C.Three-year-old healthy wild-type Total RNA from approximately 1.0 g of each tissue was extracted using TRIzol  following the manufacturer’s instructions. Three replicates were employed for each experiment. mRNA was isolated from total RNA by using Oligo (dT) magnetic beads. By mixing with fragmentation buffer, the mRNA was broken into short fragments.The short fragments were purified and resolved with EB buffer. After end repair and single base “A”addition, adapters were ligated to the cDNA molecules. To select suitable cDNA fragments for PCR amplification, we purified the sample library with the AMPure XP system . Finally, PCR products were purified, and library quality was were determined using an Agilent Bioanalyzer 2100 system  and a Qubit 3.0 fluorometer . Each cDNA library was sequenced in a single lane of the Illumina Hiseq 2500 platform.The raw reads were first filtered to exclude the reads containing adaptors or with ambiguous nucleotides (‘N’). Next, the low-quality reads having more than 20% Q < 20 bases were also trimmed. The yielded high-quality clean reads were used to the develop sequence assembly by using the Trinity software. After the removal of redundant Trinity-generated sequences by using the TGICL, clusters and unigenes were finally obtained.http://www.ncbi.nlm.nih.gov/), UniProt (http://www.uniprot.org/downloads), Pfam (http://pfam.xfam.org/), COG (http://www.ncbi.nlm.nih.gov/COG/) databases with an E-value <1e-5. Only the top hit results were extracted for each unigene. GO (http://www.geneontology.org) terms were functionally classified based on Nr annotations by using the Blast2go program (http://www.Blast2 go.de/). KEGG (http://www.genome.jp/kegg/) was used to draw metabolic maps. The KEGG analysis results included KEGG orthology (KO) numbers and enzyme commission (EC) numbers.All assembled unigenes were annotated by BLASTx analysis against the Nr  gene in Entada phaseoloides.Additional file 9. List of qRT-PCR primer sequences.Additional file 10. Melting curves of reference gene Actin for qRT-PCR amplification.


Formica polyctena belongs to the red wood ant species group. Its nests provide a stable, food rich, and temperature and humidity controlled environment, utilized by a wide range of species, called myrmecophiles. Here, we used the high-throughput sequencing of the 16S rRNA gene on the Illumina platform for identification of the microbiome profiles of six selected myrmecophilous beetles  and their host F. polyctena. Analyzed bacterial communities consisted of a total of 23 phyla, among which Proteobacteria, Actinobacteria, and Firmicutes were the most abundant. Two known endosymbionts—Wolbachia and Rickettsia—were found in the analyzed microbiome profiles and Wolbachia was dominant in bacterial communities associated with F. polyctena, M. subterraneus, L. formicetorum and P. formicetorum (>90% of reads). In turn, M. angusticollis was co-infected with both Wolbachia and Rickettsia, while in the microbiome of T. angulata, the dominance of Rickettsia has been observed. The relationships among the microbiome profiles were complex, and no relative abundance pattern common to all myrmecophilous beetles tested was observed. However, some subtle, species-specific patterns have been observed for bacterial communities associated with D. pygmaeus, M. angusticollis, and T. angulata. Myrmecophiles (or ant associates) are organisms that are dependent on various ant species, at least during part of their lifecycle. They are able to penetrate and survive inside the heavily defended ants’ nests ,2,3. A vProciphilus aphids and Rhizoecus mealybugs farming by Brachymyrmex and Lasius ants revealed a phylogenetic signal in the microbiomes of trophobionts [Buchnera, Tremblaya, Sodalis, and Serratia). That result was explained by the strict vertical transmission of endosymbionts between generations of trophobionts. In contrast, tested Brachymyrmex and Lasius microbiome was possibly shaped by host’s partnerships, and ant-bacteria associations were subject to horizontal transmission or social transmission within colonies [Megalomyrmex ants and their fungus-growing ant hosts from the genera Cyphomyrmex, Trachymyrmex, and Sericomyrmex [Acinetobacter, Wolbachia, and Pseudonocardia) and that Entomoplasmatales and Bartonellaceae can co-infect specifically associated combinations of hosts and social parasites with identical 16S rRNA genotypes. Moreover, obtained results implied that cohabiting ant social parasites and hosts may obtain functional benefits from bacterial symbiont transfer even when they are not closely related.Despite the great interest in biology and ecology of myrmecophiles, studies on the microorganisms associated with them, the potential similarities of microbiome profiles of ants and their associates, and the probable horizontal transfer of microorganisms among insects inhabiting or visiting ants nests are limited. A recent study focusing on the microbiota of several honeydew-producing hobionts . Each secolonies . Bactericomyrmex . PerformFormica genus), this group has been better recognized.Although studies by Ivens et al. (2018)  and LibeFormica are the widespread and significant component of forest ecosystems. They influence soil qualities and the presence of some plant species. Formica ants also have a strong influence on surrounding zoocenosis [Formica genus, one may distinguish the Formica rufa group . All red wood ant’s species are morphologically very similar and show high intraspecific variability [Formica [Formica representatives.The ants of the genus ocenosis . Within iability . Therefoiability ,16,17,18F. polyctena, selected myrmecophilous beetles commonly inhabiting its nest and microbial communities associated with all species tested. We selected six beetle species belonging to five different families: Thiasophila angulata , Leptacinus formicetorum  (Staphylinidae), Dendrophilus pygmaeus  (Histeridae), Monotoma angusticollis  (Monotomidae), Ptenidium formicetorum  (Ptiliidae) and Myrmechixenus subterraneus  (Tenebrionidae). All selected species are commonly found inside F. polyctena nests, but they are rather unspecialized in the level of integration into colony [Thiasophila angulata and Leptacinus formicetorum are predators, actively seeking out early developmental stages of ants (eggs and larvae) and other invertebrates inhabiting nests of red wood ants. They can also scavenge dead bodies of adult ants and other insects in the anthill [T. angulate adapts its behavior to avoid ants aggression or to defend when attacked [Dendrophilus pygmaeus (Histeridae) is carnivorous, feeding on insect remains or dead ants. It is classified as a synechthran. Convex shape and hard exoskeleton, as well as the ability to conceal its antennae and legs , allow them to avoid various ant attacks [Monotoma angusticollis (Monotomidae) is a typical scavenger that feeds on animal food, such as dead ants or ants’ prey [Ptenidium formicetorum (Ptiliidae ) is a very small beetle, which feeds on animal food and probably hunts small mites or springtails, commonly occurring in the nests of red wood ants. The last studies species, Myrmechixenus subterraneus (Tenebrionidae), is a well-known Euro-Siberian species, common in the nests of Formica ant species. It is also included in the synechthran category [In the present study, our main goal was to shed light on complex tripartite relationships among o colony ,20. Two  anthill . What isattacked ,22. Dend attacks . Monotomts’ prey . Ptenidicategory .F. polyctena using the Next Generation Sequencing of the 16S rRNA gene. The bacterial communities were screened to establish the presence of known endosymbionts and bacterial groups that may be related to the adaptations to the conditions prevailing in the nest.The presented study aimed to define the microbiome profiles of selected myrmecophiles and their host F. polyctena in the Polesie National Park (Poland). The permission for field studies has been granted by the Ministry of the Environment, Poland (permission nos. DLP-III.286.148.2016.MGr and DLP-III.286.141.2016.MGr).Samples were collected as part of a research project on the fauna of myrmecophilous beetles inhabiting nests of the red wood ant Monotoma angusticollis (n = 5), Ptenidium formicetorum (n = 17), Leptacinus formicetorum (n = 4), and F. polyctena (n = 20) were collected from the nest near the Dlugie Lake (N 51°27′04.0″ E 23°09′39.9″). Myrmechixenus subterraneus (n = 15), Thiasophila angulate (n = 6), Dendrophilus pygmaeus (n = 3) as well as the second sample of F. polyctena (n = 20) were collected from a nest near the Lipniak village (N 51°27′51.2″ E 23°06′53.5″).Specimens were collected during the summer and autumn of 2017. Approximately three liters of the nest material was taken from a depth of 20–30 cm and then densified using a standard entomological sieve. The obtained nest material, along with the live insects found, was placed in cloth pouches and transferred to the laboratory. Species were identified by the authors, according to particular insect identification keys ,28,29,30DNA was extracted from whole insects using a universal Sherlock AX Purification Kit  based on spin column technology. Insects were rinsed three times in sterile distilled water prior to DNA extraction (without soaking in ethanol). Sterile equipment was used to avoid cross-contamination of samples. The quantity and quality of the extracted DNA were evaluated by using a NanoDrop ND-1000 spectrophotometer . After extraction, the DNA was stored at −20 °C until further use.® Q5 Hotstart High-Fidelity DNA polymerase , according to the manufacturer’s protocol using Q5® Hot Start High-Fidelity 2X Master Mix , and the Nextera Index kit (2 × 250 bp). Paired-end  sequencing with a 5% PhiX spike-in was performed with an Illumina MiSeq (MiSeq Reagent kit v2) at Genomed, Warsaw, Poland, following the manufacturer’s run protocols . The automatic primary analysis and the de-multiplexing of the raw reads were performed with MiSeq with the use of MiSeq Reporter (MSR) v2.6 (16S Metagenomics Protocol).Twenty-four samples consisting of specimens from six myrmecophilous beetles and red wood ants from two nests were used for further analyses . Negative controls have not been included. The V3-V4 hypervariable regions of the bacterial 16S rRNA gene were amplified using the following primer set: 341F (CCTACGGGNGGCWGCAG) and 785R (GACTACHVGGGTATCTAATCC). Libraries were prepared with a two-step PCR protocol based on Illumina’s “16S metagenomic library prep guide” (15044223 Rev. B), NEBNextL—D. pygmaeus, LF-D—L. formicetorum, MA-D—M. angusticollis, MS-L—M. subterraneus, PF-D—P. formicetorum, TA-L—T. angulata, and FP-D and FP-L for F. polyctena. Indices D and L refer to the sampling sites .The data obtained for the independent sequencing were analyzed separately. Samples were marked as follows: DP-The samples were processed and analyzed using the Quantitative Insights Into Microbial Ecology  pipeline . Paired-Similarity percentage (SIMPER) analysis was performed to calculate the average dissimilarities in bacterial community structures between samples and to access which family was responsible for the observed differences. For this purpose, all profiles were grouped according to the taxonomic position of the host. According to the Diss/SD values, we identified families, which were primarily responsible for the observed differences among profiles (larger number means more consistently contributes to the dissimilarity between profiles). Relative abundances of those bacterial families, which were primarily responsible for differences between samples, were then used for a proximity analysis . A similarity profile (SIMPROF) test was used to identify well-defined groups of samples . FinallyThe NGS data are deposited and fully available under study accession number PRJEB31343 in ENA—the European Nucleotide Archive.T. angulata, specimen TA1-L) and 150,363 . Chao1 curves almost reached a plateau, suggesting that the sequencing was approximately deep (L specimens (D. pygmaeus), which suggests the high alpha diversity of those communities. More details of the sequence data for each species, as well as the number of observed OTUs and alpha diversity indices, are given in More than 8200 good quality sequences were obtained for each sample tested, ranging between 8253  clustered together and were characterized, for example, by less abundant Anaplasmataceae, and more abundant Streptomycetaceae and Propionibacteriaceae. Bacterial communities associated with M. angusticollis also clustered together and were characterized by more abundant Enterobacteriaceae and Tsukamurellaceae , which was congruent with SIMPROF results at lower levels of similarity of microbiota profiles (about 20%) (F. polyctena (both from Dlugie Lake and Lipniak), M. subterraneus (except MS4-L), L. formicetorum (except LF3-D), and P. formicetorum (except PF1-D) were the most similar and clustered together. Nonparametric MDS analysis (85% similarity) revealed three vectors , which allowed for distinguishing among clusters  A. Bacterclusters B.Wolbachia and Rickettsia are the most abundant genera (Wolbachia has been noticed for L. formicetorum (98.75% in LF2-D). In the case of T. angulata, Rickettsia was more abundant than Wolbachia and accounted in the range of 22.63% for TA6-L to 99.01% for TA2-L  . We could not a priori exclude that high relative abundance of = 0.001) A. That f cluster B. ValuesL. formicetorum, M. angusticollis, and F. polyctena, the average similarity of profiles within species was higher than between species pair , we were able to connect this result with higher abundance of Enterobacteriaceae in its profiles (but profiles remained similar to P. formicetorum PF1-D profile and T. angulata TA1-L profile). The profiles separation according to the locality of the host’s nest has not been identified. .In all bacterial communities associated with ants, a higher relative abundance of Lactobacillaceae and Acetobacteraceae have been identified compared to other profiles tested  B. In theSpecialized interactions of bacterial communities with insect hosts are ubiquitous and often beneficial to insects . NeverthF. polyctena and six unspecialized myrmecophilous beetle species commonly inhabiting its nests [Although many studies have been focused on revealing the bacterial communities associated with insects, especially social species  and Actinobacteria was more frequently identified than in the rest of bacterial communities tested (>29%)  . Althougromyrmex , the plaraponera , the tur rohweli  or the waragdina . Interesd (>29%) .Pseudonocardia and Streptomyces have been found  from pathogens by excretion of substances with antibiotic activity . In our en found . Those bmicrobes ,79,80. Tc fungus ,81. In ac fungus . Althougntatives ,84,85,86F. polyctena we found an increased abundance of Acetobacteraceae  have also been shown to actively colonize different insect tissues and organs and being involved in the regulation of the innate immune system homeostasis [In the case of Proteobacteria, the explanation seems to be different. Those bacteria are known to be metabolically versatile or specialized and therefore adapted to complex environments, such as the gut of social insects . Therefoteraceae B. Memberine ants ,89,90, aine ants ,92. Thateostasis .F. polyctena, all tested bacterial communities were primarily predominated by other Proteobacteria members—Anaplasmataceae and Rickettsiaceae  [Linepithema, Acromyrmex, and Solenopsis located in Latin America. However, a recent study showed that not all Wolbachia strains associated with ants have the same genetic potential for horizontal transmission [F. polyctena and associated myrmecophiles. However, this hypothesis seems likely, because all specimens share the same identified OTUs. At this stage of research, we cannot exclude both direct and indirect transfer. The direct horizontal transfer has been discussed by Dedeine et al. (2005) [Wolbachia variants infecting fly parasitoids, inquiline social parasite Solenopsis daguerrei, and their hosts , revealing identical symbiont variants shared among S. daguerrei and both its hosts. Interaction between the social parasite and its host  may provide opportunities for the transmission of Wolbachia from the host to the social parasite and the other way around [Nevertheless, the identification of . (2005)  for thresmission . Here, w. (2005)  who invey around . Myrmecoy around ,125. Mory around ,127. D. pygmaeus and T. angulata remains unsolved. Both species feed on dead ants or their brood and live in close relationships with F. polyctena, which would allow expecting that Wolbachia will be present in their microbiome profiles as well. However, the results presented here have been obtained for limited samples, and therefore, we cannot exclude that both D. pygmaeus and T. angulata are infected with Wolbachia in a smaller percentage than the other selected species. Moreover, we cannot unambiguously reject the possibility that native microbiota of these two myrmecophiles impede Wolbachia transmission. Such observations have been described previously for Anopheles mosquitoes [Although all hypotheses mentioned above seem likely, the reason for the lack of infection in squitoes .Rickettsia. Like Wolbachia, Rickettsia can also influence the biology of its insect hosts. As vertically transmitted endosymbiont, it is known to have diverse effects on hosts, ranging from influencing host fitness to manipulating its reproduction [Rickettsia-containing populations are more tolerant of heat shock than those without this endosymbiont [Rickettsia-infected species can also produce more offspring, have a greater survival rate to adulthood, develop faster, and produce a higher proportion of female offspring [Rickettsia can be linked to host protection against entomopathogenic bacteria, for example, Pseudomonas syringae [The second known endosymbiotic Proteobacteria member found here with a higher relative abundance was oduction . Recent symbiont . Rickettffspring . Moreovesyringae  or a higsyringae .Rickettsia has been identified in bacterial communities associated with six selected beetle species, comprising a different share of the microbiome depending on the beetle host. However, Rickettsia was not found in F. polyctena bacterial communities. A similar result has been obtained for another Formica ant, F. cinerea [Rickettsia abundance have been observed for T. angulata (range from 22.63 for TA6-L to 99.01% for TA2-L). In the rest of the tested microbiome profiles that endosymbiont counted for was less than 5% (M. angusticollis (MA1-D–MA3-D), P. formicetorum (PF1-D) and T. angulata (TA6-L) Rickettsia coexist with Wolbachia, which was identified previously for other beetles [In the present study,  cinerea . In the  than 5% . Interes beetles ,135,136.F. polyctena nests and belonging to the same trophic level. Such a study has been performed previously for predatory and herbivorous ant species [D. pygmaeus, M. angusticollis, and T. angulata) cluster together, but the observed similarities are rather the results of Wolbachia infection than the common microbiome pattern. This trend has been shown in D. pygmaeus, M. angusticollis, and T. angulata (Wolbachia and Rickettsia reads from the analyzed dataset revealed that all F. polyctena samples cluster together (The variation in microbiota in insects is related to many factors. The microbial community can be determined by the host phylogeny, gut morphology, and physic-chemical conditions, such as pH and oxygen availability in the insect gut ,138. The species . Neverthangulata . Filteritogether B, and fuF. polyctena and six myrmecophilous beetles inhabiting its nests. The host phylogeny does not directly affect the composition of the microbiome. Most of the analyzed microbiome profiles were clustering together, but it might be strictly correlated with a high relative abundance of Wolbachia reads. This endosymbiont has been previously identified for F. polyctena, but here, we identified its occurrence in the microbiome profiles of six myrmecophilous beetles for the first time. Furthermore, for all species except F. polyctena, the Rickettsia endosymbiosis has been identified. However, after filtering out Wolbachia and Rickettsia reads, we revealed that bacterial communities associated with F. polyctena differ from those associated with selected myrmecophiles. Nevertheless, microbiota profiles of selected myrmecophiles were not homogenous.This paper presents the results of the case study and analyses of bacterial communities associated with


Genetic interpretation of rare variants associated with arrhythmogenic cardiomyopathy (ACM) is essential due to their diagnostic implications. New data may relabel previous variant classifications, but how often reanalysis is necessary remains undefined. Five years ago, 39 rare ACM-related variants were identified in patients with features of cardiomyopathy. These variants were classified following the American College of Medical Genetics and Genomics’ guidelines. In the present study, we reevaluated these rare variants including novel available data. All cases carried one rare variant classified as being of ambiguous significance (82.05%) or likely pathogenic (17.95%) in 2016. In our comprehensive reanalysis, the classification of 30.77% of these variants changed, mainly due to updated global frequencies. As in 2016, nowadays most variants were classified as having an uncertain role (64.1%), but the proportion of variants with an uncertain role was significantly decreased (17.95%). The percentage of rare variants classified as potentially deleterious increased from 17.95% to 23.07%. Moreover, 83.33% of reclassified variants gained certainty. We propose that periodic genetic reanalysis of all rare variants associated with arrhythmogenic cardiomyopathy should be undertaken at least once every five years. Defining the roles of rare variants may help clinicians obtain a definite diagnosis. Arrhythmogenic cardiomyopathy (ACM) is a rare disorder characterized by progressive replacement of the myocardium by fibrofatty tissue, and this myocyte disorganization increases the risk for ventricular arrhythmias and sudden cardiac death ,2. TissuPKP2 being the main gene currently associated with ACM [A definite genetic variant classified as pathogenic represents a major criterion for ACM diagnosis in the TFC . Currentwith ACM . Howeverwith ACM ,12,13. Twith ACM . Accuratwith ACM .Continuous improvement in the genetic data concerning rare variants may modify previous classifications and thus alter patient diagnosis and treatment, prenatal diagnosis or pre-implantation genetic diagnosis , and scrWe reanalysed and reinterpreted 39 rare variants identified in 2016 from 39 patients that presented features of ACM . These rare variants were originally classified as pathogenic (P), likely pathogenic (LP), or variants of unknown significance (VUS) following ACMG recommendations . Variantwww.ensembl.org/) linked to RefSeq code (www.ncbi.nlm.nih.gov/refseq/) or CCDS (www.ncbi.nlm.nih.gov/CCDS/). Sequence data coordinates were based on the UCSC human genome version hg19 (NCBI GRCh37 build). Secondary, bioinformatic analysis included adaptor and low-quality base-trimming of the FASTQ files. Variant calling from the cleaned BAM files was performed with SAMtools v.1.2 and an ad hoc developed script. The final annotation steps provided information included in public databases. Non-common  genetic variants were confirmed by Sanger sequencing. Exons and exon–intron boundaries of each gene were amplified in both directions with posterior analysis, comparing obtained results with the reference sequence from hg19.Genomic DNA was extracted from whole blood, and its concentration and purity were determined. Comprehensive genetic analysis was performed, including all genes associated with ACM in 2016 and all isoforms described in Ensembl 75 (www.hapmap.ncbi.nlm.nih.gov/), the 1000 Genomes Project (www.1000genomes.org/), the Exome Variant Server (EVS) (www.evs.gs.washington.edu/EVS/) in 2016, and the Genome Aggregation Database (gnomAD) (www.gnomad.broadinstitute.org/) in 2021. All rare variants were described following HGVS (www.hgvs.org/) and consulted in ClinGen , VarSome (www.varsome.com/), the SCD-associated Variants Annotation Database (SVAD) (www.svad.mbc.nctu.edu.tw/), CardioClassifier (www.cardioclassifier.org/), InterVar (www.wintervar.wglab.org/), CardioVAI (www.cardiovai.engenome.com/ and CardioBoost (www.cardiodb.org/cardioboost/).Identified rare variants were compared with with HapMap (www.hgmd.org), ClinVar (www.ncbi.nlm.nih.gov/clinvar/intro/), the National Center for Biotechnology Information SNP database (www.ncbi.nlm.nih.gov/SNP), Index Copernicus (www.en.indexcopernicus.com), Google Scholar (www.scholar.google.es), Springer Link (www.link.springer.com), Science Direct (www.sciencedirect.com), Excerpta Medica Database , and IEEE Xplore Digital Library (www.ieeexplore.ieee.org/Xplore/home.jsp).An exhaustive review of the literature concerning each variant was performed through January 2021. Data was collected from HGMD : three in TMEM43 and one in DES : 11 in e in DES A. Most re in DES B.After a comprehensive analysis, 12 of the 39 rare variants (30.77%) were reclassified . Most ofOf the nine reclassified VUS variants, two were now classified as benign, three as likely benign (LB) and four as likely pathogenic (LP). In 2016 we identified seven likely pathogenic (LP) variants (17.94%), but only four remained LP five years after. Two of the 2016 LP variants changed to VUS and one to P .Importantly, 10 out of 12 (83.33%) reclassified variants gained certainty upon reclassification. Among likely pathogenic mutations, 37.5% (3/8) qualified as predicted null variants in a gene where loss of function is a known mechanism of disease (very strong criteria-PVS1). Only one missense variant  was classified as P due to the sum of different criteria and with several strong supporting clinical reports available (very strong criteria-PP5) . On the In our study, 39 rare variants in ACM-related genes were identified in 39 non-related Caucasian patients with features of ACM five years ago. Using currently available data, we updated the 2016 classifications of 30.77% of the rare variants. Importantly, 83.33% of these relabelled rare variants gained certainty in their significance. Our results support periodic evaluation of rare variants associated with ACM at least once every five years as this may directly impact final diagnosis as described in a previous study . They alPKP2 premature termination codon (PTC), with new global genetic studies identifying no alleles or alleles with an extremely low frequency. Haploinsufficiency in PKP2 is well established to be disease-causing in ACM [PKP2.VUS variants represented 82.05% of variants in ACM carriers in the original classification in 2016 but only 64.1% of variants in the present study. Moreover, other studies in different cardiomyopathies have similarly concluded that uncertainty is reduced after variant reclassification ,24. In tg in ACM , so noveOn the other hand, three other VUS variants in 2016 changed to LB in our reanalysis. In all these cases, massive genotyping data released in the past five years played a key role in reclassification. Therefore, the absolute number of alleles is currently higher than expected for a rare genetic disease such as ACM. Importantly, updated global frequencies allow us to identify data in 53.85% of ACM carriers compared to only 20% of ACM variants identified five years ago. In a recent study, a common reason for genetic reclassification of variants in ACM was a high minor allele frequency in the general population .DSG2_p.(Thr335Ala) [DSC2 that cause ACM [In addition to updated global frequencies, it is also important to take into account specific scientific findings regarding rare variants to improve knowledge and clarify their role in ACM pathophysiology. In this sense, all rare variants with novel available in vitro and/or family co-segregation data have been reclassified into a higher degree of certainty. In our view, complete family segregation is the most important data to clarify the role of a rare variant, especially if a recessive pattern of inheritance is suspected, as observed in r335Ala) . High vaause ACM ,32,33.PKP2_ p. variant was previously described as potentially disease-causing in Brugada syndrome (BrS) [PKP2_ p. variant has increased during these five years, suggesting a non-deleterious role. Thus, new in vitro studies are also important for reclassifying rare variants into a group with higher certainty. In our study, DSG2_ p.(Arg49His) has been reclassified as definitively P based on co-segregation [Moreover, we should be especially careful in interpreting variants, taking into account suspected disease, affected genes, and previous classification. The me (BrS) . Althougme (BrS) ,36. Howeregation  and an iregation  publisheFinally, a report has been published recently concerning the role of VUS in inherited arrhythmias and cardiomyopathies . The VUSIdentifying a definite pathogenic variant is a key point in the diagnosis of ACM. Therefore, accurate interpretation of variants identified in patients should be mandatory. An exhaustive and updated genetic clarification of rare variants associated with ACM is warranted because it has practical consequences for patients and their relatives. Most rare variants remain of ambiguous significance due to lack of functional data, conclusive family segregation, and updated global frequency. It is important to highlight this data because VUS is an uninformative genetic result in translation into clinical practice, and genetic and clinical professionals cannot make informed clinical decisions based on such a classification. We recommend reanalysing rare variants associated with ACM at least once every five years. When significant changes in classification occur, cardiologists should promptly inform interested patients and, if necessary, modify their therapeutic approach.A different interpretation of genetic data for some rare variants included in our study may induce controversy regarding their role, especially for variants classified as VUS. To minimize controversy, all authors came to a consensus regarding the final classification decision. Importantly, reclassifications in our study should be corroborated in additional larger cohorts of ACM families. Finally, we have not assessed the time or economic cost of reinterpretation.


Aspergillus strains erroneously curated as Aspergillus oryzae and Aspergillus fumigatus have been noticed in the National Center for Biotechnology Information (NCBI) genome database. Aspergillus parasiticus is one of several aspergilli that produce aflatoxin, the most potent carcinogenic mycotoxin known up to now. To ensure that valid conclusions are drawn by researchers from their genomics-related studies, molecular analyses were carried out to authenticate identities of A. parasiticus strains in the NCBI genome database.The use of genome sequences from strains authenticated to correct species level is a prerequisite for confidently exploring the evolutionary relationship among related species. A. parasiticus strains, E1365 and NRRL2999, were found to be misidentified. They turned out to be Aspergillus flavus based on genome-wide single nucleotide polymorphisms (SNPs) and genetic features associated with production of aflatoxin and cyclopiazonic acid. NRRL2999 lacked the additional partial aflatoxin gene cluster known to be present in its equivalent strain, designated as SU-1, and shared a very low total SNPs count specifically with A. flavus NRRL3357 but not with other A. flavus isolates. Therefore, the mislabeled NRRL2999 strain actually is a clonal strain of A. flavus NRRL3357, whose genome was first sequenced in 2005.Two of the nine supposedly The online version contains supplementary material available at 10.1186/s13104-021-05527-6. Aspergillus parasiticus is a saprophytic fungus found in soil and decayed plant materials. It was first isolated, from dead mealy bugs in Hawaiian sugarcane, and characterized by Speare in 1912 [A. parasiticus was originally classified as a subspecies of Aspergillus flavus, because of its morphological resemblance to A. flavus. Nonetheless, A. parasiticus can be distinguished from A. flavus based on other characteristics, such as darker green conidial heads and more rough conidium surface ornamentation [1 is the most carcinogenic mycotoxin known [A. parasiticus isolates are highly aflatoxigenic but abilities of A. flavus isolates to produce aflatoxin vary greatly. Evidence of the aflatoxin biosynthesis gene cluster, as well as its complete characterization, were revealed in A. parasiticus [A. parasiticus [A. flavus has gradually replaced A. parasiticus in studies dealing with population and genetic diversity, pathogen-host interactions, and biological control; however, A. parasiticus remains an important research subject because of its potential to co-infect crops such as corn and peanuts.Aflatoxin Bin known . Nearly asiticus . Every kasiticus . Since tA. flavus isolates and nine A. parasiticus strains have been made publicly available from NCBI (https://www.ncbi.nlm.nih.gov/genome). These genome assemblies, along with those from closely related aspergilli such as A. oryzae and A. sojae, are invaluable resources for comparative genomics studies involving the investigation of chromosomal structure, evolutionary relationship, and genetic variations in relation to aflatoxin-producing capabilities of aspergilli [Rapid advancements in massive parallel sequencing in the genomic era have accelerated resolution of genome information. Genome sequences of over 100 pergilli –8.A. parasiticus, necessitating a closer examination to (1) uncover the misidentified strains, (2) reveal their correct species identities, and (3) notify NCBI so that researchers would not risk erroneous conclusions from genomics studies involving these misidentified strains.In the course of analyzing genome sequences deposited in the NCBI genome database, discrepancies were revealed for identities of some of the strains designated as A. parasiticus and A. flavus were retrieved from the NCBI genome database (https://www.ncbi.nlm.nih.gov/genome). Table A. parasiticus and A. flavus strains along with their genome information used in the study. The program progressiveMauve of Mauve (http://darlinglab.org/mauve/mauve.html), a system for multiple genome alignments [Genome sequences of ignments , was usehttps://genomevolution.org/coge/), an online platform for retrieval and comparison of genomic information. For the determination of the norB-cypA deletion patterns in the aflatoxin gene cluster [A. parasiticus SU-1, and A. flavus NRRL3357 (L-morphotype) and AF12 (S-morphotype) were used as alignment templates for Aspergillus genome sequences. For the determination of aflatoxin and cyclopiazonic acid (CPA) gene clusters, respective accessioned gene clusters from A. flavus AF36 (GenBank Accession numbers: AY510455 and JN712209) were used in sequence alignment.Analyses of genetic features of genome sequences were performed via CoGe  used for an Aspergillus whole-genus sequencing project [A. parasiticus strains range from 30.0 to 41.5 Mb, and most fall within a range of 38.0–40.0 Mb. The extraordinarily small size of strain 68–5 (> 20% less than others) likely resulted from poor library construction and/or an inadequate sequencing read coverage. Total SNPs among Ethiopian isolates, with the exception of E1365, ranged from 227,929 to 303,881 . For 18 A. fumigants strains, there was a wide range of total SNPs observed although less than 170,000  also has been reported [A. flavus S- and L-morphotype isolates except NRRL3357  isolates in comparison had total SNPs twice that count . Strikingly, the Ethiopian and American isolates shared total SNPs counts three times that of the Ugandan and Argentinian isolates. Whether the observed evolutionary distance in terms of total SNPs resulted from geographic separation and niche adaptation, or the Ethiopian and American isolates are not A. parasiticus but very closely-related aspergilli, is not clear. With regard to NRRL2999 and the genome sequence associated with it, the source of error, whether it was due to an erroneously provided stock culture, or a mix-up of sequencing samples of Aspergillus isolates, or a mislabeling of sequence read datasets, cannot be pinpointed.Ugandan and Argentinian isolates of Additional file 1: Fig. S1. Total SNPs from paired genome sequence comparisons among 18 A. fumigatus isolates. Fig. S2. Schematic representation of the norB-cypA region in the aflatoxin gene cluster of A. parasiticus and A. flavus. Arrows indicate direction of gene transcription. Dashed lines indicate deleted sequences. Deletion patterns, type I and type II, correspond to A. flavus S- and L-morphotype isolates. Fig. S3. CoGeBlast of the CPA gene cluster sequence of A. flavus AF36 against genome sequences of E1365, NRRL2999, and SU-1.