Mycobacterium tuberculosis. Macrophages engulf the mycobacterial bacilli through bacterial cell surface proteins or secrete proteins, which activate the complement pathway. The classical pathway is activated by C1q, which binds to antibody antigen complexes. While the alternative pathway is constitutively active and regulated by properdin, the direct interaction of properdin is capable of complement activation. The lectin-binding pathway is activated in response to bacterial cell surface carbohydrates such as mannose, fucose, and N-acetyl-d-glucosamine. All three pathways contribute to mounting an immune response for the clearance of mycobacteria. However, the bacilli can reside, persist, and evade clearance by the immune system once inside the macrophages using a number of mechanisms. The immune system can compartmentalise the infection into a granulomatous structure, which contains heterogenous sub-populations of M. tuberculosis. The granuloma consists of many types of immune cells, which aim to clear and contain the infection whilst sacrificing the affected host tissue. The full extent of the involvement of the complement system during infection with M. tuberculosis is not fully understood. Therefore, we reviewed the available literature on M. tuberculosis and other mycobacterial literature to understand the contribution of the complement system during infection.The complement system orchestrates a multi-faceted immune response to the invading pathogen,  Mycobacterium tuberculosis was first discovered by the German physician and microbiologist Robert Koch in 1882, who subsequently won the Physiology or Medicine Nobel prize in 1905 (nobelprize.org). The M. tuberculosis cells are acid-fast rods and obligate aerobes with a doubling time of 22 h [M. tuberculosis is the causative agent of the chronic disease, tuberculosis, which caused 10 million new cases and 1.5 million deaths in 2018 [M. tuberculosis was classified as one of the top ten causes of deaths worldwide in 2016, and importantly, one-quarter of the worlds’ population is infected with latent tuberculosis [rculosis . The comM. tuberculosis can be separated into early and late infection. The early acute phase begins with the inhalation of infected airborne droplets from an infected patient. The bacilli enter the alveolar space and are phagocytosed by the abundant resident macrophages, which are favoured over any other cell type [M. tuberculosis in vitro [M. tuberculosis through bacterial cell surface proteins or through secreted proteins that activate the complement pathway [M. tuberculosis for recognition by macrophages through the complement receptor [M. tuberculosis cell surface ligands, lipoarabinomannan (LAM), via the mannose receptors; however, this scenario has been reported for virulent strains of M. tuberculosis [The agreed paradigm of infection with ell type . Type IIin vitro . These m pathway . The comreceptor . The absrculosis . The bacrculosis .M. tuberculosis in the macrophages and is referred to as the latent infection; factors that contribute to this can be host and pathogen derived, for example, the virulency of M. tuberculosis and host immune status. The failure of macrophages to eliminate M. tuberculosis infection results in the recruitment of monocytes, which differentiate into macrophages. This causes the further phagocytosis of M. tuberculosis in a passive manner, and the cycle continues, causing the logarithmic growth of tuberculosis infection within the macrophages. The production of chemokines recruits’ lymphocytes and neutrophils to the site of infection, which are not capable of clearing infection but increase the inflammatory effects. Eventually, macrophage-derived giant cells and lymphocytes cause the development of a caseous granuloma to compartmentalisation of the infection; however, the exact factors that drive granulomatous formation are not fully understood [M. tuberculosis antigen presentation and the development of anti-mycobacterial T cell responses after a couple of weeks of infection [M. tuberculosis [The chronic phase of infection involves the survival of derstood . The bacderstood ,10. Tissnfection . The T crculosis .M. tuberculosis infection is poorly understood. There are a handful of publications that show complement pathway activation during challenge with M. tuberculosis; however, there is very little understanding of the exact involvement of the complement cascade and its contribution to tuberculosis disease progression. Therefore, this review focuses on the evidence for the role of the complement system during M. tuberculosis infection.The role of the complement system in M. tuberculosis. Most individuals received the Bacillus Calmette-Buérin (BCG) vaccination in the UK up until 2005, so studies in the model organism, Mycobacterium bovis BCG, have shown C1q binding in the presence of Immunoglobulin G (IgG) and immune globulin M (IgM) in serum samples [M. tuberculosis and showed that incubating nonimmune human serum for less than 5 min with M. tuberculosis resulted in the cleavage of C3 to C3b, C3bi, and other fragments [M. tuberculosis [Mycobacterium kansasi showed evidence for CR3 binding [WT and CR3−/− mice upon infection with M. tuberculosis, nor was there a difference of bacterial burden in the liver, spleen, and lungs, which was confirmed histologically [Mycobacterium avium in CR3−/− mice and have shown that there is no difference in bacterial burden, susceptibility to M. avium, and granulomatous response compared to the control mice [M. avium in bone marrow-derived macrophages (BMDM) in CR3−/− mice [M. avium was sufficient for activation of the classical complement pathway independent of antibodies in vitro; the levels of C3 cleavage were measured by incubation of C1, C2, C3, and C4 with M. avium [C1q activates the classical pathway, which is the first subcomponent of the classical complement cascade. C1q is a hexameric molecule (460 kDa) with a characteristic tulip-like structure  that bin samples . The pre samples . C1q-def samples . Some ar samples . Additioragments . The antrculosis . Neutrop binding . Hu and ogically . Others Mycobacterium leprae [Patients with tuberculoid leprosy (TT) exhibit little bacilli in the skin lesions whilst lepromatous leprosy (LL) patients have greater numbers of bacilli in many skin lesions. Leprosy is associated with the development of pathologic immune reactions, reversal reaction (RR), or erythema nodosum leprosum (ENL). Transcriptomic analysis of PBMCs in RR and ENL has shown increased C1q activity, but the C1q levels in blood did not increase ,22. Thism leprae .M. tuberculosis antigen complexes. The lectin binding pathway is activated by the recognition of complex sugar moieties on the M. tuberculosis cell surface. The alternative pathway is constituently active at basal levels, and unlike the other pathways, it does not require activation. The alternative pathway is regulated by properdin and factor H. All three complement pathways lead to the activation of C3 convertases, which cleaves C3 to C3b. C3b opsonises M. tuberculosis, which is ready for recognition by macrophages. The bacilli reside within the macrophage and experience a multitude of stresses induced by the host to clear the infection. This event develops into the characteristic granuloma. The C3 convertases also activate C5 convertases, resulting in the cleavage of C5 to C5a and C5b, leading to the formation of the MAC and ultimately, mycobacterial cell lysis.A simplified cartoon representing the involvement of the three complement pathways in tuberculosis infection: classical, lectin binding, and alternative. Briefly, the classical pathway is initiated by C1q, which recognises antibody—2+ and factor D. This causes the cleavage of factor B into Bb and Ba fragments, thus forming the C3bBb convertase. The C3bBb convertase produces the opsonin C3b, which is deposited alongside C3bBb convertase, resulting in the formation of C5 convertase, which produces C5a and C5b fragments and results in cell lysis when the membrane attack complex (C5-9) is inserted in a membrane [Unlike the classical and lectin complement pathways, which require stimuli for activation, the alternative pathway is constitutively active at basal levels, and it is ready to recognise invading pathogens rapidly. The alternative pathway and its contribution to the host response during tuberculosis infection is summarized in membrane ,26.Mycobacterium bovis BCG and thus preventing uptake of M. bovis BCG by THP-1 macrophages [M. tuberculosis infection. IL-12, IL-10, and tissue growth factor β (TGF-β) are downregulated by factor H, which suppresses the Th1 response and IFN-γ, thus dampening the anti-inflammatory cytokine response [Properdin is a regulator of the alternative pathway and composed of seven thrombospondin type 1 repeats (TSR1-TSR6). TSR4 is crucial for the stabilisation of C3bBb, and TSR5 contributes to the binding of C3b demonstrated through domain deletion studies . Properdrophages ,28. Addiresponse ,30.An important component of the innate immune system is collectins, which are key pattern-recognition molecules (PRMs) that bind to invading pathogens and contribute to neutralisation and clearance . The rolCollectins are also important in influencing the subsequent adaptive immune response against these microbial infections. Among the collectins are surfactant proteins A (SP-A) and D (SP-D), mannose-binding lectin (MBL), liver collectin (CL-L1), kidney collectin (CL-K1), CL-LK (composed of CL-L1 and Cl-K1), and placenta collectin (CL-P1) . AdditioStaphylococcus aureus, Streptococcus pyogenes, Listeria monocytogenes, and non-encapsulated Neisseria meningitidis, Escherichia coli, Haemophilus influenzae, Klebsiella spp, and mycobacteria species [Mycobacterium avium [M. tuberculosis, M. bovis, M. kansasii, Mycobacterium gordonae and Mycobacterium smegmatis [M. leprae LAM in mice contributes to the deposition of membrane attack complex (MAC), resulting in myelin loss and axonal damage [M. leprae infection [M. leprae was believed to release dead bacilli, and Mycobacterium. leprae LAM can sustain persistent complement activation, resulting in chronic inflammation [As discussed, MBL is a major component of the complement system’s lectin pathway and is mainly a serum protein. The structure of MBL is similar to C1q and SP-A and can target terminal sugars on the surface of bacteria in a calcium-dependent manner . SP-A an species ,38,39. Ium avium  and mannmegmatis . The perl damage . Conversnfection . Multi-dammation ,42.M. tuberculosis [Mycobacterium bovis BCG have demonstrated complement activation of all three pathways [Other mycobacterial ligands that can be targeted by MBL include the antigen 85 (Ag85) complex of rculosis . Experimpathways . For thepathways . Patientpathways . Interacpathways . Presumapathways . It is apathways .Staphylococcus aureus infection [Pseudomonas aeruginosa [M. tuberculosis and M. leprae [Deficiency in MBL is known to increase susceptibility to microbial infection, despite most individuals being apparently healthy ,46,47. Mnfection , whilst ruginosa . MBL defruginosa ,53,54. I. leprae ,56, whic. leprae ,58,59. I. leprae , showing. leprae . However. leprae .M. tuberculosis via ManLAM but not to M. smegmatis [M. tuberculosis infection, whilst CL-LK opsonised M. tuberculosis does not alter its phagocytosis or intracellular persistence in human macrophages [The serum collectin CL-LK (composed of CL-L1 and CL-K1) can also activate the lectin pathway by via MASP-1 and MASP-2 . The sermegmatis . Furtherrophages . Intrigurophages .M. bovis BCG in THP-1 macrophages [M. bovis BCG surface or by preventing an adaptive immune response [M. bovis BCG and conglutinin and thus indicated competitive inhibition by binding to carbohydrate surface motifs [M. bovis BCG with complement from human serum caused the inhibition of M. bovis BCG with conglutinin-mediated phagocytosis [Conglutinin is a type of collectin largely found in bovine serum and is secreted from the liver, binding to iC3b. Truncated conglutinin (rfBC) exhibited bacteriostatic activity in culture and showed an inhibition of phagocytosis of rophages . The inhresponse . Maltosee motifs . The incocytosis .M. tuberculosis via its putative adhesin Apa glycoprotein [M. tuberculosis and M. avium by macrophages via the increased expression of mannose receptor [M. bovis BCG via the specific 210-kDa SP-A receptor (SPR210) in U937 macrophages and rat alveolar macrophages [Lung surfactant proteins SP-A and SP-D can also both bind to Gram-negative and Gram-positive bacteria, aggregating, neutralising, inhibiting their growth, and acting as opsonins . SP-A haoprotein . SP-A, areceptor ,71,72, wrophages ,74, leadrophages .In humans, there are two isotypes of SP-A (SP-A1 and SP-A2) that are coded for by separate genes . SeveralOne of the hallmarks of a successful tuberculosis infection is the presence of granulomas, which have been described as solid host-protective structures. However, this idea has vastly changed as a result of the advancement in microscopic technologies that show granulomas to be dynamic structures that allow cells to move in and out . The graM. tuberculosis granuloma. Briefly, the polarisation of macrophages into M1 and M2 is balanced finely by the host to manage chronic infection. These groups of macrophages are distinct; M1 macrophages promote a pro-inflammatory response using glycolytic respiration, whilst the M2 macrophages promote an anti-inflammatory response. The inflammatory markers regulate the promotion and formation of the granuloma and determines disease outcome.This is an overly simplified cartoon of the complex +, CD11c, and CD40; however, they are poor antigen-processing cells [M. tuberculosis through aerobic glycolysis and the induction of genes encoding lipid transporters and lipolytic enzymes [M. tuberculosis towards the host lipid reservoirs [Foamy macrophages are distinct to non-foamy macrophages as they express higher levels of MHCII, CD11bng cells . Infecte enzymes ,84,85. Lservoirs . FinallyOther aspects of the granuloma include necrotic areas also known as caseum, made up of neutrophils, natural killer (NK) cells, dendritic cells (DC), B and T cells. This structure is lined with epithelial cells, histocytes (mature monocytes), and lymphocytes, which play a key role in the granulomatous formation. The histiocytes are activated by T lymphocytes, which secrete IFN-γ, infected macrophages become necrosed (Ghon focus), and thus persistence of chronic infection occurs ,44,87. TM. tuberculosis have an effect on dampening the immune response and cause the increased survival of mycobacteria. Distinct types of macrophages, M1 and M2, can allow permissive or restrictive mycobacterial growth [M. tuberculosis infection by binding to the TNF receptor, which results in the activation of the nuclear factor kappa-light change enhancer of activated B cells  . M. tubeironment ,95,96. Mironment ,97,98,99piration ,100. M2 piration ,94. Blocpiration . Additiopiration . M. tubenditions . Each grnditions . InteresM. tuberculosis infection and limits its growth and spread, but conversely also is a place for the mycobacterium to persist as latent infection [M. tuberculosis-infected macrophages early on in the infection process, accelerating the recruitment of cells in the granuloma [M. bovis BCG was found to inhibit phagocytosis by macrophages but also boosted pro-inflammatory cytokines  whilst simultaneously decreasing anti-inflammatory cytokines  [M. bovis BCG [M. tuberculosis whilst promoting inflammatory conditions for the granuloma and limiting the spread of infection [M. tuberculosis and influence the inflammatory response, shaping the adaptive immunity . Indeed, Factor H expression is enhanced in monocytes by IFN-γ, and macrophages can locally produce complement proteins such as C1q, suggesting that they may reside within the granuloma [M. tuberculosis reactivation and growth, which is also advanced by CD4+ T cell activation [The maintenance of the granuloma in critical in the control of tuberculosis, and the early interactions of complement and other innate immune factors  may be essential in modulating the subsequent adaptive immune response and formation of the granuloma. The granuloma is a complex lesion made up of various immune cells that effectively cordons off nfection . Inflammranuloma . Recent ranuloma ,29. Factovis BCG . It has ranuloma . Howevertivation . Moreovetivation .M. tuberculosis growth [M. tuberculosis persistence [M. tuberculosis, facilitating pathogenesis and disease progression [M. tuberculosis, thus enhancing bacteria clearance in the early stages of infection and facilitating a protective response immune response in the form of a granuloma.The suppression of IL-10 and TGF-β may further promote mycobacterial clearance during the early stages of infection rather than promoting s growth . Indeed,sistence , and it sistence . Furthersistence , leadingsistence . The levgression . Thus, tM. marinum have shown that a virulence-associated RD1 locus plays a role in the macrophage internal environment for the development of granuloma formation [M. tuberculosis granulomas, which were subsequently dissected and cultured ex vivo; these granulomas displayed the same heterogeneity in immune cells and in immune response [Additionally, studies in zebrafish infected with ormation . RD1 locormation . Others response .M. tuberculosis granulomatous formation in mice [M. tuberculosis infected macrophages, and C5a is responsible for the transcription/translation of cytokines. Animal studies have demonstrated that A/J (C5 deficient) and C5aR−/− mice succumb to M. tuberculosis infection compared to C57BL/6 mice when treated with TDM from a failure to contain and localise the infection in granulomas [M. tuberculosis infection and enhanced the multiplication of M. tuberculosis within the macrophages [Trehalose 6,6′-dimycolate (TDM) is a glycolipid component of the mycobacterial cell wall and has been shown to mimic  in mice ,117,118.anulomas . The BMDrophages .−/− mice decreased M. tuberculosis dissemination into livers, reducing lung immunopathology and smaller granulomas [M. tuberculosis remains mostly unclear.Additionally, complement protein 7 (C7) inserts into the cell membrane and plays a vital role in the formation of the membrane attack complex, C5b-9, resulting in cell lysis . A studyanulomas . HoweverM. tuberculosis by myeloid cells via PRRs causes the activation of nucleotide-binding oligomerisation domain-like receptors (NLRs). NLR activation drives the assembly of an inflammasome consisting of oligomerised NLRs, adaptor apoptosis-associated speck-like protein containing a CARD, and caspase-1. Caspase-1 activation and cleavage enables the cleavage of IL-1β and IL-18 and pore-forming molecule gasdermin D (GSDMD). IL-1 β is released through GSDMD pores, which result in lytic cell death. M. tuberculosis infected THP-1 macrophages caused the formation of ASC specks and were only localised in dead cells, and IL-1 β secretion increased [+ efflux, NLRP3 activation, and subsequent caspase-1 mediated IL-1β release in THP-1 cells.The recognition of ncreased . The secM. tuberculosis infection can occur in non-myeloid cells. M. tuberculosis with functional type VII secretion systems and Phthiocerol dimycocerosates (PDIM) form intracellular cording phenotypes that have been reported within the human lymphatic endothelial cells (hLEC) [M. tuberculosis-infected hLEC gain access to the cytosol, whereby cording occurs. The bacilli prevent the activation of host-immune sensors, thus preventing xenophagy and the persistence of chronic infection [A subset of s (hLEC) . M. tubenfection .M. tuberculosis in hypoxic cultures and lung sections [M. tuberculosis contains a family of cell wall cleaving enzymes known as resuscitation promoting factors (Rpfs) involved in the resuscitation of differentially culturable bacteria [Anti-tuberculosis drugs have been shown to be less effective in vitro against non-replicating mycobacteria generated under hypoxic conditions . An imposections ,124. Thesections . Culturebacteria ,127. Togbacteria .The anti-tuberculosis drugs currently available for the treatment of tuberculosis not only render the bacilli defective through unique mechanisms, they also have some effect on the immune response. This section focuses on the interplay between the anti-tuberculosis drugs and the immune response.M. tuberculosis by binding to the β subunit of DNA-dependent RNA polymerase; resistance is associated with the rpoB gene. This drug has been shown to increase CD1b expression, which is found on cytokine-activated macrophages, thus boosting the T-cell response to M. tuberculosis [2 (PGE2) is involved in the activation of antigen presenting cells, regulation of T- and B- cell responses, and chronic inflammation. PGE2 production was inhibited by RIF in human alveolar cells [Rifampicin (RIF) is one of the most potent anti-tuberculosis drug and was introduced into the anti-tuberculosis drug regimen in 1968. RIF inhibits rculosis . Additiorculosis . Prostagar cells . RIF inhar cells . RIF andM. tuberculosis catalase-peroxidase KatG, which produces free radicals and in turn inhibits mycolic acid synthesis [M. tuberculosis to clear the bacteria; however, M. tuberculosis is highly resistant to ROS/NOS, and thus the phagocytic cells undergo necrosis. A study has shown INH can inhibit the oxidative stress-induced necrosis of phagocytic cells; however, this is not fully understood [+ T cells, autophagy, and phagosomal maturation in response to M. tuberculosis [Isoniazid (INH) was approved by the FDA in 1952 for treatment against tuberculosis. INH is oxidised by the ynthesis ,134. Phaderstood . Additiorculosis ,137.pncA and is only activate against bacilli in an acidic pH or inside macrophages [M. tuberculosis infected monocytes [Pyrazinamide (PZA) has been recognised as an anti-tuberculosis drug since 1952. PZA is converted to pyrazinoic acid by nicotinamidase encoded by rophages . This dronocytes .M. tuberculosis [Bedaquiline (BDQ) is a recent FDA-approved drug used for the treatment of multi-drug resistant tuberculosis (MDR-TB). This drug inactivates bacterial ATP synthase and thus depletes the pathogen of ATP . BDQ reprculosis ,142.M. tuberculosis (XDR-MTB) has shown interest in CFZ once again [Clofazimine (CFZ) demonstrated anti-tuberculosis action in the 1950s; however, extreme side effects were reported—for example, skin discolouration and mental disturbances. Thus, its use was halted. However, in the early 1960s, CFZ was approved again by WHO for the treatment of drug-resistant M. leprosy. Additionally, the emergence of extremely drug-resistant ce again . CFZ is ce again . The potce again .M. tuberculosis research field has mainly focussed on the impact of complement activation on the actively growing mycobacterial populations; very little is known about the dormant, non-replicating, and resuscitation of heterogenous sub-populations of mycobacteria that exist within granulomas.The M. tuberculosis infection, we need to continue advancing in our knowledge of the infection and immune response to M. tuberculosis. To date, we carry out macrophage infections in vitro using M. tuberculosis but do not understand fundamental questions. Why are macrophages not infected equally whilst using a homogenous M. tuberculosis culture and immortalised cell lines? Why does multiplicity of infection need to be tightly controlled in vitro, and why do a large number of bacteria overwhelm the macrophage if there are only a limited number of receptors for phagocytosis?To understand the complement cascade in M. tuberculosis infection; although poorly understand, this review sheds light on the complexity of tuberculosis infection.All three complement pathways together play a role in the pathogenesis of


Solid pseudopapillary tumors (SPTs) constitute 0.2 to 2.7% of non-endocrine primary tumors of the pancreas and comprise the majority (70%) of pediatric pancreatic neoplasms. These tumors are of unclear pathogenesis, low malignancy and favorable prognosis. Surgical resection offers an excellent chance for longterm survival, even in the presence of distant metastasis. The objective of this study is to review our experience in the management of SPT in a 12 years old girl at the pediatric hospital of the University hospital of Casablanca, in Morocco and provide an update on current management in pediatric population. Solid pseudopapillary tumors (SPTs) are rare neoplasms accounting for 2% to 3% of pancreatic tumors and 0.9% to 2.7% of exocrine pancreatic neoplasms . The firWe present our experience in the management of SPT in a 12 years old girl with no past medical history. The story of her illness began 2 months previously with abdominal pain without other associated signs. Abdominal examination revealed a palpable mass in the left hypochondrium. Ultrasound demonstrated a heterogeneous mass, having a contact with the tail of the pancreas and the spleen . On compPathologic results, combined with immunohistochemical study were consistent with the diagnosis of SPT , Table 1Epidemiology: SPTs comprise the majority of pediatric pancreatic neoplasms [eoplasms . It is aeoplasms ,7. The feoplasms . About 2eoplasms . The oriClinical features: Lee et al. demonstrated that SPTs in children presented with a palpable mass (60%), followed by abdominal pain (33.3%) [ (33.3%) . SPTs ar (33.3%) .Biologic features: pancreatic enzymes and common serum tumor markers  are consistently negative, as was seen in our patient [ patient ,5. There patient .Imaging features: SPTs may appear in any part of the pancreas [pancreas . Tumor lpancreas . SPTs arpancreas . Ultrasopancreas . The fibpancreas . The mospancreas . It helppancreas ,10. CT dpancreas . A delaypancreas . Enhancepancreas .et al. reported that solid pseudopapillary carcinoma may be differentiated from benign SPTs by its aggressive behavior, such as pancreatic duct dilation and vessel invasion, either with or without metastases. If such imaging features are present, aggressive surgical approach is mandatory and intensive follow-up is recommended [Fluid-debris levels are seen in up to 20% of tumors. Less than one-third demonstrate internal septations. Duct dilatation is uncommon even with large masses in the head of the pancreas because SPTs often compress adjacent structures rather than invading them . Solid pommended . Preoperommended ,8,9.Pathology: SPTs are a rare epithelial solid tumor of the pancreas that invariably develop significant cystic degeneration, acquiring the characteristic solid-cystic appearance.Gross appearance: macroscopically, SPTs are usually large, well circumscribed and have mixed solid whitish brown areas with foci of necrosis and hemorrhage and cystic areas containing necrotic fragments. As they increase in size, the areas of cystic-hemorrhagic degeneration grow until they get simular to a pseudocyst [eudocyst ,9.Histologic appearance: histologically, SPTs have a very characteristic appearance of solid areas composed with of poorly cohesive cells that form pseudopapillae around thin blood vessels. These polygonal epithelioid cells have medium-size, soft appearance, basophilic cytoplasms, large rounded or ovoid nuclei, with reinforced nuclear membrane and thin chromatin [hromatin ,9. FoamiImmunohistochemical: immunohistochemical study is very useful, revealing a diffuse positivity for certain histochemical markers like neuron-specific enolase (NSE), vimentin, CD-10 and béta-catenin, which is not specific [specific . α1-antispecific . Immunosspecific . Notoharspecific . Cells aspecific . Accordispecific . To datespecific .Treatment and prognosis: the current recommendations concur that children with pancreatic SPTs should undergo complete surgical resection that is dictated by tumor location and remains the treatment of choice [f choice . Distal f choice ,9. Invasf choice . Chemothf choice . Metastaf choice . There if choice . Althougf choice .Differential diagnosis: despite the technological advances, preoperative diagnosis is difficult because of the similarity of findings among other pancreatic tumors [c tumors . In chilc tumors ,6. The ic tumors .SPTs constitute the majority (70%) of pediatric pancreatic neoplasms. It is a rare exocrine pancreatic tumor that typically have the form of a well-encapsulated mass with solid and cystic components due to varying degrees of internal hemorrhage and necrosis. These tumors are of unclear pathogenesis, low malignancy and surgical resection offers an excellent chance for longterm survival, even in the presence of distant metastasis. Although surgical resection is generally curative, a close follow-up is advised in order to diagnose a local recurrence or distant metastasis and choose the proper therapeutic option for the patient.The authors declare no competing interests.


TaHsfs) were identified in the whole wheat genome and they are unequally distributed on 21 chromosomes. According to gene structure and phylogenetic analyses, the 61 TaHsfs were classified into three categories and 12 subclasses. Genome-wide duplication was identified as the main source of the expansion of the wheat HSF gene family based on 14 pairs of homeologous triplets, whereas only a very small number of TaHsfs were derived by segmental duplication and tandem duplication. Heat shock protein 90 (HSP90), HSP70, and another class of chaperone protein called htpG were identified as proteins that interact with wheat HSFs. RNA-seq analysis indicated that TaHsfs have obvious period- and tissue-specific expression patterns, and the TaHsfs in classes A and B respond to heat shock, whereas the C class TaHsfs are involved in drought regulation. qRT-PCR identified three TaHsfA2bs with differential expression in sterile and fertile anthers, and they may be candidate genes involved in anther development. This comprehensive analysis provides novel insights into TaHsfs, and it will be useful for understanding the mechanism of plant fertility conversion.Heat shock transcription factors (HSFs) play crucial roles in resisting heat stress and regulating plant development. Recently, HSFs have been shown to play roles in anther development. Thus, investigating the HSF family members and identifying their protective roles in anthers are essential for the further development of male sterile wheat breeding. In the present study, 61 wheat HSF genes ( Hsp genes as molecular chaperones to help refold, assemble, distribute, and degrade related proteins, as well as repairing damaged proteins and maintaining cell survival (527 Mb) ,53, but e et al.  and Duane et al. . Using te et al.   . These fC1.html)  and Duane et al. . The maiTaHsfs, we used the wheat HSF sequence in the plantTFDB database (http://planttfdb.cbi.pku.edu.cn/family.php?sp=Tae&fam=HSF) and NCBI (https://www.ncbi.nlm.nih.gov/) as query sequences to search for all of the wheat protein sequences. However, after verification based on Pfam (http://pfam.xfam.org/) [http://smart.embl.de/), Conserved Domain Database (CDD) (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi) [https://applbio.biologie.uni-frankfurt.de/hsf/heatster/analyse.php#menu) [TaHsfs. The gene family member identification strategy presented in this study will greatly facilitate the identification of HSF family in other species.In the present study, in addition to employing two approaches described in the method section for identifying am.org/) , SMART (psb.cgi) , MARCOILhp#menu) , the finTaHsfs are typical dose-sensitive genes because they regulate the expression of HSPs by encoding transcription factors. Thus, during the long process of evolution, wheat has retained 14 groups of complete homeologous triplet HSF gene family members in the ABD sub-genomes, and only a very small number of genes were derived by tandem duplication. This balanced state maintains the metabolic stability of the organism and it is also required for the fine regulation of mechanisms in plants. A gene family is a group of genes derived from the same ancestor, where two or more copies of the gene may be present. Whole genome duplication is a major driver of gene family expansion to increase the number of gene family members in a single species . Genes wTaHsf family and found that the 61 TaHsfs contained 1–3 exons and 0–4 introns. The lengths and positions of the TaHsf gene introns in the same subclass were relatively well conserved, but the introns varied greatly among the different TaHsf subclasses. The presence of introns allows a variety of splicing modes and the same DNA sequence can produce different protein products via alternative splicing of the gene after transcription [TaHsfs to temperature [The evolution of gene families is often accompanied by increases or losses of exons, which have played key roles in the evolution of gene families . Thus, wcription . This isperature . s are expressed in male reproductive cells during heat stress. In particular, AtHsfA2 (AT2G26150) and AtHsfA5 (AT4G13980) were identified as critical during pollen reproduction in Arabidopsis [Hsfs and three class B Hsfs are up-regulated in rice spikes [HsfA2 and HsfA3 were detected during tomato pollen development, and other Hsfs were also studied in the anthers [Hsfs in anther development are not yet known but their downstream regulated HSPs are important molecular chaperones involved in the development of male gametophytes in plants [Hsp70 antisense expression vector driven by the anther-specific promoter Osg6B was constructed to inhibit Hsp70 expression during pollen development and block male gamete development, thereby resulting in male sterility [HsfA2 at 42 °C and 36 °C in tomato microspores, respectively, and we demonstrated the high expression level of TaHsfA2 in fertile anthers cultured at 22 °C. These studies were based on heat treatment, but we hypothesize that increased temperature can induce the upregulation of HSFs to regulate the expression of downstream HSPs in the cytoplasm and increase the number of mitochondria to ensure the energy supply is adequate for pollen development. Thus, the findings obtained in the present study indicate likely roles of wheat HSF family members in anther development, and provide a new theoretical basis for exploring the mechanism responsible for male sterility in wheat. In plants, transcription factor HSFbidopsis ,68. Fivee spikes . In addi anthers ,41,50. Tn plants ,70,71. Cterility . Frank eterility  demonstrKTM3315A is a type of thermo-sensitive male sterile wheat with various advantages and it was selected by our research group in 2001. It is completely male sterile during the normal wheat-growing season, whereas its fertility can be restored in a high-temperature environment . Ten pothttps://wheat-urgi.versailles.inra.fr/Seq-Repository/Annotations) [http://pfam.xfam.org/family/PF00447) [−20. The results obtained were used to construct a wheat-specific HSF HMM profile by hmmbuild program, before searching the wheat reference genome again with an E-value threshold of 1 × 10−3. In order to avoid missing other HSF members, the HSF protein sequences for rice and Arabidopsis were downloaded from phytozome  and used as queries to BLAST the wheat reference genome, where the E-value threshold was 1 × 10−10. The results obtained were identified as wheat candidate genes and verified by online software CD-search, SMART, and pfam for presenece of protein domain. Coiled-coil structures were detected using the MARCOIL program. Protein sequences that lacked the DBD domain or coiled-coil structure were removed. All high confidence level HSF protein sequences were submitted to Expasy  to calculate the number of amino acids, molecular weight, and theoretical isoelectric point. NetNES (http://www.cbs.dtu.dk/services/NetNES/), cNLS Mapper (http://nls-mapper.iab.keio.ac.jp/cgi-bin/NLS_Mapper_form.cgi#opennewwindow), and HEATSTER were used to search for the NESs and NLSs in the TaHsfs [TaHsfs was submitted to the online site MapGene2 Chrom web V2 (http://mg2c.iask.in/mg2c_v2.0/) to map their chromosomal locations.The latest wheat reference genome and the HMM profile PF00447) of the HSF family were downloaded from the wheat genome database IWGSC  . The HSFPF00447)  and relie TaHsfs ,74,75. TArabidopsis were used for evolutionary analysis with the full-length amino acid sequences of the TaHsfs [The full-length amino acid sequences of the HSFs in rice and e TaHsfs . All of TaHsfs using McscanX software [Arabidopsis, the genome-wide CDS sequences and genome annotation information for Chinese cabbage (http://brassicadb.org/brad/datasets/pub/Genomes/Brassica_rapa/V1.0/Scaffold1.0/), sorghum (http://www.plantgdb.org/BdGDB/), maize (http://interface.maizegdb.org/gbrowse/maize_v4), and Brachypodium  were obtained, and the synteny relationships between the HSF family member in wheat and these species were determined using Mcscanx.Segmental and tandem duplications were determined for the software . Sequencsoftware . A segmesoftware . In addiTaHsfs were visualized using the online drawing tool Gene Structure Display Server 2.0  [TaHsfs were visualized using TB tools [TaHsfs and their interacting genes, which were visualized with Cytoscape (https://cytoscape.org/) [ClustalW was used to align the conserved DBD and HR-A/B domains in the TaHsfs. The gene structures of the edu.cn/) . The MEMedu.cn/)  obtainedTB tools . STRING pe.org/) ,83,84.http://www.wheat-expression.com/) to study the expression patterns of the TaHsfs. The RNA-seq data were derived from nine different growth periods and tissues in Chinese Spring under normal conditions  [TaHsfs in thermo-sensitive male sterile wheat, transcriptome data were analyzed from temperature-sensitive materials under different fertility conditions  [http://www.omicshare.com/tools/index.php/Home/help/index/html/combinemore).Part of the wheat transcriptome data was obtained from the Wheat Expression Browser powered by expVIP (e stage) , as welle stage) . In addiand AS3) . The hea–ΔΔCt method [TaHsfs sequences , respectively, according to the manufacturers’ instructions. qRT-PCR was conducted with an Applied Biosystems 7500 Real-Time PCR System and 2×RealStar Green Fast Mixture with ROX II according to the operating instructions and program default settings with three replicates per group. ROX II was added to correct fluorescence signal errors between wells during qRT-PCR reactions. The wheat Actin gene was used as an internal reference, the final relative quantitative results were calculated using the 2t method . At leasequences .


Rhincalanus gigas, one of the predominant zooplankton species of Antarctic waters. Sequence analyses identified possible adaptation strategies adopted by the organism to cope with cold environments. Among those, we identified in R. gigas transcriptome three predicted genes encoding for antifreeze proteins and gene duplication within the glutathione metabolism pathway. This new molecular resource, provided here, will be useful to study the physiology, ecology, and biology of R. gigas and it increases the information available for polar environments.Compared to more accessible sites, organisms inhabiting Antarctic waters have been poorly investigated. This study provides the first molecular resource  for the eucalanoid copepod Rhincalanus gigas, one of the predominant zooplankton species of Antarctic waters. This transcriptome represents also the first molecular resource for an eucalanoid copepod. The transcriptome is of high quality and completeness. The presence of three predicted genes encoding antifreeze proteins and gene duplication within the glutathione metabolism pathway are suggested as possible adaptations to cope with this harsh environment. The R. gigas transcriptome represents a powerful new resource for investigating the molecular basis associated with polar biological processes and ecology.Antarctic waters are the largest almost untapped diversified resource of our planet. Molecular resources for Antarctic organisms are very limited and mostly represented by sequences used for species genotyping. In this study, we present the first transcriptome for the copepod  Polar regions represent the largest almost untapped diversified resource of our planet. Antarctic waters can be considered an extreme environment where, in order to survive, organisms have evolved adaptations to cope with seasonal changes in day length, food availability, and ice cover, as well as to consistently cold waters .Tigriopus kingsejongensis [Euphausia superba) (KrillDB) [Limacina helicina [In the Southern Ocean, zooplankton communities are dominated by copepods in terms of numbers and biomass . These tongensis , (2) theKrillDB)  and (3) helicina .Rhincalanus gigas  is a dominant species in Antarctic and sub-Antarctic circumpolar waters [R. gigas is an epipelagic copepod, predominantly feeding on small-particle phytoplankton, mainly diatoms, and protozoans [R. gigas is still unclear. The copepod seems to have a flexible two year cycle depending on conditions, and it is not clear if, during winter, the copepod enters a period of developmental arrest (diapause) [R. gigas as the most abundant representative zooplankton taxon [R. gigas adult individuals. This reference transcriptome is also the first molecular resource for an eucalanoid copepod. The R. gigas transcriptome is a new source that can provide the basis for gene discovery or for gene expression studies to investigate changes in response to environmental stressors and to identify the molecular mechanisms of environmental adaptation to extreme environments.r waters . This cor waters ,7, with  Brady, 183 is a dr waters . R. gigaotozoans ,11. The iapause) ,9,12. Reon taxon . Thus, bThe collection of marine samples from the South Shetland Trough was approved by the Foreign and Commonwealth office. The permit (No. 51/2015 and 52/2015) was granted under Section 3, Section 6, and Section 7 of the Antarctic Act 1994 to Alan J. Jamieson (the permit holder), expedition Chief, on behalf of the University of Aberdeen. The main purpose of the permit was to collect the marine organisms from deep and cold habitats with the goal to survey the zooplankton composition of the South Shetland Trough and to compare and contrast the fauna with other trench ecosystems.Rhincalanus gigas individuals were collected on 21 and 22 December 2015, from two stations of the South Shetland Trench (SST)  during the “PharmaDEEP” cruise  [R. gigas were live sorted; six individuals were immediately preserved in RNAlater Stabilization Reagent .esults/) . ZooplanR. gigas adults using Trizol according to the manufacturer’s protocol . DNA quantity was assessed by Nano-Drop . The mitochondrial cytochrome oxidase subunit I region (COI) (fragment 518 bp) was amplified using the following primers: COI-F (5′–GGCCAAAACAGGGAGAGATA–3′) and COI-R (5′–CGGGACTCAGTATAATTATTCGTCTA–3′) [TM Gel Extraction Kit , and the fragments were sequenced. Sequence reactions were obtained by using the BigDye Terminator Cycle Sequencing technology  in automation, using the Agencourt CleanSEQ Dye Terminator Removal Kit  and a Robotic Station Biomek FX . Products were analyzed on an Automated Capillary Electrophoresis Sequencer 3730 DNA Analyzer . To confirm their identity, sequences were BLASTed  against the National Center for Biotechnology Information (NCBI) nucleotide database (nr/nt) by using the BLASTn algorithm.Total DNA was extracted from individual TCTA–3′) . ReactioR. gigas individuals using Trizol according to the manufacturer’s protocol (Invitrogen). Each individual was transferred in 500 µL of Trizol (Invitrogen) and was homogenized using the Tissuelyser  and tungsten carbide beads (3 mm)  for 5 min  at 20.1 Hz until it was completely homogenized [Total RNA was extracted from six ogenized . After cogenized ). After R. gigas individuals, confirmed from the genotyping analysis (mtCOI 98.8% identity), were processed for RNA-Seq. Total RNA samples were shipped on dry ice to the Genomix4life S.R.L. . There, using the TruSeq Stranded mRNA Sample Prep Kit , complementary DNA (cDNA) libraries were prepared with 500 ng/each of purified RNA. Libraries were quantified using the TapeStatiFon 4200 (Agilent Technologies) and Invitrogen Qubit fluorometer (Thermo Fisher Scientific), and then pooled so that each index-tagged sample was present in equimolar amounts, with a final concentration of the pooled samples of 2 nM. Each sample was indexed and paired-end sequenced (PE 75 bp) using an Illumina NextSeq 500 System (Illumina) on a single lane.Two https://www.bioinformatics.babraham.ac.uk/index.html). From each library, Illumina adapters , the first 12 bp from each read, reads < 50 bp long, and reads with an average Phred score < 30 were removed using Trimmomatic (v.2.0.0) [The quality of the two RNA-Seq libraries was assessed using FASTQC  . From eav.2.0.0)  at the Nv.2.0.0) . Additiov.2.0.0)  was usedRaw reads were submitted to the National Center of Biotechnology Information (NCBI) to the Sequence Read Archive under the Bioproject PRJNA639356.https://github.com/TransDecoder/TransDecoder/releases) (default settings) was used to identify protein coding sequences. These predicted proteins were functionally annotated using InterProScan software (v5.33) [−3 (https://www.uniprot.org/ updated at April 2020). The assembly and annotation statistics of the R. gigas transcriptome were compared with those from other calanoid copepods. Transcriptomes for Calanus finmarchicus [Neocalanus flemingeri [Labidocera madurae [TransDecoder software (v5.3.0) ( (v5.33)  (Supplem (v5.33)  using BL (v5.33)  with a marchicus , Neocalaemingeri , and Lab madurae  were selDrosophila melanogaster was used as a reference organism to identify the “key” enzymes in the glutathione metabolism pathway (map00480). The D. melanogaster “expected” enzymes were searched based on their annotation (Enzyme commission number) in the R. gigas annotated transcriptome. Similarly, the R. gigas reference transcriptome was also searched for antifreeze proteins (AFP). The identified R. gigas transcript encoding AFP proteins were then checked for the presence of the characteristic C-type lectin (CTL) or carbohydrate-recognition (CRD) (CLECT) domains using SMART program [Stephos longipes (NCBI Accession no. ACL00837 and ACL00838) [R. gigas homologs.On the basis of functional hypotheses of genes and pathways involved in cold adaptation, we investigated the glutathione metabolism pathway and antifreeze proteins. From the Kyoto Encyclopedia of Genes and Genomes (KEGG)web-accessible resource , the fru program  and aligCL00838) . HoweverR. gigas. For both individuals, mtCOI sequences were 98.8% identical (nucleotide) to the deposited R. gigas mtCOI sequence on NCBI (GenBank Acc. n. JN663356.1).Two of the six genotyped individuals were confirmed as R. gigas de novo assembly, generated by combining two RNA-seq libraries, resulted in 78,285 transcripts with an average length of 877 bp, a maximum of 10,033 bp, and an N50 value of 1143 bp  had coding regions, with 55% of these being successfully functionally annotated by scanning the SwissProt database. Over 39% of these transcripts also had assigned Gene Ontology (GO) terms, and ca. 49% of them were annotated among protein families (PFAM database) . TranscrR. gigas transcriptome is of at least similar quality and depth to the other calanoid references  with all enzymes being full-length  reduction (N00905) , 6-phosphogluconate dehydrogenase (EC 1.1.1.44), and glucose-6-phosphate 1-dehydrogenase (EC 1.1.1.49). Two enzymes (EC 1.1.1.42 and EC 1.1.1.44) were represented by two R. gigas predicted genes, while the 6-phosphogluconate dehydrogenase enzyme (EC 1.1.1.44) was represented by three predicted genes.According to the KEGG hierarchy, the pathway consisted of three networks: (1) Glutathione biosynthesis (N00899), (2) glutathione reduction (N00904), and (3) nicotinamide-adenine-dinucleotide phosphate (NADP(N00905) . The glu(N00905) . One of (N00905) . The sec protein . The lasR. gigas transcriptome  were identified in the criptome . All pres mordax   is a cryotolerant strain, while S. cerevisiae (96.2) is a thermotolerant strain [Tigriopus kingsejongensis (Antarctic) and T. japonicus, significant functional divergence of genes associated with energy metabolism, purine/pyrimidine metabolism, pentose and glucoronate interconversions, glycerolphospholipid metabolism, and oxidative phosphorylation pathways was reported [R. gigas transcriptome, several metabolic pathways such as purine/pyrimidine metabolism were among the top ten represented pathways, consistent with what is known for other organisms adapted to cold environments.Organisms that have to cope with harsh conditions  require a specific and complex reprogramming at the transcriptional, proteomic, and metabolic levels which alter communication and signaling between subcellular organelles . Temperat strain . The autt strain . Recentlt strain  also repreported . EnrichmS-transferases (GSTs) [Dissostichus mawsoni, duplication of genes involved in the antioxidant process is considered as a possible strategy to compensate for the highest vulnerability to ROS production and oxidative damage , can alter cellular homeostasis by damaging DNA, RNA, proteins, lipids, and carbohydrates, which, in worst cases, can even induce organism death ,43. Gluts (GSTs) . It alsoiewed in ).R. gigas was the fourth represented pathway. The completeness of this pathway was demonstrated by the presence of all expected enzymes in D. melanogaster, mostly encoding for full-length proteins. Evidence of possible gene duplication for several enzymes within this pathway was found, which is consistent with what has been reported for D. mawsoni. However, further investigations on gene multiplicity are required.The glutathione metabolism pathway in Stephos longipes [R. gigas possesses three predicted genes encoding antifreeze proteins, all similar to fish type II AFPs. Furthermore, the R. gigas AFPs are highly different from the sea ice copepod isoforms, confirming that these latter proteins had no homology with metazoans. This is the first evidence that AFPs are also present in copepods that do not live in ice. However, further investigations are needed in order to assess the activity of these R. gigas proteins.Antifreeze glycoproteins (AGFPs) are a class of proteins which are considered an evolutionary adaptation allowing organisms to survive at subzero temperatures ,47. AFPslongipes . Both islongipes . R. gigaRhincalanus gigas, one of the most abundant zooplankters of Antarctic waters. Our data show gene duplication within the glutathione metabolism pathway, as well as the presence of three predicted genes encoding antifreeze proteins suggesting, possible adaptation mechanisms to cope with this extreme environment. According to its high quality and depth, the R. gigas transcriptome is a powerful new resource for investigating the ecophysiology of this copepod within the context of life history strategies and ecosystem dynamics.In the last decade, the application of high-throughput sequencing has become more common for developing molecular resources for non-model species, including those of ecological relevance. Transcriptomics approaches used in gene discovery and gene expression studies can be used to investigate the biology and ecophysiology of key organisms. However, both types of studies depend on high-quality reference transcriptomes. In this study, we presented the first molecular resource for an eucalanoid copepod,


Vibrio cholerae, (p)ppGpp metabolism is regulated by the products of at least three genes relA, spoT, and relV. In this study, we identify and characterize the function of the guanosine-5′-triphosphate 3′-diphosphate pyrophosphatase A (GppA) encoding gene gppA of V. cholerae. Genomic analysis indicates that the gppA locus is conserved in vibrios and organized as a bicistronic operon along with the rhlB gene. We engineered the genome of V. cholerae to develop different mutants devoid of GppA and/or other phosphate metabolic enzymes. Our findings indicate that in V. cholerae, GppA plays an important role in the conversion of pppGpp to ppGpp during amino acid deprivation but not during glucose starvation. Quantitative analyses of the gppA transcript level reveal its differential expression pattern at different growth phases and starvation conditions. It has been observed that the GppA deficiency during amino acid starvation condition could be complemented by overexpressing the exopolyphosphatase coding gene ppx of V. cholerae. By deletion analysis, we further demonstrate that the amino and carboxy terminal sequences flanking the Ppx-GppA motif of the GppA protein of V. cholerae are also important for its enzymatic function.The stringent response, an adaptive response to nutrient limitation and exposure to xenobiotics in bacteria, is mediated by two intracellular signaling molecules, pppGpp and ppGpp, together called (p)ppGpp. The cellular level of (p)ppGpp in bacterial cells is controlled by the Rel/Spo family of proteins. In the cholera pathogen,  Vibrio cholerae faces various physicochemical stresses while living within or outside of the human intestine. Among multiple environmental stresses, nutritional stress is important for survival since it determines the growth and multiplication of the pathogen under such conditions. Like other bacterial pathogens, V. cholerae has evolved with complex gene regulatory networks to cope with various environmental stresses, among which the stringent response (SR) is important. The SR is typically characterized by strong repression of transcription of stable RNAs like transfer RNAs (tRNAs) and ribosomal RNAs (rRNAs), etc. ppGpp metabolism is controlled by two multidomain containing proteins RelA and SpoT, in V. cholerae, apart from these two enzymes, a small alarmone synthetase, called RelV, is also involved in (p)ppGpp metabolism as shown in relA gene, is a ribosome-associated protein and responsible for (p)ppGpp synthesis under amino acid starvation ppGpp hydrolase activity and weak (p)ppGpp synthetase activity under different stress conditions ppGpp synthesis under glucose and fatty acid starvations , is associated with the (p)ppGpp metabolic cycle , was identified in E. coli , and the genes encoding Ppk and Ppx are usually physically linked , etc. , upreguls), etc. , and inhs), etc. . Regulats), etc. . While iarvation . RelA isarvation . On the nditions . Interesrvations . Furtherrvations . In E. cic cycle . The mai E. coli . Ppx has coli ppGpp metabolism in V. cholerae ppGpp synthetase gene cholerae . Howevertabolism . In thischolerae . Like in.E. coli DH5α strain were used unless stated otherwise. Both E. coli and V. cholerae cells were routinely grown in Luria broth  at 37°C with aeration, and for plate culture, Luria agar  was used, which contained 1.5% (w/v) Bacto agar (Difco) as described earlier , 40 μg ml–1; streptomycin (Sm), 100 μg ml–1; chloramphenicol (Cm), 3 μg ml–1 for V. cholerae; and 30 μg ml–1 for E. coli. Bacterial strains were stored at −70°C in LB containing 20% (v/v) sterile glycerol (600) using a spectrophotometer .Bacterial strains and plasmids used in this study are described in  earlier . Antibioglycerol  and wereV. cholerae cells were made as described previously from this laboratory  cassette, was used. To construct pBADcam, the plasmid pPROTet.E  (∼1.6 kb) using the primers gppAorf-F/gppAorf-R  carrying plasmid pPpx - and/or carboxy (C)-terminal coding region deleted  primers  and geno primers  always c primers  always cV. cholerae cells under various starvation conditions was determined by radiolabeling of bacterial cells with [32P]-H3PO4  using 3-(N-morpholino) propanesulfonic acid (MOPS) medium with different supplements followed by lysis of cells and thin layer chromatography (TLC) essentially as described previously  was calculated by dividing the spot intensity of pppGpp with that of total intensity value of pppGpp + ppGpp.Intracellular accumulation of (p)ppGpp in eviously . For (p)eviously  followedV. cholerae cells were grown in LB at an OD600 value of ∼1.0 followed by extraction of total cellular RNA using TRI Reagent  essentially as described previously (Taq DNA polymerase (Invitrogen). Lack of amplification in the absence of RT confirmed that the desired PCR product was generated only from complementary DNA (cDNA). For quantitative RT-PCR (qRT-PCR) assay, total cellular RNA was prepared from bacterial cells grown in LB medium to an OD600 value of ∼0.5, 1.0, or 1.5 as mentioned above. Reactions were done using the One Step SYBR®PrimeScriptTM RT-PCR Kit II essentially as described by the manufacturer . qRT-PCR assay was done using the CFX96TM Real-Time System . The primer set gppArtm-F/gppArtm-R (R) were calculated using the equation R = 2–(ΔCTtarget–CTreference), where CT is the fractional threshold cycle. In each experiment, as an internal control, the recA-specific primers recA-F/recA-R ppGpp labeling experiment. The level of gppA transcripts was measured by qRT-PCR analysis. As an internal control, the gyrA-specific primers, gyrArtm-F/gyrArtm-R  assay, eviously . Purity ppArtm-R  was usedF/recA-R  were useyrArtm-R , were usyrArtm-R . As posiarvation . Each RT® Terminator v3.1 Cycle Sequencing Kit  essentially as instructed by the manufacturer. The samples were run on an ABI3130 Genetic Analyzer using the pop-7 polymer (Applied Biosystems Inc.). DNA sequence data were compiled and analyzed by using the Chromas 1.452. DNA sequences were obtained from J. Craig Venter Institute (JCVI)3, and protein domain information was obtained from Kyoto Encyclopedia of Genes and Genomes (KEGG)4. BLASTN and BLASTP programs were used to search for homologous nucleotide or protein sequences, respectively, in the database5. Clustal Omega software6 was used for the alignment of protein sequences and further modified through GeneDoc software version 2.7.0007. For designing primers, Primer3 software8 was used. For designing qRT-PCR primers, Primer Express 3.0 software  was used.For confirmation of plasmid constructs, DNA sequencing was done using the BigDyet-test.Where needed, pairwise comparison of data for each sample was analyzed for statistical significance using Student’s gppA gene of V. cholerae, BLASTN9 analysis of the whole-genome-sequenced V. cholerae Wt strain N16961 . Comparison of the gppA gene sequence of V. cholerae indicated 63% nucleotide sequence identity with that of E. coli by using the BLAST Global Alignment Tool. While the V. cholerae gppA gene codes for a 497 amino acid long protein, the E. coli GppA is composed of 494 amino acids (see footnote 3). BLASTP analysis of V. cholerae GppA protein shows 57% identity (279 out of 491 amino acids) and 72% similarity with that of E. coli GppA. Genetic organization of the gppA gene in the genomes of V. cholerae, other Vibrio spp., and E. coli indicated that the gppA locus is highly conserved in vibrios , and they transcribe as a bicistronic operon. KEGG analysis of the 497 amino acid long GppA protein of V. cholerae revealed that it carries a highly conserved Ppx-GppA motif of 281 amino acids long (position 23 to 303), which is similar to the Ppx-GppA motif of E. coli GppA enzyme (V. cholerae ppx gene (JCVI annotation no. VC0722) and found that it encodes 523 amino acids long exopolyphosphatase. BLASTP analysis indicated that the V. cholerae Ppx has 40.5% identity with that of GppA  cotranscribes with the rhlB gene (JCVI annotation no. VC0305), and they are arranged in a bicistronic operon. To confirm this, V. cholerae cells were grown till late exponential phase (OD600 = 1.0), and total cellular RNAs were prepared for RT-PCR analysis. When the primer pair gppASeq-F (VC0305 ORF specific) and gppArtm-R  genes are indeed arranged in an operon, and they are cotranscribed. Next, we wanted to ensure that in the gppA-deleted V. cholerae strain NΔgAK (gppA), the transcription of the neighboring gene rhlB (VC0305) was not affected. It is to be noted that during construction of the ΔgppA mutant, the closest rhlB ORF was kept intact; however, transcription of the arcB gene was not tested. For this purpose, the transcript status of rhlB in ΔgppA strain was examined by RT-PCR using the same set of primer rhlB-F/rhlB-R ppGpp labeling assay was performed using the ΔgppA mutant ppGpp spots using the ImageJ software as shown in gppA cells carrying the V. cholerae gppA gene expressing plasmid pGppA (gppA(pGppA) cells like that of the parental Wt strain (gppA(pGppA) in the absence of arabinose (gppA from the plasmid pGppA. By comparison of the profiles of pppGpp and ppGpp under amino acid starvation in V. cholerae relA+gppA+ (Wt) and relA+gppA– (ΔgppA) genetic backgrounds, it appears that the GDP is a more preferred substrate for RelA than GTP  and NΔgPx (henceforth will be designated ΔgppAΔppx), respectively ppGpp in V. cholerae under different nutritional stress conditions.Since the ppA gene , we alsoectively . While ΔpA cells . This wapA cells . To confpA cells . ApparenpA cells . When Ppfunction . DensitoV. cholerae ΔgppA mutant under glucose starvation. Interestingly, there was no difference in intracellular pppGpp and ppGpp levels between ΔgppA and isogenic Wt cells  that modulate GppA activity during glucose starvation or differential expression pattern of gppA during glucose and amino acid starvations cannot be ruled out and needs further investigation.We further examined the status of pppGpp and ppGpp in Wt cells . We exteanalysis . At presgppA gene in amino-acid-starved cells, we attempted to test its regulation, if any, during various growth phases and starvation conditions. To this end, we performed quantitative estimation of the gppA gene transcripts under various growth phases of V. cholerae cells in nutrient-rich LB medium using qRT-PCR assay. As shown ingppA transcripts level in stationary phase cells of V. cholerae Wt strain compared to that of early or mid-log phase cells. We further extended our analysis to explore the expression level of the gppA gene in V. cholerae under both amino acid as well as glucose-starved conditions with respect to non-starved condition in MOPS medium using qRT-PCR assay . Interestingly, qRT-PCR analysis indicated a significant downregulation (∼1.8-fold) of gppA expression in glucose-starved V. cholerae cells compared to non-starved bacteria (gppA expression goes up (∼2.5-fold) during amino acid starvation compared to non-starved cells  upregulation was observed in the latter condition  during amino-acid-starved condition compared to non-starved V. cholerae cells ] or pGppA1-310 [ΔgppA(pGppA1-310)], expressing truncated GppA where 184 or 187 amino acids were deleted, respectively, were able to complement the GppA function, the strain ΔgppA(pGppA1-272), ΔgppA(pGppA1-303), or ΔgppA(pBG1-308), where truncated gppA expressed 225, 194, or 189 C-terminally amino acids deleted proteins, respectively, failed to complement (gppA mutant strains carrying the plasmid pGppA13-497 [ΔgppA(pGppA13-497)] or pGppA18-497 [ΔgppA(pGppA18-497)], expressing N-terminally 12 or 17 amino acids deleted proteins, were able to complement the GppA function but not by the strain ΔgppA(pGppA23-497) or ΔgppA(pGppA20-497), expressing N-terminally 22 or 19 amino acids deleted GppA, respectively  of the nstructs  were mad pBADcam , and eacmplement . On the ectively . Finallyectively  containistructed , which sfunction . Thus, ifunction . To our gppA gene on certain virulence phenotypes of V. cholerae like cholera toxin production, biofilm formation, and motility. However, no defect in any of these virulence phenotypes was observed in the ΔgppA mutant  compared to the isogenic Wt strain during amino acid starvation but not under glucose deficient condition (V. cholerae RelA synthesizes (p)ppGpp ppGpp , while d(p)ppGpp . Therefo(p)ppGpp . In contnditions . Howeverppx gene of V. cholerae to determine whether it has any gppA-like function. This is because both Ppx and GppA contain conserved Ppx-GppA motif, which appears to be needed for their similar enzymatic functions (ppx1 gene of the enteropathogen Campylobacter jejuni leads to increase in intracellular accumulation of pppGpp as well as polyP (gppA V. cholerae cells (E. coli ΔgppAΔppkx mutant strain (GppA-Ppk_Ppx-) by overexpressing the Ppx enzyme. Very recently, the structure of the Ppx/GppA protein of the Gram-negative bacterium Helicobacter pylori has been solved ppGpp in V. cholerae.In this study, we have also characterized the unctions . Recentlas polyP . We founae cells . Similarn solved . It was to ppGpp . Thus, igppA gene in different growth phases is not available for any Gram-negative bacteria. Such analysis indicated that in V. cholerae, the gppA gene expression is constitutive, although there is modest increase in transcripts level during the stationary growth phase  and Ppx-GppA motif  in E. coli Ppx and GppA have been reported (E. coli (V. cholerae because disruption of these domains but not the C-terminal domains III and IV leads to functional loss of GppA (V. cholerae GppA protein.After establishing the function of the pA motif . However protein . It is ino acids . Our delreported . While d(E. coli . On the (E. coli . Our del of GppA . Thus, iV. cholerae gppA gene leads to accumulation of substantial amounts of pppGpp molecules during amino-acid-starved condition. At present, however, it is not clearly known which of the alarmones, ppGpp or pppGpp, is most potent in regulating the different phenotypes of SR in bacteria including V. cholerae. Since ppGpp is synthesized directly from GDP and almost in all bacteria, the level of intracellular ppGpp under nutritional stress conditions is higher than pppGpp, it is likely that ppGpp is the key player of SR in bacteria (V. cholerae. Further studies on other Gram-negative bacteria may help in establishing this unique link between RelA and GppA.In conclusion, the present study shows that the deletion of bacteria . In addiData presented in this study are included in the article/RKB and BD designed the study. DR and SD performed the experiments. DR, SD, BD, and RKB analyzed the data. DR, BD, and RKB wrote the manuscript. All authors have contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Scedosporium/Lomentospora fungi are increasingly recognized pathogens. As these fungi are resistant to many antifungal agents, early diagnosis is essential for initiating targeted drug therapy. Here, we review the microbiological tools for the detection and diagnosis of invasive scedosporiosis and lomentosporiosis. Of over 10 species, Lomentospora prolificans, Scedosporium apiospermum, S. boydii and S. aurantiacum cause the majority of infections. Definitive diagnosis relies on one or more of visualization, isolation or detection of the fungus from clinical specimens by microscopy techniques, culture and molecular methods such as panfungal PCR or genus-/species-specific multiplex PCR. For isolation from respiratory tract specimens, selective media have shown improved isolation rates. Species identification is achieved by macroscopic and microscopic examination of colonies, but species should be confirmed by ITS with or without β-tubulin gene sequencing or other molecular methods. Matrix-assisted laser desorption ionization-time of flight mass spectrometry databases are improving but may need supplementation by in-house spectra for species identification. Reference broth microdilution methods is preferred for antifungal susceptibility testing. Next-generation sequencing technologies have good potential for characterization of these pathogens. Diagnosis of Scedosporium/Lomentospora infections relies on multiple approaches encompassing both phenotypic- and molecular-based methods. Scedosporium species and Lomentospora prolificans (formerly Scedosporium prolificans) [ificans)  are incrificans) ,3. Severificans) ,4. In adificans) ,3,4,5. Cificans) ,3,4,6.Scedosporium/Lomentopsora species are intrinsically resistant to current antifungal agents [Many l agents ,8. This,l agents ,3,9, empScedosporium/Pseudallescheria has undergone a number of revisions. To begin with, two species within the genus Scedosporium, Scedosporium apiospermun  and Scedosporium prolificans were recognized till the detailed application of molecular phylogenetic studies in 2005 where Gilgado at al. [BT2,) TUB, calmodulin and the internal transcribed spacer regions (ITS1/2) of the rDNA gene cluster. They maintained two main “species” Scedosporium apiospermun (teleomorph P. boydii) as a species complex and Scedosporium prolificans. In addition, two new species S. aurantiacum and S. minutisporum were described. In a follow up study [S. apiospermum and P. boydii was crystallized, and an additional new species, S. dehoogii, described. Further new species have since been assigned, e.g., S. deficiens (closely related to S. dehoogi), S. cereisporum (closely related to S. aurantiacum) [The nomenclature of the genera o at al.  undertooup study , the disntiacum) ,13.Scedosporium” was retained at the expense of “Pseudallescheria. [Scedosporium contains at least 10 species , with S. angustum, S. apiospermum, S. boydii, S. ellipsoideum and S. fusoideum considered a species complex [S. apiospermum, S. boydii and S. dehoogii show extensive genetic diversity  definitions for invasive fungal disease (IFD) emphasize the visualization of fungal hyphae and/or culture of the fungus from an affected site as a criterion for proven fungal infection .Sceodosporium/Lomentospora DNA in clinical specimens followed, typically by DNA sequencing or barcoding to identify the causative species. The use of proteomic approaches, e.g., matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) have replaced phenotypic methods as a first line for species identification in many laboratories, backed up by molecular approaches. At present, serological methods have little role in routine diagnosis.Molecular techniques are increasingly used to directly detect Sceodosporium/Lomentospora has been issued by the European Confederation of Medical Mycology in cooperation with the International Society of Human and Animal Mycoses and the American Society of Microbiology [Finally, albeit not within scope of the present review, microbiological diagnosis should always be accompanied by imaging as appropriate to confirm diagnosis and determine the extent of infection. Recently, guidance on how to diagnose rare mold infections, including those by obiology . This doScedosporium/Lomentospora infection. Expertise is required to distinguish the features described below.Whilst the inability of histopathological methods to recognize species is acknowledged, examination by traditional stains such as the Gomori methenamie silver (GMS), hematoxylin and eosin (H & E) and periodic acid-Schiff (PAS) stains for the presence of fungal elements has good utility to provide a presumptive diagnosis of any IFD in a cost-effective manner . Many exScedosporium spp. are hyaline in appearance and when visualized, confirm the presence of fungal infection, but are non-specific, and it is not possible to distinguish these from the hyphae of other hyaline molds such as Aspergillus, Fusarium and Paecilomyces spp. Nonetheless, the hyphae, typically located within areas of necrosis, are septate, non-pigmented and may show acute-angle branching. More often than not, they appear irregular, sometimes with branches bridging two parallel hyphae to form an H-shaped pattern; the last is considered by some experts to be highly suggestive of Scedosporium and L. prolificans [L. prolificans are often, though not always, melanized [Scedosporium-specific DNA probe has been reported [Hyphae of lificans . Of noteelanized ,21. In Gelanized . Whilst reported , its utiDespite the fact that microscopy and culture are insensitive and slow (2–10 days), their application to clinical specimens remains a cornerstone of diagnosis. Further, culture yields an isolate for drug susceptibility testing.Scedosporium spp. and L. prolificans will grow in standard blood culture systems although most publications describe the recovery of L. prolificans from blood cultures . More often, however, these fungi are recovered from sputum and respiratory tract samples including bronchoalveolar lavage (BAL) fluid. Isolates from sterile sites should always be considered clinically significant. Direct microscopy of clinical specimens can rapidly suggest a diagnosis in appropriate clinical context. Yeast-to-hyphal structures may be seen on Gram stain of blood cultures. There are few data on use of fluorescent brighteners or potassium hydroxide (KOH) wet mounts, the latter of which is limited to skin and corneal scrapes.These fungi can be cultured from any clinical specimen including tissue, respiratory tract specimens, eye, skin and blood. Both Scedosporium/Lomentospora fungi will grow without difficulty on standard mycological media such as Sabouraud’s dextrose agar (SDA) or potato dextrose agar (PDA). However, their recovery especially from non-sterile respiratory tract specimens may be hampered by competing growth from other fast-growing molds, most often Aspergillus spp., leading to “missed” detection. It is also important to note that the growth of L. prolificans, but not Scedosporium spp., is inhibited by the presence of cycloheximide, if added to mycological media [al media .Scedosporium/Lomentospora spp. This involves the incorporation of an inhibitor, such as benomyl, cycloheximide (Scedosporium spp.) or dichloran  to inhibit non-Scedosporium/Lomentospora fungi into a base medium to reduce growth of other molds. Such media include Scedosporium Selective agar (SceSel+) and its newer variation Scedo-Select III which contains both benomyl and dichloran [TM agar , both of which may be purchased although the latter was found in one study to be inferior to SceSel+ agar for recovery of Scedosporium from sputum [Hence, many laboratory mycologists choose to employ the use of semi-selective or selective media to “select” for growth of ichloran ,25 whichichloran , and Mycm sputum . Most exScedosporium/Lomentopsora species are given in the tables below.Although many laboratories now rely on molecular-based methods to confirm species identification, there are phenotypic features from cultured colonies that can assist identification, briefly summarized in Scedosporium spp. and L. prolificans will be sufficiently mature in growth on PDA to allow elucation of macroscopic and microscopic characteristics. One distinguishing feature of L. prolificans from Scedosporium species is its grey-back color , a system examining various physiological reactions of these fungi [TM  [S. aurantiacum rather than for species identification. In practice, phenotypic methods are largely superseded by MALDI-TOF MS and molecular approaches.Phenotypic identification methods include growth at 40 °C and 45 °C , patternse fungi , and theCA, USA) . HoweverScedosporium species and L. prolificans with comparable accuracy to many molecular methods with the proviso that MS databases contain reference spectra [® MS v3.0  database was approved by the US Food and Drug Administration (FDA) for identification of molds in July 2017, but does not contain spectra of S. aurantiacum. Consequently, within the S. apiospermum complex, only S. apiospermum and S. boydii can be identified by the Vitek® MS v 3.0 system [Scedosporium/Lomentospora when relying on their database alone [Although multi-locus DNA sequencing is considered the reference method (see later), MALDI-TOF MS offers good potential for fast, and relatively economical identification of  spectra . The sta spectra . The Vit0 system . The othse alone ,35,36. Ase alone ,37.n = 17) in the identification of Scedosporium/Lomentospora isolates  after the Bruker Filamentous Fungi Library v1.0  was supplemented with an in-house database. Although L. prolificans was represented in the Bruker database, only one of four strains was identified, yet all four were identified using the enhanced database, suggesting test performance also relies on the inclusion of spectra from local strains in the database. In another study, only one of 21 Scedosporium spp. isolates were correctly identified to genus level using the same Bruker library. The inclusion of 24 reference spectra from S. boydii, S. apiopsermum and L. prolificans to an in-house database allowed 76% (16/21) of isolates to be identified to species level but with scores of 1.65–1.9 [Sleiman et al.  reported1.65–1.9 .L. prolificans and five morphologically indistinguishable species—S. boydii, S. apiosperum, S. aurantiacum, P. minutispora and S. dehoogii for use on the Andromas platform. The database was evaluated against 64 isolates previously identified by multi-locus sequence type (MLST) analysis, and all isolates were unequivocally identified in minutes , demonstrating the power of MALDI-TOF MS with a comprehensive database. However, a similar study [S. apiospermum complex.Of note, Sitterle et al.  built a ar study  using reScedosporium/Lomentospora [S. boydii and S. ellipsoidea  [β-tubulin gene (βT2) may be required to resolve species. Of central importance is a reliable reference sequence database such as those of the Westerdijk Institute (http://www.westerdijkinstitute.nl/Collections/BioloMICSSequences.aspx) or the International Society for Human and Animal Mycology (http://its.mycologylab.org) for sequence pairwise alignment.DNA sequence-based analysis is the current “gold standard” for species identification of all fungi. Sequencing of the primary fungal DNA barcode, the ITS region is able to identify the main pathogenic species of ntospora ; howeverspecies) , amplifiTM System (bioMerieux) enabled not only the identification of a number of Scedosporium species but also identified genotypes within species [S. apiospermum and S. boydii whilst all S. aurantiacum isolates shared the same profile. Rep-PCR profiles were concordant with their MLST (see below).Other than DNA sequencing, the analysis of repetitive DNA sequences of isolates (rep-PCR) using the Diversilab species ; of a toS. apiospermum, S. boydii and S. aurantiacum (https://mlst.mycologylab.org) [To this end, species identification may also be undertaken by MLST as inferred in lab.org) . HoweverScedosporium species, Zhou et al. distinguished between L. prolificans and S. apiospermum/S. boydii [Finally, rolling circle amplification (RCA) which uses a simple isothermal amplification format can differentiate between sequences to a single nucleotide level . Using a. boydii . The advScedosporium/Lomentospora fungi directly from clinical specimens. Such approaches include broad range PCR followed up by DNA sequencing and species-specific multiplex PCR in various formats including real time PCR and oligonucleotide arrays. Each has its relative strengths and uptake of technology is dependent on laboratory resources. There are no standadized commercial assays.The high resolving power together with increasing access to molecular biology technologies in clinical laboratories have enhanced the laboratory’s capacity to rapidly detect and identify Broad range or panfungal PCR is increasingly employed in clinical laboratories to identify fungi directly from fresh and formalin fixed paraffin-embedded (FFPE) tissue as well as from other types of clinical specimens including BAL fluid and cerebrospinal fluid (CSF). Multiple assays have been published ,45,46. Mn = 60). Assay sensitivity was similar across different specimen types  [L. prolificans in tissue, was 97% when applied to fresh tissue and 68% when performed on FFPE tissue [In one report, using a panfungal real-time PCR, test performance was best when used on sterile body fluids  100% and positive predictive value (PPV) 86%)) but performance was considerably lower when performed on BAL fluid  . Gomez eor FFPE) . These tor FFPE) ,52 whereor FFPE)  made theE tissue . For 20 E tissue . Under tS. apiospermum complex, as no DNA barcoding gap is present, due to the relatively large degree of genetic variation found in this species complex. The use of alternative loci, such as the βT2 gene, TUB and elongation factor 1 alpha genes may be necessary  patients; the test specificity was 97.2% with a PPV of 78.3% and NPV of 94.1% [There are relatively few reports on the use of multiplex PCR for the specific detection of of 94.1% . InsuffiScedosporium species in routine diagnostics is limited. Castelli et al. developed two separate ITS-targeted PCR assays for species identification of L. prolificans and S. apiospermum and tested their assay in infected mice. In mice infected with L. prolificans, the assay sensitivity was 95.5% for lung tissue, 85% for serum and 83.3% for whole blood. For S. apiospermum infected mice the sensitivity of the assay was 97.2%, 81.8% and 54.5% for lung, serum and blood, respectively [Mulitplex PCRs may also be in real time format using fluorescent-labelled primers, probes or dyes. Experience with such PCR assays to detect and identify Scedosporium in the sputum of CF patients. For 57 sputa, results were compared with those of culture. In more than half the cases (n = 33 samples), the array detected a more diverse range of fungi than culture. Scedosporium fungi were identified but not at species level. Disadvantages include the high costs for set up and need for batch testing for efficiencies.Bouchara et al.  were oneScedosporium/Lomentospora-specific markers. However, large scale studies on their diagnostic utility remain sparse. The best studied panfungal marker is 1,3-β-D-glucan (BDG) which is found in the cell wall of fungi and can be detcted in the blood of patients with IFDs except in Mucorales and Cryptococcus infections. The clinical utility of this test is well summarized by meta-analyses [Scedosporium and L. prolificans infections and is supported by the detection of BDG in serum in reports of Scedosporium brain abscess and L. prolificans fungemia [TM assay .Diagnosis by serological approaches have encompassed the study of both panfungal biomarkers as well as analyses ,57 and dfungemia ,59,60. TScedosporium or L. prolificans-specific serological tests. Nonetheless, Thornton and colleagues have develoepd a number of Scedosproium-Lomentopsora specific monoclonal antibodies (Mabs) that enable species identification. One targets an extracellular 120-kDa antigen located in the spore and hyphal walls of S. apiospermum and S. boydii whilst the other tragets the enyme tetrahydroxynaphthalene reductase which plays a role in the biosysnthesis of melanin in L. prolificans [Scedosporium proteins such as cytosolic catalase have been studied as markers of infection as have various metabolites and siderophores [S. apiospermum complex infection demonstrated antibodies to catalase A1 of S. boydii by ELISA whilst those colonized or infected with Aspergillus fumigatus did not [There are no comemrcial lificans ,62. Certrophores ,64 of th did not . Study iScedosporium/Lomentospora fungi are resistant to many antifungal agents currently available. As there are species-specific susceptibility patterns, it is imperative the causative species is accurately identified. Further within species, minimum inhibitory concentrations (MICs) to some antifungal drugs vary between isolates, rendering it advisable to determine drug MICs to all clinically significant isolates. The methodology chosen should be an established method such as the reference broth microdilution methods of the Clinical Laboratory Standards Institute (CLSI), USA and the European Committee on Antimicrobial Susceptibility Testing . Where the commercial Sensititre® Y010 panels are used .), comparative evaluations are recommended but in general, similar MIC results have been obtained [obtained ,66,67.Scedosporium species and L. prolificans are resistant to amphotericin B , 5-flucytosine (MIC90 > 16 mg/L), fluconazole (MIC90 > 16 mg/L) and itraconazole (16—>16 mg/L) although a number of studies have observed that S. apiospermum complex isolates may exhibit lower MICs to itraconazole (MIC 0.5–1 mg/L) [L. prolificans being the most “resistant’ (MICs 4–16 mg/L) [S. apiopermum and S. boydii (GM MIC 1.64 and 0.75 mg/L respectively) with similar observations for posconazole. Notably, L. prolificans is pan-antifungal resistant [All –1 mg/L) ,67. In aesistant ,68.L. prolificans is also resistant to isavuconazole , [S. apiospermum complex strains using both the EUCAST E. Def. 9.3 (GM MIC 1.22 mg/L) and the Etest  [S. apiospermum [L. prolificans infections, it demonstrates in vitro synergy with voriconazole and to a lesser extent with posaconazole and is used in combination typically with voriconazole [4 mg/L), ,66,68,6908 mg/L) . Terbinaospermum . AlthougGM MIC 1.2 mg/L anconazole ,72. The conazole ,72.Scedosporium and L. prolificans infections. In due course, it could be argued that clinical laboratories will consider the inclusion of these agents in their antifungal susceptibility testing menu.It is pertinent to briefly touch on a number promising novel antifungal drugs for treatment of S. apiospermum (n = 28), S. aurantiacum (n = 7) and L. prolificans (n = 28) revealed that MICs were at least 10-fold lower than found for standard antifungal drugs including voriconazole [90 values for fosmanogepix against both S. apiospermum and L. prolificans were 0.12 mg/L [Scedosporium and L. prolificans agent is Olorofim (previously F901318). Olorofim is a first-in-class antifungal drug that inhibits dihydroorotate dehydrogenase, an enzyme central to fungal pyrimidine biosynthesis [L. prolificans strains having MICs at the upper end of the range [N-chlorotaurine (NCT) has broad spectrum antimicrobial killing activity due to its transhalogenated nature, which markedly enhances its in vivo activity. Clinical phase I and II studies have demonstrated good tolerability of topical 1% NCT in aqueous solution including during inhalation [Fosmanogepix (formerly APX001 and E1211) is a new first-in-class antifungal with broad spectrum activity which targets the inositol acylation step in the biosynthesis of the glycosyl phosphatidyl inositol (GPI) anchor, which in turn anchors proteins to the cell wall . In vitrconazole . The MEC.12 mg/L . Anotherynthesis . Biswas mum n = 2, S. aurahe range . Finallyhalation . WhetherScedosporium spp. and L. prolificans. To re-emphasize, imaging procedures are essential parallel investigations. The Global guideline for the diagnosis and management of rare mold infections refer to the strength of recommendations for each diagnostic option and give the rationale behind these recommendations [Given the many options for laboratory diagnosis, a systematic guide to assist laboratories with diagnosis is helpful. Whether a diagnostic approach is performed will be partially dependent on access, resources and on the size and nature of the laboratory . ndations .Scedosporium/Lomentospora infections, the publishing of the draft genomes, using next generation sequencing (NGS) methodology, of all four major pathogenic species is welcomed [L. prolificans, S. boydii, S. apiospermum and S. aurantiacum, respectively) [L. prolificans contains at least 7, and possibly 11 chromosomes [Scedosporium/Lomentospora isolates using these draft genomes as a “reference” is ongoing.Given the increasingly emergent nature of welcomed ,79,80,81welcomed  have beectively) ,79,80,81omosomes . Of noteL. prolificans isolates from infections occurring over an 8-month period a single hospital unit [As such, there are few data on the use of NGS to study genomic relationships of clinical strains and in the tracking of infection. One small study investigated a possible link between four tal unit . There wScedosporium and L. prolificans infections. With wider access to molecular detection and identification methods, it is tempting for laboratories to reduce focus on culture and conventional identification methods as these are time consuming and require trained personnel. However, in many resource-limited settings, culture and phenotypic approaches remain mainstay of diagnosis; in this context, access to MALDI-TOF MS and molecular-based methods are a priority. It should be noted, however, that basic biology and morphology is still required for characterization of fungi and represents a criterion for the lodging of authenticated fungal strains in a recognized and curated culture collection. Hence it is important for laboratories to maintain an understanding of all diagnostic approaches.In conclusion, the microbiology-pathology laboratory has good potential to assist with diagnosis of The role of NGS genomics is currently still largely research based. Improved and standardized approaches to bioinformatic analysis of data would help position this technology into being translated into routine use.