Plectranthias perchlets are described, collected from mesophotic coral ecosystems in French Polynesia and the Republic of the Marshall Islands, in the tropical Central Pacific. Plectranthiaspolygoniussp. nov. was collected at a depth of 105 m in Tahiti, French Polynesia, and 120 m in Maloelap Atoll, Republic of the Marshall Islands. It was also observed in Moorea and Rangiroa (French Polynesia), and at Majuro and Erikub Atolls, Republic of the Marshall Islands. Plectranthiashinanosp. nov. was collected at a depth of 90–98 m in Tahiti, French Polynesia, and observed in Moorea. The barcode fragment of the cytochrome oxidase I gene of Plectranthiaspolygoniussp. nov. does not closely match any published sequence of Plectranthias, with approximately 15% uncorrected divergence from several species. Plectranthiaspolygoniussp. nov. can be distinguished from all of its congeners by coloration and morphology. The barcode fragment of the COI gene of Plectranthiashinanosp. nov. is closest to Plectranthiasbennetti, with 5.4% uncorrected divergence. Plectranthiashinanosp. nov. is also distinguished from all of its congeners by morphology, and a coloration that includes two indistinct black spots along the base of the dorsal-fin, and transparent yellow dorsal and anal fin membranes. With this publication, the genus Plectranthias now comprises 58 valid species, with representatives from tropical to temperate waters of the Atlantic, Pacific, and Indian oceans. These two new discoveries add to the growing body of research highlighting the rich biodiversity of mesophotic ecosystems.Two new species of  Plectranthias Bleeker, 1873, currently comprises 56 valid species from tropical to temperate waters in the Atlantic, Pacific, and Indian oceans (The anthiadine genus n oceans . Most ofn oceans . In genen oceans . Due to n oceans .MCEs) are coral reef habitats found at depths of 30–150 m, and are also known as the coral reef “twilight zone” , unless otherwise noted. Values in parentheses represent ranges for paratypes, when different from the holotypes. The holotypes were deposited at the California Academy of Sciences ichthyological collection (CAS), and the paratypes were deposited at the Smit hsonian Institution, National Museum of Natural History ichthyological collection (USNM).All specimens were collected with hand nets while diving on mixed-gas closed-circuit rebreather (Hollis Prism 2) in French Polynesia  and Micronesia . Specimens were collected and immediately transported to a field laboratory, where they were photographed, tissues sampled, fixed in 10% formalin, and preserved in 75% ethanol. The preserved specimens were later measured and x-radiographed at the California Academy of Sciences. Measurements were taken with digital calipers to the nearest 0.01 mm and rounded to one decimal place, following the conventions described in COI) DNA was sequenced and analyzed for the new species. DNA extraction and PCR amplification of the COI gene were performed following protocols detailed in Plectranthias species available in GenBank . Alignments of DNA sequences were done using a standard Geneious global alignment with free end gaps and 65% similarity in the program Geneious Prime 2020.0.3 ; width of body 2.7 in depth (2.3); head length 3.2 in SL (3.1); snout length 3.4 in HL (3.6); bony interorbital width 1.6 in snout length (1.3); orbit diameter 2.8 in HL; post-orbital head length 4.8 in SL (4.7); least depth of caudal-peduncle 2.5 in HL; caudal peduncle length 3.6 in HL (4.0).Scales ctenoid; lateral line complete and broadly arched over pectoral fin following body contour; 30 (27) tubed scales; scales above lateral line to origin of dorsal fin 2; scales above lateral line to base of middle dorsal spine 2; scales below lateral line to origin of anal fin 9 (10); diagonal rows of scales on cheek 5; scales on top of head extending anteriorly to vertical from anterior margin of pupil; no scales on chin, maxilla, or snout; circumpeduncular scales 10 (11); gill rakers 5+13 (6+13); vertebrae 10+16; supraneurals 3; anterior supraneural-dorsal ray-pterygiophore-neural spine interdigitation pattern: 0/0+0/2/1+1/1.Mouth large and terminal, slightly upturned; lower jaw protrudes slightly; maxilla expanded posteriorly, extending to below the posterior edge of eye; dorsal profile of head almost straight; upper jaw with one fixed, stout canine on either side of symphysis; upper canines flanked internally by villiform band with four or five rows of depressible, smaller, sharp-tipped teeth; inner rows become progressively longer, innermost row with largest teeth; lower jaw has pair of fixed, short stout canines on either side of symphysis followed by smaller, depressible, sharp-tipped conical teeth in a villiform band of 3–5 rows; lower teeth become progressively longer on inner rows; vomer roughly V-shaped band of two rows of similarly sized, sharp-tipped, conical teeth; palatines with one row of small, sharp-tipped conical teeth; tongue small, slender, pointed, and without teeth.Opercle with three spines, the middle spine the longest; preopercle with 14 (17) small spines (serrae) along posterior margin; antrorse spines lacking on ventral margin; interopercle with no spines; subopercle smooth, with no spines; anterior nostrils positioned halfway between snout and eye, each with a small rounded flap rising from anterior rim; posterior nostrils an elliptical opening at anterior border of orbit.Body: overall white with two rows of bright orange rhomboid-shaped polygons, four to six in each row, arranged in an irregular grid along lateral midline of body; uppermost row of orange polygons proceeds from behind eye to dorsal third of caudal peduncle; lower row starts just dorsal to origin of pectoral fin and continues to ventral half of caudal peduncle; throat and belly white. Head: dorsal third of head orange and bottom two thirds pinkish white with two yellow stripes, both originating from the tip of the upper lip. The first extending horizontally across orbit, bifurcating past posterior edge of pupil to approximately edge of opercle. The second from tip of upper lip, tracing obliquely along maxilla and extending to ventral edge of preopercle. Preopercle region with a yellow triangular patch, from lower mid-orbit expanding in width to edge of preopercle with bifurcations to horizontal edge of operculum and pelvic fin bases respectively; orbit mostly orange-red; iris outlined in silver-grey to black with horizontal yellow stripe through middle of anterior portion; posterior portion of iris with two yellow stripes arising from a bifurcation of the anterior yellow stripe; pupil black. Fins: first three membranes of spinous portion of dorsal fin mostly orange with yellow highlights; third spine with yellow and white membrane; remaining membranes of spinous portion mostly yellow, with hyaline tips; yellow coloration continues on lower third of soft portion of dorsal fin, with upper two-thirds mostly hyaline; some yellow and pale orange on tips of soft dorsal and membranes of last four to five rays; caudal fin hyaline with white and orange rays; anal fin mostly yellow, with white margin; pelvic fins mostly white distally, with yellow rays proximally; pectoral fins hyaline. Living specimen photographed in the Marshall Islands exhibits more yellow coloration on head, within rhomboid-shaped polygons on lateral midline, and on first three membranes of spinous dorsal fin ; canine teeth on the lower jaw (lacking in P.inermis); and 14–17 spines on the posterior edge of the preopercle . The new species differs from P.altipinnatus by having X, 16 dorsal-fin rays , a shallower body , smaller head , and a larger eye .The general body shape, color, and prolongation of the third dorsal-fin spine in tus Fig. ; howeverTaxon classificationAnimaliaPerciformesSerranidaeACE3522C-C99C-5170-B48C-BDE5B44115EDhttp://zoobank.org/84A5C4AA-577B-4D79-BE21-8973EFDF7BE9Tahiti, French PolynesiaMN922329. 49.6 mm SL, Tahiti, French Polynesia, 17°29'27"S, 149°28'01"W, depth of collection 98 m, collected with hand nets by B Shepherd, HT Pinheiro, TAY Phelps, MV Bell, and LA Rocha, 03 March 2019 ; width of body 2.1 in depth (2.4); head length 2.4 in SL (2.5); snout length 3.0 in HL (3.2); bony interorbital width 3.5 in snout length (2.3); orbit diameter 3.7 in HL (3.5); post-orbital head length 5.4 in SL (5.6); least depth of caudal-peduncle 3.7 in HL (3.4); caudal-peduncle length 4.0 in SL (4.1).Scales ctenoid; lateral line complete and broadly arched over pectoral fin following body contour; 29 (30) tubed scales; scales above lateral line to origin of dorsal fin 3; scales above lateral line to base of middle dorsal spine 2; scales below lateral line to origin of anal fin 12; diagonal rows of scales on cheek 4; scales on top of head extending anteriorly to vertical from center of eye; area on top of head between eyes with scales; no scales on chin, maxilla, or snout; circumpeduncular scales 12; gill rakers 8+13 (7+12); vertebrae 10+16; supraneurals 3; anterior supraneural-dorsal ray-pterygiophore-neural spine interdigitation pattern: 0/0+0/2/1+1/1.Mouth large and terminal, slightly upturned; lower jaw protrudes slightly; maxilla expanded posteriorly, extending to below the posterior edge of pupil; upper jaw with one fixed, stout canine on either side of symphysis; upper canines flanked internally by villiform band with 7–9 irregular rows of depressible, smaller, sharp-tipped teeth; inner rows become progressively longer, innermost row with largest teeth; lower jaw has one large, fixed canine on either side of lower jaw, roughly at midpoint, followed by smaller, depressible, sharp-tipped conical teeth in a villiform band of 4–6 irregular rows, innermost teeth same size as outer rows; vomer roughly V-shaped band of two rows of similarly-sized, sharp-tipped, conical teeth; palatines with one row of small, sharp-tipped conical teeth anteriorly, two rows posteriorly; tongue small, slender, pointed, and without teeth.Opercle with three flat spines, the middle spine the longest; preopercle with 22 (12) small spines (serrae) along posterior margin; ventral (inferior) margin of preopercle with three antrorse spines; interopercle with no spines; subopercle smooth, with one spine on inferior margin; anterior nostrils positioned closer to eye than to snout, each with a small rounded flap rising from anterior rim; posterior nostrils an elliptical opening at anterior border of orbit.Body: overall light red in color; chest and belly mostly light red to pink; dorsal portion of body darker red with yellow along scale margins and lateral line; series of 8–12 pink to white incomplete bars on the body, originating just behind orbit and continuing to base of caudal fin; bars are approximately 20 degrees off of vertical anteriorly, becoming near vertical as they approach base of caudal fin; black spot, almost twice the diameter of orbit, at base of spinous dorsal-fin spines 7–10 and soft rays 1–5, continuing slightly more than halfway up membrane of spinous dorsal; black spot is interrupted by the near-vertical white bar originating below soft rays 1 or 2; smaller second black spot, slightly smaller than orbit, located at base of soft dorsal rays 11–15. Head: snout, throat, anterior portion of lower lip, maxilla, and operculum mostly light pink; yellow stripe originating at snout, proceeding across maxilla, below orbit to preopercle; lattice-like network of indistinct yellow stripes radiating outward from pupil across iris, between eyes, across top of head, from ventral margin of orbit to origin of lateral line and lower margin of operculum; iris mostly pink with yellow splotches radiating outward from pupil; pupil black and teardrop-shaped, pointed anteriorly. Fins: spinous portion of dorsal fin predominantly translucent yellow, with upper half of black spot on membrane between spines 7–10; lower third of soft dorsal fin mostly translucent yellow, upper two-thirds hyaline; caudal-fin membranes mostly hyaline with some regions a faint transparent yellow, fin rays white with red margins; pelvic fins hyaline with faint yellow on rays; anal fin mostly translucent yellow with hyaline margins; pectoral fins hyaline with rays outlined in pink; base of pectoral fins yellow. Freshly dead specimens exhibit similar coloration, with slightly more yellow on head, body, and fins . Morphologically, it can be distinguished from P.bennetti by having a longer snout (3.0–3.2 in HL vs. 4.4 in P.bennetti), a larger orbit (3.5–3.7 in HL vs. 4.1 in P.bennetti), in the number of circumpeduncular scales (12 vs. 14 in P.bennetti), the number of gill rakers , and in coloration .The most similar barcode fragment of the mtDNA COI gene to Plectranthias appear to be common and conspicuous inhabitants of MCEs, and we have regularly observed them in highly complex, rocky habitats with abundant coralline algae, sponges, and black corals (Fig. Anthiadinae, the genus Plectranthias is in need of revision, as it is not currently defined on the basis of synapomorphies, and there is high variation within many of the defining characters (Plectranthias will continue to expand with further MCE exploration, and that future sampling, comparative work, and genetic analysis will unravel some of the taxonomic confusion within this genus.Species within the genus als Fig. . They araracters . Hence, More than 70% of all research on MCEs has been published in the past seven years , and undPlectranthiasinermis. CAS 241326. Philippines, Batangas, 28 April 2015.P.japonicus. CAS 33555, 1. Philippines, Batangas, 23 June 1966.P.longimanus. CAS 213185, 3. Fiji, Viti Levu Island, 31 May 1999.P.sagamiensis. CAS 235596, 1. Philippines, Luzon Island, 02 June 2011.P.winniensis. CAS 219169, 1. Fiji, Vanua Levu Island, 27 May 2003.


Escherichia coli (STEC) is an important cause of diarrheal disease, most notably hemorrhagic colitis, and post-diarrheal sequela, such as hemolytic-uremic syndrome (HUS) [Globally, Shiga toxin-producing me (HUS) . Cattle me (HUS) . Preventme (HUS) . Despiteme (HUS) . Many qume (HUS) ,5,6,7, vme (HUS) ,9,10, anme (HUS) ,12,13 ofme (HUS) ,15,16 anme (HUS)  of ShigaA systematic review of STEC in Brazil found no data for 44% of the Brazilian states, highlighting the need for expansion of epidemiological monitoring to the entire country and alignment of food safety standards with that of international bodies ,6. AlthocsgDEFG and csgBAC in E. coli O157:H7 stably conferred biofilm formation, epithelial cell invasion, and persistence in cattle [E. coli O45:H2 is a close relative, phylogenetically, to E. coli O103:H2, sharing a high degree of homology in virulence factors, such as Stx prophages; however, it is distinct from E. coli O45:H16, suggesting that serotype O45:H2 may share virulence characteristics of O103:H2, which is frequently associated with severe illness [E. coli O157:H7 secretes EF-Tu and L-asparaginase II, with the latter inhibiting T-lymphocyte proliferation [E. coli O157:H7 adhesion to human epithelial cells [Several studies addressing virulence factors provided novel information about how they influence carriage in reservoir hosts and disease in human patients. A single base pair A to T transversion, intergenic to the curli divergent operons n cattle . E. coli illness . E. coliE. coli O157:H7 causing attaching–effacing lesions in the uroepithelium and the first evidence of the utility of the gnotobiotic piglet as a model for studies of the pathogenesis of STEC-induced urinary tract infections [Other studies, using a variety of methods and approaches, extend our knowledge of the pathogenesis of STEC infections. A comparison of the transcriptomic and phenotypic responses of host cells infected with STEC O113:H21 strains from a HUS patient or bovine feces having similar virulence factor profiles, found that the former induced greater and earlier host cell global gene expression with excessive inflammatory and apoptotic responses, which may explain its enhanced virulence . AnotherE. coli outer membrane and loading with functional Stx2a was described [Studies utilizing top-down proteomic analysis and a protocol for preparation of outer membrane vesicles (OMVs) provide further new information on Stx, and position scientists for future studies to address the effects of Stx on cells. Top-down proteomic analysis of the B-subunit successfully identified both Stx1a and Stx2a in STEC strains with results consistent with whole genome sequencing, validating the utility of this analytical method to distinguish types and subtypes of Stx . A studyescribed . The OMVescribed .A study evaluating three immunological diagnostic assays for STEC  found that all were highly sensitive and specific, indicating that robust tools for the diagnosis of STEC infections are available . Collect


Reactive oxygen species (ROS) are a chemically defined group of reactive molecules derived from molecular oxygen. ROS are involved in a plethora of processes in cells in all domains of life, ranging from bacteria, plants and animals, including humans. The importance of ROS for macrophage-mediated immunity is unquestioned. Their functions comprise direct antimicrobial activity against bacteria and parasites as well as redox-regulation of immune signaling and induction of inflammasome activation. However, only a few studies have performed in-depth ROS analyses and even fewer have identified the precise redox-regulated target molecules. In this review, we will give a brief introduction to ROS and their sources in macrophages, summarize the versatile roles of ROS in direct and indirect antimicrobial immune defense, and provide an overview of commonly used ROS probes, scavengers and inhibitors. This group consists of two subclasses: (I) highly reactive free radicals, including the superoxide anion (O2●−), the hydroxyl radical (●OH), alkoxyl (●OOR) and peroxyl radicals (●OOH) [2O2), singlet oxygen (1O2) [3), and the hypochlorite anion (OCl−) [2●−, the common precursor of all ROS produced by cells, is produced by single electron transfer to O2. O2●− quickly dismutates to H2O2, either spontaneously in the presence of water or catalyzed by superoxide dismutases [The term reactive oxygen species (ROS) describes a group of molecules with at least one oxygen atom and with higher reactivity than molecular oxygen (Os (●OOH) ,2,3 and en (1O2) ,5, ozonen (OCl−) ,7. ROS an (OCl−) ,10,11,12smutases ,14  [2●− levels is associated with oxidative stress and cellular damage [2●− can irreversibly inactivate proteins and thereby contribute to cellular signaling [Os (VDAC) . An incrr damage ,17,18,19r damage ,21,22, ar damage  and DNA r damage  or lipidr damage ,26,27. Mignaling .2O2, as a diffusible and relatively stable molecule, is more suitable as a cellular signaling factor than O2●− [2O2 is more diffusible than O2●−, its diffusion over membranes is limited [2O2 to induce signaling pathways without regarding the compartment of ROS production [2O2 over membranes and add another regulatory level to ROS-mediated signaling [2O2-mediated signaling is based on the oxidation of cysteine residues [−), which are susceptible to oxidation in dependency of their pKa [2O2-dependent signaling occurs at nanomolar concentrations (≈100 nM), leading to reversible oxidation of the thiolate group to a sulfenic group (Cys-SOH). Protein oxidation by H2O2 can lead to allosteric changes that alter binding affinity for substrates or promote or inhibit enzymatic function [2O2 production, however, leads to further oxidation of the sulfenic group (Cys-SOH) to sulfinic (Cys-SO2H) and sulfonic groups (Cys-SO3H), a process that is irreversible and results in protein malfunction [Hhan O2●− ,31,32,33 limited ,35. Thisoduction ,37. Aquaignaling ,39. The residues ,43,44,45heir pKa ,47. H2O2function ,49,50,51function ,40,52. Sfunction ,54. Excefunction .In cells, ROS levels are in a dynamic but stable equilibrium that consists of the sum of all producing and eliminating factors. ROS are produced as byproducts of oxidative metabolic processes ,57 and i2●− to H2O2 and catalase, which detoxifies H2O2 to H2O and O2 [The cellular antioxidant defense system balances ROS production. The most prominent components are the three superoxide dismutases SOD1, SOD2 and SOD3, which catalyze the dismutation of OO and O2 ,65,66,67O and O2 ,68. VariO and O2 ,71,72,73O and O2 ,66,74.ROS levels that exceed the capacity of the cellular antioxidant defense system induce oxidative stress ,75. ExceThe enzyme family of NADPH oxidases (Nox) ,83 and t2, thereby generating O2●−. In fact, generation of ROS is the sole purpose of all Nox isoforms. Nox1, Nox2, Nox3, and Nox4 depend on a common subunit, p22phox [phox. They are directly activated through calcium binding to their EF-hand calcium-binding domain [The Nox enzyme family consists of the isoforms Nox1, Nox2, Nox3, Nox4, Nox5, and the dual oxidases Duox1 and Duox2. All isoforms are integral membrane proteins and transfer electrons from NADPH to O p22phox ,88,89. Ng domain ,90,91  are mainly produced by complexes I and III as O2●−. Complex I produces O2●− into the mitochondrial matrix where it is dismutated by SOD2 to H2O2 [Mitochondria produce ROS during respiratory activity ,57. Elec to H2O2 ,84,85,86Complex II is also capable of matrix mtROS production ,103,104 2●− into the matrix but also directly into the intermembrane space (IMS) [2O2, which reaches the cytosol via diffusion or aquaporins [Complex III, as exception to complexes I and II, not only produces Oce (IMS) ,120. Froce (IMS) ,121 or iuaporins ,125,126.uaporins ,128,129.uaporins ,131. We For a long time, production of mtROS was described as an unavoidable byproduct of respiratory activity and increased mtROS levels viewed only as causing oxidative distress ,134,135.This review focuses on mitochondria and Nox enzymes as the most prominent and best characterized ROS sources in macrophages during antimicrobial responses. However, also other ROS sources are present in macrophages, which are discussed in this section.2O2 is generated as a byproduct during two oxidation steps of the enzymatic reaction, marking XO as a potential ROS source. XO has important physiological functions [Xanthine oxidase (XO) is a well characterized enzyme ,138, maiunctions ,142,143,unctions , regulatunctions  and defeunctions .2●− and, subsequently, H2O2 are generated as byproducts [Drosophila melanogaster [Peroxisomes are cytosolic organelles present in nearly all eukaryotic cells. Due to their oxidative metabolism of fatty acids and amino acids, they are in close contact and communicate with mitochondria ,148,149.products . With hiproducts ,151,152 products . Moreoveproducts ,155 and products , which bproducts ,158. Perproducts ,160,161.products ,163,164.nogaster .2 is consumed as cosubstrate and H2O2 is generated. In the second step, prostaglandine-G2 is reduced to prostaglandine-H2 by peroxidase activity and the previously generated H2O2 is used as cosubstrate. COX enzymes are therefore crucial for the biosynthesis of prostaglandins, which exert many physiologic functions, such as vasodilation, generation of eicosanoid hormones, thermoregulation, digestive regulation, and induction of fever [The arachidonic acid (AA)-metabolizing enzyme families of cyclooxygenases (COX) and lipoxygenases (LOX) both generate ROS as a byproduct. The two COX isoforms, COX1 and COX2, are membrane-bound enzymes that catalyze the conversion of AA in a bifunctional reaction . In the of fever ,168,169.of fever ,171, wheof fever ,173. Nevof fever ,177,178.of fever ,181,182 of fever ,184. Howof fever  and matrof fever  were sugof fever ,188. In of fever ,190. Theof fever ,192,193.2 and a variable electron donor. In the active center of CYP enzymes, several radical molecule species can be generated as byproducts, including ROS [2●−, which then quickly dismutates to H2O2 [2O2 is directly formed through reduction of O2●− by addition of one H atom [Cytochrome P450 (CYP) enzymes are heme monooxygenases  and nameding ROS ,208. ROSding ROS . The fir to H2O2 . During e H atom . While ee H atom , so far e H atom ,213 and e H atom ,215. Expe H atom , howevere H atom ,218 and Macrophages often are the first immune cells that encounter invading pathogens ,220,221.Recognition of bacteria by macrophages leads to ROS production in different cellular compartments, where they fulfill different antibacterial functions. One of the first functional roles described for ROS produced by macrophages was the inactivation of phagocytosed bacteria by the oxidative burst generated by Nox2. Recognition of invading bacteria induces a fast and robust production of ROS into the extracellular space and the phagosomal lumen (extracellular ROS) see  30,58,2,234,235.Listeria monocytogenes (L.m.), a food-borne pathogen, specializes in escaping from the phagosome via its pore-forming toxin listeriolysin O and two phospholipases PlcA and PlcB [, Nox2-derived ROS do not seem to be listericidal by themselves [phox in binding gp91phox [Escherichia coli (E.c.) coupled to the ROS probe CellROX , is controversial. This pathogen is resistant to antibacterial mechanisms of phagocytes and can use them as a replicative niche [2●− to H2O2. Unfortunately, ROS levels in S.a.-containing phagosomes were not determined, e.g., with bacteria coupled to a ROS probe [In contrast to the clear role of Nox2-dervied ROS in antilisterial activity of macrophages, the role of Nox2 during infection with another Gram-positive pathogen, ve niche . An in vve niche . While Nve niche . Additiove niche . Howeverve niche ,250,251 ve niche . While ove niche ,246,253 ve niche ,61, but OS probe ,99,254, OS probe  excludinMycobacterium tuberculosis (Mtb) uses phagosomes of macrophages as proliferative niche. Mechanisms to survive in phagosomes are the inhibition of fusion with lysosomes and avoiding to be targeted by xenophagy [Like S.a., enophagy . Throughenophagy ,256. Sinenophagy ,257. Mtbenophagy ,128,129,enophagy . They alenophagy ,131. HowSalmonella typhimurium (S.t.), a pathogen responsible for severe food-borne illness and a major cause of diarrheal diseases [− generation via myeloperoxidase (MPO) is clear-cut [diseases , also cadiseases . The impdiseases . The antdiseases ,263,264,diseases . While tlear-cut , macrophlear-cut ,267.Beyond Nox2, mitochondria were introduced as new players for antibacterial ROS production by West and colleagues . They deGaraude et al. also investigated the rearrangement of ETC complexes during infection with Gram-negative bacteria . In BMDMA recent study also investigated the role of mitochondrial FGR in macrophages in the context of obesity . In contWhile Nox2 and mitochondria so far are the only ROS sources identified to be activated by macrophages upon bacterial challenge, several other Nox enzymes were suggested to be involved in defense against infection with protozoan parasites.Toxoplasma gondii (T.g.) is such a protozoan parasite and responsible for the disease toxoplasmosis, affecting one third of the human population [Toxoplasma parasites to detoxify phagosomal ROS was demonstrated with the evolutionary related Toxoplasma cruzii in phagosomes of J774A.1 macrophage-like cells (J774) and BMDM [2●− in the parasitic cytosol and the phagosomes were performed in WT and Nox2-deficient J774 cells. The superoxide dismutase Fe-SODB in the parasitic cytosol was identified as the main defense mechanism against antiparasitic ROS. Fe-SODB is conserved among Toxoplasma parasites suggesting that also other Toxoplasma strains may possess this ability, questioning the still controversially discussed direct antiparasitic role for ROS in T.g.-infected macrophages.pulation ,275. Macpulation . While apulation ,278, thepulation . T.g. inpulation . Nox4 wapulation ,281,282,and BMDM . No ROS Leishmania cause the tropical disease Leishamaniasis [Leishmania parasites [Leishmania subspecies, Leishmania major, actively impairs recruitment of Nox2 to phagosomes, which leads to reduced extracellular ROS production and reduced activation of LAP [Leishmania subspecies are capable of reducing direct antiparasitic ROS production through inhibition of Nox2 recruitment. Leishmania amazonensis infection in J774 cells induced an increase in Nox2 protein levels and total cellular ROS levels [Protozoan parasites of the genus maniasis . Neutroparasites ,286. Macarasites ,288. At n of LAP ,289. It S levels . HoweverWhile many studies focused on bacterial and parasitic infections, studies investigating the direct role of ROS during viral infection of macrophages are rare.98 in a negative feedback loop and therefore dampened the antiviral response. This study showed that Nox2-derived ROS had detrimental consequences for the host by negatively influencing the antiviral response. Accordingly, in vivo Nox2 inhibition protected mice from influenza A virus infection. Moreover, several other studies also described a rather exacerbating role for Nox-derived ROS during virus infection in various cell types and disease settings [A conclusive study demonstrated a role for Nox2 during viral infection of macrophages . The autsettings ,294,295.A role for mtROS during virus infection in vivo was investigated in a study from the same group . AdminisSurprisingly, studies investigating a direct antiviral role for ROS in macrophages, e.g., by induction of oxidative damage to the viral genome  or by moThe role of ROS as signaling molecules in general ,305 and 2●− was scavenged with mitoTEMPO and H2O2 was removed by overexpression of catalase in the mitochondrial matrix but in both cases neither cytosolic ROS levels nor cytokine secretion were altered. Infections with pathogens, which cause mitochondrial damage also cause matrix mtROS production [While the direct antimicrobial role of mtROS has been intensively investigated, their role in the proinflammatory response of infected macrophages remained an open question and the underlying molecular mechanisms through which mtROS mechanistically regulate proinflammatory signaling remained elusive. In a recent study, we have elucidated for the first time the role of mtROS in proinflammatory signaling of macrophages during bacterial infection . Infectioduction ,131 but oduction ,308, L.moduction  indicatioduction ,131,310.54 and Cys374, [Of note, our study provided a mechanistic target for mtROS-mediated proinflammatory signaling, namely the IκB kinase γ/NF-kappa-B essential modulator (IKKγ/NEMO) subunit of the IKK complex, which is essential for initiation of the proinflammatory pathways leading to cytokine secretion see  311,312312. A pr Cys374,  and, indphox subunit showed cytokine secretion similar to Nox2-deficient macrophages, nicely showing that Nox enzymes were dispensable for cytokine secretion after LPS stimulation. Not surprisingly, the NLRP3 inflammasome was, with exception for IL-1β, not involved in cytokine secretion as NLRP3-, Caspase-1- and Caspase 1l-deficient macrophages showed normal secretion of TNF and IL-6 , recruitment and autocatalytic activation of caspase-1 and finally release of IL-1β and IL-18. Initiation of pyroptosis, a regulated lytic cell death pathway, can be a consequence of inflammasome activation but is not obligatory. Five receptor proteins leading to inflammasome assembly were identified. The canonical inflammasomes are activated by the nucleotide-binding oligomerization domain (NOD) of the leucine-rich repeat (LRR)-containing protein (NLR) family members NLRP1, NLRP3, NLRC4, NLRP6, NLRP7, NLRP9b, the absent in melanoma 2 (AIM2)-like receptor (ALR) AIM2 and Pyrin ,325,326.2O2. High concentrations of H2O2 (10 mM) disrupted the interaction of the thioredoxin-interacting protein (TXNIP) with thioredoxin leading to its interaction with NLRP3 resulting in caspase-1 activation and finally IL-1β cleavage. Phagocytosis of uric acid crystals, as inflammasome-activating stimulus, also led to increase in total cellular ROS levels  from patients with CGD were analyzed. The studies covered patients lacking Nox2 subunits, e.g., gp91 samples  or isola samples ,336,337 es [phox  and by h species . While t species ,341 and  species  , a key enzyme for fatty acid oxidation. However, no mechanism for the role of Nox4-derived ROS was shown. Location of Nox4 to mitochondria was demonstrated by Western blot analysis of cellular fractions and Nox4-deficient BMDM showed reduced matrix mtROS production after stimulation with LPS and nigericin. However, if and how Nox4-dependent matrix mtROS production regulates inflammasome activation in a CPT1-dependend manner was not investigated. Therefore, in mice, it also remains elusive if Nox-derived ROS directly activate the inflammasome in infected macrophages.In contrast to humans, a role for Nox4 in activation of the NLRP3 inflammasome was suggested in mice during infection . Nox4-deWhile the role for Nox-derived ROS for NLRP3 inflammasome activation in infected mouse macrophages still needs further investigations, a subsequent study from Zhou et al. identified mitochondria as source of the ROS that are required for NLRP3 inflammasome activation in human and mouse macrophages . Zhou anFrancisella tularensis [The ROS-mediated regulation of the NLRP3 inflammasome in macrophages is extensively investigated, but nearly nothing is known about the source and the role of ROS for activation of other inflammasomes. One study showed that matrix mtROS production was also crucial for activation of the AIM2 inflammasome in BMDM infected with larensis . Global Like in nearly all fields of ROS-mediated immunity in infected macrophages, the general role of ROS for inflammasome activation is unquestioned. While identification of ROS sources took momentum and mitochondria as main ROS source for inflammasome activation became evident, only two studies so far identified a redox-dependent target necessary for NLRP3 inflammasome activation, namely TXNIP ,334. FutA plethora of ROS probes are available and with ongoing development the choices are frequently widened. Most precise ROS measurements concerning compatibility and specificity for ROS subspecies can be achieved with genetically modified cells, which express the ROS probe of choice in the cellular compartment of choice. Examples are the HyPer reporter family and reduction-oxidation sensitive green fluorescent protein 2- OSBP Related Protein 1 (roGFP2-Orp1) ,350,351.The listed ROS probes either emit chemiluminescence via a ROS-consuming reaction or emit fluorescence after reaction with ROS. All ROS probes listed are usable in a plate reader and therefore, in terms of used media, one must consider that every type of pH indicator  or serum  leads to falsification of both, luminescence and fluorescence signals and must be avoided.Diffusible ROS probes are not retained or targeted to a specific cellular compartment, however, some show specificity for ROS subspecies. Therefore, with the ROS probes listed below, only total cellular ROS levels in cells can be measured see .Luminol is a luminophore that reacts with all types of ROS. In the presence of an endogenous or exogenous peroxidase, such as horseradish peroxidase (HRP), and ROS, luminol is oxidized to 3-aminophtalate. This molecule is in an energetically excited state and emits light when returning to ground state. The generated chemiluminescence represents a quantitative value for the amount of ROS generated ,354. Sin2●− detection [2●−, but instead with cytochrome c and XO [Lucigenin was originally described as probe for Oetection . Howeverc and XO , increasc and XO . Since a2DCF-DA) is a cell-permeable derivative of fluorescein and one of the most commonly used ROS probes. After its diffusion into the cytosol, the two acetate groups are cleaved by cellular esterases generating 2′,7′-dichlordihydrofluorescein (H2DCF). This molecule can now be oxidized by ROS to generate the highly green fluorescent 2′,7′-dichlorofluorescein (DCF). H2DCF-DA is often referred to as intracellular ROS probe. However, even after cleavage of the acetate groups, it remains diffusible and therefore does not only remain in the cytosol but also reaches cell organelles and diffuses back into the extracellular space. Therefore, like with luminol, only total cellular ROS levels can be detected with this probe  is a red fluorescent probe that rather specifically reacts with cellular Othe cell . Therefotion see .CellROX oxidative stress reagents are different cell-permeable fluorescent probes that are mainly used as indicators for oxidative stress. The different CellROX probes are diffusible and cannot differ between cellular compartments and ROS subspecies, but vary greatly in usage conditions, like live cell compatibility, resistance to detergents, fixation capabilities, and emitted fluorescence spectra. However, since it can be coupled to bacteria, compartment-specific ROS measurement of phagocytosed bacteria in the phagosome can be performed .In contrast to the previous listed ROS probes, the following list contains either cell-impermeable ROS probes or probes targeted or retained to a specific cellular compartment. Some but not all probes additionally detect specific ROS subspecies. Therefore, these ROS probes offer a more detailed and specific approach to analyze ROS production in cells see .Isoluminol is a derivative of the chemiluminescent luminol with the amino group placed on a more exposed position on the molecule. This modification makes isoluminol cell-impermeable. This makes it ideal for exclusive measurement of extracellular ROS, which also includes ROS produced into the lumen of endosomes and phagosomes ,353,361.2O2 [2O2, Amplex Red is oxidized to red fluorescent 10-acetyl-3,7-dihydroxypenoxazine [2O2.Amplex Red also is cell-impermeable but, in contrast to isoluminol, detects specifically H2O2 . In the noxazine . In cons2DCF-DA that contains two additional carboxyl groups that enhance its hydrophilicity and therefore strongly increase its retention in the cytosol. Therefore, in contrast to H2DCF-DA, which detects total cellular ROS levels, the green fluorescent 5/6-Carboxy-DCF can be used to specifically detect cytosolic ROS levels (see 5-(and -6)-Carboxy-2′,7′-dihydrochlorofluorescein-diacetat (5/6-Carboxy-DCF) is a derivative of Hvels see  [30,99,32●− and emits red fluorescence after the reaction (see MitoSOX Red (MitoSOX) is a modified DHE analog attached to a triphenylphosphonium (TPP) group, which leads to accumulation of the probe in the mitochondrial matrix ,366. Liktion see  [30,99,3tion see  [30,270,2●−), but can also scavenge nonradicals (like H2O2) or more than one ROS subspecies in dependency of the chemical structure of the scavenger. Moreover, this should not be mistaken with the term “Antioxidant”, which chemically defines molecules or atoms that can in general reduce an oxidizing substance and are not limited to ROS [The term “ROS scavenger” broadly describes any chemical or biological molecule, which is capable of detoxifying one or more ROS subspecies by different mechanisms defined by the chemistry of the ROS scavenger and the ROS subspecies . Therefod to ROS ,368. By 2S and sulfane/sulfur production [N-acetyl cysteine (NAC) is a commonly used global ROS scavenger, which works extra- and intracellularly. It was long assumed that NAC scavenges oxidants directly through its thiol group and by its ability to act as a source of cysteine for increased GSH synthesis. However, some studies observed that NAC did not restore decreased GSH levels  and in voduction . As a gl2●− into H2O2. The term ROS scavenger therefore is not accurate, since one ROS subspecies is converted into another. However since O2●− is removed, its cellular role can be investigated with this substance [2●− levels everywhere in the cell [Tempol (4-Hydroxy-Tempo) is a SOD mimetic and catalyzes the dismutation of Oubstance ,372. Of the cell ,374.2●− scavenger [Tiron  is a vitamin E analog, cell-permeable and acts as a global Ocavenger ,376,377.Trolox , like Tiron, is a water-soluble derivative of vitamin E and globally scavenges ●OOH and ●OOR ,379.2O2 and ONOO− [Ebselen is a heterocyclic seleno-organic compound and exerts glutathione reductase activity. It effectively removes Hnd ONOO− ,381,382.All of the probes mentioned above are highly diffusible. Therefore, they can be used to determine whether ROS in general or specific ROS subspecies do play a role in a process of interest. They cannot be used to identify the specific compartment in which the ROS exert their function, though. For this, compartment-specific removal of ROS is required, which is possible with the substances described below.+ cotransporters in the plasma membrane. While a number of studies have demonstrated extra- and intracellular ROS scavenging activities of vitamin C in different cell lines [Vitamin C (ascorbic acid and its anion ascorbate) acts as water-soluble reducing agent , donatinll lines ,387, we ll lines . Cytosol2O2 and does not scavenge ROS in hydrophilic cellular compartments such as the lumen of cell organelles or the cytosol [Vitamin E is a lipophilic molecule that is exclusively integrated into biological membranes, where it efficiently scavenges radical ROS, mainly ●OOR and therefore acts a major defense against lipid peroxidation in cells ,389,390. cytosol .2●− to H2O2 in the mitochondrial matrix [MitoTEMPO and MitoQ both are targeted to and accumulated in mitochondria due to their TPP group. MitoTEMPO is a combination of the antioxidant TEMPO attached to the TPP group and hence dismutates Ol matrix ,391, whel matrix ,315.2●−.In sum, NAC is optimal for a first investigation to analyze, if ROS play a role in the experimental setting at all and we recommend for further experiments Vitamin C for extracellular ROS scavenging, Vitamin E for general and MitoQ for mitochondrial membrane protection against radical ROS and mitoTEMPO for removal of matrix-located OWhile ROS scavengers directly remove ROS from the system, but optimally do not influence the sources of ROS production, the use of ROS source inhibitors is an option to block ROS production and analyze possible ROS sources when genetic modifications are not an option. Like in the case of ROS probes and ROS scavengers, there are a lot of ROS source inhibitors commercially available and we will focus on the most commonly used substances observed in many studies so far. For a broader overview, we refer to excellent reviews solely examining this topic ,393,394.Apocynin was long considered as specific Nox2 inhibitor. However, several studies have shown that this is indeed not the case. Instead, apocynin directly scavenges ROS due to its antioxidant capacities ,397,398.DPI (diphenyliodinium), like apocynin, was long considered as specific Nox2 inhibitor. However, due to its chemistry, it unselectively inhibits all flavin-containing enzymes of the cell ,394,399.VAS2870 was the first published inhibitor that resulted from a systematic screening effort for selective NADPH oxidase inhibitors by Tegtmeier and colleagues . VAS2870−, a RNS [GKT136901 and GKT137831 were identified by Laleul and colleagues during a screen for Nox4 inhibitors . Both co−, a RNS  and ther−, a RNS  highligh−, a RNS ,413,414.Rotenone acts as a strong inhibitor of complex I of the ETC ,416,417.2 to generate ROS, which reach the cytosol [Antimycin A blocks the Qi site of complex III of the ETC ,422, the cytosol ,125,423.Myxothiazol and stigmatellin, like antimycin A, block electron flow to complex III. However, they do so at the Qo site of the complex, which results in the opposite effect of the one observed with antimycin A. Blockade of complex III with stigmatellin  and, witusage of only one type of ROS probe without explanation of the rationale behind the choice, such as compartment or ROS subspecies specificity or no specificity at all ;usage of unspecific inhibitors of ROS production or usage of only globally working ROS scavengers;no genetic evidence for the ROS source ;inconsistent use of different stimuli and macrophage cell types for experiments in one study.The important and versatile functions of ROS in macrophage-mediated immunity are unquestioned and have been demonstrated by many studies. However, only a few studies have performed in-depth ROS analyses, and even fewer have provided mechanistic insights into the redox-regulated targets. Experimental setups that commonly lead to misinterpretation of ROS measurements in macrophages are:In consequence, the highly complex and structured production of ROS in macrophages is often reduced to the phrase “ROS levels in macrophages”.Instead of assuming that ROS are omnipresent in and around the cell once produced, studies aiming to provide new mechanistic insights into direct or indirect antimicrobial functions of ROS in macrophages have to carefully consider the parameters delineated in this review, such as macrophage type, ROS-inducing stimulus, ROS sources, ROS subspecies and their specificity and selectivity for their targets, the compartmentalization of ROS production and the corresponding ROS probes, scavengers and inhibitors.


Purpose: The aberrant of fibroblast growth factors and their receptors (FGF/FGFR) is an emerging target in the treatment of solid tumors. This study aimed to explore the landscape of FGF/FGFR alterations in a large cohort of cancer patients.Material and Methods: The formalin-fixed paraffin-embedded specimens of cancer patients who have underwent next-generation sequencing (NGS) from 2017 to 2019 in 3DMed Clinical Laboratory Inc. were included in this study.Findings: Of 12,372 Chinese cancer patients with more than 20 tumor types , genomic alterations in FGF, FGFR, and both were observed in 895 (7.2%), 862 (7.0%), and 186 (1.5%) patients, respectively. The highest prevalence of FGF/FGFR mutations fell in esophagus cancer  and urinary tract cancer . The most common pathway-level mutations were FGFR single nucleotide variants  and FGF amplifications . The microsatellite instability status was negatively associated with amplifications (p=0.0017).Conclusion:FGF/FGFR alterations were widely occurred in cancer patients, and the mutational landscape may contribute to the further study design and development of FGF/FGFR inhibitors. Fibroblast growth factor (FGF) receptors (FGFRs) are highly conserved transmembrane tyrosine kinase receptors (RTKs), which are crucial in a variety of physiological process, such as the regulation of development, differentiation, survival, and migration of cells. Upon the binding of FGF, FGFR kinases will be dimerized and autophosphorylated, thus activating multiple transduction pathways, including the Ras/mitogen-activated protein kinase (MAPK) pathway, the canonical and non-canonical Wnt pathway, and the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)-AKT pathway, which all play importantly roles in the cancer biological process FGFR2 or FGFR3 genetic alterations based on an ORR of 32.2% from BLC2001 study, a phase 2 trial (NCT02365597) FGFR isoforms 1, 2 and 3 got approval for FGFR2+ cholangiocarcinoma later line treatment upon the data from the FIGHT-202 study with an ORR of 36% FGF/FGFR alterations in a large cohort of Chinese cancer patients through next-generation sequencing to aid the future study design and development of FGF/FGFR-inhibiting drugs.In tumor cells, aberrant FGFR signaling can be resulted from the amplification, fusion or missense mutations of FGF or FGFR family members, which are considered as promising targets for cancer treatment The Formalin-Fixed Paraffin-Embedded (FFPE) specimens of solid tumor patients who have underwent next-generation sequencing (NGS) from 2017 to 2019 in 3DMed Clinical Laboratory Inc. were included. The diagnosis of the specimens was confirmed by hematoxylin and eosin (H&E) staining by an independent pathologist. The specimens were required to have a percentage of tumor cells over 20% and a size ≥1mm for further analysis.FGF genes included FGF3, FGF4, FGF6, FGF10, FGF14, FGF19, and FGF23. FGFR genes included FGFR1, FGFR2, FGFR3, and FGFR4. Gene alterations of other cancer signaling pathways, including RTK/RAS pathway, PI3K pathway, and cell cycle pathway, were also identifiedDNAs extracted from the FFPE tumor specimens were sequenced in NGS platform with a well-designed 381 cancer gene panel on Illumina Nextseq 500 to > 500X coverage as previously describedP values were two-sided, and P < 0.05 was considered statistically significant unless otherwise specified.Data were analyzed with GraphPad Prism  and R . All reported Table 1). The median age was 58  and 7,440 (60%) were male patients. NGS was conducted with the FFPE tumor specimens of the patients through surgical resection , biopsy , or other approaches .A total of 12,372 Chinese cancer patients with more than 20 tumor types were included in the study, including 3,557 (29%) lung cancer, 1,433 (12%) liver cancer, 1,310 (11%) colorectal cancer (CRC), 960 (8%) biliary tract cancer, and 758 (6%) and gastric cancer (GC), etc. . Amongst all, there are 1,032 (8.3%) patients with SNVs, 930 (7.5%) with amplifications, and 116 (0.9%) with fusions in FGF/FGFR genes. The alteration frequencies between FGF and FGFR were mostly comparable in each tumor type. FGF/FGFR mutations were widely occurred across all involving tumor types, with a prevalence ranging from 61.6% (98/159) in esophagus cancer, 52.7% (145/275) in urinary tract cancer, 32.3% (83/257) in head and neck carcinoma to 7.4% (7/95) in prostate cancer, 7.1% (45/635) in pancreatic cancer, and 4.4% (24/548) in kidney cancer . When stratified by the mutational types, the most common pathway-level abnormalities fell in FGFR genes SNVs , FGF genes amplifications , and FGFR genes amplifications . The top frequent gene-level alterations were amplifications in FGF19 , FGF4 , FGF3 , and FGFR1 , followed by SNVs in FGFR2 , FGFR3 , FGFR4  and FGFR1 . The somatic mutation maps of FGFR1/2/3/4 were shown in Figure .Of all patients, FGF/FGFR alterations were observed in 1,943 (15.7%) patients, including 895 (7.2%) with alterations in FGF, 862 (7.0%) in FGFR, and 186 (1.5%) in both FGF and FGFR . On the other side, most of the other FGF/FGFR genes were mutually exclusive, for example, the alterations of FGFR1, FGFR2, and FGFR3 (p<0.001). The mutual exclusivity and co-occurrence mutational pattern was mostly consistent when constrained to gene amplifications, with the co-occurring FGF3, FGF4, and FGF19 amplifications, and the widely observed mutual exclusivity across the other genes (data not shown). As the top frequent alterations in the overall population, amplifications in FGF3/4/19  occurred the most in esophagus cancer , head and neck carcinoma , and urinary tract cancer , and were the least in pancreatic cancer , sarcoma , and ovarian cancer .The relationships between genetic alterations were investigated among FGF/FGFR genes. In brief, alterations in Figure ). Significantly higher mutational rates of PI3K pathway , WNT pathway , and cell cycle pathway  were observed in patients harboring FGF/FGFR alterations compared to the wild-type patients, while such trend was not found in RTK/RAS pathway .In addition, the occurrence of alterations in different pathways were investigated (The status of microsatellite instability (MSI) was available in 10,680 (86.3%) of the patients. In overall, 2.4%  patients showed a high MSI (MSI-H) status. Amongst all, MSI-H was found to be positively associated with the FGF/FGFR alterations (p<0.0001), with 124 (48.1%) of the MSI-H patients harbored at least one genetic alteration in FGF/FGFR. However, MSI-H was negatively associated with FGF/FGFR amplifications (p=0.0017), with only seven (2.7%) patients with MSI-H harbored FGF/FGFR gene amplifications.This is the first study that comprehensively depicting the landscape of FGF/FGFR aberrations in Chinese cancer patients. Our study showed that FGF/FGFR alterations were common across various histologies in Chinese cancer patients.FGF/FGFR in various cancer, non-selective FGF/FGFR inhibitors have been approved for cancer treatment, especially for tumors harboring aberrant FGFR2 or FGFR3 activations FGFR1, 2, and 3 in 22 cholangiocarcinoma patients with FGF/FGFR genetic alterations other than FGFR2 translocation from the interim results of flight-202 study FGF3, FGF4, and FGF19), which was found to be negatively correlated with MSI-H in this study, was reported to be potentially associated with hyperprogression upon immune checkpoint inhibitors as observed in three out of four patients with lung cancer or esophageal adenocarcinoma The recurring oncogenic FGF/FGFR mutations were detected in 15.7% cancer patients, including 7.2% with alterations in FGF genes, 7.0% in FGFR genes, and 1.5% in both FGF and FGFR genes, which is comparable to the previously reported 7% of FGFR alterations in the Caucasian cancer population There are several limitations in this study. Firstly, given the cross-sectional setting of this study, no treatment or survival data were available for the studied subset, and thus the association between FGF/FGFR alterations with clinical outcome cannot be assessed. Secondly, the cancer subtype was not further identified in the current study, which may lead to potential bias. Collectively, our study has provided valuable clues to the future clinical development, and further investigations are warranted for the clinical application of cancer treatment upon molecular profiling.


SLC12A3 and CLCNKB mutations, respectively. We described a 48-year-old male patient with fatigue, carpopedal spasm, arthralgia, hypokalemic alkalosis, mild renal dysfunction, hypomagnesemia, hypocalciuria, hyperuricemia, normotension, hyperreninemia and chondrocalcinosis in knees and Achilles tendons. His parents are first cousin. Genetic analysis revealed simultaneous homozygous mutations in SLC12A3 gene with c.248G>A, p.Arg83Gln and CLCNKB gene with c.1171T>C, p.Trp391Arg. The second younger brother of the proband harbored the same simultaneous mutations in SLC12A3 and CLCNKB and exhibited similar clinical features except normomagnesemia and bilateral kidney stones. The first younger brother of the proband harbored the same homozygous mutations in CLCNKB and exhibited clinical features of hypokalemia, normomagnesemia, hypercalciuria and hyperuricemia. Potassium chloride, spironolactone and potassium magnesium aspartate were prescribed to the proband to correct electrolytic disturbances. Benzbromarone and febuxostat were prescribed to correct hyperuricemia. The dose of potassium magnesium aspartate was subsequently increased to alleviate arthralgia resulting from calcium pyrophosphate deposition disease (CPPD). To the best of our knowledge, we are the first to report an exceptionally rare case in an inbred Chinese pedigree with simultaneous homozygous mutations in SLC12A3 and CLCNKB. GS and BS type III have significant intrafamilial phenotype heterogeneity. When arthralgia is developed in patients with GS and BS, gout and CPPD should both be considered.Gitelman syndrome (GS) and Bartter syndrome (BS) type III are both rare, recessively inherited salt-losing tubulopathies caused by  SLC12A3 gene, encoding the thiazide-sensitive sodium-chloride cotransporter (NCC) apically expressed in epithelial cells of the distal convoluted tubule (DCT). BS type III is caused by loss-of-function mutations in the CLCNKB gene encoding the chloride channel ClC-Kb [SLC12A3 and CLCNKB gene. To the best of our knowledge, this is the first report on simultaneous homozygous mutations in SLC12A3 and CLCNKB gene.Gitelman syndrome (GS) and Bartter syndrome (BS) type III are both rare, recessively inherited salt-losing tubulopathies (SLTs), characterized by hypokalemic metabolic alkalosis with low or normal blood pressure, despite secondary hyperreninemia and aldosteronism . GS is cl ClC-Kb . Howeverl ClC-Kb . Arthritl ClC-Kb . Unlike l ClC-Kb  and, theOn May 28th, 2017, a 48-year-old male patient (II-2) was admitted to our hospital due to fatigue for 45 years and intermittent carpopedal spasm for 30 years. From the age of three years old he suffered from paroxysmal fatigue, salting craving, polydipsia and polyuria. Laboratory exams revealed hypokalemia, and he had been taking oral potassium chloride irregularly. No palpitation, constipation or physical and mental retardation was reported. Episodic carpopedal spasm developed at the age of 18 years. Intense pain, redness, warmth and swelling in the proximal interphalangeal (PIP) joint of the right index finger developed at the age of 36 years. Bilateral knees and heels were also involved later. The arthralgia was initially diagnosed as gout and was partially resolved following acetaminophen and benzbromarone administration. However, the arthralgia flared frequently. On physical examination, blood pressure was 128/75 mmHg. There was no joint deformity or tophus in any joint. Serum biochemistry revealed hypokalemia, hypomagnesemia, hyperuricemia and mildly renal dysfunction. Arterial blood analysis showed decompensated metabolic alkalosis. Urinary electrolytes analysis demonstrated renal potassium, magnesium wasting and hypocalciuria. Renin activity was elevated . His parSLC12A1, KCNJ1, CLCNKA, CLCNKB, BSND, CaSR, HNF1B and SLC12A3. To identify the exonic deletions of HNF1B, capture copy-number variants (CapCNV) analysis was applied. WES, detecting all exon regions of over 20,000 genes, was applied in patient III:1. The enrichment libraries were sequenced on Illumina HiSeq X ten platform or paired-end read 150bp. After sequencing, bioinformatics analysis was used to select the potential pathogenic mutations. Variants were further annotated by ANNOVAR ) and associated with multiple databases, including 1000 genome, ESP6500, dbSNP, EXAC, Inhouse , HGMD, and predicted by SIFT ), PolyPhen-2 ), Mutation Taster ), GERP++ ). Filtered candidate variants were confirmed by Sanger sequencing. The coding exons that contain the detected mutations were amplified with Ex Taq DNA polymerase . Purified polymerase chain reaction (PCR) samples were sequenced on an ABI 3730 Genetic Analyzer . Sequence traces were analyzed using Mutation Surveyor . The mutations of family members were confirmed by the same procedures. II-2 and II-4 harbored homozygous mutations  in SLC12A3, I-1, I-2 and III-1 harbored heterozygous mutations  in SLC12A3  in CLCNKB  was predicted to be probably-damaging by PolyPhen-2, disease-causing by Mutation Taster, and damaging by SIFT. This variant has been reported by Vargas-Poussou, et al [CLCNKB(p.Trp391Arg) was predicted to be probably-damaging by PolyPhen-2, disease-causing by Mutation Taster and damaging by SIFT . The amplified DNA was captured with a Panel/Whole exome sequencing (WES) probe  following DNA Library Preparation. The panel applied in the proband (II-2) consisted of 62 known SLT related genes, including  SLC12A3 . II-2, In CLCNKB . II-2 an by SIFT . This va by SIFT . Pedigred CLCNKB  mutationPotassium chloride (KCl) (2.0 g/day), spironolactone (40 mg/day) and potassium magnesium aspartate (8 tablets/day) were prescribed to II-2 to correct electrolytic disturbances. Benzbromarone (50 mg/day) was prescribed to correct hyperuricemia. On Sep 26th, 2019, serum chemistry analysis showed uric acid (UA) at 761 μmol/L, Creatinine (Cr) 145 μmol/L, K 2.9 mmol/L, and Mg 0.41 mmol/L. The arthralgia persisted. Febuxostat (50 mg/day) was, therefore, added to further lower UA. The arthritis in the PIP joint was completely resolved, and the arthralgia in bilateral knees and heels was slightly improved. X-rays of bilateral hands, shoulders, hips, knees and ankles revealed chondrocalcinosis (CC) in knees and Achilles tendons . The artSLC12A3 and CLCNKB. II-2 and II-4 with simultaneous homozygous mutations in SLC12A3 and CLCNKB have phenotypes of mixed cBS and GS, and II-3 with one homozygous mutation in CLCNKB has phenotypes of cBS.We described an exceptionally rare case in an inbred Chinese pedigree with simultaneous homozygous mutations in −) ions to the basolateral side [− exit, NaCl reabsorption through the Na-K-2Cl cotransport (NKCC2) and subsequently calcium reabsorption [− via basolateral Cl− channels, results in the cell to hyperpolarize. This, in turn, stimulates entry of calcium into the cell via luminal voltage-activated Ca2+ channels. In addition, the lowering of intracellular Na+ concentration facilitates Ca2+ exit via a basolateral Na+/Ca2+ exchanger [− exit in the DCT decreases NaCl reabsorption via the NCC, accounting for the GLS phenotype, including hypocalciuria in BS type III patients with impaired ClC-Kb function in the DCT [CLCNKB mutation was in the TAL instead of DCT. II-2 and II-4 have an additional SLC12A3 mutation besides the CLCNKB mutation. DCT is distal to TAL, and thus the increased urinary calcium resulted from CLCNKB mutation in the TAL is then reabsorbed in the DCT due to the SLC12A3 mutation. Finally, hypocalciuria is generated in II-2 and II-4. Accordingly, the GLS phenotypes of II-2 and II-4 are generated by the SLC12A3 mutation rather than the CLCNKB mutation.The ClC-Kb channel is expressed in the thick ascending limb (TAL), distal convoluted tubule (DCT) and collecting duct, where it transfers chloride  whose first symptoms appeared in infancy and childhood, respectively. Their serum magnesium and urine calcium excretion rate fluctuated, ranging from normal to low, indicating they had inconsistent hypomagnesemia and hypocalcinuria. Mutation analysis identified the simultaneous presence of heterozygous and compound heterozygous mutations in the SLC12A3 and CLCNKB genes, respectively. They were diagnosed as cBS [SLC12A3 and CLCNKB were frameshift and missense, respectively. The brother developed proteinuria and had growth retardation due to growth hormone deficiency. The sister also had growth retardation. They both had normal renal function. Another female patient was reported to have growth retardation with persistent hypokalemia, hypomagnesemia, hypocalciuria, hypochloremic alkalosis and elevated levels of plasma renin and aldosterone. Her younger brother, father and paternal grandmother all had histories of mild-to-low levels of plasma potassium, which were rectified by potassium-rich foods. Gene sequencing revealed this patient carried a paternally inherited heterozygous mutation in SLC12A3 and maternally inherited heterozygous variants in both CLCNKB and CLCNKA. Based on clinical phenotypes, genetic evidence of the pedigree and previous reported studies, this case of GS indicates a digenetic inheritance of SLC12A3 and CLCNKB that resulted in renal tubular dysfunction, perhaps due to a genetic double-hit mechanism. The putative pathogenicity of the CLCNKB p.L94I variant requires confirmation [SLC12A3 and a missense heterozygous mutation in CLCNKB. His phenotype was consistent with severe GS [SLC12A3 and CLCNKB mutations could cause SLT. I-1 also carried both heterozygous mutation in SLC12A3 and CLCNKB, which may explain his hypokalemia. However, I-2 and III-1, both of whom carried the same mutations as I-1, had no electronic disorders.To the best of our knowledge, no cases with simultaneous homozygous mutations in the irmation . A 19-yeevere GS . In concCLCNKB gene and the concomitant presence in the kindred of GS and cBS phenotypes. These findings demonstrated intrafamilial heterogeneity [SLC12A3 and CLCNKB, II-2 had hypomagnesemia, gout and CPPD, whereas II-4 had normal magnesemia and kidney stones. Both of their phenotypes are consistent with mixed cBS and GS. It is hypothesized that variation in expression and/or function of any one of the channels or transporters participating in Cl− transport in the TAL and the DCT may modify variable degrees of impairment in ClC-Kb function, thereby influencing the disease phenotype. Such a modifying effect could occur at the cellular/regulatory level, whereby one or more transport mechanisms are recruited to compensate for impaired ClC-Kb function or, alternatively, may be determined at the level of modifier gene [Both GS and Type III BS have significant intrafamilial phenotype heterogeneity ,4 Zelikoogeneity . For theier gene .CLCNKB, missense mutations were more frequent in patients with less severe phenotypes. On the contrary, severe mutations  were more frequent in patients with earlier onset and severe phenotypes [CLCNKB and has a mild phenotype of cBS. Therefore, his phenotype is consistent with his type of mutation in CLCNKB. II-2 and II-4 carry dual homozygous mutations in SLC12A3 and CLCNKB. II-4 has renal dysfunction and very severe hypokalemia (serum K+ 1.8 mmol/L), which both indicate his phenotype is severe. II-2 has renal dysfunction, suffering from gout and CPPD. He has severe hypokalemia and moderate hypomagnesemia despite potassium and magnesium supplementation. Accordingly, his phenotype is also severe. The phenotypes of II-2 and II-4 are more severe than that of II-3 due to additional mutations in SLC12A3. GS is a milder disease compared with BS [SLC12A3 that were associated with a severe presentation were, at least, the combination for one allele of a missense mutation that resulted in a nonfunctional intracellular retained protein or, even more frequent, a missplicing that resulted in a short transcript [SLC12A3 that was identified here is located within the amino-terminal domain of NCC, which is not involved in defining affinity for ions and thiazides [SLC12A3 that was identified here results in mild dysfunction of NCC. Taken together, the simultaneous mutations in CLCNKB and SLC12A3 result in severe rather than very severe phenotypes, as patients take low dose of thiazide and loop diuretics rather than large dose of them.BS type III has a high phenotypic variability with clinical presentations ranging from very severe salt-losing nephropathy with marked hypokalemia to almost asymptomatic presentation . The typenotypes . II-3 caanscript . The mishiazides . The cenhiazides ,17. TherIn some patients with GS rather than BS, CPPD might develop due to long-term and profound hypomagnesemia . CPPD maSLC12A3 and CLCNKB gene are exceptionally rare. Consanguineous marriage should be strictly discouraged. GS and BS type III have significant intrafamilial phenotype heterogeneity. Both gout and CPPD should be considered and differentiated if arthralgia is observed in patients with GS and BS.Simultaneous homozygous mutations in


Genomic analysis could contribute to a better understanding of the biological determinants of the evolution of multiple myeloma (MM) precursor disease and an improved definition of high-risk patients. To assess the feasibility and value of next-generation sequencing approaches in an asymptomatic setting, we performed a targeted gene mutation analysis and a genome-wide assessment of copy number alterations (CNAs) by ultra-low-pass whole genome sequencing (ULP-WGS) in six patients with monoclonal gammopathy of undetermined significance and 25 patients with smoldering MM (SMM). Our comprehensive genomic characterization highlighted heterogeneous but substantial values of the tumor fraction, especially in SMM; a rather high degree of genomic complexity, in terms of both mutations and CNAs, and inter-patient variability; a higher incidence of gene mutations and CNAs in SMM, confirming ongoing evolution; intraclonal heterogeneity; and instances of convergent evolution. ULP-WGS of these patients proved effective in revealing the marked genome-wide level of their CNAs, most of which are not routinely investigated. Finally, the analysis of our small SMM cohort suggested that chr(8p) deletions, the DNA tumor fraction, and the number of alterations may have clinical relevance in the progression to overt MM. Although validation in larger series is mandatory, these findings highlight the promising impact of genomic approaches in the clinical management of SMM. On the contrary, genomic analysis is proving increasingly important for better understanding the biological determinants of the evolution of MM precursor clinical entities [The recent progress in next-generation sequencing (NGS) techniques applied to multiple myeloma (MM) has prompted comprehensive characterizations of genomic alterations in malignant plasma cells (PCs) through clinical-grade approaches ,2. Diffeentities ,4,5 and entities ,7. Whileentities ,10,11,12+ cells could be undermined by the presence of normal PCs in the sample. To address this issue, we estimated the tumor fraction by ichorCNA analysis of ULP-WGS data for each patient, and correlated it to the number of variants identified. No correlation was identified  and ultra-deep targeted NGS of a 56-gene panel were performed at an average coverage across all samples of 0.1× and 3062×, respectively. Our targeted panel identified a median of one mutation per patient in MGUS (range: 0–4) and two in SMM (range: 0–9), without a statistically significant difference between the distributions of the two groups a. Approxentified b, and map = 0.012) involving different genes; the most extensively mutated patient was ID#143, for whom we identified nine variants in seven genes. In terms of the mutation type, variants were mostly missense variants. The KRAS gene was mutated in 19.4% of patients (6/31); NRAS and SP140 in 12.9% (4/31 each); and DUSP2, FGFR3, and IGLL5 in 9.7% (3/31 each). Out of the 66 mutations, 31 were clonal and 35 subclonal . Notably, mutated samples more often carried co-occurring clonal and subclonal variants, even within the same gene. Of note, KRAS was targeted by multiple mutations in three patients. In particular, we found clonal Q61H and subclonal K117N and Q22K in ID#99; G12C, G12D, and Y64D, all at subclonal levels, in ID#143; and clonal G13D and Q61H and subclonal G12V in ID#153. Interestingly, each of these three patients carried one or more additional mutated genes belonging to the mitogen-activated protein kinase (MAPK) pathway, i.e., NRAS  and BRAF  (Our targeted NGS approach identified mutations in 68% of patients (21/31) and in half of the genes included in the design . Positiv ID#143) . Altogetp = 0.04). Interestingly, we found instances of bi-allelic events in ZNF292 in ID#103, DIS3 in ID#68, TP53 in ID#144, and CYLD in ID#060, which is a finding often associated with advanced stages [2 copy ratios for genomic windows of 1Mb [TP53 gene  (p = 0.02) (p = 0.011) (p = 0.027)  had a significantly shorter time to progression (TTP)  a. Concer = 0.02) b. The pe= 0.011) c. Lastly= 0.027) d.Monoclonal gammopathy of undetermined significance (MGUS) and SMM represent intermediate steps in the development of overt MM. MGUS progresses at a constant and low rate of 1%/year . SMM is KRAS and NRAS being the two top-ranking genes in terms of mutational incidence. An SMM sample also carried a BRAF V600E mutation. Furthermore, our in-depth analysis highlighted examples of convergent evolution that included multiple mutations within the same gene or pathway found in the same patient: among these, there were three cases harboring MAPK mutations, and one patient with two TP53 mutations, similar to what has been reported in the symptomatic setting [TP53 and CYLD, which were frequently hit by bi-allelic events, something previously described in advanced disease. Furthermore, interstitial deletions in chr(1p) not detected by FISH could be identified by ULP-WGS, further confirming how genomic approaches can outperform FISH [Our series of asymptomatic cases was characterized by heterogeneous but substantial values of the tumor fraction, especially in SMM. This was independent of the number of mutations identified in each sample, which ensures the reliability of our data and supports the use of NGS approaches for an in-depth genomic characterization of these patients. Overall, our analyses of asymptomatic forms revealed a rather high degree of genomic complexity, in terms of both mutational and chromosomal aberrations, and inter-patient variability. The median number of variants was one in MGUS and two in SMM patients. SMM patients presented a higher incidence of CNAs and specific aneuploidies in hyperdiploid samples, confirming the ongoing evolution in these asymptomatic stages, as reported in preliminary reports based on FISH . More re setting ,25,26,27orm FISH . Althougorm FISH . Interes® Cell Separation Media, Cedarlane, Canada). CD138+ PCs were isolated from BM MNCs by an immunomagnetic method with anti-CD138 monoclonal antibodies (mAbs) conjugated with microbeads . For the purification of genomic DNA, we used the AllPrep DNA/RNA/miRNA isolation kit , following the manufacturer’s protocol.The study was based on a series of six patients with MGUS and 25 with SMM admitted to our institution. MGUS and SMM were diagnosed according to the International Myeloma Working Group (IMWG) revised criteria  and SMM +-purified BM PCs. The targeted resequencing gene panel included coding exons and splice sites of 56 genes that emerged as drivers from a recent analysis of whole genome/exome data in more than 800 MM patients [ACTG1, BCL7A, BHLHE41, BRAF, BTG1, CCND1, CDKN1B, CYLD, DIS3, DTX1, DUSP2, EGR1, FAM46C, FGFR3, FUBP1, HIST1H1B, HIST1H1D, HIST1H1E, HIST1H2BK, IGLL5, IRF1, IRF4, KLHL6, KMT2B, KRAS, LCE1D, LTB, MAX, NFKB2, NFKBIA, NRAS, PABPC1, PIM1, POT1, PRDM1, PRKD2, PTPN11, RASA2, RB1, RFTN1, RPL10, RPL5, RPRD1B, RPS3A, SAMHD1, SETD2, SP140, TBC1D29, TCL1A, TGDS, TP53, TRAF2, TRAF3, XBP1, ZNF462, ZNF292). Ultra-deep NGS was performed on MiSeq  using the CAPP-seq library preparation strategy , as previously described [mpileup2cns was used to identify non-synonymous somatic mutations, and a stringent bioinformatic pipeline was developed and applied to filter out sequencing errors (detection limit 3 × 10−3). Details of the experimental procedures are given in Supplementary Methods. For ULP-WGS, libraries were prepared using the Kapa HyperPlus kit  with SeqCap Library Adapters starting with 400 ng of gDNA. Up to 24 libraries were pooled and sequenced using 200 bp paired-end runs on a MiSeq (Illumina). In order to estimate the quality and presence of a tumor, we performed ULP-WGS with an average genome-wide fold coverage of 0.1×. We analyzed the depth of coverage to estimate large-scale CNAs and the fraction of tumor DNA using ichorCNA [We collected tumor gDNA from CD138patients   was calculated from diagnosis to the first occurrence of any myeloma-defining event (event) or to last follow-up (censoring). TTP analyses were performed using the Kaplan–Meier method. The statistical significance of associations between individual variables and TTP was calculated using the log-rank test.Overall, our findings demonstrate that, starting from a limited amount (400 ng) of DNA from purified BM PCs of asymptomatic patients, a highly informative genomic analysis is feasible. To entirely replace FISH, in the future, more sophisticated targeted designs could be conceived, including chromosomal regions of recurrent CNAs and the IGH locus, to enable the detection of IGH translocations. Preliminary studies have proven promising in this setting ,10,11,12